International Review of Cytology: A Survey of Cell Biology, Volume 154

VOLUME 154

SERIES EDITORS Geoffrey H. Bourne James F. Danielli Kwang W. Jeon Martin Friedlander Jonathan Jarvik

1949-1988 1949-1984 19671984-1 992 1993-

ADVISORY EDITORS Aimee Bakken Eve Ida Barak Howard A. Bern Robert A. Bloodgood Dean Bok Stanley Cohen Rene Couteaux Marie A. DiBerardino Donald K. Dougall Charles J. Flickinger Nicholas Gillham Elizabeth D. Hay Mark Hogarth Anthony P. Mahowald M. Melkonian Keith E. Mostov

Audrey Muggleton-Harris Andreas Oksche Muriel J. Ord Vladimir R. Pantic M. V. Parthasarathy Thomas D. Pollard Lionel I. Rebhun L. Evans Roth Jozef St. Schell Manfred Schliwa Hiroh Shibaoka Wilfred Stein Ralph M. Steinman M. Tazawa Yoshio Watanabe Alexander L. Yudin

Edited by

Kwang W. Jeon Department of Zoology The University of Tennessee Knoxville, Tennessee

Jonathan Jarvik Department of Biological Sciences Carnegie Mellon University Pittsburgh, Pennsylvania

VOLUME 154

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Copyright 0 1994 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

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PRINTED IN THE UNITED STATES OF W C A 94 95 9 6 9 7 98 9 9 E B 9 8 7 6

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Contributors .......................................................................................

ix

Intermediate Filament Proteins: Cytoskeletal Elements with Gene-Regulatory Functions? Peter Traub and Robert L. Shoeman I. II. 111. IV.

V. VI.

Introduction ................ .......................... Cytoskeletal Functions of Int .......................... Potential Nuclear Functions of Intermediate Filament Proteins ........................... Intermediate Filament Proteins as Potential Gene-Regulatory Elements in ............................................. Differentiation Systems

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............................................. ............................................. ........................................................ .

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Secretion and Endocytosis in the Male Reproductive Tract: A Role in Sperm Maturation Louis Hermo, Richard Oko, and Carlos R. Morales I. II. Ill. IV. V. VI. VII.

Introduction ............ ..................................... Sertoli Cell Structure and Function ..... Germ Cells ....................................................... ........ Intermediate (Terminal) Region of the Seminiferous Tubule .... Structure and Function of the Rete Testis and Efferent Ducts . . Eljididymis: Cell Types and Functions ........ .......................... Vas Deferens: Secretion and Endocytosis by Epithelial Principal Cells ...................

V

126

146 177

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CONTENTS

VIII. Modification of the Sperm Membrane during Epididymal Transit ........................ IX. Concluding Remarks ..................................................................... References ...............................................................................

178 184 184

Evolution of Mammalian Sex Chromosomes and Sex-Determining Genes Jennifer A. Marshall Graves and Jamie W. Foster ........... Introduction ....................................................... ........... Organization and Evolution of Sex Chromosomes ............... ........... Dosage Compensation and X Chromosome Inactivation ........... Gonadal Differentiation and Sexual Dimorphisms . . ......................... ......................... V. The Search for the Testis-Determining Factor ...... VI. Identification and Characterization of the Mammalian Testis-Determining Factor SRY . . VII. Conclusions ............................. ......................... References . .............................. ......................... I. II. 111. IV.

191 191 206 216 222 233 244 248

Organization of Replication Units and DNA Replication in Mammalian Cells as Studied by DNA Fiber Radioautography Natalia A. Liapunova I. II. 111. IV. V. VI. VII. VIII.

Introduction ............................ ...................................... Organizaton of Mammalian Chromosomes for Replication ......................... DNA Fiber Radioautography as a Method for Replicon Analysis ......................... Sizes of Replication Units ............. ...................................... Rate of Replication Fork Movement ... ...................................... Termination of Replicons .............. ......................... Replicon Model for DNA Replication in Mammalian Chromosomes ...................... Conclusion ............................ ...................................... References ............... ......................................

261 263 265 277 286 292 294 301 302

Instability of the Homogeneous State as the Source of Localization, Epigenesis, Differentiation, and Morphogenesis Yoram Schiffmann I. Introduction ................................................................................ II. Antithesis between Preformation and Epigenesis .........................................

309 313

CONTENTS

111. IV. V. VI. VII. VIII. IX. X. XI.

Improbability of the Turing Couple and of Biological Coherence ......................... Dorsoventral and Terminal Systems in Drosophila ....................................... Spontaneous Endogenous Electrophoresis . . . . . . . . . . . . . . . . . ..................... Localized Activity instead of Localized Distribution of Pump Sufficiency of Child's Results ............................... Reduction Fields ........................................................... The Metabolic Field and Cytoskeleton Localization ....................................... Metabolism of Proliferation versus Metabolism of Differentiation and Morphogenesis . . . Concluding Remarks: The Reducibility of Development to Molecular Genetics .......... References .......................................................................

Index ..............................................................................................

vii 322 335 340 343 346 347 356 361 363 368 377

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CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors' contributions begin.

Jamie W. Foster (191), Department of Geneticsand Human Variation, LaTrobe University, Melbourne, Victoria 3083, Australia Louis Hermo (105), Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada H3A 292 Natalia A. Liapunova (261), Institute of Human Genetics, Medical Genetics Research Center, Russian Academy of Medical Science, Moscow 1 15478, Russia Jennifer A. Marshall Graves (191), Department of Genetics and Human Variation, La Trobe University, Melbourne, Victoria 3083, Australia Carlos R. Morales (1 05), Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada H3A 2B2 Richard Oko (105), Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada H3A 282 Yoram Schiffmann (309),Department ofApplied Mathematics and Theoreticalfhysics, University of Cambridge, Cambridge CB3 9EW, United Kingdom Robert L. Shoeman (1) , Max-Planck-lnstifutfur Zellbiologie, 0-68526 LadenburglHeidelberg, Germany

Peter Traub (1), Max-Planck-lnstitut fur Zellbiologie, 0-68526 LadenburgNeidelberg, Germany

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Intermediate Filament Proteins: Cytoskeletal Elements with Gene-Regulatory Function?’ Peter Traub and Robert L. Shoeman Max-Planck-Institut fur Zellbiologie, D-68526 LadenburglHeidelberg, Germany

1. Introduction

Research in the fields of cell and molecular biology has furnished an impressive knowledge of the regulation of gene expression in cell differentiation and pattern formation in eukaryotes. Although the basic reactions of these complex processes occur in the nucleus in close association with the nuclear matrix, there are indications that the cytoskeleton and the extracellular matrix are somehow involved. However, the precise role of cytoarchitecture in developmental processes and in the maintenance of terminal differentiation is far from being understood in detail. There appears to be a continuum of proteinaceous, filamentous structures, beginning with the extracellular matrix, extending through the cytomatrix, and terminating in the nuclear matrix, that is presumed to constitute a framework for the coordination of the interactions and functions of a multitude of cellular substructures and organelles, and for the transduction of intraand extracellular signals. Whether elements of this framework operate passively as signal conductors or whether they actively engage in regulatory processes by establishing direct contacts with nuclear target sites is largely unknown. One constituent of the cytomatrix, the intermediate filaments (IFs), poses a particularly serious problem in this respect. IFs probably represent one of the last functionally important macromolecular protein assemblies of the eukaryotic cell whose biological role has not yet been unveiled. In this chapter, we have developed a unifying hypothesis on the cellular function of IFs, which is based on some characteristic reactivities of

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Dedicated to Dr. Harold F. Deutsch on the occasion of his 75th birthday.

lnrcrnurionol Rruirn, of CVI<J/ORV. Vo/. 154

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Copyright 0 1994 by Academic Press. Inc. All rights of reproduction in any form reserved.

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PETER TRAUB AND ROBERT L. SHOEMAN

their constituent protein subunits with nuclear components in uitro. The structure and function of the IF proteins and their oligo- and polymeric forms will be compared with a variety of gene-regulatory protein factors and nuclear matrix elements and shown to have many features in common with these compounds. Each coincidence in itself may seem to be fortuitous, but seen in perspective the manifold parallels give a picture of IFs and their protein subunits that is completely different from that generally accepted and which we believe may indeed come close to reality. Our model describes IF proteins as a class of regulatory DNA binding factors which, in accordance with their developmentally and tissue-specifically regulated expression, may play an important role in cell differentiation as one of their major biological functions.

II. Cytoskeletal Functions of Intermediate Filaments

Since their first morphological description in developing skeletal muscle by Holtzer and co-workers (Ishikawa et al., 1968), IFs have been thought to participate, together with microfilaments and microtubules, in the construction of the cytoskeleton. Because of their characteristic three-dimensional distribution in the cytoplasm, their rigidity of structure, their relatively low dynamics, and their association with a large variety of subcellular structures, they are envisaged as mechanical integrators of cellular space (Lazarides, 1980; Traub, 1985; Steinert and Roop, 1988; Klymkowsky et af., 1989; Carmo-Fonseca and David-Ferreira, 1990; Skalli and Goldman, 1991 ; Albers and Fuchs, 1992). A typical example is the mechanical integration of all contractile actions of a muscle fiber via lateral interconnection of individual myofibrils at their Z discs and linkage of the latter to the transverse-tubular membrane and to other membrane-bounded organelles by desmin-type IFs (Lazarides er al., 1982). In addition, a collection of other diverse, complex, cellular activities, such as formation of cell shape, cell-cell interaction, adherence of cells to the substratum, cell locomotion, intracellular particle and vesicle transport, cell division, and protein synthesis are also presumed to rest, to varying extents, on the manifestation of organized IF systems. For instance, perturbation of the three-dimensional organization of keratin filament networks in the course of various genetic skin diseases appears to have a devastating effect on the mechanical integrity of epidermal keratinocytes (Fuchs and Coulombe, 1992). An alternative view of IF function relates the existence of a physical continuum of IF-like structures spanning the space between the plasma

INTERMEDIATE FILAMENT PROTEINS

3

membrane and the center of the nucleus to the transmittance of mechanical as well as molecular information from the cell surface to the nucleus and vice versa (Traub, 1985; Traub et al., 1987; Skalli and Goldman, 1991). In this context, the IFs presumably take advantage of their dynamic nature, a feature which was greatly underestimated in the past (Skalli et al., 1992). 1Fs may be composed of one or more of more than 40 individual subunit proteins, which were grouped into 6 distinct classes on the basis of their gene organization, their cellular localization, and their tissue or developmental occurrence. The one common characteristic of all I F proteins is a long central rod domain with a high propensity to form an a-helix and which is the major determinant in higher order associations leading to I F formation. The non-a-helical end domains (which are extremely variable in size and composition among the various subunit proteins) play a modulating role in I F assembly and are thought to be arrayed primarily on the surface of the filament. The need for distinct classes of IFs during cell differentiation is explained by the assumption that the intracellular organization of differentiation-specific subcellular structures requires filaments with well-defined surface structures that are composed of the nonconserved, non-a-helical head and tail domains of the respective I F subunit proteins (Steinert et al., 1985). However, this merely cytoskeletal concept of IF function became suspect upon the discovery of a number of cell lines which are totally devoid of cytoplasmically extended I F networks (Venetianer et af., 1983; Giese and Traub, 1986; Hedberg and Chen, 1986; Roser et al., 1991) and with the observation that the collapse of preexisting IF systems following microinjection of I F protein-specific antibodies into in v i m cultured cells has no apparent effect on the morphology and physiology of the microinjected cells (Gawlitta et al., 1981; Lin and Feramisco, 1981; Klymkowsky et al., 1983). Also, disassembly of preexisting desminhimentin IFs by the expression of a dominant-negative, carboxy-terminally truncated desmin following transfection of myogenic cells with cDNA (Schultheiss et al., 1991), or their segregation into meandering cables that greatly distort the spatial relationships of IFs with all cell organelles by treatment of cells with colcemid (Bischoff and Holtzer, 1968), had no effect on the assembly of normally aligned, striated, spontaneously contracting myofibrils. Moreover, nerve growth factor-elicited outgrowth of neurites from cultured rat pheochromocytoma cells, a differentiation-specific phenomenon thought to depend on the action of peripherin filaments, was totally unaffected by drastic depletion of the I F protein in response to cell treatment with the appropriate antisense oligonucleotides (Troy et al., 1992). This apparent dispensability of a cytoplasmically extended network of IFs for activities of single cells was interpreted to mean that IFs may

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PETER TRAUB AND ROBERT L. SHOEMAN

function in cell-cell interaction and communication at the level of tissues, organs, or whole animals (Klymkowsky et al., 1989). Yet, even at this level IF networks appear to be of limited essentiality, at least during mammalian early development. Mammalian eggs and embryos contain unique structural elements referred to as sheets, which are arrays of IFs aligned side by side and held tightly together by sheet-associated proteins. In this form, they are not recognized by antibodies (Bement et al., 1992; Capco et al., 1993). Indeed, microinjection of IF protein-specific antibodies into early embryos did not hinder normal development through the blastocyst stage (Emerson, 1988). Another interesting example in this context is the observation that, despite the disruption of both alleles of the mouse keratin K8 and therefore the complete absence of keratin IFs, embryonic stem stells differentiate to both simple and cystic embryoid bodies with ultrastructurally and functionally normal epithelia (Baribault and Oshima, 1991), whereas in mice a targeted null mutation in the keratin K8 gene causes midgestational lethality. Yet, the fact that a few mutant animals survive into adulthood clearly shows that once the respective embryos have somehow managed to pass through the critical phase dominated by keratin 8, development and growth proceed in the total absence of this IF protein (Baribault et al., 1993). However, the most convincing demonstration of the dispensability of a particular IF network for cellular function was provided with the creation of a neurofilament-deficient mutant of Japanese quail that, except for mild generalized quivering, showed no severe symptoms in either its general behavior or physical fitness throughout its complete life cycle (Ohara et al., 1993).

111. Potential Nuclear Functions of Intermediate Filament Proteins

The lack of unequivocal evidence in support of the cytoskeletal function of IF proteins and the fact that differentiation processes that were previously claimed to be dependent on the existence of well-organized, cytoplasmic I F networks can indeed go on in the absence of these subcellular structures necessitates the development of alternative views of the biological role of IF proteins. One promising strategy, employed extensively in our laboratory, is to search for cellular constituents for which IFs and their protein subunits show high affinities in uitro and to correlate these binding activities or interactions with the physiology and the differentiation state of intact cells.

INTERMEDIATE FILAMENT PROTEINS

5

A. Interaction of Intermediate Filament Proteins with Nucleic Acids and Histones in Vitro

In pursuing this strategy, it was found that all nonepithelial IF proteins tested (including vimentin, desmin, glial fibrillary acidic protein, neurofilament triplet proteins) are nucleic acid binding proteins with a preference for single-stranded (ss) polynucleotides of high guanine content (P. Traub et al., 1983, 1985; Traub and Nelson, 1983; Vorgias and Traub, 1986; Shoeman et al., 1988; Shoeman and Traub, 1990b). In general, IF proteins exhibit a higher binding affinity for DNAs than for RNAs. The observation that the relative vimentin-binding potentials of close to 75 naturally occurring and synthetic DNAs and RNAs of varying base composition and secondary structure differ by more than 2.5 orders of magnitude indicates that the association is not based on a merely electrostatic and therefore possibly unspecific interaction. The employment of proteolytic digestion products of vimentin identified the arginine-rich, non-a-helical head domain of the protein as the active binding principle (Shoeman er al., 1988; Traub et al., 1992a). Although not all IF proteins were subjected to this kind of analysis, the fact that they all contain a structurally different but arginine-rich head piece (Geisler et al., 1983) suggests that the same mechanism may govern their reaction with polynucleotides. Chemical modification of amino acid side chains of the isolated N-terminus of vimentin, and spectroscopic analysis of its complexes with nucleic acids revealed that a series of aromatic amino acids make important contributions to the binding reaction, presumably via an intercalation mechanism (Traub et al., 1992a). In this respect, the N-terminus of vimentin is astonishingly similar to some prokaryotic ssDNA-binding proteins, especially to the gene 5 protein (G5P) of bacteriophage fd and related viruses. A conserved, partial amino acid sequence of the N-terminus can even be folded into the same antiparallel P-sheet structure, which in the form of a nucleic acid binding wing mediates the interaction of G5P with ssDNA (de Jong et al., 1989) (Fig. 1). Biochemical and electron microscopic analysis of reaction products formed from several topological variants of pBR322 DNA and vimentin revealed a very efficient, cooperative interaction of the filament protein with the supercoiled form of the DNA, but no reaction with its relaxed or linearized forms. The vimentin-superhelical plasmid DNA complexes resembled circular (i.e., relaxed) DNA molecules uniformly coated with protein. Thus, vimentin can recognize, bind to, and relax superhelical plasmid DNA, presumably by local unwinding of the DNA double helix. All other nonepithelial IF proteins tested showed the same reactivity. Occasionally, vimentin formed globular or longitudinally extended,

6

PETER TRAUB AND ROBERT L. SHOEMAN INA-binding wing

T

D- Ladder

turn region

15

3 KeG5P

K E

OR 30

25

Vim N T

S N S G LO

15

fdG5P

T T O N 30

FIG. 1 Folding of a partial amino acid sequence of the N-terminus of vimentin into the ploop structure of the DNA-binding wing of GSP of bacteriophages IKe and fd. Boxes with a continuous outline mark amino acid residues conserved in either two or all three partial sequences, and boxes with a broken outline indicate conservative substitutions. Amino acid residues shown or thought to be in close contact with DNA and constituting one side of the p-ladder are shaded. (Used with permission from Traub er al., 1992a.)

scaffold-like aggregates to which supercoiled plasmid DNA bound with a loop-like configuration at high density (Kiihn e f al., 1987).The structure of these complexes was reminiscent of that of a rosette-enriched euchromatic fraction from interphase mouse fibroblast nuclei (Ascoli et al., 1988;Glazkov, 1988a), of histone-depleted metaphase chromosomes (Paulson and Laemmli, 1977), and of histone-depleted interphase nuclei (Hancock and Huges, 1982). In extension of these findings, vimentin was shown to interact with nucleic acids also in its filamentous form, with the one major difference that the filaments preferentially bind double-stranded (ds) nucleic acids (Traub et al., 1992b). This peculiarity is a function of the structure of IFs in which the filament core is made up of the a-helical rod domains of the protein subunits and the surface is coated with the corresponding non-ahelical head and tail domains. Due to their linear extension, flexibility, and protein-induced bending, double-stranded nucleic acids can bring a maximum number of binding sites in register with the surface-exposed Ntermini of the filaments. However, single-stranded nucleic acids generally possess a random coil configuration at physiological ionic strength and

INTERMEDIATE FILAMENT PROTEINS

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thus are capable of only a punctiform interaction with the filament surface. The interaction of dsDNA with vimentin filaments was exploited to search for physiologically relevant sequences among mouse genomic DNA fragments ( X . Wang and P. Traub, unpublished results; see later discussion). In addition to their affinities for a large variety of nucleic acids, IF proteins also show strong reactivities with histones (Traub et ul., 1986). Since most IF proteins have isoelectric points in the acidic region, electrostatic interactions between the reactants should be expected. Surprisingly, the very basic histone species HI is not accepted by the IF proteins as long as a mixture of core histones is present. Among these, the argininerich histones H3 and H4 yield the most stable reaction products. This binding involves the a-helical rod domain of the IF proteins and the globular, central regions of the histones. The interactions have a characteristic stoichiometry in that one tetrameric protofilament binds 16 core histone molecules, which, because of its symmetry, makes it attractive to speculate that they may be in the form of two histone octamers as they occur in nucleosomes. These results suggest that IF proteins possess reactivities that are eminently suited to fulfill nuclear functions in addition to their activities as a component of the cytoskeleton. This notion is supported by the finding that cytoplasmic IF proteins are very similar in their gross nucleic acid and histone binding properties to steroid hormone receptors, which as gene regulatory DNA binding proteins definitely carry out their tasks in the nucleus (P. Traub et af.,1983; Traub and Nelson, 1983; Ueda et al., 1989). In the following sections, an attempt is made to relate the in uitro activities of IF proteins to currently held views on the structure of the nucleus and its role in gene expression. B. Organization of the Nucleus as a Basis for Activities of Intermediate Filament Proteins

The DNA of most eukaryotic cells is highly compacted and organized at several, hierarchical levels: it is packed by histones, via the 10-nm nucleosome chain and the 30-nm solenoid filament, into a higher order chromatin structure which, in the form of topologically independent and differentially controlled loop domains capable of sequestering negative superhelical constraints, is firmly anchored to the proteinaceous nuclear matrix (Cook, 1989, 1991; Garrard 1990; Berezney, 1991; Georgiev et al., 1991; D. A. Jackson, 1991; Pienta et af., 1991; Boulikas, 1992; Laemmli et af., 1992; Stuurman et af., 1992; Zlatanova and van Holde, 1992a). These loops constitute discrete DNA replication and transcription units

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PETER TRAUE AND ROBERT L. SHOEMAN

whose boundaries are determined by distinct DNA sequences, the nuclear matrix-associated regions (MARs) (Boulikas, 1993a). MARs are usually AT-rich single-copy sequences 200- 1000 bp in length and contain clusters of topoisomerase I1 consensus sequences. While the replication units (replicons) are delineated by permanent MARs (replication origins, potential enhancers), the transcription units consisting of one or a group of coordinately regulated genes are bordered by facultative or transient MARs in a seemingly differentiation-specific and developmentally regulated arrangement. The activation of potential replication and transcription units appears to be tightly linked (Goldman, 1988). In some cases, a large chromatin domain representing a single replicon can be divided into smaller looped DNA domains (Cockerill, 1990). In general, MARs lie close to or overlap with promoter elements or transcription regulatory sequences such as enhancers, and co-map with the boundaries of chromatin domains that, when active, are characterized by a disrupted nucleosome structure and elevated sensitivity to DNAase I (Phi-Van and Stratling, 1988). Enhanced expression of transfected genes was demonstrated when they were linked to MAR sequences (Blasquez et al., 1989; Stief et al., 1989). The coincidence of some chromosomal breakpoints with MAR sequences suggests that these sequence elements also serve as cis-regulatory DNA regions in recombination processes (Sperry et al., 1989). The binding of the MARs to the nuclear matrix is mediated by discrete sets of nonhistone proteins whose composition varies characteristically with the differentiation state of the cell (Fey and Penman, 1988; Dworetzky et al., 1990; Stuurman et al., 1990). However, in different species these interactions are remarkably constant, suggesting a strong evolutionary conservation of function (Cockerill and Garrard, 1986; Phi-Van and Stratling, 1988), even though no strictly conserved MAR consensus sequence elements have been found so far. MARs are composed of subdomains that individually exhibit reduced binding affinity for the nuclear matrix but attach strongly to the protein scaffold when present in a repetitive arrangement extending over several hundred base pairs (Opstelten et al., 1989; Mielke et al., 1990; Romig et al., 1992). This could mean that MAR binding proteins are not strictly sequence-specific DNA binding proteins, but rather proteins that .recognize characteristic DNA structures with which they associate in a highly cooperative fashion (Laemmli et af., 1992). To be expressed, the genes of tissue-specific proteins must be in a transcriptionally competent state, a condition that appears to be fulfilled by the organization of the nuclear genome in distinct three-dimensional configurations through a hierarchy of functionally different, nuclear matrix-associated, regulatory DNA binding proteins operating in a cascade type mechanism (van Wijnen et al., 1993; Jackle and Sauer, 1993).

INTERMEDIATE FILAMENT PROTEINS

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The nuclear matrix can be divided into two interconnected subnuclear structures-the peripheral nuclear lamina consisting of lamins A, B, and C as major protein components; and the internal fibrogranular network of complex protein composition. In support of the conjecture that the nuclear lamina is important for the organization of interphase chromatin (Gerace and Burke, 1988), this fibrillar structure and its various lamin subunit proteins were shown to interact with mitotic chromosomes (Glass et al., 1993) and chromatin material in uitro (Bouvier et al., 1985; Hoger et al., 1991; Yuan et al., 1991). After UV irradiation of intact cells, a variety of DNA fragments containing repetitive sequences and displaying homology to introns and/or flanking regions of different genes could be covalently linked to the nuclear lamina (Galcheva-Gargova and Dessev, 1987; Christova et al., 1992), whereas ionizing radiation firmly bound transcriptionally active gene sequences to the lamins (Chiu et al., 1986). Cross-linkage of presumably repetitive DNA sequences to the nuclear lamina was also achieved by UV irradiation of isolated nuclear matrices (Boulikas, 1986). The observation that the lamina protects a relatively small number of about 10-kbp long, AT-rich satellite DNA segments from nuclease digestion was interpreted as a demonstration of its involvement in the fixation of chromosomes to the nuclear envelope (Razin, 1987; Glazkov, 1988b; Georgiev et al., 1991). The segments may be related to multiples of a 41-42-bp tandemly repeated sequence isolated from DNasetreated nuclear envelopes of chicken erythrocytes (Matzke et al., 1990). Possibly, lamin B is one of the active principles governing this association, since in vitro assays identified it as a MAR binding protein (Luderus et al., 1992). Lamins A/C did not show this property. Because purified lamins have a preference for G-rich polynucleotides and lamin B has a considerably lower affinity for nucleic acids in general than lamins A/C (Shoeman and Traub, 1990b; Hakes and Berezney, 1991a),the interaction of lamin B with MARs is probably restricted to an as-yet-unidentified, limited subset of sequences and/or structures conserved in MARs and would thus be of high specificity. In contrast to the traditional view that in interphase somatic cells the fibrillar network of the nuclear lamina surrounds the chromatin as a continuous shell interrupted only by the nuclear pore complexes, it was recently shown to have a discontinuous distribution in the nuclear periphery, occupying no more than about half of the surface area of the nucleus. Only 15 to 20% of the peripheral chromatin appears to be in direct, physical contact with the lamina (Paddy et al., 1990). If these associations are indeed specific and involve the aforementioned DNA sequences, they may contribute to the spatial organization of peripheral chromatin and thereby to regulation of gene expression during development and the cell cycle (Paddy et al., 1992). Along with the discontinuities in the peripheral

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PETER TRAUB AND ROBERT L. SHOEMAN

lamina, extensive internal lamin foci and cables occur in the nucleoplasm of proliferating human dermal fibroblasts, predominantly in the early G I phase of the cell cycle. They were seen to be associated with heterochromatin regions and therefore it was suggested that they play a role in chromatin organization (Bridger et al., 1993). Similar nucleoplasmic lamin accumulations and subsequent lamin translocation to the peripheral lamina were observed after microinjection of biotinylated lamin A into the cytoplasm of mouse 3T3 cells (Goldman et ul., 1992). A careful comparison of various optical and electron microscopy methods for studying lamin B distribution in the nuclear envelope of interphase CHO cells has both confirmed and clarified the discontinuous distribution of lamin B (Belmont er al., 1993). In these cells, a more or less continuous lamin B network was observed with accumulations or concentrations of additional lamin B molecules unevenly distributed over a scale (about 0.5 pm) similar to that found by more conventional techniques (Paddy er af., 1990). What is of particular interest is that the accumulations of lamin B were highly correlated to the underlying chromatin distribution, suggesting that the nuclear lamina “caps” the surface of the envelope-associated, large-scale chromatin domains (Belmont et af., 1993). The protein composition of the internal nuclear matrix is still obscure. Recently, a number of its constituents were isolated and characterized as DNA binding proteins (matrins) with a preference for ssDNA and low affinities for dsDNA and RNA (Hakes and Berezney, 1991a; Nakayasu and Berezney, 1991). This is in accordance with the observations that DNA regions associated with the nuclear matrix expose single-stranded sites after deproteinization (Probst and Herzog, 1985) and that MARS stably unwound under superhelical strain exhibit a strong affinity for the nuclear scaffold (Bode er al., 1992; Kay and Bode, 1994). Since a particular MAR showed preference for only a limited number of the total matrix binding sites, it was proposed that the nuclear matrix contains different classes of DNA binding sites, each with a separate sequence specificity (Hakes and Berezney, 1991a; Dworetzky et al., 1992). Like the nuclear lamins, the nuclear matrins are common to nuclear matrices from a variety of mammalian cells and therefore their function at least has been highly conserved during evolution. While one member of this protein class, matrin FIG, employs two overlapping zinc finger motifs as a putative DNA binding domain (Hakes and Berezney, 1991b), another member, matrin 3, possesses an arginine-rich, positively charged, amino-terminal region with multiple serine and threonine residues as in the lamins and several non-lamin IF proteins (Belgrader et al., 1991). Both matrins have potential sites for phosphorylation and 0-glycosylation. Another set of internal matrix proteins is distinguished by the possession of a long a-helical core domain with the capability to form coiled-coil dimers like the IF proteins.

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INTERMEDIATE FILAMENT PROTEINS

Since their non-a-helical tail pieces are highly positively charged owing to the presence of large numbers of basic, mostly arginine residues, they may be endowed with DNA binding activity (C. H . Yang et al., 1992; Compton et al., 1992; Mirzayan et al., 1992). In this context, it is pertinent to point out that the structural framework of the nuclear matrix seems to be built generally from subunit proteins capable of forming a-helical coiled-coil rope structures. While the lamins were identified as true members of the IF protein family (McKeon et al., 1986; Fisher et al., 1986), another class of intranuclear filaments was demonstrated to be morphologically indistinguishable from cytoplasmic IFs (Jackson and Cook, 1988; He et al., 1990; Wang and Traub, 1991; Nickerson and Penman, 1992). They have a diameter of 10 nm and an axial repeat of 23 nm, structural features normally resulting from the lateral and staggered alignment of protofilaments made up of two staggered, antiparallel-oriented, coiled-coil dimers. An indirect hint of the close structural and functional relationship between these intranuclear and cytoplasmic IFs was provided by the observation that certain cell types totally devoid of cytoplasmic IFs have also lost their potential to build a saltstable, intranuclear filament system; this can be detected, for instance, in the nuclei of fibroblasts (Wang and Traub, 1991).Nonchromatin fibers of 10 nm were also detected within the macronucleus of the ciliated protozoan Euplores eurystomus, where they appear to be continuous with the nuclear lamina. Probably consisting of proteins with molecular masses similar to those of cytoplasmic IF proteins and nuclear lamins of higher eukaryotes, they were suggested to constitute a framework for organizing the multitude of macronuclear minichromosomes by interacting directly or indirectly with telomeres (Olins and Olins, 1990).Also, IF-related components within the nuclei of plant cells were shown to react with monoclonal antibodies raised against nuclear matrices from purified plant nuclei (Beven et al., 1991). Furthermore, the yeast nuclear DNA-binding protein REPl, which copurifies with the karyoskeletal protein subfraction and shows in its ahelical, C-terminal half a strong sequence homology to vimentin and nuclear lamins A/C of higher eukaryotes, seems to promote partitioning of 2-pm plasmid circles to progeny cells via intercalation of its coiled-coil rod domain into the nuclear lamina; it thus provides anchorage sites for the attachment of plasmid molecules (Wu et al., 1987). Finally, coiled-coil proteins with positively charged C-termini that are related to a-helical nuclear matrix proteins were tentatively identified as major components of the transverse filaments occurring in the synapsed segments of meiotic prophase chromosomes (Meuwissen et al., 1992; Sym et al., 1993). A monoclonal antibody directed against the synaptonemal complex and probably recognizing these or closely related proteins labeled the cytoplasm of fibroblasts in a pattern reminiscent of that of vimentin

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PETER TRAUB AND ROBERT L. SHOEMAN

filaments (Moses et al., 1984). By analogy, two large heptad-repeat regions capable of coiled-coil formation provide the structural framework for the functioning of the yeast RADSO gene product in chromosome synapsis and recombination during meiosis, and in repair of DNA damage during vegetative growth (Alani et al., 1989). It is still unclear whether the intranuclear meshwork of protein filaments is directly responsible for the loop organization of chromatin or whether it only represents a passive stage for the accomodation of chromatin (MAR) binding proteins such as topoisomerase 11, histone H1 (Laemmli et af., 1992), the attachment region binding protein (ARBP; von Kries et al., 1991), SP120 from rat brain (Tsutsui et al., 1993), and others. In the first case, it is conceivable that the filament system changes its subunit composition in response to differentiation directives just as the nuclear lamina (Nigg, 1989) and the cytoplasmic IF system (Steinert and Roop, 1988; Klymkowsky et al., 1989) alter their protein compositions upon supply of developmentally determined, environmental stimuli. Alternatively, it may be equipped with surface elements which recognize relatively constant or conserved protein domains that are shared by whole sets of differentiation-specific nuclear protein factors of distinct but different chromatin binding specificity. In any case, the arrangement of the chromatin binding proteins in a filamentous, scaffold-like form implies a regular succession of surface-exposed binding sites which may match those of the repetitive sequence elements in the MARS and effect their strong and cooperative, multisite attachment to the proteinaceous matrix (Opstelten et af., 1989). By contrast, transcription factors of general functionality interact individually with short DNA consensus sequences. The cooperativity of MAR binding results from the formation of direct protein-protein interactions in addition to nucleic acid-protein interactions, and is a function of the tendency of MAR binding proteins to self-polymerize. It is conceivable that under appropriate ionic conditions many of the nuclear matrix-localized DNA binding proteins can be assembled into filamentous aggregates, in a homo- as well as heterologous fashion, as indicated, for instance, in the case of the SAF-A protein from HeLa cells (Romig et al., 19921, the zeste gene product of Drosophila (Chen et al., 1992) or the retinoblastoma susceptibility gene product (Rb) (Hensey et al., 1994; Mancini e? al., 1994). The Rb protein is especially interesting since it becomes hypophosphorylated and associates with the nuclear matrix only during the early G1 phase of the cell cycle in wild type cells, whereas mutant Rb in tumor cells does not associate with the nuclear matrix (Mancini et al., 1994). Another striking example with a high similarity to I F proteins is provided by the core proteins of nuclear ribonucleoprotein (hnRNP) particles, which, in the form of heterotetramers, interact with single-

INTERMEDIATE FILAMENT PROTEINS

13

stranded nucleic acids in a highly cooperative manner and polymerize, in the absence of nucleic acids, into very salt-stable, regular filaments (Lothstein et al., 1985; Barnett et al., 1991; Casas-Finet et al., 1993). It was speculated that such proteins as well as their association products with nuclear RNA participate in the construction of the nuclear matrix (Nickerson et al., 1989; Patriotis et al., 1990; He et al., 1991). A liverspecific protein with a strong sequence homology to hnRNP-C was shown to interact with a hepatitis B viral enhancer element (Tay ef al., 1992). C. Relationships between Intermediate Filament Proteins, Transcription Factors, and Nuclear Matrix Proteins

Having set the stage with this brief outline of some structure-function relationships essential to the nucleus, we now are in a position to ask the question: Can cytoplasmic IF proteins perform on this stage and, if so, how? In our opinion, this is possible because of the close structural and functional relationships of the IF proteins to elements of the nuclear matrix and associated transcription factors. In response to differentiation directives, IF proteins may bind to or even incorporate into preexisting filaments of the karyoskeleton and thus modulate its activities in a variety of DNA-based nuclear events. Alternatively, cytoplasmic IFs as such may enter the nucleus and direct, in complementation of other nuclear matrix elements, the distribution of chromosomes and organization of chromatin.

1. Similarities between Intermediate Filament Proteins and Transcription Factors A comparison of IF proteins with a variety of gene regulatory factors reveals that all these proteins have a series of principal, structural, and functional features in common. Most characteristic is their bipartite construction from a dimerization motif and an adjacent DNA-binding region (Jones, 1990; Lamb and McKnight, 1991). While the coalignment of two transcription factor molecules, mostly via formation of two-stranded coiled-coils from parallel-oriented a-helical sequence elements, permits the recognition of dyad-symmetric DNA sequence motifs, the neighboring DNA-binding regions impart sequence specificity to these interactions. IF proteins most closely resemble transcription factors of the bZIP (Cohen and Curran, 1990; Kerppola and Curran, 1991) [GCN4 (O’Shea et af., 1991; Ellenberger et al., 1992) and members of the C/EBP (Landschulz et al., 1989; Williams et al., 19911, Fos-Jun (Turner and Tjian, 1989;Pathak and Sigler, 1992) and ATF-CREB (Brindle and Montminy, 1992) groups] and bHLH (Garrell and Campuzano, 1991; Kadesch, 1992) [e.g., the myo-

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PETER TRAUB AND ROBERT L. SHOEMAN

genic factors MyoD and myogenin (Tapscott and Weintraub, 1991; Edmondson and Olson, 1993) or Myc (Blackwood and Eisenmann, 199111 protein families in that they all possess a coiled-coil dimerization interface flanked on the N-terminal side by a normally unstructured cluster of basic amino acid residues harboring the DNA binding site. Interestingly, the heptad repeat of the N-terminal half of the a-helical core domain of vimentin reveals a leucine zipper motif which matches three to four out of five leucines (nonleucine positions are occupied by hydrophobic amino acids) of the zipper domains of members of the CREB and Fos-Jun protein groups. There is an additional structural similarity of 50 to 70% between the basic domain of the DNA binding region of Fos, Jun, and CREB with part of the N-terminal half of helix l b of vimentin (Capetanaki et a / . , 1990). Moreover, a region encompassing the middle of the a-helical rod domain of the small neurofilament triplet protein (NFPL) shows a 20% identity and 40% similarity in primary structure and excellent agreement in predicted secondary structure with a segment of the DNA binding product of the retinoblastoma susceptibility gene (Rb protein; amino acids 307-435) (Hong and Lee, 1991). It seems likely that this segment plays a role in the oligomerization of the Rb protein and in its in uitro polymerization into filaments, since removal of the N-terminal region up to amino acid position 379 blocks these activities (Hensey et al., 1994; Mancini et al., 1994). Another relationship between the I F proteins and the Rb protein is shown by the ability of the Rb protein to bind to lamins A and C in uitro (Mancini et al., 1994). In fact, it is a fundamental property of many transcription factors to form heterooligomers with members of their own or related protein groups on the basis of coiled-coil interactions. Aligned heptad leucines, however, are not absolutely required for protein dimerization (Chang et al., 1990; Paluh and Yanofsky, 1991). Heterooligomerization is exploited by transcription factors to expand their repertoire of regulatory potential (Jones, 1990; Lamb and McKnight, 1991) and may have a structural and functional equivalent in the general capacity of I F proteins to produce heterodimers (Quinlan and Franke, 1982, 1983; Hatzfeld and Weber, 1990; Steinert, 1990; Traub et al., 1993). However, while dimerization among transcription factors of the bZIP and bHLH type is relatively promiscuous, that among IF proteins is less so. This is probably due to the longer a-helical coiled-coil region of I F proteins and has a correlate in the observation that C-terminal extension of the dimerization domain of bHLH proteins by a ZIP motif substantially increases their capability to discriminate between different dimer partners (Hu et al., 1990; Beckmann and Kadesch, 1991). Simultaneously, such extended factors acquire the property to tetramerize, although they still bind to DNA as dimers (Dang et al., 1989; Fisher et al., 1991). In this

INTERMEDIATE FILAMENT PROTEINS

15

connection, it would be intriguing to know whether IF proteins, which normally exist as tetramers, dissociate into dimers upon interaction with DNA as well. It also remains to be seen whether in IF proteins the spacing between the coiled-coil dimerization interface and the adjacent basic region is conserved, a requirement which in the case of bZIP and bHLH transcription factors must be fulfilled to symmetrically position the basic region for specific DNA binding (Pu and Struhl, 1991; Vinson and Garcia, 1992). The conservation of a 20 to 25-residue-long sequence with the potential for a-helix formation immediately in front of the a-helical core domain of nonepithelial IF proteins (Geisler and Weber, 1983; Capetanaki et a l . , 1990) is indeed suggestive in this respect. Another interesting phenomenon based on the oligomerization potential of bZIP and bHLH factors is that of dominant-negative regulation of transcription factor activity. Derivatives of these factors missing the basic region adjacent to the dimerization domain assemble normal factor molecules into heterooligomers that no longer bind to DNA (Jones, 1990; Lamb and McKnight, 1991). They can therefore serve as general antagonists of differentiation programs, as exemplified by the performance of the MyoD/ Id system in myogenesis (Benezra et al., 1990). IF proteins may mimic this situation in that their derivatives lacking the DNA binding region are still capable of oligomerization with intact protein molecules (Traub et al., 1993). There are also similarities in the DNA binding domains of IF proteins and transcription factors although they are less pronounced. Often, these regions are highly positively charged owing to the presence of a large number of basic amino acids, particularly arginine residues. The positive charge confers a nonspecific, electrostatic binding mode on protein-DNA interaction and facilitates target location, a process which is in equilibrium with a specific binding mode (von Hippel and Berg, 1989; O’Neil er al., 1990). Whereas structurally well-defined DNA binding motifs, like the helix-turn-helix or zinc finger motif (Latchman, 1990; Harrison, 1991), were assigned to selected families of transcription factors, the polypeptide chain of the DNA binding domain of bZIP and bHLH proteins is disordered in its free state but attains a-helix configuration upon interaction with DNA containing a specific recognition sequence (O’Neil et al., 1991; Ellenberger et al., 1992). Unfortunately, the configuration of the N-terminal polypeptide of IF proteins in association with nucleic acids is unknown. However, since the isolated, non-a-helical N-terminus of vimentin increases its a-helix content significantly upon interaction with the two-dimensional lattice of anionic phospholipid bilayers (Perides et al., 1987a), it may respond similarly to the one-dimensional lattice of negatively charged polynucleotides. In this connection, it is appropriate to mention that many Z-DNA binding

16

PETER TRAUB AND ROBERT L. SHOEMAN

proteins were actually shown to be phospholipid binding proteins (Krishna et al., 1990). On the assumption that as a single-stranded and supercoiled DNA-binding protein vimentin also shows affinity for Z-DNA, it would belong to the same category of “promiscuously binding” proteins that owe their affinity for negatively charged lattices to the amphiphilic character of their cognate binding regions. Alternatively, I F proteins may make use of a totally different binding motif for the specific component in their interaction with DNA. Sequence comparison of the N-terminus of vimentin with the ssDNA-binding G5P of bacteriophage fd and related viruses identified a DNA binding wing of antiparallel p-sheet structure as the potentially active principle (Traub et al., 1992a). This mode of interaction would not be exceptional since an increasing number of binary systems are being detected that employ a two-stranded antiparallel p-sheet as the DNA interaction motif (Knight et al., 1989; Phillips, 1991). Prokaryotic repressors and members of the prokaryotic HU family in particular, but also the TATA box binding transcription factor TFIID (Lee et al., 1991; Starr and Hawley, 1991) appear to insert antiparallel p-ribbons into the major and minor groove of the B-DNA helix, respectively, to make sequence-specific contacts with the bases of the DNA. In addition, TFIID has a proposed ssDNAbinding activity that may be needed to maintain the factor’s position when DNA melts during the initiation of transcription (Horikoshi et al., 1989). Another interesting relationship exists between cytokeratins, which are also ssDNA-binding proteins (unpublished observations), and a series of single-stranded nucleic acid binding proteins that play a role in RNA metabolism but interact with ssDNA as well. Nucleolin, fibrillarin, and hnRNP proteins (Kumar et al., 1990; Kiledjian and Dreyfuss, 1992, and further references therein) are representative examples. They share with the non-a-helical end domains of many cytokeratin species the feature of having quasi-repetitive, glycine-rich peptide sequences with regularly spaced aromatic amino acid residues and occasionally interspersed, positively charged, mostly arginine residues (Steinert et al., 1991). In general, the terminal domains of the cytokeratins exhibit a lower net positive charge than the glycine-rich regions of the RNA binding proteins. Whereas the arginine residues contribute nucleic acid binding functions going beyond a mere electrostatic interaction, the aromatic amino acid residues certainly contribute to hydrophobic stacking interactions with the nucleic acid bases. Owing to the high flexibility of the glycine-rich regions in the end domains of the cytokeratins, these may adopt ordered conformations upon interaction with nucleic acids that allow specific contacts between their arginine and aromatic amino acid residues and certain nucleotide sequences. The hnRNP proteins also bind to single-stranded vertebrate telomeric repeats (McKay and Cooke, 1992) which, as a conse-

INTERMEDIATE FILAMENT PROTEINS

17

quence of their high G content, can form a number of unusual structures, including the four-stranded G-quartet. Thus, the hnRNP proteins share another functional similarity with IF proteins, which also specifically bind to telomere oligonucleotide models (Shoeman et al., 1988; Shoeman and Traub, 1990b). Although ssDNA-binding proteins are generally viewed as ligands associating nonspecifically and cooperatively with single-stranded nucleic acids, IF proteins nonetheless may exhibit sequence specificity in their binding to nucleic acids, including dsDNA. This is indicated by the strong influence of the base composition of single- and double-stranded polynucleotides on their affinity for IF proteins (P. Traub et al., 1983; Traub and Nelson, 1983; Vorgias and Traub, 1986) and supported by several parallels detected in eukaryotic replicational and transcriptional regulation systems. For instance, the yeast factor ACBP [ARS (autonomously replicating sequence) consensus-binding protein], which is thought to play a role in an early step of the initiation of DNA replication, binds the T-rich single strand of the MAR-like ARS consensus sequence in a sequencespecific manner, but also, though in a less stable interaction, the doublestranded ARS consensus. The latter activity, however, may be a reflection of the capability of the protein factor to melt the DNA helix at the binding site (Hofmann and Gasser, 1991). Similarly, the myogenic determination factors MF3 and MyoD exhibit potent sequence-specific, ssDNA-binding activities in addition to their capacities to interact with dsDNA (Santaro et al., 1991). Subsequent studies showed that MyoD specifically binds to helical structures formed by stacks of G residues in square planar arrays (Sen and Gilbert, 1991) as they occur with low abundance in the ssDNA of the noncoding strand of an E-box element from the muscle-specific creatine kinase enhancer. MyoD also binds to a G-quartet structure formed by self-association of the G residues of a single-stranded Tetrahymena telomere probe (Walsh and Gualberto, 1992). These activities are closely paralleled by those of vimentin, which also shows high affinity for G-rich ssDNA in general and telomere oligonucleotide models in particular (Shoeman et al., 1988; Shoeman and Traub, 1990b). Since runs of G residues are common in immunoglobulin switch regions (Leung and Maizels, 1992) and gene regulatory sequences (Tazi and Bird, 1990), vimentin and its relatives may activate them by sequencespecific interaction, in analogy to the performance of transcription factors. In this case, the protein-DNA complexes formed may serve to maintain DNA in a single-stranded configuration induced by release of torsional strain from supercoiled DNA or produced as the result of the potential helix-unwinding activity of the IF proteins. It should be noted, however, that there are a variety of regulatory DNA binding proteins which can

18

PETER TRAUB AND ROBERT L. SHOEMAN

definitely interact also with ssDNA elements in a sequence-specific manner (Bergemann and Johnson, 1992; Muraiso et al., 1992: Nordstrom et al., 1993; Quinn and McAllister, 1993, and further references therein). Because of its specificity for the single-stranded, G-rich pentameric repeats of immunoglobulin switch regions, the Spbp-2 protein compares particularly well with IF proteins (Fukita et al., 1993). 2. Potential Role of Intermediate Filament Proteins in Transcription Initiation There is substantial evidence that, although the bulk of DNA in nuclear chromatin is not significantly torsionally strained, the transcriptional process increases the local superhelical density of DNA (Freeman and Garrard, 1992). Major contributions to local supercoiling come from the breakdown of higher chromatin structure, loss of nucleosomes from nuclease-hypersensitive sites (unfolding of nucleosomes), histone acetylation, and the passage of the transcribing RNA polymerase. How could IF proteins contribute to these processes with their supercoiled and ssDNA- and histone-binding properties? One possibility could be, for instance, their involvement as transcription factor-like agents in the initiation of transcription of genes whose cis-acting elements are incorporated into nucleosomes (Kornberg and Lorch, 1991; Felsenfeld, 1992; Hayes and Wolffe, 1992; van Holde et al., 1992; Adams and Workman, 1993; Svaren and Horz, 1993; Workman and Buchman, 1993). It can be imagined that in this concerted, multistep process the first step encompasses the recognition by IF proteins of the enhancer and/or promoter sequences on individual nucleosomes in chromatin regions that are decondensed by removal of linker histone HI (Zlatanova and van Holde, 1992b; Bresnick et al., 1992) and by acetylation of the N-terminal domains of the core histones (Turner, 1991; Ausio, 1992; Garcia-Ramirez et al., 1992). Sequence recognition would be facilitated by the fact that the minor grooves of runs of G / C face outward on the surface of the nucleosomes (Powers and Bina, 1991; Thoma, 1992), which are partially unfolded as a result of histone acetylation (Oliva et al., 1990). Interestingly, short chromatin fragments containing CpG-rich DNA, which are found upstream of many genes, contain highly acetylated core histones (Tazi and Bird, 1990). These primary contacts would lead to reorientation of the nucleosomal DNA and thus to labilization of the DNAhistone core interaction. Supported by the superhelical strain generated by chromatin decondensation (Freeman and Garrard, 1992), the properly arranged, multiple arginine residues surrounding the DNA binding sites in the structurally flexible N-termini of the IF protein dimer might displace the tripartite histone core with its centrally located, arginine-rich

INTERMEDIATE FILAMENT PROTEINS

19

(H3-H4), tetramer (Arents et a l . , 1991) from the DNA. While the DNA consolidates its interaction with the DNA binding domains of the IF protein dimer by attaining a supercoiled configuration, the (H3-H4)2histone tetramer transfers to the coiled-coil domain of the protein dimer with which it interacts via the central, globular regions of its constituent protein subunits (Traub et al., 1986). The interaction would be strengthened by contributions from the acetylated N-terminal domains of histones H3 and H4, in accord with the observation that acetylated histone H4 molecules facilitate promoter activation (Durrin et a / . , 19911, Throughout the histone core rearrangement, the two H2A/H2B dimers may remain in loose association with the (H3-H4), tetramer. Like the glucocorticoid receptor (Archer et al., 1991) and the PH05 promoter-activating transcription factors PH02/PH04 (Schmid et al., 1992), IF proteins may induce in this way the formation of nucleosomefree, DNAse I-hypersensitive regions (Gross and Garrard, 1988)and clear the way for the binding of additional transcription factors. That transcription activators are capable of displacing core histones from nucleosomes on cis-acting elements was recently demonstrated by the in uitro production of a metastable ternary complex from the yeast regulatory protein GAL4 and nucleosomes consisting of core histones and DNA carrying multiple GALCbinding sites and transfer of the core histones onto nonspecific, competing DNA. The displacement of histones was seen to be mediated primarily by the nature of the DNA-binding domain of the regulatory factors and it was speculated that in the nucleus other components, such as acidic chromatin assembly factors (nucleoplasmin), function as transient histone acceptors in place of nonspecific DNA (Workman and Kingston, 1992). In a purified in uitro system, nucleoplasmin could indeed be shown to stimulate the binding of transcription factors, such as GAL4 or SPl, to nucleosome-reconstituted DNA. It specifically removed histones H2A and H2B from the nucleosomes and enhanced the subsequent transfer of the histone (H3-H4), tetramers onto competing DNA (Chen et al., 1994). In the case of IF proteins, the destabilization and transfer reactions would be mediated by one and the same protein dimer, which combines the required DNA and histone binding domains in one component. Higher order oligomers of IF proteins may overcome nucleosome position effects more easily by binding to repetitive recognition sequences, similar to the facilitated, cooperative binding of multiple GAL4 dimers to nucleosome cores containing multiple binding sites for the transcription factor (Vettese-Dadey et al., 1994). Once bound to cis-acting elements, IF proteins may transduce positive regulatory signals to the promoter-associated general transcription factors and RNA polymerase directly or via mediators or coactivators (Martin, 1991; Gill and Tjian, 1992). Certain members of the IF protein family, like

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PETER TRAUB AND ROBERT L. SHOEMAN

the neurofilament proteins, could make use of their unusually glutamic acid-rich C-terminal extensions, in analogy to a large variety of transcription factors with an acidic activation domain (Earnshaw, 1987; Struhl, 1991; Hahn, 1993). Such IF proteins would meet the hypothetical demand for activator proteins that relieve the inhibition of transcription by histones and stimulate initiation of complex formation in a single mode of action (Kornberg and Lorch, 1991). Since neurofilament proteins are highly susceptible to phosphorylation in their C-terminal domains (Shihag and Nixon, 1989; Geisler et al., 1987), their hypothetical function as transcription activators may be regulated in a way similar to that of, for example, the nucleolar transcription factor mUBF (O’Mahony et al., 1992; Voit et al., 1992) or of the phosphoprotein of vesicular stomatitis virus (Takacs et al., 1992). Both the C-terminal extensions of the neurofilament proteins (Link et al., 1992) and these transcription factors are phosphorylated by casein kinase 11-type enzymes. Regarding the suggestion that the acidic activation domains exert their functions by dislodging histones from nucleosomes (Peterson and Herskowitz, 19921, it is interesting to note that the small neurofilament triplet protein NFP-L binds 16 core histone molecules per dimer, in addition to the eight molecules normally associating with the coiled-coil rod domain (Traub et al., 1986).

3. Potential Role of Intermediate Filament Proteins in Transcription Elongation The simultaneous occurrence of two DNA and histone core binding sites in IF protein tetramers in rotational symmetry would make these, in addition, ideal catalysts of transcription elongation (Morse, 19921, fulfilling the requirements of the twin supercoil domain model of chromatin transcription (Liu and Wang, 1987; Wu et al., 1988; Giaever and Wang, 1988). In this model, the transcribing RNA polymerase induces local DNA domains of positive supercoiling ahead of it and regions of negative supercoiling behind it. It was hypothesized that, because nucleosomes assembled on positively supercoiled DNA are thermodynamically less stable than those formed on negatively supercoiled DNA, histone octamers are sequentially transferred from the region in front of the polymerase to the region behind during transcription (Clark and Felsenfeld, 1991). Since in an in uitro competition experiment performed under irreversible binding conditions at physiological ionic strength, histone octamers were deposited equally on positively and negatively supercoiled plasmids, their exchange between regions in front of and behind the polymerase in transcribed chromatin was speculated to be facilitated by a catalyst, such as an octamer-binding protein, or by histone acetylation. However, it was subsequently shown that a histone octamer assembled on a distinct seg-

INTERMEDIATE FILAMENT PROTEINS

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ment of a plasmid was displaced from its original site during transcription and shifted to a new site within the same plasmid molecule, with some preference for the nontranscribed region behind the promoter (Clark and Felsenfeld, 1992). Nonetheless, the activation energy for histone octamer dissociation and transfer might be too high in uivo so that a catalyst would be necessary to reduce it to a tolerable value. Given their binding properties, IF protein tetramers or higher oligomers seem to be predestined to catalyze histone octamer transfer. On the assumption that they bind equally well to positively and negatively supercoiled DNA (Kuhn et a[., 1987), they may be able to form a protein bridge between DNA regions immediately ahead and immediately behind the transcribing polymerase and to transport, like a conveyor belt, histone octamers across their a-helical rod domains as the DNA is threaded through the transcription complex located at the apex of the DNA loop formed (Fig. 2). Since IF proteins both saturate and seem to unwind supercoiled DNA without regard to sequence (Kuhn et al., 1987), the flexible, arginine-rich N-termini of the tetramers will slide smoothly along the advancing DNA molecule of the unfolding chromatin strand, possibly relieving its superhelical strain by unwinding it in front of the polymerase. Owing to the long distance between the actual DNA binding region and the a-helical core domain of IF proteins, and the lack of specific secondary structure in the intervening amino acid sequence, the latter could absorb some of the superhelical tension released from the supercoiled DNA. Higher oligomers of IF proteins with their interconnected rod domains would be equally suited for octamer transfer. Possibly, other nuclear proteins possessing arginine-rich DNA binding regions adjacent to long coiled-coil domains, such as some of the constituents of the nuclear matrix, may function in the same way. This, however, would imply an at least loose attachment of the transcription complexes to solid, subnuclear structures, in concord with a hypothesis put forward previously (Cook, 1989; Dickinson et af., 1990; Dubochet, 1993;Jackson and Cook, 1993).Immobilization of the transcriptional machinery on the karyoskeleton would also allow the storage and transfer of thermodynamic energy, an aspect recently considered regarding the biological significance of MAR-nuclear matrix associations. Matrix-bound MARS also tend to become unbase-paired in relieving superhelical strain generated in chromatin loop domains (Bode et al., 1992). These activities are reflected by the equal potential of both isolated nuclear scaffolds (Tsutsui et al., 1988) and IF protein aggregates (Kuhn et al., 1987) to discriminate among various topological forms of plasmid pBR322-DNA, with a high preference for the supercoiled (and singlestrand) variant. It cannot be excluded, however, that the superhelical tension in such cases is dissipated by the production of other stress-

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PETER TRAUB AND ROBERT C. SHOEMAN

Nucleosomes

@

C) (2

FIG. 2 Model of the catalytic function of IF protein oligomers in transferring histone octamers from positively to negatively supercoiled DNA during transcription elongation. The shorter arrows on the left indicate the movement of the DNA relative to the nuclear matrix-bound RNA polymerase (RNAP). The longer arrows in the middle of the drawing indicate the pathway taken by histone core particles after their displacement from chromatin (top) by the head domains of the IF proteins (IFP). The histone core particles move along the rod domain of the IF protein oligomer and finally reassociate with DNA downstream of the RNA polymerase. The concept embodied in this model would also describe the potential function of IF proteins in transcription initiation, except that after the removal of histone octamers from promoter and enhancer elements the initiation complexes would be stalled due to metastable binding of the N-termini of the IF protein oligomers to their recognition sequences.

absorbing, non-B structures, such as cruciforms, triple helices, and ZDNA (Kladde et al., 1993), to which transcription factors, nuclear matrix elements, and IF proteins may bind specifically. It is of interest that histone acetylation is not found uniformly throughout actively transcribed chromatin but is restricted to defined sites, possibly in the nontranscribed flanking regions. It was speculated that histone acetylation facilitates the assembly of the initial transcription (and replication) complexes, rather than being required for the passage of RNA polymerase through nucleosomes (Turner ef al., 1990; Sommerville et al., 1993). Relative to the hypothetical function of IF and related proteins in transcription, these findings could mean that the processes of initiation and elongation involve different mechanisms of histone octamer activation. The lack of sufficient torsional strain may make it necessary to acetylate the histones, particularly H3 and H4, in the initiation steps, to allow their transfer to the coiled-coil rod domains of acceptor (IF) proteins.

INTERMEDIATE FILAMENT PROTEINS

23

However, during elongation, the positively superhelical tension generated downstream of the transcribing RNA polymerase may be sufficient to labilize the nucleosome structure and prepare the histone octamer for transfer. In any case, an increase in DNase I sensitivity of both hyperacetylated nucleosomes (Simpson, 1978; Ausio and van Holde, 1986) and nucleosomes exposed to positively superhelical tension (Lee and Garrard, 1991), possibly resulting in an enhanced dissociability of histone H2A/ H2B dimers from both structures ( V . Jackson, 1993, and further references therein), points in this direction. Actually, the introduction of hydrophobic acetyl residues into the Ntermini of core histones would probably provide an obstacle to the smooth and continuous exchange of octamers across the IF protein bridges on transcribed nucleosome chains, whereas because of the more static nature of initiation complexes, the opening of nucleosome-masked enhancer and promoter regions would profit from such a modification. A model was proposed which relates the association of specifically acetylated isoforms of histone H4 with particular chromatin types to the generation of an array of markers on the nucleosome surface that are potentially able to interact with nonhistone proteins that respond sensitively to changes in the acetylation state of histone H4 (Turner et al., 1992). This may also explain the negative influence of histone H4 on promoter activation as a result of the removal of the N-terminus (with its acetylation sites) (Durrin et al., 1991). Finally, the general requirement of transcription for IF and structurally related proteins would provide a plausible explanation for the observation that in uitro systems lacking such components are rather inefficient in transcription elongation along arrays of canonical nucleosomes (Izban and Luse, 1991). 4. DNA Loop Formation by Higher Oligomers of Intermediate Filament Proteins as an Essential Feature of Their Potential Gene-Regulatory Function If they took advantage of their tendency to tetramerize and to form higher oligomers, IF proteins could further extend their regulatory potential. There is a striking structural similarity between IF protein tetramers and the lac repressor (Chakerian and Matthews, 1992). The latter is also a tetramer of identical (38-kDa) subunits in which the sequence-specific DNA binding activity is confined to a small N-terminal domain while each of the subunit dimers is held together through the interaction of two long a-helices in a coiled-coil conformation at the dyad axis (Alberti et al., 1993). In the same way that pairs of the non-a-helical head domain of IF proteins are positioned at opposite ends of the protofilaments by antiparallel alignment of two parallel-oriented dimers, pairs of the N-terminal DNA

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PETER TRAUB AND ROBERT L. SHOEMAN

binding region of the lac repressor subunit also become located on diametrically opposed sides of the tetramer by the opposition of two subunit dimers. Just as the lac repressor employs its inherent capacity to oligomerize and to recognize operator DNA for the formation of DNA loops, whereby the stability of the repressor-operator complex is enhanced significantly by DNA supercoiling, tetramers of IF proteins may exert regulatory functions by bringing spatially distant, cis-acting elements in close proximity through a protein bridge with DNA loop formation of the intervening sequence (Matthews, 1992). The capability of IF proteins to assemble heterotetramers from different dimers (Quinlan and Franke, 1982; Hatzfeld and Weber, 1990; Steinert, 1990; Traub et al., 1993)would provide this mode of action with considerable regulatory flexibility, whereas the formation of dominant-negative regulators from dimers of intact and N-terminally truncated subunit proteins (Traub et d . , 1993), respectively, would suppress specific gene expression. This is seen in the case of Id dimers inhibiting the DNA-binding activity of various homo- and heterotypic HLH protein dimers (Fairman et a f . , 1993). The efficiency of loop formation and loop stability could be further increased by the cooperative interaction of multiple DNA recognition sequences in repetitive arrangement with the corresponding number of stacked IF protein tetramers. Since nuclear matrix-associated, genomeregulatory proteins, possibly including IF proteins, appear to recognize characteristic DNA structures rather than strictly specific DNA sequences (Laemmli et al., 1992), a certain sequence degeneracy in the DNA target sites would not be detrimental to such a mechanism. DNA looping via homotetramerization of transcription factors bound to remote cis-acting elements in conjunction with synergistic activation of transcription was observed in the case of steroid hormone receptors (Theveny et al., 1987; O’Malley, 1991), homeodomain proteins (Beachy, 1990),the E2 transactivator protein of bovine papilloma virus (Knight et al., 1991), the GC-boxbinding factor SP1 (Su et al., 1991) etc. Higher oligomerization of SP1 through stacking of tetramers to multiple adjacent and distantly located GC-rich elements further increases transcription factor concentration at the loop juncture (promoter site) and thus transcription activation (Pascal and Tjian, 1991; Mastrangelo et al., 1991). 5. The zeste Gene Product of Drosophila as a Model for the Potential Role of Intermediate Filament Proteins as Matrix Elements in Gene Expression

An excellent model showing how higher oligomers of IF proteins may exert regulatory functions in gene expression is provided by the zeste gene product of Drosophila (Pirrotta, 1991). This protein recognizes DNA

INTERMEDIATE FILAMENT PROTEINS

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sequences at many sites on the polytene chromosomes. As a transcription factor with an additional transvection activity, it is thought to juxtapose enhancer and promoter elements of multiple genes on the same or on homologously paired chromosomes with looping out of the intervening DNA sequences. A common feature of its binding sites in these cis-acting elements is that they are composed of two or more hexamer core consensus sequences (containing two degeneracies) in either direct or inverse orientation at distances between 16 and 55 nucleotides, with imperfect consensus sequences in the immediate vicinity of the true consensus. Whereas there is no binding to a single cloned copy of the core consensus sequence, the interaction becomes increasingly stronger with an increasing number of tandem copies, an effect which also extends over longer distances between independent binding sites. This cooperative effect rests on the strong tendency of the zeste gene product to form large, salt- and nonionic detergent-resistant aggregates which measure hundreds of monomers in size and which are capable of binding DNA and stimulating transcription. While the N-terminal half of the protein with its homeodomain-like DNAbinding region has a limited capacity to oligomerize, the major determinants of protein aggregation reside in the C-terminal half. Because this part consists of a long a-helical domain with heptad repeats, including a leucine zipper, it is almost certainly engaged in coiled-coil interactions. Mutations that delete the a-helical C-terminus or diminish its dimerization competence drastically reduce the regulatory potential of the zesteencoded protein (Bickel and Pirrotta, 1990; Chen et a f . , 1992; Chen and Pirrotta, 1993b). All these properties suggest the zeste gene product is a nuclear matrix protein, and it is therefore not surprising that typical nuclear matrix preparations from Drosophifa nuclei contain the majority of the total zeste-encoded protein found in the cell. The zeste gene product is thought to interact with the nuclear lamina or other nuclear DNA binding proteins possessing helical domains. Although I F proteins are structurally different from the zeste product, they fit this model nearly perfectly. They can be isolated in the form of stable, filamentous aggregates with surface-exposed DNA binding regions in close association with the nuclear matrix (Fey et al., 1984; Katsuma et a f . , 1987; French et al., 1989; Wang et al., 1989; Bastos et al., 1992). Their N-termini harboring the DNA binding motif show some tendency to oligomerize (Saeed and Ip, 1989; Traub et al., 1992a), whereas the remaining C-terminal parts rich in a-helical repeats and therefore prone to form coiled-coil rope structures represent the actual aggregation domains (Steinert and Roop, 1988). Minor mutational changes in sensitive regions of the coiled-coil rod domains also effect major losses of oligomerization potential (Hatzfeld and Weber, 1991; Letai et a f . , 1992; Rothnagel et a f . , 1992).

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Exactly as the aggregates of the zeste-encoded protein, higher oligomers of IF proteins or even filaments readily bind DNA in an apparently sequence-specific way (Traub et al., 1992b), although there seems to be some degeneracy of the DNA at the primary sequence level. Affinity binding of mouse genomic DNA fragments to vimentin filaments, followed by cloning and sequencing of the most strongly bound fragments, made it possible to identify a number of repetitive sequences that are not all identical or homologous (X. Wang and P. Traub, unpublished results). Several of them are quite similar to sequences that flank exons of actively expressed genes, both 5' upstream elements as well as introns. Our previous inability to specify a true consensus nucleotide sequence for vimentin binding may be a function of either the small sample size of tightly bound sequences analyzed and/or vimentin may recognize and bind to specific secondary or higher level structures which are not immediately apparent from the primary sequence. This, however, does not weaken the analogy of the potential mode of action of vimentin to that shown to be operative for the zeste gene product. Indeed, the recognition of a multitude of related sequences may be a prerequisite for gene regulation on a global scale. It also should be pointed out that, regarding their general sequence characteristics, some of the sequences of the cloned DNA fragments most strongly bound to vimentin filaments were rather similar to the DNA regions covalently cross-linked to the nuclear lamina by UV irradiation of intact cells (Christova et al., 1992). Since both sets of fragments originated from Ehrlich ascites tumor cells, it is possible that owing to the close association of large quantities of vimentin filaments with the nuclear envelope in these cells, some of the genomic DNA cross-connected to the lamina was actually linked to vimentin filaments (Galcheva-Gargova and Dessev, 1987). A similarity was also detected between the isolated genomic DNA fragments and a novel class of MARS with alternating GAand CT-rich motifs as well as GT boxes (Boulikas and Kong, 1993). D. Physical Association of Intermediate Filaments with the Nucleus

When compared with the structural and functional properties of known transcription factors and nuclear matrix constituents, the features of cytoplasmic IF proteins would easily fulfill the requirements for labeling the various aggregation forms of the cytoskeletal components as gene regulatory elements, if they had a nuclear localization. Can the cell indeed make use of these reactivities to regulate its activities at the gene level? One absolute prerequisite for cytoplasmic IF proteins to exert a direct influence on gene activity is that they enter the nucleus and establish physical

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contact with chromatin. However, until recently, no cytoplasmic IF proteins could definitely be localized within the nucleus by immunofluorescence and immunoelectron microscopy, a circumstance that gave rise to the dogma that they are strictly separated from their nuclear relatives by the nuclear membrane. This notion seems to be strengthened by the fact that, in contrast to most intranuclear proteins, cytoplasmic I F proteins do not possess a nuclear localization signal. However, it should be considered that immunological techniques do not furnish absolutely reliable results and that the failure to detect certain antigens cannot be taken as proof that they are not present in aparticular compartment. In the nucleus, IF proteins may be masked by intimate interaction with other nuclear constituents or hidden in locations that are not accessible to antibodies. For instance, the nuclear matrix protein HlB2 is completely masked in immunofluorescently stained interphase cells and only after removal of chromatin or after redistribution of the nuclear matrix during mitosis is the protein epitope uncovered (Nickerson et al., 1992). 1. Mechanisms of How Cytoplasmic Intermediate Filament Proteins May Traverse the Double Nuclear Membrane Since cytoplasmic IF proteins lack a nuclear localization signal, they have to draw on alternative mechanisms to overcome the barrier of the double nuclear membrane. That such mechanisms exist is indicated by the occurrence of large amounts of other cytoskeletal proteins in the interphase nucleus, such as actin, myosin, and tubulin (Douvas et al., 1975;Armbruster et al., 1983; Valkov et al., 1989; Lafarga et al., 1993; Milankov and De Boni, 1993). There are four major possibilities for how I F proteins may gain access to the interior of the nucleus: (1) IFs as such cross the perinuclear cisternae through the nuclear pore channels; (2) after posttranslational modification and release from IFs, activated IF protein oligomers are targeted to the nucleus by karyophilic carrier proteins; (3) posttranslationally modified and thus activated IF proteins or IF fragments are enclosed, along with other nuclear constituents, by the nuclear envelope during telophase of mitosis; (4) IFs traverse the double nuclear membrane directly. The often-observed enrichment of IFs immediately around the nucleus would certainly promote the translocation process. A fifth, almost trivial explanation exists and has been ignored by most investigators for years. The N-terminal domain of the IF proteins is readily released from the filaments as a result of proteolysis, both in vivo and in v i m (Shoeman and Traub, 1990a; Chen et al., 1993). While not all of the liberated N-terminal fragments would be expected to retain their complete DNA binding properties or an ability to dimerize, the largest fragments would. Since they are no longer immobilized on the surface of filaments

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and have a relatively small size (the full length of the vimentin N-terminus is 104 amino acids), they could not only diffuse from their point of creation (presumably near the nuclear envelope) but also enter the nucleus unrestricted through the nuclear pores. In the larger context of this chapter, these N-terminal fragments of IF proteins would probably function as (competitive) inhibitors of either their intact cognate IF proteins or perhaps of other unrelated DNA binding proteins. Thus, the function of I F proteins as filaments may include the sequestering of the DNA binding domain, the N-terminal head piece. At present, there is very little experimental evidence in favor of any one of the first four possibilities, yet the potential association of IF proteins with carrier proteins and their incorporation into subnuclear structures during telophase of mitosis deserves special consideration. In the course of nuclear disassembly, many of the nuclear matrix proteins are solubilized by post-translational modification, especially phosphorylation, and distributed in the cytoplasmic compartment (Nigg, 1992; Marugg, 1992). At the same time, cytoplasmic IFs are also subjected to extensive phosphorylation, causing their dramatic rearrangement or even disassembly (Evans, 1989; Robson, 1989; Chou et al., 1990; Eriksson et al., 1992b). It is conceivable that, as a consequence of this activation process, phosphorylated or otherwise modified protein dimers or protofilaments are released from the filaments to become associated with cytoplasmically dispersed nuclear proteins for which they show affinity because of similar structure. During telophase, the nuclear constituents are re-collected around the condensed chromosomes, bringing with them associated I F proteins, reassembled into nuclei, and enclosed by the nuclear envelope. One nuclear matrix protein endowed with such a carrier function could be lamin B, which interacts stably and specifically with the C-terminal tail domain of vimentin (Georgatos and Blobel, 1987a,b), desmin (Georgatos et al., 1987),peripherin (Djabali et al., 1991),and apparently also cytokeratin D (Bastos et al., 1992). In prometaphase-arrested cells, vimentin filaments were found to serve, in a phosphorylation-dependent manner, as a transient docking site for mitotic inner membrane vesicles carrying lamin B on their surface (Maison et al., 1993). Another potential carrier may be the nuclear matrix protein NMP 125, which in interphase cells is confined to a nuclear substructure with a granular aspect, whereas during mitosis it is freed into the cytoplasm to become associated with the vimentin filament network. In late mitotic stages, the protein forms punctate aggregates which are transported by the vimentin filaments toward the newly forming telophase nuclei (Marugg, 1992). It is possible that during nuclear reassembly vimentin molecules are channeled by NMP 125 or related proteins into the interior of the nucleus and deposited on the karyoskeleton.

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The possibility of a direct penetration of the nuclear membrane by cytoplasmic IFs is not as far-fetched as it first seems given that, first, bundles of IFs are frequently intimately associated with the nuclear surface (Franke, 1971;Goldman et al., 1986; Carmo-Fonseca and David-Ferreira, 1990; Aumuller et al., 1992)and second, the filaments have an amphiphilic character which allows them to not only establish tight contacts with but also to perturb lipid membranes (Perides et al., 1986a,b, 1987a; Ouellet et al., 1988; Asch et al., 1990). For instance, in thin sections of HeLa cells, the I F bundles were often seen to merge with the nuclear envelope, rendering it rather indistinct by obscuring the normal cisternal double membrane character at the attachment sites. Clear nuclear envelope outlines were observed in the immediate vicinity of these nuclear surface regions of indistinct membrane appearance. Intriguingly, the nucleoplasm underlying the filament attachment sites was found to be almost totally euchromatic and poorly developed or often totally lacking (Franke, 1971). It appears as if these changes were caused by a direct transmembrane attachment of the filaments to the nuclear lamina-chromatin complexes. In agreement with this view, the interaction between nuclei and IFs withstands cell extraction with nonionic detergents and high salt (Fey et al., 1984; Goldman et al., 1986; Katsuma et al., 1987; French et al., 1989; Willingale-Theune et al., 1989; Carmo-Fonseca and David-Ferreira, 1990; Bastos et al., 1992). In whole-mount preparations of the resulting karyocytoskeletal frameworks, the IFs are seen to be interwoven with the lamina filaments of the residual nuclei. Possibly these associations are mediated by the interaction of the tail domains of the IF subunit proteins with lamin B, but the involvement of other components of the nuclear periphery, such as further elements of the nuclear matrix or chromatin, cannot be excluded. The mechanism that allows the IFs to traverse the double nuclear membrane is not clear but may be intelligible on the basis of the capacity of IF proteins to interact with negatively charged lipid bilayers (Perides et al., 1986b). As in the case of the binding of IF proteins to nucleic acids, their interaction with lipid membranes is determined by their positively charged, N-terminal head pieces (Perides et al., 1987a; Ouellet et al., 1988). The N-terminus of vimentin, and also that of the mouse type I1 keratin Endo A (the mouse homolog of human keratin No. 8) (Ouellet et al., 1988), was shown to be an amphiphilic polypeptide able to adopt some a-helical conformation upon interaction with artificial membranes prepared from anionic phospholipids. In addition to this, the a-helical rod domain of I F proteins shows a high affinity for neutral lipids, including long-chain fatty acid esters and diglycerides (Traub et al., 1987). Owing to the specific properties of its head and rod domain, vimentin in its protofilamentous as well as filamentous form is capable of causing aggrega-

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tion, leakage, and very likely also fusion of negatively charged phospholipid vesicles (Horkovics-Kovats and Traub, 1990). In the presence of excess filament material, the vesicles are completely destroyed and the filaments themselves are heavily fragmented (Perides et al., 1986b). Although the N-terminus is highly essential for filament formation and stability, it appears from the above data to be available for interactions with negatively charged macromolecular assemblies. This should also be of direct relevance to the interaction of vimentin filaments with the nuclear envelope, particularly if it is considered that the alignment of multiple, positively charged, amphiphilic N-termini on the filament surface and the combined action of many filaments as filament bundles will foster the formation of negatively charged membrane microdomains, thereby promoting further membrane perturbance and vimentin translocation into the nucleus. In this connection, it is pertinent to refer to the close similarity between the N-terminal head domains of some IF proteins and the signal peptides of mitochondrial precursor proteins. Mitochondria1 presequences can translocate into protein-free phospholipid vesicles in an electrical potential-dependent manner (Maduke and Roise, 1993), perhaps by a partial disruption of the bilayer structure (Roise et al., 1986). This inherent ability of the charged and hydrophilic polypeptides to pass through a hydrophobic lipid barrier was envisioned as an essential activity for sorting of precursors destined for mitochondria. Since a potential difference was also described to exist across the nuclear membranes (Matzke and Matzke, 1991), it seems possible that the amphiphilic head domains of I F proteins channel the molecules in a similar way through the nuclear envelope into the nucleus. The translocation process might involve IF protein-induced interbilayer contacts between the outer and inner nuclear membranes in analogy to such contacts produced between cardiolipin-containing unilamellar phosphatidylcholine vesicles by mitochondrial presequences (Leenhouts et al., 1993). Electron-opaque material in the perinuclear cisternae of various cell types connecting the inner and outer nuclear membrane faces (as well as the outer nuclear membrane and the outer mitochondrial membrane) may represent intermediate membrane links in this translocation process (Franke et al., 1973). An intensification of the membrane-perturbing effect of the IFs should occur if the C-terminal tail domains, which, owing to their net negative charge, partially compensate the effect exerted by the N-terminal head domains, are removed from the filament surface. Indeed, transfection of cells with cDNA coding for C-terminally truncated vimentin (Eckelt et al., 1992) or cytokeratins 8 and 18 (Bader et al., 1991) caused massive accumulations of the tail-less IF proteins in the nucleus. The same observa-

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tion was made after transient expression of a C-terminal deletion mutant of the small neurofilament triplet protein in cultured cells (Gill et a / . , 1990). The displacement of the mutant proteins to central areas of the nucleus may be explained because, since they lack the C-terminally located lamin B interaction sequence, they are not retained in the nuclear periphery by the nuclear lamina. It should be noted, however, that tail-less cytokeratin mutants also lacking their head domains accumulated in the interior of the nucleus as well. In these cases, a carrier-mediated transport of the rod domains employing a-helix-rich nuclear matrix proteins or proteins of the NMP125 type as vehicles may be operative. The specification of an all-encompassing general mechanism of IF protein translocation into the interior of nuclei becomes even more difficult in view of the finding that ectopic synthesis of the epidermal cytokeratin KI in pancreatic islet cells of transgenic mice under the control of the rat insulin promoter produced fibrous or granular accumulations of the K1 protein in the nucleus, but, strangely enough, not of its identically expressed partner, cytokeratin K10 (Blessing et a / . , 1993). In any event, these data show that cytoplasmic IF proteins are not absolutely or obligatorily excluded from the interior of the nucleus and that there are ways in which such material, possibly only in structurally modified, activated forms, can overcome the nuclear membrane to establish contacts with intranuclear structures and constituents. In support of this, IF proteins like vimentin and cytokeratins were found covalently cross-linked to DNA in normal cells (Cress and Kurath, 1988) and cells exposed to UV light or chemical cross-linking agents (Galcheva-Gargova and Dessev, 1987; Wedrychowski et af., 1986a,b). Moreover, an increased accumulation of several IF-like DNA binding proteins in the nuclei of regenerating optic nerves of the goldfish were detected and hypothesized to change the transcriptional activity of chromatin (Gossels et al., 1992). Constraints imposed on chromatin motion (nuclear rotation) by IFs in interphase nuclei of murine dorsal root ganglion neurons (Hay and De Boni, 1991), and the observation that agents altering the rate of chromatin motion also change gene expression (De Boni, 1988),can be cited as further support of physical contact between IFs and chromatin. The detection of substantial amounts of vimentin in the histone-depleted protein cores of Chinese hamster metaphase chromosomes is suggestive of the participation of the IF protein in the construction of the interphase nuclear scaffold, but they also may be contaminants picked up by the chromosomes during their isolation (Gooderham and Jeppesen, 1983). There are several reports on immunofluorescence microscopic detection of IF proteins in the nucleus, including monoclonal antibody detection of a vimentin epitope in chromatin structures and chromosomes (Fidlerova et a / ., 1992).

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2. Potential Interaction of Intermediate Filaments with Repetitive DNA Sequences in the Nuclear Periphery If cytoplasmic IFs protruding into the nucleus through the nuclear membrane were indeed prevented from advancing farther into the interior by the nuclear lamina, then they must necessarily fulfill their regulatory functions in the nuclear periphery. They possibly produce stable associations with chromatin through interaction with extended, repetitive DNA sequences; fix these sequence elements in the nuclear periphery; and thus dynamically modify chromosome distribution and chromatin organization. These reflections are relevant in light of the cell type-specific expression of IF proteins and the nonrandom arrangement of chromosomes and chromosomal domains in the interphase nuclei of differentiated cells (Manuelidis and Borden, 1988; Manuelidis, 1990; Billia and De Boni, 1991). We postulate that, depending on the differentiation state of cells and tissues, the various cytoplasmic IF systems participate in the maintenance of defined chromosome distributions and chromatin configurations, which in turn permit or even drive the expression of distinct sets of cell- and tissue-specific proteins. In this case, similar to the function of nuclear matrix (lamina) proteins, IFs may stabilize the arrangement of tandem repetitive sequence blocks in locus-specific superstructures whose potential genetic function is not determined by the primary DNA sequence composition of the blocks but largely by their ability to develop and maintain specific chromatin folding structures (Vogt, 1990, 1992). Depending on the nature of the repetitive DNA sequences, these will be incorporated by the IFs either into open, transcriptionally active or into condensed, transcriptionally inactive chromatin complexes, as substantiated by the opposition of nucleus-associated, cytoplasmic IFs to eu- and heterochromatic regions underneath the nuclear envelope on electron micrographs (Franke, 1971). Although substantial fractions of the nuclear chromatin may thus be inactivated through condensation in the extreme nuclear periphery, they can nevertheless exert a long-range, activating influence on chromatin regions located more toward the interior of the nucleus. Operating, for instance, as repressors, IFs might interact via their surface-exposed N-termini with the G-rich, repetitive sequences of chromosome telomeres, which are often seen to be localized adjacent to the nuclear envelope (Manuelidis and Borden, 1988; van Dekken et al., 1989; Billia and De Boni, 1991; Gilson et al., 1993; Wang et al., 1993) and for which their subunit proteins show high affinity (Shoeman et al., 1988; Shoeman and Traub, 1990b). This would be consistent with the observation that the telomeres are attached to the nuclear matrix via their G-rich

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repeats (De Lange, 1992). However, since nuclear envelopes of mammalian origin were found not to be enriched for telomeric DNA, the lamins can be ruled out as major telomere receptor molecules. Is it possible that instead of lamins, cytoplasmic IFs interwoven with the lamina are at least partially responsible for the associations observed? We believe the answer to this question may be yes, given that vimentin exhibits a 10 times higher affinity for oligonucleotide telomere models than isolated lamins A/C; lamin B is virtually inactive in binding to telomere sequences (Shoeman et al., 1988; Shoeman and Traub, 1990b).Since the affinity of vimentin for telomere sequences was measured employing rather short oligonucleotide telomere models and low ionic strength, at which the IF protein prevails in its tetrameric form, a much stronger interaction should be expected for extended telomere repeats as they occur in uiuo and large vimentin polymers. The composition of mammalian telomeres of closely spaced nucleosomes (Makarov et al., 1993) should not interfere with their association with IFs since their constituent core histones could be deposited after unfolding, if necessary, on the a-helical rod domains of the IF proteins. Such telomere-IF interactions could potentially be of wide-ranging physiological relevance in that IFs might (1) affect telomere elongation and degradation activities, (2) control the organization of chromosome regions into transcriptionally inactive heterochromatin or heterochromatin-like structures, and (3) be involved in telomere clustering (“bouquet” formation) near the nuclear membrane. All these activities would be analogous to those of several yeast protein factors. For example, the repressoractivator and telomere-binding protein RAPl plays a role in telomeretelomere interaction and in telomere length regulation (Conrad et al., 1990; Kyrion et al., 1992), the latter function apparently being related to transcriptional silencing by this factor at the silent mating-type loci HML and HMR (Sussel and Shore, 1991). It is assisted by several SIR gene products that also act in transcriptional repression of the silent matingtype loci as well as at telomeres (Aparicio et al., 1991). Because one of these factors, the SIR4 gene product, possesses in its C-terminus a long, a-helical heptad repeat region related to the central dimerization domain of human lamins A/C (Diffley and Stillman, 1989), but apparently does not bind to DNA directly (Buchman et al., 1988), it was proposed to be a factor that brings telomeres up to the nuclear lamina via interaction with RAPl (Aparicio et al., 1991; Gilson et al., 1993). In the absence of the SIR4 protein, RAPl no longer aggregates at the nuclear envelope (Klein et al., 1992). The dependence of the efficiency of this telomere position effect on the size of the telomere tract favors either a multisite occupation of the chromosome by multiple RAPl molecules or other telomere-binding proteins in a cooperative fashion or the formation of higher order structures,

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including DNA loops (Kyrion et al., 1993). Through a similar, but direct association with telomere sequences, vimentin filaments and possibly other IFs anchored to the nuclear lamina could bring about a metastable telomeric position effect (Aparicio et al., 1991; Sandell and Zakian, 1992) by repressing euchromatic genes placed near telomeric, heterochromatic regions via a DNA looping mechanism. This would result in an effect similar to position effect variegation. Thus, IFs could achieve semistable inactivation of large chromatin regions at different stages of development and complement lamin B in its proposed function as a MAR-binding protein that silences genes in heterochromatic, peripheral regions of the nucleus (Luderus et al., 1992). Mammalian telomere repeats occurring at chromosome-internal sites have to be excluded from these considerations since they do not attach to the nuclear matrix (De Lange, 1992) and do not influence gene expression (Gottschling et al., 1990), although they appear to be incorporated into (pericentric) regions of constitutive heterochromatin (Meyne et al., 1990). Nuclear protein factors acting at the telomere level related to I F proteins of higher eukaryotes are exemplified by two homologous telomere-binding proteins from the ciliated protozoans Stylonychia mytilis (Fang and Cech, 1991) and Oxytricha noua (Raghuraman and Cech, 1989). Heterodimers consisting of a and /3 subunits recognize G-rich, single-stranded telomere repeat sequences and mediate the formation of salt-resistant, telomeretelomere interactions. On the basis of this self-association of the telomeric protein-DNA complexes, it was speculated that in uiuo the telomere binding proteins might self-oligomerize or interact with nuclear lamins and thereby mediate chromosome-chromosome interactions or attachment of chromosomes to the nuclear envelope (Fang and Cech, 1991). Interestingly, both subunit proteins possess a degenerate leucine zipper which may mediate their interaction in the heterodimer, and the a-subunits exhibit significant sequence similarity with the entire coiled-coil rod domain of the ssDNA- and telomere-binding protein vimentin. Although secondary structure prediction did not reveal longer a-helix or coiled-coil structures in the vimentin-related region, the two telomere-binding proteins and vimentin seem to be of the same principal structure: they have nearly the same molecular weight and the almost identically long region of similarity is flanked by a highly positively charged N-terminal head piece with interspersed serine, threonine, and tyrosine residues, and by a shorter and neutral C-terminal tail region. Possessing separate DNA binding and protein-protein interaction domains, the a-subunits of both telomere-binding proteins resemble a number of transcription factors in their general structure (Fang et al., 1993). Very surprisingly, and against all expectations that only GC-rich DNA

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segments bind to vimentin filaments with high affinity, the latter also selected with great efficiency AT-rich sequences highly homologous to centromeric satellite DNA sequences from a mixture of mouse genomic DNA fragments (Wang and Traub, unpublished results). In conjunction with the observation that in certain cell types centromeres are preferentially localized to the nuclear periphery or the nucleolar surface in cell type-specific, nonrandom patterns that relate to the differentiation state of the cells (Spector, 1993; Haaf and Schmid, 1991), the potential interaction of nuclear lamina-anchored vimentin filaments with centromere regions may serve to repress active genes by packaging them into heterochromatic areas, thereby exerting a centromeric position effect. In addition to their interaction with telomere and centromere sequences, IFs could make use of their potential affinities for cis-acting elements of developmentally or tissue-specifically regulated genes to control gene expression. Among the mouse genomic DNA fragments binding tightly to vimentin filaments, those containing (GA), and (GT), repeats (X. Wang and P. Traub, unpublished results) are of particular interest. Both repeats are recognized in their single-stranded forms and, presumably in doublestranded, topologically restricted DNA, by a variety of DNA-binding proteins (Aharoni et al., 1993) and are highly interspersed in eukaryotic genomes, but significantly underrepresented in heterochromatin regions (Wong et al., 1990; Stallings et al., 1991). The absence of (GA), tracts from centromeric regions and their highly conserved, nonrandom distribution to the chromosomal arms of a variety of species from fish to human indicates an organizational and functional role for this repeat class in the control of gene expression. Purine tracts are known to occur frequently within regulatory regions of active genes and near recombination hotspots (Wells ef a/., 1988; Wong et al., 1990; Vogt, 1990). For instance, purine-rich stretches of nucleotides are important sequence control elements in the promoters of several genes expressed in rat pancreatic p-cells and in that of the c-myc oncogene, and are apparently recognized by a series of transcription factors in a more global fashion in transcriptional regulation (Kennedy and Rutter, 1992). Enhanced accessibility of the DNA to the recombination machinery seems to be facilitated by nucleosomal loss following nearby DNA-transcription factor interactions (Shenkar et al., 1991), a condition which could easily be achieved on the basis of the histone binding and other transcription factor-like activities of IF proteins. It was proposed that there might be a general tendency for recombination to occur preferentially in structural gene regions of the eukaryotic genome (Thuriaux, 1977). The structural and functional importance of purine-rich sequences is also strongly suggested by the identification of proteins that have affinity for the comple-

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mentary (CT), tracts occurring in gene promoters (Gilmour et al., 1989; Kolluri et al., 1992; Lu et al., 1993). A Drosophila protein, the GAGA factor, has been shown to bind to 4 regions of the Ubx promoter containing one or more sequences similar to a GAGAGAGC consensus sequence (Biggin and Tjian, 1988). The GAGA factor binds to a wide variety of housekeeping and developmental genes and has been colocalized at several heat shock promoters with the heat shock transcription factor HSF. The GAGA factor has also been shown to disrupt nucleosomes at a heat shock promoter in an ATPdependent fashion (Tsukiyama et al., 1994). These results are consistent with the prediction that the GAGA factor and HSF act in concert at many chromosome loci, with the GAGA factor setting up chromatin structure and the HSF exploiting it (Lu et al., 1993). Accordingly, the existence of a previously unidentified functional class of proteins-the chromatin modifying factors-has been proposed. These proteins, which serve to alter chromatin structure, would be expected to be of moderate abundance and to bind to many sites in chromatin (van Holde, 1994). The similarities between the GAGA factor and vimentin with respect to the binding of (GA),-tracts are immediately obvious, making it attractive to speculate that IF proteins belong to this new class of proteins: the chromatin modifying factors. The positions of (GT), repeats are also conserved in the genomes between closely related mammalian species, but to a lesser extent between distantly related species (Stallings et al., 1991; Wintero e f al., 1992). The findings that (GT), repeat sequences can enhance transcriptional activity of genes in plasmid constructs (Hamada et al., 1984) and that they are of the same nature and distribution in 30% of genomic sites in evolutionarily distant species such as rodents and humans (Stallings et al., 1991), point to a regulatory function for these sequences in the genome. In accordance with this notion, (GT), blocks were found in the 5’ upstream regions of many genes (Vogt, 1990; Naylor and Clark, 1990; Kingsley and Winoto, 1992) and are components of MARS of the human /3-globin gene cluster (Boulikas, 1993b). In addition to the selection of perfect (GT), repeats by affinity binding of genomic DNA fragments to vimentin filaments, imperfect (GT), sequences with short interruptions and compound repeats of the (GA), (GT), type were isolated (X. Wang and P. Traub, unpublished results). Some of these sequences are similar to GT boxes of enhancer-promoter regions recognized by DNA binding proteins of the SPl multigene family (Hagen et al., 1992; Kingsley and Winoto, 1992). Like (GA), blocks, (GT), repeats were also proposed to be a necessary, but not sufficient, condition for recombinational hotspots and to be the determining factor in what distin-

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guishes constitutive heterochromatin from euchromatin or facultative heterochromatin (Stallings et al., 1991). Their interaction with IFs may allow reversible condensation-decondensation of the respective chromatin regions. With respect to the potential involvement of IF proteins in recombination, it is significant that repeated sequences capable of forming Z-DNA were found to enhance genetic exchange in yeast and mammalian cell culture systems (Shenkar et al., 1991). Z-DNA formation, in turn, might be promoted by the release of core histones from chromatin regions containing (GT), tracts onto acceptor proteins, for example, IF proteins, a process which would be further facilitated by the reduced capacity of ZDNA to form nucleosomes (Nickel et al., 1982). Given the high affinity of I F proteins for G-rich DNAs, extended clusters of nonmethylated CpG in GC-rich islands come into consideration as additional gene elements being localized to the nuclear periphery by the lamina-anchored I F protein aggregates. While such islands are most often associated with the promoter regions of housekeeping genes and occupied by ubiquitous transcription factors, up to 40% of vertebrate genes with tissue-specific or limited tissue distribution may contain CpG islands in their 5' upstream and, in part, also in their 3' downstream ends (Bird, 1986; Gardiner-Garden and Frommer, 1987; Edwards, 1990; Larsen et af., 1992). Since the nucleotide sequences of the different islands are unrelated and present only once or a few times per genome, only a distinct selection of those sequences would interact with certain IF protein species. The islands generally occur in the same position relative to the transcription unit of equivalent genes in different species (GardinerGarden and Frommer, 1987), implying their selective recognition by evolutionarily conserved, tissue-specific DNA binding proteins (e.g., IF proteins). It was noted that due to the large size of the islands compared with proteins, multiple factor molecules are bound to the respective sequences in a cooperative manner (Bird, 1986), conditions that could comfortably be fulfilled by the large IF protein aggregates. Exploiting their high affinity for GC-rich DNA and core histones, the aggregates could disintegrate nucleosomes in the nonmethylated CpG islands, which is in agreement with the observation of only very low amounts of histone H1, strong acetylation of histones H3 and H4, and nucleosome-free regions in CpG island chromatin (Tazi and Bird, 1990). In addition, acting through their extended, scaffold-like structure, the IF protein aggregates could juxtapose the promoter regions of the CpG islands to G-rich enhancer elements, for instance (GA) or (GT) boxes, via loop formation, and thus activate gene expression.

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E. Gene Regulation by Intermediate Filaments and the “Reverse Transformation” Model

A literature search for evidence in support of the presumed function of cytoplasmic IFs as regulators of gene expression at the nuclearcytoplasmic border was successful insofar as it brought to light the extremely intriguing observations published by Theodore Puck and coworkers on the role of the cytoskeleton in genome regulation and cancer (Puck and Krystosek, 1992). These investigators used nick translation to study the compartmentalization of actively transcribed chromatin in interphase nuclei of cultured cells as a function of their differentiation state. Employing the same technique, previous studies had revealed a preferential localization of DNase I-sensitive sequences, that is, of actively transcribed chromatin, at the periphery of interphase nuclei from a series of actively growing cell species (Hutchison and Weintraub, 1985;De Graaf et al., 1990).

1. Role of the Cytoskeleton in Reverse Transformation of Malignant Cells Puck and co-workers extended these observations by showing that reverse transformation of malignant cells with CAMPleads to a massive exposure of transcriptionally active chromatin in a rim about the nuclear periphery; earlier, this fraction of chromatin had been sequestered in a largely inactive form within the nucleus. At the same time, the transformed cells regain the phenotype of differentiated cells. This involves normalization of cell morphology, the cytoskeleton, and cell membrane structure; density control of cell growth and elimination of growth in suspension are also initiated and new patterns of protein synthesis and processing are established. These results were interpreted to indicate that (1) each separate state of cell differentiation is characterized by a unique set of genes exposed in the nuclear periphery and maintained in this position by interaction with specific regulatory DNA binding proteins; (2) through exposure in the nuclear periphery, differentiation-specific genes are committed to be expressed via interaction with ubiquitous transcription factors, for example, members of the CREB multigene family (de Groot and Sassone-Corsi, 1993), and other effector molecules; (3) dedifferentiation of cells results from the sequestration of differentiation-specific genes, including growth control genes, in the interior of the nucleus; (4) in the malignant state, the primary lesion in genome exposure is located at the nuclear rim, at the convergence of the nuclear matrix and lamina and the nuclear membraneassociated cytoskeleton. It was also proposed that housekeeping genes are clustered around the nucleoli and are exposed in both normal and

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malignant cells; nucleoli are thought to act in the same way as the nuclear surface in allowing newly synthesized gene products to exit from the nucleus (Krystosek and Puck, 1990; Puck and Krystosek, 1992). Additional support for transcriptionally active chromatin in the nuclear periphery and the nuclear-nucleolar border region comes from an immunofluorescence microscopic study of linker histone distribution in rat kidney interphase nuclei. It revealed a collection of intensely labeled, irregular spots on or near the nuclear surface upon staining with anti-H1" antibody, as well as brightly fluorescent regions surrounding the nucleoli, denoting the location of active or potentially active genes (Breneman et al., 1993; Parseghian et al., 1993). This is in agreement with the preferential distribution of active RNA polymerase I1 in the nuclear periphery (Clark et al., 1991). Moreover, an investigation of the cell-specific transcription of neurohormone genes as well as of housekeeping genes in supraoptic neurons in response to osmotic stimulation showed, concomitant with a partial decondensation of chromatin clusters and activation of transcription, characteristic changes in the distribution of eu- and heterochromatin-specific nuclear particles. These alterations were initially restricted to the nuclear periphery and then progressively proceeded to the nuclear interior with increasing times of osmotic stimulation (Garcia-Segura et al., 1993). Taking mitotic chromosome condensation as a model, the gene sequestration process was envisaged as being induced by the attachment of specific filaments of the cytoskeleton to conserved, repetitive DNA sequences of the genome. Nonhistone proteins exhibiting affinity for both the respective chromatin regions and the various filament systems were considered as mediators of these associations. The prevention of reverse transformation by colcemid as well as by cytochalasin B suggested that microtubules and microfilaments are involved in genome exposure (Krystosek and Puck, 1990; Puck and Krystosek, 1992). Since, however, these cytoskeletal elements are interconnected with IFs (Schliwa et al., 1982; Hollenbeck et al., 1989; Zamansky et al., 1991; H.-Y. Yang et al., 19921, the effects observed also might have been due, at least in part, to distortion of the IF network. In fact, phosphorylation of vimentin followed by reorganization of the vimentin filaments from a perinuclear mass to a well-spread, cytoplasmically extended network was one of the earliest responses of the malignant cells to CAMP treatment, long before the other aspects of reverse transformation became evident. It was concluded from these results that in fibroblasts, specific gene expression is partially under the control of a normal vimentin filament system and, moreover, that in other differentiated cells or tissues, specific transcription is analogously regulated by the other types of IFs through their attachment to corresponding key repetitive DNA sequences (Chan et al., 1989). This hypothesis is supported by the observation that in

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cultured rat hepatocytes epidermal growth factor-induced DNA synthesis and cell division are preceded by selective phosphorylation of a 55-kDa cytokeratin and reorganization of the tonofilament system from a cell peripheral to a predominantly cytoplasmic distribution (Baribault et al., 1989). The role of IFs in the proposed mechanism of reverse transformation is strikingly similar to the hypothetical gene-regulatory function of such material derived from its in uitro affinities for a variety of nuclear constituents. Both views consider the interaction of repetitive DNA sequences with IFs in the nuclear periphery as essential components of differentiation-specific gene expression. However, whereas the reverse transformation model envisages the participation of accessory proteins as mediators of these interactions, we believe the IFs themselves could fulfill these functions directly by virtue of their DNA- and histone-binding activities. Even actin (microfilaments) and tubulin (microtubules) may exert direct effects on chromatin organization in interphase nuclei. Actin was shown to be a MAR-binding protein (Ivanchenko and Avramova, 19921, within cross-linking distance to DNA (Miller et al., 1991), a component of the nuclear matrix (Nakayasu and Ueda, 1986; Valkov et al., 1989), and to play a role in transcription (Scheer et al., 1984). In addition, it is transferred into the nucleus in response to treatment of cells with the differentiation-inducing agent dimethyl sulfoxide (Sanger et al., 1980)and its intranuclear occurrence appears to be cell type-specific (Comings and Harris, 1976). The potential capacity of tubulin to directly act upon nucleic acids is indicated by its transcription factor-like activity in RNA synthesis for the negative-strand viruses, Sendai virus and vesicular stomatitis virus (Moyer et al., 1986), as well as measles virus (Moyer et al., 1990). However, microtubule-DNA interactions preferentially occur through the agency of microtubule-associated proteins (MAPs) which are also DNA binding proteins. An evolutionarily conserved interaction between MAPs and repetitive DNA sequences of unusually high GC content was demonstrated, in addition to the binding of MAPs to successive, short dA,/dT, runs in satellite DNA (Marx and Denial, 1992). Similar to IF proteins, tubulin shows a high affinity for core histones (Mithieux et al., 1984) and it also interacts with chromatin and isolated oligonucleosomes (Mithieux et al., 1986). Considerable amounts of microtubules appear in the nucleus after frog virus 3 infection of fibroblasts (Tripier-Darcy et al., 1980) or of paracrystals of tubulin after treatment of cells with Vinca alkaloids (Somosy et al., 1976).As a result of either cytodifferentiation or increased metabolic activity, an increasing incidence of intranuclear rod-like inclusions consisting of microfilaments and microtubules was found in developing neurons (David and Nathaniel, 19781, an observation also made after inhibition

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of protein synthesis in osmotically stimulated supraoptic neurons (Lafarga et al., 1993). Moreover, administration of cAMP analogs to neuronal tissues led to the intranuclear accumulation of actin and tubulin (Seite et al., 1977). Occasionally, the rod-like structures bridged the nuclear envelope (Masurovsky et al., 1970) or were seen to be connected to chromatin granules, occasionally penetrating dense areas of chromatin (Scheuermann er a/., 1988).

2. Activation of Intermediate Filaments by Post-translational Modification as a Prerequisite for Their Interaction with Intranuclear Structures The reorganization and functional modulation of cytoskeletal elements in response to cAMP treatment of cells is certainly the consequence of posttranslational modifications, most likely phosphorylation, of their protein constituents. Actin, tubulin, and their associated proteins are all substrates for diverse protein kinases. IFs particularly respond sensitively to phosphorylation by a variety of enzymes; the modification is largely restricted to the N-terminal head domains of their subunit proteins (Robson, 1989; Inagaki et al., 1989; Hisanaga et al., 1990; Yano et al., 1991). Since the N-termini are highly essential for filament formation and stability (Traub and Vorgias, 1983; Kaufmann et al., 1985; Hofmann and Herrmann, 1992; Raats and Bloemendal, 1992), their phosphorylation causes labilization or even disassembly of filament structure. The introduction of just one phosphate residue into a distinct site in the N-terminus of glial fibrillary acidic protein seems to be sufficient to provoke disassembly of glial filaments (Y. Nakamura et al., 1992). Limited, site-specific phosphorylation of vimentin and other IF proteins may similarly exert labilizing effects on their IFs (Geisler et al., 1989; Hisanaga el al., 1990).It is therefore conceivable that relaxation of filament structure by phosphorylation-induced loosening of the N-termini from the filament core will render these more reactive with negatively charged macromolecular assemblies such as lipid bilayers and nucleic acids. In this context, it is important to state that the putative multisite interaction of IF proteins with repetitive DNA sequences in the nuclear periphery requires the maintenance of extended, macromolecular IF protein assemblies, a prerequisite that seems to be fulfilled by the observation that, for example, vimentin filaments are impaired in their stability but not directly disassembled upon phosphorylation (Geisler et al., 1989). This balance between the effects of post-translational modification of the N-terminus on filament stability and filament interaction with other cellular constituents is possible because the N-terminus is essential for filament formation and stabilization but not over its full length. Vimentin

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mutants with considerable internal deletions in the head region are not significantly affected in their capacity to form filaments, as long as the gaps do not encroach upon an approximately 25-residue-long sequence at the very beginning of the vimentin molecule and the total length of the non-a-helical N-terminal polypeptide does not fall below a critical value (S. Kiihn and P. Traub, unpublished results). The initial 25-residue peptide contains a short consensus sequence which is fairly well conserved since it is also found, as more or less divergent variants, in the same position of other cytoplasmic IF proteins (Herrmann et al., 1992).Through interaction with the carboxy end of the rod domain of another IF subunit, it probably determines the staggered, antiparallel arrangement of two parallel-oriented IF protein dimers in the protofilament (Traub et al., 1992~).Through its interaction with the amino end of the rod domain, it may also be involved in the staggered and lateral alignment of the resulting tetrameric building blocks in the course of filament formation (Herrmann et af., 1992). Phosphorylation of this sequence element has a strong, inhibitory effect on filament assembly and stability. On the other hand, the deletable, N-terminal polypeptide region probably loops out from the filament assembly to become particularly susceptible to differential, post-translational modification and available for interactions with other cellular constituents. Among these, the association with negatively charged lipid bilayers and the binding to nucleic acids may be of particularly great physiological relevance. In any case, this region contains a large part of the antiparallel @sheet, nucleic acid-binding wing, and its modifications should not be of great influence on the stability of IFs. Phosphorylation of IF proteins may be counteracted by 0-glycosylation, another highly dynamic form of post-translational protein modification (Haltiwanger et al., 1992). 0-GlcNAc residues apparently block phosphorylation sites on proteins, so that there seems to be a reciprocal relationship between both reactions (Kearse and Hart, 1991). So far, among IF proteins, cytokeratins 13 (King and Hounsell, 1989), 8 and 18 (Chou et d., 1992) and the neurofilament proteins NFP-L and NFP-M (Dong et ul., 1993) were found to be 0-glycosylated; however, the inverse relationship between phosphorylation and 0-glycosylation could not be demonstrated for the cytokeratins (Chou and Omary, 1993). Since the respective glycosyl transferases use serinehhreonine-rich polypeptide regions as attachment sites (Haltiwanger et al., 1992) and such sites occur frequently in the Ntermini of many IF proteins, O-glycosylation could be a general means to modulate the functions of IF proteins (Chou and Omary, 1993). Indeed, the neurofilament proteins were found to be 0-glycosylated predominantly in their N-terminal head domains (Dong et al., 1993). 0-glycosylation is of special interest in the scope of IF protein function since many eukaryotic

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transcription factors, such as the GC box binding protein S P l , are considerably more efficient at activating transcription in their glycosylated than in their nonglycosylated forms (Jackson and Tjian, 1988; Reason el al., 1992). This may explain the preferential association of glycoproteins with the euchromatin regions of mammalian nuclei (Kan and Pinto da Silva, 1986). Concanavalin A-reactive forms of nuclear lamins found in several tissues (Ferraro et al., 1989) represent another example of glycosylated IF proteins in the nuclear periphery. They were suggested to be capable of particular interactions with other lamina or chromatin proteins, and to influence the anchorage of chromatin in the nuclear periphery. An identical function can be postulated for nucleus-associated, glycosylated, cytoplasmic IF proteins, in accord with the hypothesis that 0-glycosylation of proteins plays a role in nuclear targeting (Schin,dlerer al., 1987). Recently, sugar-dependent nuclear import of glycosylated proteins lacking the conventional nuclear localization signals was reported and suggested to depend on shuttling proteins like nuclear lectins or to make use of a so-farundeciphered sugar-dependent translocation mechanism (Duverger et al., 1993). A dynamic mode of post-translational modification of IF proteins intermediate between that of phosphorylation and 0-glycosylation may be that of ADP-ribosylation. Arginine-specific ADP-ribosyl transferase was shown to catalyze the modification of 2 to 3 moles of arginine residues per mole of soluble desmin, resulting in its total incompetence to polymerize into filaments. Much less incorporation of ADP-ribose was achieved employing preassembled desmin filaments. The fact that ADP-ribosylation inhibited phosphorylation by the catalytic subunit of CAMP-dependent protein kinase (Huang et al., 1993) localizes the site of modification to the N-terminal head piece of the IF protein. It is possible, therefore, that ADP-ribosylation of the N-termini of IF proteins contributes to the activation of IFs to allow their penetration through the nuclear membrane and association with chromatin regions in the nuclear periphery. This would add another class of proteins to the numerous chromatin constituents whose ADP-ribosylation was implicated in DNA replication and repair, gene expression, cell differentiation and transformation, or modification of chromatin structure (Ueda and Hayashi, 1985; Gaal and Pearson, 1986). Employing an ADP-ribosyl transferase isolated from cytoskeletonassociated mRNP particles, ADP-ribosylation and its negative effect on CAMP-dependent phosphorylation could also be demonstrated for neurofilament proteins. The same type of neurofilament protein modification was observed in isolated neurons deprived of their axons and dendrites (Jesser et al., 1993). Finally, partial relaxation and therefore activation of IFs could be

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achieved via limited truncation of their surface-exposed N-termini by Ca2+-activated, neutral proteinase (Perides et al., 1987b). Although such shortened I F proteins have a diminished capacity to form and maintain filaments, they still bind to nucleic acids (Traub and Vorgias, 1984) and also may largely retain their membrane-active properties due to their amphiphilic nature. The activation of IF proteins by post-translational modification would affect nuclear activities most directly and efficiently if it occurred in close neighborhood to the nucleus where the substrate concentration is highest and from where the distance to the potential nuclear target sites for the modified products is shortest. In fact, all the post-translational modifications of IF proteins and other cytoskeletal elements mentioned above could be the consequence of the active lipid metabolism taking place in the nucleus and its envelope along the nuclear polyphosphoinositide pathway (Cocco et al., 1990; Irvine and Divecha, 1992). Regarding the induction of reverse transformation of malignant cells by CAMP, it may be of relevance that in B lymphocytes a CAMP-generating signal transduction system or treatment with cAMP analogs activates translocation of protein kinase C (PKC) from the cytosol to the nucleus and hydrolysis of polyphosphoinositides (Cambier et al., 1987). Moreover, in various other agonist-stimulated, differentiated cell systems, cAMP analogs enhance the production of inositol phosphates (Baukal et al., 1990), and multiple mechanisms by which CAMP-dependent protein kinases potentiate inositol- 1,4,5-triphosphate [Ins( 1,4,5)P3]-induced Ca2+ mobilization are operative in permeabilized hepatocytes (Hajnbczky et al., 1993). Of course, cAMP and its analogs can provoke the phosphorylation of cytoskeletal proteins directly by activating cytoplasmic CAMPdependent protein kinases and of nuclear proteins via transfer of the respective catalytic and regulatory subunits to the nucleus (Ally et al., 1988; Meinkoth et al., 1990; Trinczek et al., 1993). In support of this, the association of the catalytic subunit of CAMP-dependent protein kinase with neurofilaments (Trinczek and Schwoch, 1990; Dosemeci and Pant, 1992) and keratin filaments (Joachim and Schwoch, 1988) was demonstrated. However, because of considerable cross-talk between the different signal transduction pathways (Houslay, 1991)and the high affinity and susceptibility of IF proteins and other cytoskeletal elements to substrates and enzymes of the polyphosphoinositide signaling system (Burn, 1988; Isenberg, 1991;Surridge and Burns, 1992),the activation of these elements may also be brought about by means of the latter pathway. Vimentin, for instance, is distinguished by an increasingly strong reactivity with phosphatidyl serine (PtdSer), phosphatidyl inositol (PtdIns), PtdIns(4)P, and Ptd1ns(4,5)P2,the latter two compounds being 40 times stronger in their influence on filament formation and stability than the

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potent ionic detergent, sodium dodecyl sulfate (Perides et al., 1986b). IF proteins also interact efficiently through their a-helical core domains with diacyl glycerol (DAG). DAG actually has a stimulating effect on the binding of vimentin to negatively charged phospholipid vesicles (Traub et al., 1987). Since during their interaction with the nucleus, which possibly involves receptor proteins, vimentin filaments bring a multitude of Ntermini in contact with the nuclear membrane in confined surface areas, they very likely will induce the formation of negatively charged membrane microdomains (Edidin, 1992; Glaser, 1993) with gradient distributions of the various anionic phospholipids that correspond to their reactivities with vimentin and other membrane-associated proteins. Once activated, possibly via a G-protein- or tyrosine kinase-mediated regulation system (Irvine and Divecha, 1992;Cockcroft and Thomas, 1992),certain phospholipase C isozymes, due to their high affinity for PtdIns(4,5)P2 (Rebecchi et al., 1992), will associate with the microdomains and cause in a Ca2+dependent reaction the production of DAG and Ins( 1,4,5)P,. Ins( 1,4,5)P, will then cause a rise of the free Ca2+ concentration in the immediate vicinity of the nuclear surface by release of Ca2+from the nucleus (Malviya et al., 1990; Nicotera et al., 1990; Matter et al., 19931, whereas DAG will induce the translocation and binding of cytoplasmic PKC to the nuclear envelope (Leach et al., 1989; Hocevar and Fields, 1991). The stable binding and activation of PKC is dependent on PtdSer, DAG, and Ca2+(Nishizuka, 1988) and also involves other phospholipid cofactors, for example, the polyphosphoinositides (Lee and Bell, 1991)and protein receptor molecules (RACKS), whereby these receptors may serve as substrates of the enzyme (Mochly-Rosen et al., 1991). In addition to this reversible and Ca2+-dependentPKC-nuclear membrane complex, a permanently active, Ca2+-independentform of the enzyme was described to be irreversibly inserted into the nuclear membrane (Nelsestuen and Bazzi, 1991; Buchner et al., 1992). In the present context, it is pertinent to note that in in uitro cultured baby hamster kidney (BHK) cells, certain PKC isozymes are associated with cytoplasmic filament networks, particularly vimentin filaments and microfilaments, and focal contacts; microtubules are free of PKC in these cells (Murti et al., 1992). Yet, association of PKC with microtubules was found in adult rat optic nerves, and here the decoration of neuro- and glial filaments with PKC was also prominent (Komoly et al., 1991).Moreover, a colocalization of PKC and phosphorylation-dependent immunoreactivity of neurofilaments was detected in intact, decentralized, and axotomized rat peripheral neurons (Roivainen et al., 1993). Most striking was the accumulation of phosphorylated neurofilaments and PKC in neuronal perikarya after axotomy or deafferentiation, with a concentration of the enzyme at the nuclear membrane and punctate nuclear staining detectable

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with phosphorylation-sensitive neurofilament antibodies (Shaw et al., 1988; Roivainen et al., 1993). This phenomenon is in contrast to the absence of phosphorylated neurofilaments from the perikarya and dendrites of normal neurons and may be interpreted as an attempt by the affected neurons to regenerate the injured axon by reprogramming their nuclear activities. Multiple PKC isozymes have been localized to the cytoskeleton and to the nuclear periphery in rat cardiac myocytes (Disatnik et al., 1994). That the association of PKC with IFs and their phosphorylation is a general phenomenon was demonstrated by the detection of the enzyme also on cytokeratin filaments in epithelial cells (Omary et a/., 1992). This may be a function of the affinity of the enzyme for the arginine-rich Ntermini exposed on the surface of the filaments (Leventhal and Bertics, 1993). Being excellent substrates of PKC, IFs and other cytoskeletal elements will be preferentially phosphorylated at those intracellular locations where the requirements of enzyme activation are optimally fulfilled, namely in direct association with membrane systems, such as the plasma and nuclear membrane. However, all the polyphosphoinositide-dependent reactions thought to proceed on the nuclear envelope also appear to take place in the interior of the nucleus, as evidenced by the phosphorylation of lamin B and other nuclear proteins by PKC (Fields et al., 1988; Hornbeck et al., 1988; Beckmann et al., 1992; Hocevar et a/., 1993). As pointed out above, 0-glycosylation may limit the phosphorylation of IF proteins, but in addition, the introduction of only carbohydrate residues into their N-termini may provide them with specific affinities for other cellular, preferentially nuclear, constituents. The fact that shortterm activation of T lymphocytes causes a rapid and transient decrease in the levels of GlcNAc in cytosolic proteins and a concomitant increase in the glycosylation of nuclear proteins (Kearse and Hart, 1991), strongly suggests that this reaction cycle is governed by a regulatory system similar to that controlling stimulus-induced phosphorylation of cellular proteins. The much higher turnover of 0-GlcNAc residues on cytokeratins 13, 8, and 18 in comparison to that of the respective protein backbones (Chou et al., 1992) could prove these proteins to be components of regulatory circuits. It remains to be seen whether 0-glycosylated proteins, including IF proteins, are components of signal transduction pathways comparable to those regulating phosphorylation, and whether these pathways are confined to strategically important membrane systems. The release of Ca2+from the nucleus in the process of polyphosphoinositide signaling will also activate Ca2 -dependent neutral thiol proteinases. The low Ca2+-requiringform of the enzyme is positively regulated by polyphosphoinositides (Saido et a/., 1992), in the same order as these show affinity for phospholipase C and vimentin, and they act as phospholipid +

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cofactors in PKC activation. In a series of transformed cells, it was found predominantly associated with the perinuclear area, whereas in another cell line it was primarily located within the nuclei (R. D. Lane er al., 1992). Significant quantities of the proteinase were also detected in the nuclei of rat skeletal muscle cells (Kumamoto er al., 1992). A particularly interesting distribution was observed in transformed Schwann cells where fluorescent antibodies against the proteinase labeled, in addition to diffuse immunostaining in the cytoplasm, a sharp ring around the nuclei indistinguishable from that obtained by immunofluorescence staining of the nuclear lamina. Isolated primary Schwann cells showed the same enzyme distribution (Banik et al., 1991). Furthermore, imrnunoelectron microscopy of proerythroblastic human leukemia cells induced to differentiate by phorbol ester revealed dense deposits of enzyme-antibody complexes in the karyoplasm and particularly on the outer nuclear membrane (M. Nakamura et al., 1992). On the other hand, in rat brain and spinal cord, reaction products were observed by the same technique in association with microtubules and glial filaments (Perlmutter et al., 1988). In accordance with the “activation of the membrane” theory (Suzuki and Ohno, 1990), the unusually high concentrations of Ca2+-activated proteinase around and in the nucleus and in part in association with cytoskeletal structures, may provoke limited proteolysis and thereby activation of the filament- and filament-associated proteins as soon as optimal concentrations of free Ca2+ have been attained at the nuclear-cytoplasmic border. Among cytoskeletal proteins, the N-termini of IF proteins (Nelson and Traub, 1983) and microtubular MAPS belong to the most sensitive substrates (Shoeman and Traub, 1990a), whereas among the constituents of the nuclear periphery, the lamins are selectively cleaved by a proteinase intimately associated with the nuclear scaffold and with functional properties very similar to those of Ca2+-activated neutral thiol proteinase. However, this enzyme was identified as a protein with a predominantly chymotryptic-like substrate specificity (Clawson er al., 1992). Because of the structural relationship between IF proteins and certain nuclear matrix proteins and transcription factors, it is noteworthy that treatment of purified rat liver nuclei with Ca2+ -activated thiol proteinase at low Ca2+ concentrations caused degradation of a number of matrix proteins and release of large complexes of these compounds, including a histone HI kinase. DNase I eliminated this effect, and it was shown that autoproteolysis of the enzyme to a low Ca*+-requiringform is dependent on a complex interaction of DNA with the proteinase and the nuclear protein substrates (Mellgren, 1991). In uitro-translated transcription factors c-Jun and c-Fos were also efficiently cleaved by the enzyme (Hirai et al., 1991). An extremely low Ca2+-requiringform of thiol proteinase, which undergoes autolysis rapidly after translation in skeletal muscle and

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which is localized on the cytoskeleton, at the nuclear membrane and in the nuclear interior, was suggested to be imported into the nucleus in order to regulate the levels of short-lived regulatory proteins such as transcription factors (Sorimachi et al., 1993). In conclusion, the joint occurrence of IFs and a variety of enzymatic post-translational modification systems in association with the nuclear envelope may provide the basis not only for the establishment of direct contacts between the IFs and intranuclear structures but also for their specificity. Owing to the heterogeneity of the family of IF proteins, the susceptibility of their N-terminal head domains to a wide spectrum of posttranslational modifications, the makeup of the various modifying enzyme systems of multiple tissue-specific isoforms of differential responsiveness to intra- and extracellular signals, and the multifaceted lipid metabolism and Ca2+ regulation in and on the nuclear envelope, eukaryotic cells possess myriad possibilities for regulating their DNA-based nuclear activities alone through their IF proteins. This situation is best exemplified by the phosphorylation state of IF proteins, which responds quickly and in a highly dynamic fashion to the activities of a multitude of protein kinases (see, e.g., Yano et al., 1991; Eriksson et al., 1992b) and phosphoprotein phosphatases (Eriksson et al., 1992a; Lee et al., 1992). Whereas it is conceivable that limited phosphorylation of the N-termini of IF subunits at variable sites, in conjunction with other IF-activating modifications, enables the IFs to penetrate the double nuclear membrane, specific phosphorylations at distinct sites in the N-termini may enable the IFs to select certain chromatin regions for transcriptional regulation. Changes in regulatory activity induced by phosphorylation, particularly site-specific phosphorylation, are a generally observed phenomenon among transcription factors, including effects on the regulation of nuclear translocation, DNA binding, and transactivation (S. P. Jackson, 1992; Hunter and Karin, 1992).

IV. Intermediate Filament Proteins as Potential Gene-Regulatory Elements in Differentiation Systems

Our attempt to fit the in uitro reactivities of I F proteins with other cellular components into current concepts of structure-function relationships governing gene expression and its regulation indeed revealed clear hints as to the involvement of these cytoskeletal elements as regulatory DNAbinding agents in differentiation processes. Suggestive as they are, they

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of course demand experimental support and proof. In this respect, it is appropriate, also for historical reasons, to refer particularly to the myogenic differentiation system, that is, to the cellular system that was employed for the first detailed description of the display and structure of IFs in relation to actin and myosin filaments (Ishikawa et al., 1968) and that during the past 25 years has furnished a large body of information on structural as well as functional aspects of IFs. A. Role of Desmin in Muscle Differentiation

In the course of their transition from replicating presumptive myoblasts to postmitotic mononucleated myoblasts and multinucleated myotubes, developing muscle cells change their IF protein complement by gradually replacing vimentin with desmin. Both proteins were considered to be functionally equivalent in constituting a mechanically integrating matrix which, during the assembly of striated, contractile myofibrils, organizes the lateral linkage of individual fibrils at their Z-discs and the connection of the Z-discs to the sarcolemma and other membranous organelles, including the nucleus (Lazarides et al., 1982; Lockard and Bloom, 1993). However, massive reorganization of IFs into thick cables induced by longterm treatment of myogenic cells with colcemid did not have any significant effect on the assembly of contractile myofibrils and the formation of multinucleated cells (Bischoff and Holtzer, 1968). Furthermore, destruction of the I F system by transfection of differentiating postmitotic myoblasts and multinucleated myotubes with cDNA that codes for desmin lacking a portion of the a-helical rod domain and the entire non-a-helical tail region still allowed the myoblasts and myotubes to assemble and laterally align normal striated myofibrils in a manner indistinguishable from contracting control myogenic cells (Schultheiss et al., 1991). These results clearly demonstrate that the IFs in developing and terminally differentiated muscle cells fulfill functions beyond or different from those of an integrating matrix, IFs as such might even be completely irrelevant for myogenic development. An indication as to a possible biological role of IF proteins was provided by analysis of the temporally differential synthesis of IF- and myofibrillar proteins during myogenesis. While the majority of early replicating presumptive myoblasts were found to be vimentin+/desmin- /myofibrillar protein-, all postmitotic mononucleated myoblasts and multinucleated myotubes were without exception vimentin+/desmin+. Interestingly, immediately after their coming into being, a number of postmitotic myoblasts were seen to be desmin+/myofibrillar protein-, but at this stage and thereafter there were never cells which were desmin- /myofibrillar pro-

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tein+. After fusion of these myoblasts into multinucleated myotubes and concomitant with their maturation, vimentin synthesis was drastically downregulated (Schultheiss er al., 1991). These results suggest that the synthesis of myofibrillar proteins is not only preceded by that of desmin but that it may even be under the control of the IF protein. This conjecture received further support from the characteristics of desmin and myofibrillar protein expression upon transfection of nonmuscle cells with cDNA encoding the myogenic transcription factor MyoD. Forced expression of MyoD induced primary fibroblasts and a number of immortilized and/or transformed cell lines to express several myofibrillar proteins and to fuse into multinucleated myosacs (Weintraub er al., 1989). Similarly, MyoD converted a series of cell types with decreasing affiliation to the skeletal myogenic lineage. Primary dermal fibroblasts, chondroblasts, smooth muscle, and retinal pigmented epithelial cells were converted into striated myoblasts and multinucleated myotubes in various nonmuscle environments consisting of different combinations of foreign extracellular matrix molecules (Choi er al., 1990). In the latter cases, the transfected cells were indistinguishable from normal skeletal muscle cells with respect to the localization of desmin and myofibrillar proteins in striated myofibrils and their ability to contract spontaneously. Concomitant with the differentiation to muscle cells, the endogenous differentiation program of the transfected cells was shut off. The most interesting result of these studies in regard to a potential nuclear function of IF proteins was again the difference in time when desmin and myofibrillar proteins were synthesized. At early time points after MyoD-cDNA transfection, the number of mononucleated desmin + / myofibrillar protein- cells greatly exceeded the number of mononucleated desmin+/myofibrillar protein+ cells, whereas in older cultures the myofibrillar protein+ cells predominated. As in normal myogenic cell populations, desmin-/myofibrillar protein+ cells were never observed. Also, the MyoD-converted cells withdrew from the cell cycle prior to the accumulation of myofibrillar proteins independent of the level of exogenous mitogens (Choi et al., 1990). It is clear from these observations that the synthesis of desmin is an early event in myogenesis (Li and Paulin, 1991), and it is possible, particularly on the basis of the temporally differential expression of desmin and myofibrillar proteins, that the IF protein somehow controls events lying farther downstream in the myogenic program and participates in the downregulation of the endogenous differentiation program of the transfected cells. Withdrawal from the cell cycle is, however, directly regulated by MyoD (Tapscott and Weintraub, 1991). A further important step forward in understanding the cellular function of desmin in myogenesis was furnished by the demonstration that the differentiation program can be interrupted by the prevention of desmin

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synthesis. This was achieved by transfection of differentiating myoblasts with DNA constructs designed to express antisense desmin RNA. In some cases, the program of cell fusion and myotube formation was totally suppressed, whereas in other cases of partial inhibition of desmin expression, this process was only delayed. The prevention of desmin synthesis had no effect on the expression of vimentin (Choudhary and Capetanaki, 1990). However, desmin is not capable of executing the terminal myogenic differentiation program on its own. In transgenic mice, desmin expression under the control of the 5' flanking sequences of the vimentin gene did not result in detectable developmental, morphological, or physiological abnormalities in vimentin-positive nonmuscle cells (Pieper et al., 1989), indicating that the potential gene regulatory function of desmin may have an effect only when the myogenic differentiation program has been initiated at earlier time points by higher ranking, muscle-specific transcription factors (such as MyoD). To successfully interact with their corresponding genomic DNA regions, these higher ranking factors in turn seem to depend on chromatin structures that are in part predetermined by vimentin (filament s). Taken together, these results lend strong support to the notion that desmin is directly involved as a control element in the process of muscle differentiation. Along the regulation cascade of myogenesis, heterodimers of MyoD or related transcription factors of the bHLH protein family and the protein product of the ubiquitously expressed bHLH gene E2A (Lassar et al., 1991) also transactivate the desmin promoter to initiate desmin expression (Li and Capetanaki, 1993; Li and Paulin, 1993). Desmin, in turn, may exert its regulatory function via interaction with a hierarchically subordinate group of cis-acting DNA sequence elements of muscle-specific protein genes and thus commit the cell to go through the terminal steps of the myogenic program. In contradiction t o the reverse transformation model (Puck and Krystosek, 1992), however, desmin apparently must not exist as a cytoplasmically extended filament network which is claimed to be maintained by sitespecific phosphorylation of its constituent I F protein molecules. Nevertheless, there is some indication that desmin filaments must be phosphorylated since blockage of PKA-catalyzed desmin phosphorylation by antibodies directed against the N-terminal phosphorylation sites inhibits incorporation of mononucleated myoblasts into multinucleated myotubes (Tao and Ip, 1991). Alternatively, this inhibition can also be explained by an interference of the microinjected antibodies with the interaction between the IFs and their corresponding repetitive DNA sequences in the nuclear periphery due to blockage of the N-terminal DNA-binding regions. In any event, these inhibition studies demonstrate a clear requirement of muscle cell development for an at least partially normal desmin display.

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The requirement of reverse transformation and maintenance of cell differentiation for a cytoplasmically extended IF network is probably not absolute and of secondary importance in the scope of the formation of cell shape and intracellular transport processes. While their precise configuration may be relatively unimportant, the presence of large, activated IF protein aggregates in association with the nuclear surface seems to be highly essential for these processes. What is required is the DNA-binding activity of the desmin N-terminus, which may be organized into either typical IFs or be present as large aggregates (like the zeste gene product of Drosophila). This point can only be addressed by the production of transgenic animals in which the expression of the appropriate endogenous IF proteins is completely shut off, rather than by the production of various dominant-negative mutant transgenic systems that have altered IF distributions. In the latter case, the DNA binding domains are still expressed in the cell and may still function sufficiently well to allow normal differentiation to take place.

6. Role of Vimentin in the Differentiation of Leukemia Cells

A similar involvement of an IF protein in differentiation processes was described for human promyelocytic and monocytoid leukemia cells induced to differentiate with dimethyl sulfoxide or phorbol ester along either granulocytic or monocytic-macrophage differentiation pathways (Bernal and Chen, 1982). The transitions were characterized by the appearance of several new cytoskeleton-associated proteins, one of which was vimentin, and the disappearance of several differentiation-sensitive proteins from the cytoskeleton in an orderly fashion: vimentin was synthesized in large amounts prior to changes in cellular morphology and the induction or loss of other cytoskeletal proteins. The cells produced lamins A/C as additional differentiation markers (Paulin-Levasseur et al., 1989). In extension of the suggestion that vimentin may play a role in the reorganization of the cytoskeleton to support the process of differentiation, it is hypothesized here that the I F protein exerts this function via interaction with specific, repetitive DNA sequence elements in the nuclear periphery. In related mouse plasmacytoma cells also induced to differentiate and express vimentin de nouo by treatment with phorbol ester, the I F system was found to make direct contact with the lipid-depleted nuclear envelope. In these cells, the differentiation process was accompanied by a halt in cell proliferation (Wang et al., 1989). However, differentiation of monocytoid leukemia cells along the monocytic-macrophage pathway can also proceed in the absence of vimentin (Taimi et al., 1990).

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C. Role of Glial Fibrillary Acidic Protein and Vimentin in the Extension of Cellular Processes

Differentiation in the form of extension of cell-type specific processes of astrocytes and neuroblastoma cells has been shown to be dependent on the presence of the IF proteins, glial fibrillary acidic protein (GFAP), and vimentin, respectively (Weinstein et al., 1991; Shea et al., 1993). Astrocytes, both in vivo and in in vitro model systems, respond to the presence of neurons in a characteristic fashion. When the astrocytes stop dividing, they produce cytokines essential for neurons and they differentiate, producing long cytoplasmic extensions. In an astrocytoma cell line stably expressing a GFAP antisense RNA, the ability of these cells to elaborate cellular extensions in response to neurons was dramatically reduced (about 28-fold), while the withdrawal from the cell cycle and the production of cytokines was unaffected (Weinstein et al., 1991). GFAP plays an important role in this process, although it is not sufficient, since the control astrocytoma cells contain normal GFAP IFs but do not produce cell processes unless stimulated by neurons (Weinstein et al., 1991). While this study did not address the mode by which GFAP participates in this developmental process, the possibility was raised that a neuron-induced, post-translational modification of GFAP, such as phosphorylation, represents the trigger that starts the cascade of events leading to the formation of stable astrocytic processes (Weinstein el al., 1991). The terminal development of neurons is characterized by a cascade of expression of IF proteins and neuron-specific proteins (reviewed by Liem, 1993 and van de Klundert et al., 1993) that accompanies the extension of cellular processes (axons and dendrites) and the development of cellular polarity. During the development of most neuronal cells, vimentin is initially- and then coexpressed with one or more neuron-specific I F proteins (such as a-internexin, peripherin, and nestin). As differentiation proceeds, neuron-specific I F proteins such as the N F proteins become abundant and vimentin synthesis is turned off. Details of this process have been studied using undifferentiated neuroblastoma cells in culture (Shea et al., 1993). These cells continuously extend and retract short neurite extensions. Upon treatment with dibutryl CAMP, a transient increase in vimentin expression is observed as stable neurite extension begins, followed by a concomitant down-regulation of vimentin and up-regulation of the N F proteins (Shea et al., 1993 and references therein) in a recapitulation of the changes seen during normal neuronal development. In neuroblastoma cells in which the vimentin IF network was perturbed by the microinjection of antibodies or the steady-state level of vimentin reduced by application of antisense oligonucleotides, the initiation of neurite outgrowth was inhibited (Shea et al., 1993).No effect was seen on the elonga-

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tion or stability of existing neurite extensions. This is in marked contrast to a similar approach employing an anti-tau antibody and antisense oligonucleotides (Shea et al., 1992)in which no effect was seen on the extension of these processes, or with antibodies to tau, tubulin, MAP1, and NFPH (Shea and Beermann, 1991; Shea et a / . , 1991,1992),in which the existing neurite extensions became labile and were retracted. Likewise, other studies have shown that peripherin and NFP IFs do not play a role in the elaboration of neurites (Shea et al., 1992; Troy et al., 1992). Thus the neurite-specific proteins are necessary for the stable maintainance of the neurite processes but do not appear to be involved in the initiation of neurite development. For both the astrocytoma and neuroblastoma cell lines, discussion is made of the possible structural role that GFAP and vimentin IFs may play in the production of cellular extensions, In both systems, differentiation normally occurs only in response to an extracellular signal (i.e., contact with neurons or treatment with a CAMPanalogue). This signaling presumably results in modulation of the activity of various cellular enzymes, such as kinases. It therefore appears as if the main result of these studies has been the demonstration of a formal requirement for a modified IF protein at the beginning, as an inducer, of a multi-step process. This is exactly the observation predicted if the real function of these proteins in these cells is, when activated by posttranslational modification, to turn on a group of genes whose products are necessary for the observed morphogenesis. D. Role of Cytokeratin Filaments in Imparting Mechanical Integrity t o Keratinocytes

If cell differentiation depends on normally extended IF networks or at least on large, cohesive IF protein aggregates in association with the nucleus, perturbation of these assemblies should cause an impairment of cell structure at a bare minimum and perhaps even alterations in function and development. Such disturbances in the display of cytoplasmic IFs are observed in certain pathological conditions of stratified squamous epithelia (Fuchs and Coulombe, 1992;Epstein, 1992).For instance, epidermis bullosa simplex (EBS) is an autosomal human skin disease which manifests itself in several variants characterized by more or less severe blistering of the epidermis upon mild physical trauma. The various forms, which are caused by point mutations in the highly conserved boundary peptides of the a-helical rod domain of keratins K5 (E. B. Lane e t a / . , 1992) and K14 (Coulombe et a / . , 1991a), have equivalents in the phenotypes of

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transgenic mice expressing dominant-negative mutants of these cytokeratins. In transgenic basal cells producing K14 mutant proteins with deletions in their N- and/or C-terminal regions, including boundary sequences of the rod domain, mechanical stress produces cell rupture in a narrowly defined zone midway between the nucleus and the hemidesmosomes, that is, in that region where the organization of the filaments is most perturbed and basal cells, because of their columnar shape, are most vulnerable to shearing forces. A strong correlation exists between the severity of the pathobiological effect in transgenic mice and the extent to which the antimorphic mutant proteins disrupt the keratin filament network in situ, perturb filament network formation in cultured transgenic and transfected keratinocytes, and affect keratin filament elongation and stability in uitro. To generate the observed phenotype, a critical ratio between the mutant and wildtype protein appears to be necessary. On the basis of these observations, the keratin filaments are thought to impart cell shape-dependent mechanical integrity to basal epidermal cells as a major biological function (Vassar et al., 1991; Coulombe eta!., 1991b). Another human hereditary skin disorder characterized by skin blistering, epidermolytic hyperkeratosis (EH), was traced to a defect in the stability of the keratin Kl/KlO network in the suprabasal cells. It is also caused by point mutations in the conserved end regions of the a-helical rod domain of the respective keratin species (Cheng et al., 1992; Rothnagel et al., 1992) as well as in a subdomain immediately in front of the central, ahelical region (Chipev et d., 1992). Elegant transgenic mouse experiments, employing a mutant K10 gene, essentially reproduced the EH phenotype (Fuchs et al., 1992). Although suprabasal cells are not columnar in shape but of more flattened appearance and therefore less susceptible to shearing forces, both mouse and human cells expressing the mutant keratin(s) display clear signs of cytolysis. In addition to keratin filament clumping, the affected cells exhibit perinuclear halos of clear cytoplasm, surrounded by shells of keratin filaments, and significantly distorted nuclei (Cheng et al., 1992). Such nuclear aberrations and the formation of perinuclear filament rings clearly separated from the nuclei are also seen, though to a lesser extent, in basal epidermal cells in EBS. In EH, enhanced mutant keratin expression is paralleled by an increase in all these abnormalities and by a gradient of general cell degeneration from lower to upper suprabasal cells (Cheng et al., 1992). The reason we have gone to great lengths to introduce this topic is that we believe the keratins are doing more in this system than providing structural integrity. Intriguingly and for unknown reasons, the perfectly stable K5/K14 keratin network of basal cells is replaced in the suprabasal

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cells by de nouo synthesis of a new network of keratins K1 and K10. Since the ultimate fate of the suprabasal cell layer is one of programmed cell dehydration and death as the cells are displaced toward the surface of the epidermis, this last-minute shift in keratin phenotype remains completely enigmatic from a structural standpoint. Thus, it is attractive to speculate that this shift in keratin phenotype reflects a requirement for an alteration in keratin function. As nucleic acid binding proteins in intricate association with the nucleus, keratins may, following the general concept outlined in this chapter, directly participate in the realization of the genetic program of epithelial cells by interacting with specific, repetitive DNA sequences in the nuclear periphery. This interaction very likely requires the regular arrangement of the N-terminal nucleic acid binding regions of the protein subunits on the keratin filaments or aggregates in order to generate a cooperative, multisite association of the cytoplasmic and nuclear macromolecular assemblies. Any severe disturbance of the keratin aggregates that results in the labilization or disorganization of subunit interaction and the aggregates’ association with the nucleus should impair the correct expression of the genetic program. Since the pathological effects characteristic of the above genetic skin diseases are caused by point mutations in regions of the affected cytokeratin species that are essential for IF assembly, the zeste gene product of Drosophila may again serve as an appropriate model to illustrate the molecular mechanism possibly underlying the disorders observed. The zeste protein engages in extensive protein-protein interactions through incorporation of its a-helical C-terminal region into coiled-coil structures. Parallel, staggered assembly of the subunits into theoretically indefinitely long structures, on which the N-terminal DNA binding domains are exposed, was suggested to be the molecular basis for aggregate formation. Point mutations that disrupt the hydrophobic ridges of the helices greatly reduce the extent of aggregation and thereby abolish the gene-regulatory activity of the protein factors. Hyperaggregation of the zeste protein has a similar, disadvantageous effect (Bickel and Pirrotta, 1990; Chen et al., 1992; Chen and Pirrotta, 1993a,b). It is conceivable that point mutations in critical regions of the oligoand polymerization motifs of cytokeratins provoke similar losses in generegulatory capacity. In this context, it is also noteworthy that, regarding the involvement of the nuclear lamina in the organization of interphase chromatin and hence in gene expression, assembly-negative phenotypes of lamins A/C were produced by point mutations at the ends of their ahelical rod domain (Heald and McKeon, 1990). In analogy to studies performed on cytokeratins (Bonifas et a!., 1991; Letai et a!., 1992), muta-

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tions in the non-a-helical head and tail regions or in the internal regions of the rod had no obvious effect on assembly. On the basis of these considerations, the morphological and physiological alterations induced by mutant keratin expression in suprabasal (and basal) epidermal cells on their way to terminal differentiation may be, at least in part, attributed to the changes taking place in and around the nuclei of the affected cells. At early stages of terminal differentiation, the relatively low amounts of assembly-incompetent keratin synthesized in the cells of the lower suprabasal layers cause only minor deviations from the normal structure and distribution of the keratin filaments or aggregates and their association with the nucleus. However, as the cells move outward toward the skin surface, thereby replacing the original K5/K14 keratins characteristic of basal cells with keratins Kl/KlO, and accumulating increasing quantities of the mutant proteins, the keratin aggregates gradually lose their normal subunit organization and stability and therefore abandon their interactions with structures of the nuclear periphery. In cells of the upper suprabasal cells, the communication between the keratin aggregates and the nucleus is totally interrupted in that these retreat into a perinuclear ring separated from the nucleus by a halo of clear cytoplasm. The detachment of the keratins from the nuclear envelope may also be the reason why the nuclei undergo changes in their shape. Their often binucleate appearance may be due to the existence of lobed structures, since the lobes are sometimes uneven in size and shape. On the other hand, according to the proposed mechanism of reverse transformation (Puck and Krystosek, 1992), suspension of the interaction of cytoskeletal elements with the nuclear genome may lead to sequestration of growth control genes into the interior of the nucleus, and to uncontrolled DNA replication and the formation of binucleate cells. In any event, changes in the organization of chromatin might be expected to cause alterations in gene expression and thus exert effects on the structure and stability of the cell and its organelles, including the cell membrane and the membrane-associated cytoskeleton. This also may apply to those structures in the cell periphery with which keratin filaments usually interact to establish and stabilize a cytoplasmically extended filament network. In this way, perturbation of the keratin filament network would exercise a direct as well as indirect influence c n the cells’ mechanical integrity. The unsuccessful attempts to culture transgenic keratinocytes expressing mutant cytokeratins and the aberrant keratinization of transgenic skin with its unprogrammed synthesis of keratin K6 in the suprabasal cells (Vassar et al., 1991)would make it intriguing to determine the general protein synthesis pattern of the diseased tissues and thus to detect possible changes in gene expression induced by the mutant keratins.

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E. Role of Intermediate Filament Proteins in Cultured Cells, Transgenic Animals, and Mammalian and Amphibian Early Development

The identification of a number of cell lines that do not express any of the classic cytoplasmic IF proteins (for references, see Section 11) shows that these are not absolutely essential for cell viability. However, we would be remiss if we failed to mention a point that referees repeatedly make: in these rare cases, caution is advised since the cells may contain either small quantities or cryptic forms of IF proteins that are not detectable by conventional analytical techniques, or even entirely new species of IFs that are not detected with the conventionally employed antibodies. On the other hand, the lack of cytoplasmic IF proteins is not in contradiction to their hypothetical role as differentiation-specific, gene-regulatory DNA binding factors since without exception these cells are transformed cells; that is, they are cells in which differentiation is by definition out of control. The observation that tissue cells adapted to cell culture express vimentin in addition to their cell-specific IF proteins (Franke et al., 1979; Virtanen et al., 1981; U. Traub et al., 1983) may be explained as a consequence of a partial dedifferentiation process. Since during development the expression of vimentin usually precedes that of the IF proteins typical of terminally differentiated cells, the additional vimentin synthesis may be considered a partial reversion of the cells’ original differentiation program. This, of course, also holds true for transformed cell lines, which commonly express vimentin. Microinjection of IF protein-specific antibodies into cultured cells or other manipulations of the IF network apparently do not cause significant changes in the pattern of gene expression (for references, see Section 11). The reason for the maintenance of the genetic program may be that those IF protein molecules or aggregates that are in tight association with the nucleus and therefore are masked, escape attack by the antibodies or other cell manipulations or even, as we have speculated before, there may be no requirement for an orderly network of IFs throughout the cytoplasm; a collapsed aggregate of IF proteins may still retain its DNA binding propensities. Alternatively, the regulatory function of the IF proteins may be taken over by other (related) protein factors as a consequence of a redundancy phenomenon (see later discussion). The coexpression of vimentin with cell-specific IF proteins in cell culture is neither overly disturbing nor completely foreign to most cells since an overlapping synthesis of the various types of IF proteins is also observed during normal development in situ (Traub, 1985; Steinert and Roop, 1988). Similarly, transgenic expression of desmin under the control of cis-acting elements of the vimentin gene in all kinds of normally vimentin-positive

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cells has been reported to have no effect on their morphology and physiology, with the exception of cataract formation in the eye lens (Pieper et al., 1989). However, this phenomenon is also observed upon overexpression of vimentin (Capetanaki et al., 1989)or the small neurofilament triplet protein (Monteiro et al., 1990) in transgenic mice. It may be due to a cytochemical effect of the overexpressed IF protein material on cellular membrane systems during eye lens formation rather than to an influence on gene expression, and may be caused by perturbation of the enucleation process in the lens fibers and of fiber fusion (Dunia et al., 1990). As in the case of transgenic expression of desmin in nonmuscle cells (Pieper et al., 19891, the synthesis of the neurofilament protein in other nonneuronal cells of transgenic mice is not accompanied by overtly different phenotypes. The apparent absence of such changes in cell structure and behavior is comprehensible on the assumption that the neurofilament protein exerts its gene-regulatory function only in those cells that have been committed to terminal differentiation through trans-acting protein factors that occupy higher positions in the gene regulation cascade of the neurogenic system. The same considerations may apply to the absence of any effect of ectopic lamin A expression on the differentiation state or potential of normally lamin A-deficient embryonal carcinoma cells. Here also, the indifference of the cells may be attributed to the possibility that they are in a state of noncommitment owing to the lack of other developmentally controlled factors (Peter and Nigg, 1991). On the other hand, transient expression of lamin A in normally lamin A-deficient chick embryonic muscle cells resulted in moderate and transient increases in the levels of several musclespecific proteins (Lourim and Lin, 1992). A follow-up report has described abnormal incisor tooth differentiation in transgenic mice expressing the desmin gene under control of the vimentin promoter (Berteretche et al., 1993). A totally different situation, however, was encountered when pancreatic islet cells of transgenic mice were forced to ectopically synthesize the epidermal keratins KI and/or K10, under the control of the rat insulin promoter, in addition to their endogenous keratins K8 and K18 (Blessing et al., 1993). The mice developed insulin-dependent diabetes mellitus whenever they expressed keratin K1 in the islet cells, but remained unaffected by keratin K10 when this was expressed alone. Histological examination of the pancreatic islands of keratin K1-positive mice showed disorders of tissue and cell structure, with a striking diminution of the number of insulin-containing secretory vesicles and extensive alterations in the IF system. Most prominent, and unexpected, was the accumulation of large quantities of filament bundles and unstructured aggregates consisting of keratins Kl and K18 (or K10) in the cell nuclei. Ectopic keratin K10 exhibited a normal, filamentous distribution in the cytoplasm and exerted

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no obvious influence on tissue and cell structure in the absence of keratin K1. The behavior of keratin KI in islet cells was surprising insofar as it showed a normal filament assembly competence and cytoplasmic distribution when transiently expressed, in the absence or presence of its natural partner, keratin K10, in a variety of cell lines. At present, there is no explanation for the differential, intracellular distribution of keratin K1 and K10 in islet cells and for the toxic, diabetogenic effect of keratin K1, although it is plausible that these phenomena rest on differences and peculiarities in the structure of both keratin species. A comparison of their N- and C-terminal extensions reveals a significantly higher net positive charge of both end domains in keratin K1 owing to the presence of multiple arginine residues. These features give the surface of filaments containing keratin K1 a particularly high positive charge which, together with a potentially amphiphilic character of the end domains, may manifest itself as a potent lipid bilayer-perturbing activity. Once having penetrated through the nuclear envelope into the interior of the nucleus, keratin K l and its partner protein may execute their nucleic acid- and histone-binding activities and change cell structure and function through their influence on chromatin organization and gene expression. However, this response of the islet cells appears to be unspecific and fortuitous since it results from the normally unprogrammed synthesis of a keratin species which, due to its peculiar chemical properties, is not compatible with the differentiation-specific structure of the islet cell nuclear envelope. It is possible that the IF-binding capacity of the nuclear lamina of islet cells, in contrast to that of in v i m cultured cells, is insufficient to retain keratin KI aggregates in the nuclear periphery. An uncommitted state with respect to the function of IF proteins is also characteristic of the omnipotent cells of mammalian early development. Mammalian eggs and very early embryonal cells are undifferentiated, and their IFs, which occur in large quantities in these cells, correspondingly exist as compact paracrystalline arrays with no obvious cellular function(s) (Bement et al., 1992; Capco et al., 1993). The dense IF masses, commonly referred to as sheets, probably represent an inert storage form (Plancha et al., 1989) of proteins that with progressing differentiation of the embryonal cells take over important roles at the genomic level and as information-transmitting cytoskeletal elements. Thus, mammalian early embryonal cells in a way resemble the IF protein-deficient cells of a number of dedifferentiated cell culture lines, at least with respect to the unavailability of IF protein systems for cellular functions. The IF sheet networks reorganize at key developmental transitions, such as egg fertilization, compaction, and blastocyst formation, in that they increasingly splay apart into individual IFs and make contacts with

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the plasma membrane and probably other cellular organelles. At the blastocyst stage, the sheets are still present in the cells of the inner cell mass but have been largely transformed into extended I F networks in the cells of the trophectoderm, the first differentiated cell type of the mammalian embryo (Bement et a f . , 1992; Coonen et a f . , 1993). The dispersion of the filament sheets may thus be a prerequisite for the establishment of contacts between IFs and the nuclear genome, and for further differentiation. If this speculation is true, then why did the microinjection of keratin-specific antibodies into 2-cell embryos not impair the initial stages of trophectoderm differentiation that results in blastocyst formation (Emerson, 1988)? Direct experimental support in favor of our hypothesis is the observation of associations of cytokeratin filaments with the nucleus already in 2-cell embryos of the hamster (Plancha et al., 1989); thus it may be that the antibodies cannot disturb the preformed, important interactions of the keratins with the nucleus (chromatin) or, alternatively, the function of the I F proteins is substituted for by that of redundant regulatory factors. Since the distribution of I F proteins in oocytes, eggs, and early embryos of amphibians is different from that in their mammalian counterparts-the IFs show a dynamic and asymmetric, open arrangement (Godsave et af., 1984; Klymkowsky et al., 1987; Bernent and Capco, 1990)-different effects of their manipulation on differentiation processes should be expected. However, the forced synthesis of excess amounts of vimentin mutants in fertilized Xenopus laevis eggs, mutant proteins that lacked either a large part of the arginine-rich, non-a-helical amino terminus or essential residues of the carboxy end of the rod domain together with the whole non-a-helical tail piece, did not cause any detectable morphological or developmental abnormalities, at least through the tail bud stage (Christian et a f . ,1990). While the combined expression of N-terminally truncated and wild-type vimentin obviously left the preexisting IF system intact, but nonetheless must have produced protein aggregates consisting of diand tetrameric entities with a proposed dominant-negative function at the transcription factor level, the C-terminal deletion mutant caused total destruction of the preexisting vimentin filaments. These data show that embryogenesis in Xenopus can proceed, at least during the initial stages, in the absence of an intact vimentin filament system as well as vimentin factors with an undisturbed gene-regulatory function. On the other hand, antisense depletion of maternal keratin mRNA in Xenopus oocytes prior to fertilization (Heasman et af., 1992)or microinjection of antikeratin monoclonal antibodies into fertilized eggs (Klymkowsky et al., 1992)led to abnormalities in gastrulation. Since transcription begins from the midblastula transition to the beginning of gastrulation, the impairment observed may well rest on disturbances in I F protein-regulated gene

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expression, in addition to the proposed effects on mechanically coupled cellular reorganization by disruption of the integrity of the keratin filament systems. That physiological and developmental cellular processes previously supposed to depend on cytoplasmically extended IF networks can proceed in the total absence of the cognate IF proteins was clearly demonstrated by employing cellular as well as organismal systems. Targeted inactivation of both mouse keratin K8 alleles in embryonic stem cells did not prevent the formation of a polarized and functional epithelium, the visceral endodermal layer, although keratin filaments are thought to provide epithelial cells with a stabilizing and organizing cytoskeletal network (Baribault and Oshima, 1991). To a limited extent, even whole vertebrate organisms get along fairly well without one or the other of the classic cytoplasmic I F proteins, as evidenced by the existence of mice lacking keratin 8 (Baribault et al., 1993) and of a neurofilament-deficient mutant of Japanese quail (Ohara et al., 1993). Upon targeted inactivation of both keratin 8 alleles, 6% of homozygous mouse embryos escaped the largely lethal embryonic defect characterized by an impaired integrity of fetal liver and growth retardation at the midgestational stage. The survivors developed to the adult stage without apparent histological abnormalities in those epithelia where keratin 8 is normally expressed. In addition, immunofluorescence microscopic analysis of such tissues with antibodies directed against the partner keratins 18 and 19 revealed the absence of these proteins from hepatic tissues and almost all intestinal epithelial cells. This effectively excludes functional redundancy with other keratin species as the basis for the survival of the mutant mice. Similar observations were made in the case of several embryonic and extraembryonic epithelia such as liver and visceral endoderm. Since vimentin filaments were also absent from these tissues, IF proteins seem to be dispensable for the formation of the trophectoderm and other extraembryonic epithelia. The dispensability of cytokeratin filament systems for the proper performance of intestinal epithelial cells is surprising in view of the extensive physical trauma these cells are exposed to. It is therefore conceivable that either the cytoskeletal function of cytokeratin filaments is taken over by other filament systems such as microfilaments and microtubules in combination with special, associated proteins, or the individual IF subunit proteins fulfill predominantly noncytoskeletal functions with a great potential for redundancy (see later discussion). Provided that the IF proteins act as gene-regulatory elements at the chromatin level, the probability that their inactivation or absence is compensated by non-IF protein factors would be tremendously increased owing to the myriad transcription factors cells can express. Similar considerations apply to the neurofilament-deficient mutant of

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Japanese quail insofar as neurofilaments are believed to provide neurons with structural stability and functional integrity. This system is of particular interest because the side-effects observed at the molecular level may allow some insight into the functioning of I F proteins in transcriptional regulation. The deficiency within the nervous system of the mutant animal in the small neurofilament triplet protein, NFP-L, is due to a nonsense mutation at amino acid position 114 in the 1A region of its coiled-coil rod domain. Although the animal cannot produce any NFP-L competent for filament assembly, it will certainly accumulate reasonable amounts of the mutant protein, which consists of the N-terminal head domain attached to three residual heptads of the 1A rod helix. The residual polypeptide includes two leucines of an originally longer leucine zipper (that is, in the intact, wild-type protein) together with about 20 amino acid residues that are located immediately in front of the rod domain of type I11 and type IV I F proteins and which are capable of a-helix formation (Geisler and Weber, 1983; Capetanaki et al., 1990). Both elements should allow the C-terminally truncated NFP-L polypeptide to dimerize. As a dimer of relatively low molecular weight, it may migrate into the nucleus and bind through its N-terminally located nucleic acid binding site to selected chromatin regions. In line with the previous suggestion that the expression of N F proteins is subject to a feedback control mechanism (Schlaepfer, 1983; Traub, 1985), the dimer may recognize preferentially cis-acting control elements in the NFP-L gene. Since, however, the polypeptide has lost its capacity to juxtapose enhancer elements to the promoter region via DNA looping and is also devoid of a transactivation domain but is still capable of binding and therefore blocking cis-acting sequence elements, it must necessarily repress transcription of the NFP-L gene. These considerations provide, at least in part, a reasonable explanation for the experimentally observed, -95% reduction in the amount of NFP-L mRNA and possibly also for the decrease in the concentrations of the NFP-M and NFP-H mRNAs. The cis-acting elements recognized by the truncated NFP-L dimer may be identical or similar to the CpG islands occurring in the 5’ flanking regions of all NF triplet protein genes (Bruce et al., 1993). Since the N F proteins are thought to have evolved as a single large subunit (Mencarelli et al., 1991), the CpG islands in the regulatory DNA sequence elements of the two larger NF proteins are very likely structurally related to those of the small N F protein and are probably recognized, though with lower efficiency, by its N-terminal polypeptide. In this respect, it is again noteworthy that IF proteins generally prefer G-rich nucleic acids for binding. Another example of potential feedback regulation of gene expression by IF proteins is the coordinate regulation of expression of polymerization partners of keratin IF proteins. In mouse cells, the suppression of mouse

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Endo B (a type I keratin) expression by antisense RNA technology resulted in the inhibition of expression of Endo A (Endo B’s normal polymerization partner) with a lowering of the level of stable Endo A mRNA (Trevor et al., 1987). In a somewhat more complicated follow-up experiment, mouse keratinocytes were transfected with an expression plasmid for human keratin K 18, the human homologue of mouse Endo B. In these cells, the total amount of stable type I keratin protein (endogenous Endo B plus human K 18) equaled that of the endogenous type I1 Endo A protein and the excess type I protein was degraded (Kulesh and Oshima, 1988). The stable proportion of the Endo B and human K 18 was not simply a function of the competition of the individual proteins for the partner Endo A keratin, rather a reduction in the rate of synthesis of Endo B was observed in these cells expressing the human K 18. In neither of these two sets of experiments was the level of gene expression at which the effect occurs identified: it may have occurred at the level of transcription or posttranscriptionally by affecting mRNA stability. Nonetheless, these results clearly are compatible with the proposition of a direct interaction of the IF proteins with the transcriptional machinery.

F. Redundancy of Intermediate Filament Protein Function

How does the dispensability of IF proteins for cellular functions fit into the concept of their role as regulatory factors in differentiation processes? Following previous suggestions (Christian et al., 1990; Baribault and Oshima, 1991), it may be that other (still unknown?) members of the IF multigene family or even more distantly related protein factors compensate for the loss or absence of function of a certain IF protein species and thus carry on the regulation cascade of the differentiating system. Functional redundancy between similar proteins is a common phenomenon among transcription factors. Two systems repeatedly referred to in this chapter for comparative purposes provide excellent examples. First, inactivation of MyoD in mice leads to upregulation of Myf-5, another member of the myogenic bHLH transcription factor family, and results in apparently normal muscle development (Rudnicki et al., 1992). Even in newborn mice homozygous for a targeted mutation in the gene encoding the related, muscle-specific bHLH transcription factor myogenin, a protein essential for the development of functional skeletal muscle and survival of neonates, some, albeit less abundant, differentiated muscle fibers with a normal sarcomeric organization can be detected (Hasty et al., 1993). Moreover, obliteration of the bHLH proteins El2 and E47, ubiquitous transcription factors that heterodimerize with tissue-specific bHLH proteins like MyoD, Myf-5, and others in mouse embryonic stem cells, leaves

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terminal differentiation of cardiac and skeletal muscle, erythrocytes, neurons, or cartilage unaffected (Zhuang et al., 1992). While, in a sense, the functional redundancy of I F proteins can be compared with that of families of highly homologous transcription factors that perform essentially the same function (Emerson, 1993), they also share features among themselves, with only partly overlapping activities that are integrated with other transcription factors into networks of combinatorial interactions. Because of its structural and functional similarity to I F proteins, the zeste gene product of Drosophila may serve as a representative of this class of factors. Null mutations of zeste still allow the development of fertile flies, although developmental difficulties arise at high frequency (Goldberg et al., 1989; Pirrotta, 1991). The parallels to the impaired development of seemingly normal but keratin %deficient mutant mice (Baribault et al., 1993) and to the formation of an intact suprabasal cell layer in spite of mutant keratin-based disturbances in the structure of the basal layer during epidermal differentiation (Vassar et al., 1991) are obvious. Despite the fact that the zeste protein is expressed in most cells of the embryo and is detected at more than 60 loci in polytene chromosomes, it does not act as a general activator that raises the level of transcription uniformly in all cells. Rather, it represents a strong, promoter-selective transcriptional activator which operates differently in different cells, obviously in redundancy or in concert with other transcription factors (Laney and Biggin, 1992). Even though a redundancy clearly exists, the function of the zeste gene product must be quite important given the high degree of evolutionary conservation of the sequence of the zeste gene among highly diverged Drosophila species (Chen et al., 1992). The same circumstances apply to I F proteins, as evidenced by their nonessentiality for a series of differentiation processes and their conservation during evolution. For instance, the homology between the vimentin complements of various mammalian species (mouse, hamster, pig, human) ranges from 95 to 99%. Even in the N-terminal head domain, the least conserved part of the vimentin molecule, the degree of identity is still 90 to 96%, with complete conservation of the sequence of the p-sheet nucleic acid binding wing. An interesting example of dispensability of elements of a transcription factor network, which is relevant to this discussion of redundancy of I F protein function, was described for an in v i m system for initiation of transcription by RNA polymerase I1 (Parvin and Sharp, 1993). Efficient initiation from a superhelical DNA template was observed in a minimum reaction containing only the TATA binding protein, TFIIB, and RNA polymerase. In this system, initiation from a linear DNA template required an additional five transcription factors (TFIIA, TFIIE, TFIIF, TFIIH, and TFIIJ). The function of these factors was proposed to be the promotion of unwinding of the promoter, an activity that in their absence may be equally

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well driven by the superhelical strain in the DNA template. Thus, at least two alternative (but functionally equivalent) pathways lead to successful transcription initiation with a minimal set of transcription factors: one employs accessory protein factors and the second uses a specific DNA configuration, that is, superhelical DNA. It is conceivable that the binding of IF proteins results in a DNA configuration that enables a minimum subset of transcription factors to initiate transcription. In the absence of I F proteins, other additional proteins would be required for transcription initiation. At this point it is appropriate to definitely state our basic concept of the role of IF proteins in the global regulation of gene expression. One must distinguish between a transcription factor being required for cell viability and that factor being involved in the expression of one or more genes, such as in cascades leading to differentiation of cell lineages. We propose that there is no formal requirement for either the expression or the DNA binding activity of the various IF proteins in the survival of a cell, since such a fundamental process is probably severalfold redundant. Basal (housekeeping) gene expression can probably be driven by many transcription factors, including I F proteins if they are present. On the other hand, we feel that expression of specific groups of genes, which leads to terminally differentiated cells, requires the presence and DNA binding activity of those cell types’ cognate IF proteins. These proposals fit with the experimental observations that cells lacking detectable cytoplasmic IF proteins are relatively dedifferentiated in morphology and function and with reports of blockage of differentiation as a consequence of expression of IF protein antisense RNA or oligonucleotides (Choudhary and Capetanaki, 1990; Weinstein ef al., 1991; Shea et al., 1993). This claim of a requirement for IF protein expression and correct, global, gene expression is clearly testable, for example, via the aforementioned antisense technology, by gene inactivation to eliminate endogenous I F protein expression, or by introduction (perhaps by recombinant ge.ne technology) of one or the other IF protein gene into cells not producing any known IF proteins, but of course expressing the other, higher ranking elements in the gene expression cascade.

V. Evolutionary Aspects

In the past, it was repeatedly argued that the adherence of cytoplasmic IF networks to the nuclear surface during the preparation of detergentresistant, residual cell structures may be an artifact resulting from the collapse of the IFs onto the nucleus upon delipidation of its outer double

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membrane. However, electron microscopic and biochemical observations as well as theoretical considerations based on in v i m interactions of IFs with structures in the peripheral, intranuclear zone support the notion that these associations also exist in the presence of the perinuclear cisternae. As far as assemblies consisting of I F proteins are concerned, the nuclear lamina seems to play a central role in the events taking place at the border between karyo- and cytomatrix, structurally as well as functionally. Through its lamin B component, it establishes contact with the cytoplasmic IF system and it is also connected to the 10-nm core filament network of the nuclear interior, although the chemistry of this interconnection is not yet understood. Functionally, the nuclear lamina is believed to interact in a developmentally regulated and differentiation-specific manner with distinct domains of interphase chromatin and thus to make an important contribution to the control of gene expression. A similar role might be assigned to the nuclear core filaments, whereas the potential of the cytoplasmic IFs to bind to chromatin could not yet be experimentally substantiated. Nevertheless, the structural and functional similarities that have so far been detected between the IF proteins of the nuclear lamina and those of the karyo- and cytoskeleton strongly suggest that the latter have derived as specializations from a lamin-like precursor to allow the evolving eukaryotic cell to cope with its increasing demands and tasks in a morphological as well as physiological respect. Indeed, the determination of the amino acid sequences of invertebrate and vertebrate nuclear lamins and cytoplasmic IF proteins, and studies on the intron-exon patterns of their genes clearly demonstrated that the cytoplasmic IF proteins are descendants of an archetypal, lamin-like molecule (Dodemont et al., 1990; Weber er al., 1991). The transition from nucleo- to cytoskeletal proteins was achieved by removal of two signal sequences characteristic of nuclear lamins, the nuclear localization signal and the isoprenylation site, both of which are located in the non-a-helical, C-terminal extension of the lamins. While in invertebrate cytoplasmic IF proteins the additional six amino acid heptads of the a-helical rod domain of the lamin-like precursor were retained, this region was also excised during evolution, leading to the vertebrate cytoplasmic IF proteins. The removal of the nuclear localization signal, of course, allocated the I F proteins a cytoplasmic distribution, whereas the extirpation of the isoprenylation sequence freed them from their membrane anchor. Whether similar evolutionary relationships exist between nuclear lamins and constituents of the internal nuclear matrix of vertebrates is at present unknown. However, because of the near morphological identity of nuclear and cytoplasmic 10-nm filaments, it is conceivable that a similar process has taken place, with the one major difference being that the internal nuclear matrix

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proteins lost only the isoprenylation site and the additional six core heptads typical of lamins, but retained their nuclear localization signal. Confirmation of this contention has to await the molecular and genetic characterization of the subunit proteins of the nuclear 10-nm core filaments. In addition to the structural changes the nuclear lamin-like precursor went through in its C-terminal tail domain during its transformation into cytoplasmic IF proteins, it also experienced striking alterations in its corresponding N-terminal head region. While the N-termini of the nuclear lamins are rather short, they considerably increased in size during the evolution of cytoplasmic IF proteins. Simultaneously, they acquired not only a high net positive charge due to the incorporation of multiple arginine residues but also a great potential for post-translational modification. The introduction of several phosphorylation and possibly 0-glycosylation and ADP-ribosylation sites is most remarkable. These changes made the Nterminal extension the most reactive part of cytoplasmic IF proteins. It must therefore be assumed to fulfill important functions not only in filament assembly but also in the interaction of IFs with other cellular constituents. For reasons of symmetry, we speculate that the subunit proteins of the intranuclear 10-nm core filaments acquired features most similar to those of the cytoplasmic IF proteins, probably as a result of co- or parallel evolution. That is, the split off in the IF protein family tree of the nuclear lamins and the other IF proteins occurred first and the separation of the nuclear and cytoplasmic 10-nm filament proteins is a relatively recent event. During the evolutionary transition from lamin-like precursor to cytoplasmic IF protein, the nucleic acid binding site shifted from the C- to the N-terminal extension, simultaneously substantially increasing its affinity for polynucleotides. Vimentin, for instance, has a 10 times higher affinity for synthetic oligonucleotide telomere models than the lamins A/ C. If the lamins indeed interact with polynucleotides, they probably employ their C-terminal tail domains (Collard et al., 1990; Hoger et al., 1991). They have an appropriate distribution of positively charged amino acid residues and enough structural flexibility to arrange these together with other important residues into the configuration of a reasonably efficient nucleic acid binding site. The non-a-helical head domains of the lamins are too short and of too low a net positive charge for this purpose. On top of that, they are involved in filament formation through head-to-tail interactions with the globular tail regions of other coiled-coil lamin dimers (Nigg, 1992). Binding of polynucleotides to the head domains would definitely disrupt these interactions. This function of the N-terminus in filament assembly was preserved during the transition of the lamin precursor to cytoplasmic IF proteins. However, the molecular mechanism of the polymerization reaction changed significantly in that the acceptor site for

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the N-terminus on a neighboring protein dimer was shifted from the Cterminal extension to the 0-helical rod domain of the subunit proteins. Simultaneously, and as already pointed out, the N-terminus was endowed with additional, internal sequence elements that allow it to interact with other cellular constituents without causing labilization of filament structure. The common evolutionary origin of nuclear and cytoplasmic I F proteins may have another manifestation at the level of intracellular localization and transport of ribonucleoprotein particles. A great part of the macromolecular material leaving the nucleus consists of various forms of RNA, such as mRNA, rRNA, tRNA, complexed by a large variety of proteins. Intranuclearly, this material seems to be attached during and after its processing to the 10-nm core filaments of the nuclear matrix and is possibly transported on them to the nuclear pore complexes according t o the gene gating model (Blobel, 1985; Spector, 1990; He et a / . , 1991; Carter et al., 1993; Xing et al., 1993). In the cytoplasm, its directed transport into peripheral zones of the cell appears to be taken over by the cytoplasmic IFs. For instance, cytoplasmic IFs were shown to carry prosomes, subcomplexes of untranslated, nonpolyribosomal mRNP particles (OlinkCoux et al., 1992) that are probably functional in the anisotropic distribution of mRNA in the cytoplasm (Singer et al., 1989), or RNP particles containing the Ro antigen (Carmo-Fonseca and David-Ferreira, 1990). That RNA-containing constituents of the protein-synthesizing machinery in general may be translocated along cytoplasmic IFs is supported by the capacity of in v i m reconstituted vimentin filaments to bind large quantities of ribosomal subunits (Traub et al., 1992b). In fact, in Go-arrested human skin diploid fibroblasts, the bulk of ribosomes and of elongation factor eEF-2 has been shown to have a distribution very similar to that of vimentin filaments (Shestakova er al., 1993). Even the complex reaction cycles of RNA virus replication apparently proceed in association with cytoplasmic IFs (Murti et al., 1988), suggesting a general requirement of nucleic acid metabolism and transport for a supporting, filamentous matrix. Concluding this section, we would like to briefly comment on some structural and functional parallels and evolutionary relationships between prokaryotic DNA binding proteins and eukaryotic IF proteins. At first glance, members of the two categories often appear to have similar charge distributions with a positively charged N-terminal half and a negatively charged C-terminal half. While the net positive charge of the N-terminal part serves to facilitate target location by the protein on negatively charged lattices, a partial (consensus) sequence in this region determines DNA binding specificity. The fact that organisms as far apart as phage-infected bacteria and vertebrates produce proteins, for example, G5P and vimentin,

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respectively, which use a nearly identical, antiparallel p-sheet structure for specific nucleic acid binding, suggests convergent evolution of this principle. However, there is one major structural difference between the prokaryotic DNA binding proteins and eukaryotic IF proteins: the latter contain in the middle of the molecule, separating the basic from the acidic region, a long a-helical domain with the potential to form coiled-coils. Since this a-helical rod domain is capable of binding core histones with characteristics very similar to those of the interaction of histones with DNA, we believe that both activities are functionally somehow connected and that the capacity of DNA binding proteins to also bind histones emerged during the evolution of the eukaryotic cell. While in prokaryotic cells DNA and RNA polymerases carry out their tasks, assisted by DNA binding proteins, on largely naked DNA, in eukaryotic cells the function of the polymerases is severely impeded by incorporation of the genetic information into chromatin by histones. Consequently, at least some of the accessory DNA binding proteins of eukaryotes must be equipped with core histone binding regions, such as the a-helical rod domain of IF proteins, that dislodge histones from nucleosomes and thus clear the way for the polymerases or other enzymes. Structurally similar proteins possessing a long a-helical domain capable of coiled-coil formation and flanked by a basic nucleic acid binding region appear to be common constituents of the nuclear matrix of eukaryotic cells.

VI. Summary and Perspectives The subunits of IFs represent a class of proteins which in v i m show high affinity for GC-rich nucleic acids and also react with a characteristic stoichiometry with core histones: one IF protein tetramer binds 16 histone molecules, possibly in the form of 2 histone octamers. While the nucleic acid binding site resides in the non-a-helical N-terminus of the cytoplasmic IF subunit proteins, the a-helical core domain is responsible for the interaction with histones. IF proteins resemble transcription factors in that members of both groups are composed of an a-helical dimerization sequence capable of parallel coiled-coil formation and a neighboring basic domain determining DNA binding specificity. Therefore, it is plausible that IF proteins may be able to operate as trans-acting factors in the recognition and activation of nucleosome-masked promoter and enhancer sequences and thus to participate in transcription initiation. In fulfilment of this function, they may take advantage of their tendency to form tetramers and higher oligomers and of the inherent capacity of these aggregates to

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engage in DNA loop formation by juxtaposing distant DNA sequence elements. All these activities would be promoted by the general potential of IF proteins to interact with supercoiled DNA. On the other hand, as oligomeric protein factors that combine supercoiled DNA and core histone binding sites in the same structural entity, IF proteins could serve as catalysts in transcription elongation in that, via their coiled-coil regions, they transfer histone octamers from positively supercoiled DNA in front of transcribing RNA polymerase to negatively supercoiled DNA behind it. Since the karyoskeleton possesses a large variety of coiled-coil proteins with a dimerization domain flanked by a basic DNA binding region, this might represent a general principle of facilitated, matrix-associated transcription elongation in interphase chromatin. Affinity binding of fragmented genomic DNA to vimentin filaments, and cloning and sequencing of the most strongly bound fragments identified a series of GC-rich, repetitive DNA sequences that are similar in overall sequence organization to DNA regions flanking genes on their 5' upstream side. Vimentin filaments also bind with high efficiency GC-rich telomere and, surprisingly, AT-rich centromere sequence elements. The failure to assign a true consensus DNA sequence to the binding sites of vimentin filaments may be because the interaction of IFs with DNA is dependent on DNA structure rather than on strict sequence specificity. The multisite attachment of tandem arrays of degenerate, near-consensus sequences to extended IF protein aggregates would nevertheless be stable and allow for gene regulation on aglobal scale (Zuckerkandl and Villet, 1988; Serrano et af., 1993). We suppose that, in accordance with the reverse transformation model developed by Puck and co-workers (Puck and Krystosek, 1992), the repetitive DNA sequence elements are located in the periphery of the nucleus, in direct contact with the IFs or other specifically organized IF protein aggregates associated with the nucleus. The interaction is mediated by the N-terminal DNA binding regions of the protein subunits that are exposed on the filament surface, whereas the C-terminal extensions prevent the filaments from penetrating deeper into the nucleus through interaction with the lamin B component of the nuclear lamina. It thus appears as if the accumulation of IFs often seen to occur around the cell nucleus provides a cytoplasmic scaffold for the organized interaction of differentiation-specific, regulatory DNA binding proteins with nuclear lamina-matrix-chromatin complexes localized just underneath the inner nuclear membrane. Through the interaction with specific, repetitive DNA sequences, which vary with the cell and IF type, the scaffold complements the nuclear lamina-matrix in maintaining a certain chromosome distribution and chromatin organization that are characteristic of the differentiation state of the cell. In this way, IFs may set the stage for the action of subordinate protein factors of the regulation cascades of developing

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and terminally differentiated cells, as well as for transcription factors of more general functionality. They may therefore be regarded as commitment proteins. Depending on the structural features of the repetitive sequences, IFs could act as repressors, for instance through incorporation of euchromatic genes into neighboring heterochromatic regions via a telomeric or centromeric position effect, or as activators by interaction with enhancer and promoter elements of genes to be transcribed. To be able to penetrate the double nuclear membrane, the IFs must be in an activated state, a condition dependent on the membrane-active character of their subunit proteins and maintained by post-translational modification mainly of their N-termini via a continuously operating signaling system. Since the N-termini of IF proteins are multireactive polypeptide chains, they possess a great potential for post-translational modification and therefore can be subject to multiple modes of activation in response to intra- and extracellular signals. While a particular IF system may determine a particular cellular differentiation state in a global fashion, fine tuning of gene expression could be achieved by modulation of the above-mentioned, continuously operating signaling system, for example, by phosphorylation of the N-termini in different sites by different protein kinases. The membranes of the perinuclear cisternae, particularly their lipid domains, seem to actively participate in these modifications. They contain the required IF-modifying enzyme-substrate systems and define the geometry and ionic makeup of the reaction space at the convergence of the nuclear envelope and cytoplasm. In this complex process, the IF proteins can accomplish their 2-fold task, that is, to break through the nuclear membrane and to interact cooperatively with extended, repetitive DNA sequence elements, in a coordinate fashion only if they operate in the form of large, cohesive, molecular assemblies. According to the reverse transformation model, unscheduled disturbances in the signaling systems extending between the plasma membrane and the interior of the nucleus may give rise to severe impairment of the interaction between the IF protein assemblies and peripheral structures of the nucleus, and thus to malignancy of the cell. The possibility should not be dismissed, however, that I F proteins may fulfill their nuclear functions also as smaller structural entities that, mediated by a nuclear carrier system, are in equilibrium with the IF protein complement of the cytoplasm. IFs represent fairly dynamic protein assemblies continuously exchanging in a nonpolar mode dimeric or tetrameric building blocks with a small pool of soluble or newly synthesized proteins. Nuclear matrix proteins synthesized during interphase or other carrier proteins could therefore pick up, due to their structural relatedness and affinity to IF proteins, soluble IF protein oligomers on their way to the nucleus, and deposit them on the nuclear matrix. Since the size of

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the soluble IF protein pool is not only a function of the inherent aggregation properties of the original IF subunits but also of their post-translational modification state, and this in turn varies with the cell’s physiological condition and differentiation state, the flux of IF proteins into the nucleus and transcriptional activity could be dictated by environmental as well as developmental stimuli. On the other hand, nuclear import of IF proteins may be temporally limited to mitosis, when both nuclear matrix and IF proteins are maximally activated by post-translational modification and, after their complexation, incorporated into the daughter nuclei during telophase. IF proteins may thus take part in gene expression in a way similar to that of normal, dimeric and tetrameric transcription factors, for example, steroid hormone receptors (Getzenberg and Coffey, 1990), in addition to performing their global role in chromosome distribution and chromatin organization directed from peripheral regions of the nucleus. Given the close evolutionary and structural relationships existing between the nuclear lamina on the one hand and the intranuclear and cytoplasmic IFs on the other, it seems reasonable to speculate that nuclear core filament and cytoplasmic IF proteins have not lost all their ability to play roles in DNA-based nuclear events, but rather have acquired additional functional properties as divergent specializations of their progenitor, the lamin-like precursor. The nuclear lamina in the primordial eukaryotic cell may well have been a protein scaffold mainly in charge of membrane attachment and replication and segregation of DNA (as a relic of its early, prokaryotic times) (Cavalier-Smith, 1988; Funnell, 1993), whereas its descendant intranuclear core filament network has assumed tasks that range from organization of chromatin to transcription, processing, and transport of RNA. Having expanded from a shell-like organization to a space-filling, three-dimensional distribution in the interior of the nucleus, the filament network has increased its functional capacity tremendously. The provision of extra filament material further enhances its capacity to be functionally modulated by changes in its subunit composition and supplementation with protein factors in response to physiological and developmental stimuli. It is possible that, owing to the close structural relatedness of the protein constituents, the lamina is in direct contact with the intranuclear core filament system and therefore is capable of influencing its spatial organization in addition to that of interphase chromatin. On the other hand, in a functional sense, the nuclear lamina has also expanded into the cytoplasmic compartment via its connections with the cytoplasmic IFs. The cytoplasmic IF subunit proteins have evolved special structural features which prevent them from reentering the nucleus on the normal transportation route for nuclear proteins, but nevertheless

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allow them to establish contact with the lamina and other substructures of the peripheral, intranuclear zone directly through the double nuclear membrane. We speculate that this was mainly achieved by changes in the structure of the N-terminal extension of the lamin-like precursor. The resulting polypeptides became highly activatable by post-translational modification and acquired a membrane-active, amphiphilic character that enables them to distort the lipid bilayers of the perinuclear cisternae and thus to clear the way for the cytoplasmic IFs to break through the nuclear envelope. Since at least some of the cytoplasmic I F proteins have a much higher nucleic acid binding potential than the lamins, the nuclear lamina may take advantage of this in order to increase its capacity to organize interphase chromatin and control gene expression in response to diverse physiological and developmental conditions. Being in direct association with the nuclear lamina, the cytoplasmic I F networks can be considered as sensory systems that extend into the cytoplasm, make contact with organelles and the plasma membrane-extracellular matrix and, employing their high susceptibility to post-translational modification, keep the nucleus informed about the situation prevailing in the cytoplasm and the cellular environment (Ben-Ze’ev, 1992; Lin and Bissell, 1993). A nice example illustrating these structural-functional relationships among elements of the extracellular, cytoplasmic and intranuclear compartment is provided by the observation of a conformational change in the C-terminus of lamin A in response to treatment of cultured cells with phorbol ester and blockage of this effect by drug-induced collapse of the I F system (Collard and Raymond, 1992). Propagating signals between the cell surface and the nuclear envelope, cytoplasmic IFs may thus exert an influence on the structure of the nuclear lamina-matrix and, through the interaction of these karyoskeletal protein assemblies with chromatin, on gene expression and DNA replication. Very likely, the structural changes in the I F (protein)s induced by the various signaling systems are primarily meant to provoke alterations in intranuclear organization rather than to induce a redistribution of the cytoplasmic I F networks for cytoskeletal purposes, although the coordination of both effects may be highly advantageous to the cell under certain physiological conditions. From electron microscopic observations and the evolutionary, structural, and functional relationships among intranuclear, nuclear lamina, and cytoplasmic I F proteins described earlier, it appears that the intracellular space is occupied by a continuum of IF-like protein assemblies extending between the plasma membrane and the center of the nucleus. Through its interconnection with both the intranuclear and cytoplasmic networks, the nuclear lamina may serve a relay function in that it transmits signals it is receiving from extranuclear space to the interior of the nucleus. This continuum can be regarded as a kind of cellular ground substance on

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which the exchange of basic, hierarchical information takes place without restriction by the nuclear membrane. We consider the nuclear membrane to be superimposed, through specific association with the nuclear lamina, on the filament continuum to (1) concentrate the genetic material in a small volume and protect it from physical damage; (2) physically separate DNA-based processes such as replication, recombination, repair, and transcription as well as RNA processing, from metabolic reactions taking place in the cytoplasm; (3) construct through its capacity to establish ionic gradients and electrical potentials a compartment to regulate and maintain the concentrations of ions and low-molecular-weight metabolites required for the macromolecular processes described; and (4)create through the insertion of nuclear pore complexes a second communication route between the nuclear interior and the cytoplasm through which molecular information is exchanged in both directions. In conclusion, we believe we have presented a collection of data and discussion of dogmas from the seemingly diverse fields of IFs and the structure-function relationships involved in the regulation of eukaryotic gene expression. The manifold reactivities of IF proteins and their parallels with other model systems force us to answer the question posed as the title of this contribution “Intermediate filament proteins: cytoskeletal elements with a gene-regulatory function?” with a definitive yes.

Acknowledgments We thank Mrs. Christel Fabricius for the artwork, Mrs. Annegret Gawenda for the photographs, and Mrs. Lolita Horak for secretarial work. We also thank Dr. Theodore Puck for stimulating discussions and for sharing his ideas on the participation of the cytoskeleton in regulating gene expression.

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Secretion and Endocytosis in the Male Reproductive Tract: A Role in Sperm Maturation Louis Hermo, Richard Oko, and Carlos R, Morales Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada H3A 2B2

1. Introduction

It is generally agreed that late step 19 spermatids released into the lumen of the seminiferous tubules have all the structural attributes of mature spermatozoa but are infertile. In order to acquire fertilization capability, and in this sense become mature spermatozoa, they must transit through the efferent ducts and at least the proximal portion of the epididymis, that is, the caput epididymidis. During their transit through the excurrent duct system, the immature spermatozoa are subjected to a continually changing environment regulated by the secretion and endocytic activities of the lining epithelial cells. Thus, as some molecules are secreted in a given portion of the excurrent duct system, other molecules are being endocytosed. Ultimately then, it could be argued, it is the coordinated activity of secretion and endocytosis along the excurrent duct system that influences the final maturation outcome of the spermatozoa. One of the objectives of this chapter is to clarify this concept by providing examples from our own work and that of others on the origin and fate of various proteins associated with spermatozoa in the excurrent duct system. In this context we attempt to morphologically characterize the secretory and endocytic compartments of various epithelial cells lining the excurrent duct system (i.e., Sertoli cells, germ cells, rete epithelial cells, nonciliated cells of the efferent ducts, principal cells of the various epididymal regions, and clear cells), and some of the factors regulating their activities. As will become evident in this chapter, the excurrent duct system secretes a large number of related and unrelated proteins. With the exception of a few, such as immobilin, forward mobility protein, and acrosomestabilizing factor, the exact role of most of the known secretory proteins in Inrernationd Reuien, of Cvrology. Vol. 154

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sperm maturation is still speculative or unknown. In the case of secretory proteins that have counterparts with defined roles in other tissue systems, we attempt to relate these roles to sperm maturation. Another unsolved problem which will become evident in this chapter is the nature of the association of these proteins with the spermatozoa. Evidence is beginning to appear that some of these associations are receptor mediated while others are enzymatic. It is also evident that some of the secretory proteins condition the sperm medium or create the appropriate environment for sperm maturation. Finally, the concept of sperm membrane modification will be dealt with. Three potential ways by which the sperm membrane can acquire new glycoconjugates are presented.

II. Sertoli Cell Structure and Function

During the past decade, the Sertoli cell has attracted the attention of numerous structural and molecular biologists, with the result that the functions of this cell are beginning to be unraveled. In addition to its supporting role for germ cells (Sertoli, 1865), one of the first functions attributed to the Sertoli cell was its capacity to phagocytose and lyse residual bodies which detach from late spermatids at the time of spermiation (Regaud, 1901). Since then, the Sertoli cell has been implicated in a variety of functions that maintain spermatogenesis (Fawcett, 1975; Clermont et al., 1987; Griswold et al., 1988; Morales and Clermont, 1993). These include the secretion of glycoproteins that may be functionally grouped as transport proteins, growth factors, proteases, protease inhibitors, and basement membrane proteins (Bardin et al., 1988; Griswold, 1993). Although the phagocytosis of residual bodies and particulate matter by the Sertoli cell was convincingly demonstrated in earlier studies (Dietert, 1966; Clegg and MacMillan, 1965; Carr et al., 1968; Soares Pessoa and David-Ferreira, 1980), the exact mode of residual body elimination or the precise contribution of the lysosomal apparatus to this process remained to be clarified. More recently, the Sertoli cell was shown to be active in fluid-phase, adsorptive, and receptor-mediated endocytosis (Morales et al.,1985; Clermont et al., 1987; Morales and Clermont, 1993), and the targeting of some lysosomal enzymes and saposins to phagocytozed residual bodies was analyzed (Igdoura and Morales, 1991). These points will be examined in some detail in the following sections.

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A. Ultrastructure

The Sertoli cell is a tall, columnar cell having apical and lateral processes extending to the tubular lumen and between germ cell layers, respectively (Fig. 1). The Golgi apparatus of the Sertoli cell (Fig. 1) is a single, large, continuous network consisting of stacks of saccules (saccular regions) connected to each other by intersaccular tubular connecting regions; an elaborate trans Golgi network (trans tubular network) is present on the trans face (Fig. 2), as demonstrated by Rambourg et al. (1979). Kerr (1988) calculated that the volume of the Golgi apparatus is highest at stages 11, VIII, and XIII-XIV of the cycle of the seminiferous epithelium. We have noted that the endoplasmic reticulum in the basal region of the Sertoli cell is extremely elaborate from stages V to VIII, where it appears predominantly as a close-meshed, highly anastomotic, tubular network (Fig. 3), while in stages IX to XIV it appears as a very loose network made up of dilated scale-like elements connected by short, thin, strands (Fig. 4). These stage-dependent changes in organelle structure may be significant in stage-related changes occurring in germ cells. The Sertoli cell synthesizes and secretes a number of glycoproteins that vary greatly in concentration during the cycle of the seminiferous epithelium (Griswold et al., 1988; Griswold, 1993; Sylvester, 1993; Parvinen, 1993). With the use of [3H]fucose and quantitative electron microscopic (EM) radioautography (Lalli et al., 1984), glycoprotein synthesis was analyzed in adult rats to determine the secretion of glycoproteins on the one hand and production of lysosomal hydrolases, most of which are glycoproteins (Schacter, 1981), on the other. The results indicated that the uptake of [3H]fucose in the Golgi apparatus and lysosomes of these cells was constant throughout the cycle of the seminiferous epithelium (Lalli et al., 1984). However, despite these findings, the elaborate nature of the Golgi apparatus of the Sertoli cell, and its established secretory role (see also Section II,D), typical electron-dense secretory granules as evidenced in other secretory cells have not been found (Fawcett, 1975; Rambourg et al., 1979). Indeed, adjacent to the Golgi apparatus on its cis, lateral, or trans aspect, only small vesicles are present, with no discernible content (Fig. 5 ) , and EM immunocytochemical studies attempting to identify the secretory vesicles of these cells have been unsuccessful. Membrane-bounded bodies that are variable in appearance, number, and size are present in Sertoli cells at different stages of the seminiferous epithelium cycle. They are usually apparent in distinct clusters (Figs. 5-7). Some of them are large and dense or moderately dense and may have membranous profiles (Figs. 5,6). They are often seen in proximity to late residual bodies (Fig. 5 ) , are acid phosphatase positive, and become

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FIG. 1 Electron micrograph of the seminiferous epithelium at stage I1 of the cycle. The Sertoli cell is seen as a thin, attenuated cell extending from the basement membrane toward the lumen. It has numerous dense secondary lysosomes (arrowheads), and a Golgi apparatus (G). The nucleus (N) is large, irregular in outline, and lightly stained. The head of a step 16 spermatid (arrow) is embedded in the Sertoli cell. SP, 2 step 2 spermatids; asterisk, myoid cell. x 5400.

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labeled when tracers are introduced into the lumen of the seminiferous tubules; on this basis they are referred to as secondary lysosomes (Morales et al., 1985; Clermont et al., 1987). Their origin and role with respect to residual bodies is discussed later. Within these clusters are numerous small, spherical or C-shaped, membrane-bounded vesicles or tubular elements, some of which appear empty or have a moderately dense or dense content (Fig. 6). Some smaller vesicles similar in appearance and size to those seen next to the Golgi apparatus are also present (Fig. 6). All of these membranous elements are acid phosphatase negative and have yet to be assigned a functional role. These clusters also contain occasional multivesicular bodies (MVBs) with a pale or dense matrix as well as endosomes which, as will be seen later, play a role in endocytosis. From stages IX to I, such clusters are present predominantly in the apical and supranuclear Sertoli cell cytoplasm, next to the heads of the elongating spermatids (Fig. 7), while from stages I1 to VIII they are present at the base of the Sertoli cell (Hermo et al., 1978).

B. Phagocytosis of Residual Bodies

1. Mechanism of Phagocytosis Prior to the release of step 19 spermatids into the lumen of the seminiferous tubule, the bulk of their cytoplasm, referred to as residual bodies, detaches from these cells. These bodies contain large vacuoles, clusters of ribosomes, condensed mitochondria, remnants of cisternae of ER, granular bodies, lipid droplets, and so on, but show no acid phosphatase activity (Morales et al., 1985). Initially, they are partially enveloped by the apical processes of the Sertoli cell but soon become completely embedded within the cell (Fig. 8; Morales et al., 1985; Clermont et al., 1987). Two morphological features indicate that phagocytosis of residual bodies may be mediated by ligand-receptor interactions. First, numerous thin, veil-like processes of Sertoli cells are seen closely apposed to the surface of newly forming residual bodies. Second, and more important, is the fact that at the time of spermatid release, while the Sertoli cell processes recognize and adhere firmly to the membrane of residual bodies, preventing their loss from the surface of the seminiferous epithelium, the heads and proximal part of the tail of the step 19 spermatids disengage from the Sertoli cell processes to be released into the tubular lumen (Morales et al., 1985). Therefore, it appears that the retention and eventual phagocytosis of residual bodies may be selective and is the result of recognition of their plasma membrane by that of the Sertoli cell.

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FIG. 3 Electron micrograph of the basal region of a Sertoli cell at stage VII of the cycle of material impregnated with Ur-Pb-Cu (ThiCry and Rambourg, 1976). The endoplasmic reticulum is moderately stained and consists of tubular elements (arrowheads) interconnected to form a loose, highly elaborate anastomotic network. A few flattened sheets of endoplasmic reticulum cisternae are present (asterisks). rn, mitochondria; N , nucleus. x 8460.

2. Processing of Residual Bodies

At the beginning of stage IX of the seminiferous epithelium cycle, residual bodies, soon after their internalization, show no modification of their content and their plasma membrane remains distinct from the enveloping plasma membrane of the Sertoli cell (Fig. 8). Residual bodies migrate from stages IX to XI from the apex to the base of the Sertoli cell. During this migration they fuse with secondary lysosomes, which is followed by

Electron micrograph of the basal region of a Sertoli cell at stage I11 of the cycle of material impregnated with uranyl acetate, lead citrate, and copper sulfate (Ur, Pb, Cu) (ThiCry and Rambourg, 1976). The mitochondria (m) are heavily impregnated. The ER (arrowheads) is composed of moderately stained, irregular branching elements interconnected t o form a loose anastornotic network. The Golgi apparatus has stacks of saccules (S), connected to each other by tubular elements (arrows). In frontal view the saccules appear a s fenestrated sheets. On the trans face (T), a highly anastomotic tubular network is apparent and is referred to as the trans Golgi network. Sc, spermatocytes; Lip, lipid droplet: asterisk, myoid cell. x 22,500. FIG. 2

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FIG. 4 Electron micrograph of the basal region of a Sertoli cell at stage XI1 of the cycle of material impregnated with Ur-Pb-Cu (ThiCry and Rambourg, 1976). The endoplasmic reticulum (arrowheads) consists of spheroidal, scale-like elements connected by thin bridges and is much less elaborate than at stage VII. The nucleus (N) of the Sertoli cell is lightly stained and in frontal view the nuclear envelope (asterisks) is highly porous. S, Golgi saccules; m, mitochondria; Sc, spermatocyte nucleus; BM, basement membrane. x 8460.

FIG. 5 Electron micrograph of a Sertoli cell at stage X of the cycle. A cluster of dense or

moderately dense secondary lysosomes (L) of variable sizes and content is present next to the head (nucleus, N and acrosome, A) of a step 10 spermatid. Also evident are several late residual bodies surrounded by a single membrane and containing membranous profiles. Several secondary lysosomes (large arrows) are seen in close proximity to these bodies. The more-or-less spherical elements of the endoplasmic reticulum (arrowheads) are widely dispersed in the cytoplasm. Saccules of the Golgi stacks (S) are associated with small vesicular profiles (small arrows). Sc, spermatocytes; m, mitochondria, RB, residual body. X

11,550.

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FIG. 6 Electron micrograph of the supranuclear cytoplasm of a Sertoli cell at stage XI1 of the cycle. A cluster of membrane-bound bodies is apparent and consists of several large, dense, secondary lysosomes (L), a pale multivesicular body (M), small, dense core granules with a halo (small arrows), small vesicular elements with or without a content (arrowheads), and tubular elements (arrow). The endoplasmic reticulum (asterisks) is lightly stained and dilated. Note intimate relationship between the trans Golgi network and a cisterna of endoplasmic reticulum (curved arrow). G, Golgi elements. X 19,400.

a gradual disintegration and lysis of their components (Morales et al., 1985). By the use of a specific marker for residual bodies, that is, the Yo subunit of glutathione S-transferase, it has recently been shown that residual bodies are no longer present at stage XI1 (Veri et al., 1994). In cytochemical studies to demonstrate acid phosphatase activity, the presence of cytidine monophosphatase and arylsulfatase was observed in many secondary lysosomes of Sertoli cells throughout the 14 stages of the seminiferous epithelium cycle. Beginning at stage IX, a gradient of reactivity to cytidine monophosphatase and arylsulfatase reactivity was found for the residual bodies. While the apically Located (early) residual bodies were consistently unreactive, the deeper the residual body was incorporated into the Sertoli cell, the more reactive it became (Morales et al., 1985). Since images of fusion between acid phosphatase-positive secondary lysosomes and residual bodies become increasingly apparent with depth of migration, it appears that secondary lysosomes are directly involved in the elimination and degradation of residual bodies from the seminiferous epithelium (Fig. 8).

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Electron micrograph of the supranuclear region of a Sertoli cell in which several heads of step 12 spermatids (nucleus, N and acrosome, A) are embedded. Clusters of membrane-bounded elements are present next to these heads. Such clusters contain large, dense, secondary lysosomes (arrows) and small, dense core bodies (small arrows) as well as C-shaped and spherical empty-looking vesicles (arrowheads). The endoplasmic reticulum (asterisks) is large and more-or-less spheroidal in appearance. x 19,400. FIG. 7

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FIG. 8 Diagrammatic representation of phagocytosis and endocytosis in the Sertoli cell. When fluid-phase tracers are administered into the lumen of the rete testis, they appear with time in the endocytic apparatus of the cell, which includes endocytic vesicles (EV), endosomes (E, 2-5 min), light (L, 15 min) and dense (D, 30 min) multivesicular bodies (MVB), and secondary lysosomes (L, 1 hr and later). At the time of spermiation, the bulb of cytoplasm of step 19 spermatids referred to as the residual body is enveloped by processes of the Sertoli cell. Thereafter the residual body is taken up by this cell and referred to as a phagosome (Ph); it is still encompassed by two unit membranes. Upon fusion of secondary lysosomes with the phagosome, one of the unit membranes disappears and the resulting structure, referred to as a phagolysosome (PL), begins to undergo degradation. Thus the event of phagocytosis of residual bodies appears to be integrated with fluid-phase endocytosis that leads to the formation of secondary lysosomes destined to fuse with and contribute to the lysis of phagosomes. Also represented is the hypothetical traffic of SGP-I (70 kDa) presumably via small vesicles from the Golgi apparatus to the adluminal compartment and of prosaposin (65 kDa) via small vesicles from the Golgi apparatus to the endocytic apparatus (LMVB and DMVB). RB, residual body.

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C. Endocytosis by Sertoli Cells

1. Fluid-Phase and Adsorptive Endocytosis at the Apex of the Cell Sertoli cells internalize a variety of electron-dense tracers introduced into the lumen of seminiferous tubules (Morales and Hermo, 1983; Morales et al., 1985). Native ferritin (NF) and horseradish peroxidase or albumin coupled with colloidal gold (HRP-G, Alb-G) were used to test fluid-phase endocytosis. Cationic ferritin (CF) and concanavalin A-ferritin (Con AF), which bind electrostatically to anionic groups and a-D-mannose and a-D-glucose residues respectively, which are present at the surface of the plasma membrane, were used to analyze adsorptive endocytosis. Because specific ligands and their corresponding receptors have yet to be identified on the adluminal plasma membrane of Sertoli cells, receptor-mediated endocytosis has not been analyzed. Following injection of C F or Con A-F into the tubular lumen, these tracers bound to the plasma membrane of Sertoli cells facing the lumen but were not found in endosomes, MVBs, and secondary lysosomes in the supranuclear region of the cell at any of the time intervals analyzed, up to 24 hr. On the other hand, NF, HRP-G, and Alb-G, which did not bind to the plasma membrane, were internalized by Sertoli cells at all stages of the cycle of the seminiferous epithelium. At 5 min after injection, the tracers were found in large spherical or C-shaped endocytic vesicles seen in the Sertoli cell apical process (Fig. 8). Fifteen and 30 min later, the tracers accumulated in endosomes and pale MVBs, while at 1 hr they were present in dense MVBs and occasional secondary lysosomes (Fig. 8); at later intervals, more secondary lysosomes became labeled. During stage VIII of the cycle, early residual bodies, just phagocytosed by Sertoli cells but still delimited by their own plasma membrane, did not contain either of the three fluid-phase markers analyzed. However, at stage IX, late residual bodies, now delimited solely by the plasma membrane of the Sertoli cell and showing signs of degradation, contained NF, HRP-G, and Alb-G. These results correlate well with morphological and cytochemical data demonstrating that during stage IX of the cycle, secondary lysosomes merge with late residual bodies. Thus, at this stage, the Sertoli cell integrates two distinct processes-fluid-phase endocytosis and phagocytosis (Fig. 8). In order to determine if fluid-phase endocytosis varied during the cycle of the seminiferous epithelium, NF was injected into the rete testis of rats, and seminiferous tubules infused with the tracer were collected 6 hr later and prepared for electron microscope analysis. As a result of internalization of the tracer by Sertoli cells, the label was found in 12-66%

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of the secondary lysosomes, depending on the stage of the cycle (Morales et af., 1986). A Zeiss Mop-3 instrument was used on electron micrographs to morphometrically measure a number of parameters. Applying appropriate formulas and a computerized program, it was possible to determine the absolute number of labeled and unlabeled secondary lysosomes per Sertoli cell for each of the 14 stages of the cycle. The percentage of ferritin-labeled secondary lysosomes, regarded as an index of the endocytic activity of Sertoli cells, remained low from stages I1 to VIII, increased abruptly at stage IX, stayed high from stages X to XIV, and decreased abruptly at stage I. During stage IX, there was a sharp drop in the total number of secondary lysosomes per Sertoli cell, which most probably resulted from their fusion with residual bodies at this particular stage (Morales et al., 1986).

2. Synthesis and Targeting of Sulfated Glycoprotein-1 (Prosaposin) to Secondary Lysosomes of Sertoli Cells Sulfated glycoprotein-1 (SGP-1) is a major glycoprotein secreted by rat Sertoli cells (Sylvester et al., 1989). Pulse-chase labeling showed that SGP-I is synthesized as a cotranslationally glycosylated 67-kDa form which is post-translationally modified to a 70-kDa form before secretion (Collard et al., 1988). Although the function of this protein is still unknown, immunocytochernical studies revealed that SGP- 1 is secreted apically into the lumen of the seminiferous tubule and is capable of binding preferentially to the tails of late spermatids (Sylvester et af., 1989; see Section II,D, 1). Sequence analysis demonstrated that SGP-1 shares a substantial sequence similarity with human prosaposin, a ganglioside binding and transport protein (Hiraiwa et af.,1992). Prosaposin is the precursor molecule of four small heat-stable proteins required as activators for the hydrolysis of sphingolipids by specific lysosomal hydrolases (O’Brien et af., 1988). These activators, referred to as saposins (10-15 kDa), are derived by proteolytic processing of prosaposin (Morimoto et al., 1989; O’Brien and Kishimoto, 1991). Each of these saposins consists of about 80 amino acids and shares several characteristic features, including the location of an N-glycosylation site and cysteine and proline residues. The suggestion that the saposins are located in the lysosomal compartment came from Mraz el af. (1976), who found saposin B (sulfatide activator protein-1) activity associated with liver subcellular lysosomal fractions. However, the first morphological evidence that these proteins were unequivocally located in lysosomes was obtained by Tamaru et af. (1986) and Sylvester et al. (1989). Unlike human prosaposin, rat SGP-1 was initially thought to be secreted

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exclusively into the adluminal compartment of the seminiferous tubule without being processed into smaller molecules. SGP- 1 contains a prolinerich sequence (31 amino acids) located between saposin-like C and D regions (Collard et a/., 1988) that is not present in human prosaposin. Since proline-rich proteins are chiefly secretory (Ziemer et al., 1984), it has been suggested that the proline-rich sequence of SGP-1 is responsible for the different trafficking pattern of this protein (Collard et al., 1988; O’Brien et al., 1988). EM immunocytochemistry localized SGP- 1 to secondary lysosomes of Sertoli cells (Sylvester et a / . , 1989; Morales and Clermont, 1993). This localization suggested that SGP-1-derived saposins occur in secondary lysosomes (Sylvester et al., 1989). Since SGP-1 is also secreted into the lumen of the seminiferous tubule and secondary lysosomes arise from fluid-phase endocytosis, we tested whether SGP- 1 was internalized from the lumen and hence appeared in Sertoli cell secondary lysosomes (Morales et d., 1993). However, since no immunolocalization was observed in endocytic vesicles or endosomes at the apex of Sertoli cells, it appears unlikely that secreted SGP-I is taken in from the luminal fluids. When the isolated secondary lysosomes from adult rat Sertoli cells were submitted to a purification step by immunoaffinity chromatography, two bands of 65 kDa and 15 kDa were observed (Igdoura and Morales, 1994). This finding was in agreement with other investigations showing that the 65-kDa protein is the lysosomal precursor of the 15-kDa saposins. Moreover, further analysis of the 15-kDa proteins by reverse-phase highperformance liquid chromatography (HPLC) revealed the presence of four peaks which were consistent with the presence of the four lysosomal saposins. The fact that these four highly hydrophobic proteins were in the 15-kDa range and were cross-reactive to anti-SGP-1 antibody by enzymelinked immunosorbent assay (ELISA) analysis demonstrates that Sertoli cell secondary lysosomes contain saposins which are probably derived from the proteolytic processing of a 65-kDa precursor molecule (Igdoura and Morales, 1994). These results are consistent with the existence of a targeting route of the 65-kDa precursor of the saposins to secondary lysosomes that is independent of the secretory route for the 70-kDa form of SGP-1 (Fig. 8). Immunogold labeling with anti-SGP-1 detected the presence of saposins and their precursor (65 kDa) only in late residual bodies and not in early residual bodies prior to their fusion with secondary lysosomes. This finding suggests that the 65-kDa precursor is targeted to late residual bodies by secondary lysosomes (Igdoura and Morales, 1994). In summary, these results constitute the first demonstration of the presence of a 65-kDa saposin precursor and its proteolytically derived 15-kDa saposins in secondary lysosomes of Sertoli cells. They also suggest that

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these proteins reach the secondary lysosomes directly from the Golgi apparatus (Fig. 8). Finally, the results provide evidence for the lysosomal release of saposins and other lysosomal enzymes into late residual bodies, a process that may play a significant role in the hydrolysis of sulfoglycolipids and other glycolipids which are components of the membranous elements within these bodies.

3. Receptor-Mediated Endocytosis at the Base of the Cell In contrast to the adluminal plasma membrane, no large endocytic invaginations or endocytic vesicles are seen along the basal plasma membrane of Sertoli cells. On the other hand, small coated and uncoated pits and vesicles are seen (Fig. 9). The presence of these structures at the base of Sertoli cells suggests that receptor-mediated endocytosis takes place at this pole of the cell. Confirming this idea, Morales ef al. (1987a,S)demonstrated that Sertoli cells are involved in the biosynthesis and secretion of testicular transferrin and that the initial uptake of iron by Sertoli cells from the blood serum takes place by way of receptor-mediated endocytosis of serum transferrin (Morales and Clermont, 1986). These experiments were accomplished by infusing '251-labeledtransferrin (TF) into the interstitial space of the testis. Light microscope radioautographs demonstrated a strong labeling of the basal aspect of the seminiferous epithelium 5 min after injection. This labeling was prevented by simultaneously injecting a 200-fold excess of cold TF, thus proving the presence of high-affinity binding sites. Furthermore, these results were consistent with antitransferrin serum labeling of the basal aspect of the Sertoli cell (Morales and Clermont, 1986). In EM radioautographs of seminiferous tubules collected at 5 min after an interstitial injection of radioactive TF, the basal plasma membrane of Sertoli cells was labeled. Silver grains were frequently seen overlying coated pits and coated vesicles. At 15 and 30 min after injection, silver grains were seen within endosomes and pale MVBs adjacent to the basal plasma membrane. At later intervals, no labeling of these structures was observed and at no time interval were secondary lysosomes labeled. Evidence was also obtained to indicate that TF was recycled intact into the interstitial space. This was demonstrated by exposing isolated seminiferous tubules or Sertoli cells in culture to radioactive TF at 4°C and then rinsing and reincubating them in a label-free medium at 37°C for various periods before determining the amount of radioactive proteins in the incubation medium. The results demonstrated that TF is internalized by receptor-mediated endocytosis, reaches endosomes and returns, presumably as apotransferrin within small carrier vesicles, to the basal plasma

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FIG.9 A diagram of receptor-mediated endocytosis (lower inset square) of serum diferric transferrin (solid triangles) at the base of the Sertoli cell and of the transport of testicular diferric transferrin (open triangles) synthesized by the Sertoli cell to the adluminal compartment. Serum difemc transferrin binds to the transferrin receptor and is internalized within endosomes (En) where, upon encountering an acidic pH, the iron (black dots) dissociates from transferrin. The apo transferrin bound to its receptor is then recycled back to the interstitium. The dissociated iron binds to newly synthesized testicular transfemn and is transported, presumably via vesicles, into the adluminal compartment. There the testicular diferric transfenin is internalized via receptor-mediated endocytosis (upper inset square) by adluminal germ cells in a fashion similar to that existing at the base of the Sertoli cell. RB, residual body; PI, preleptone; Sptc, spermatocyte; sptd, spermatid; G , spermatogonia. (Reproduced from R. Petrie and C. R. Morales, Cell and Tissue Research, 267,45-55 with permission of Springer-Verlag.)

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membrane where it is delivered to the interstitial space (Fig. 9) (Morales and Clermont, 1986). It was also of interest to determine whether after the simultaneous injection of N F into the lumen of the seminiferous tubule and radioactive TF into the interstitial space, they would be found together in the same endosome. Such double labeling studies showed that the endosomes at the base of the cell were labeled only with TF, while those at the apex only with NF; at no time interval were endosomes labeled with both. These results suggest that Sertoli cells are capable of performing receptormediated endocytosis of serum transferrin at their basal pole and that the endocytic apparatus at this pole is distinct from that present in the apical-supranuclear region of the cell (Morales and Clermont, 1986). D. Secretory Functions of Sertoli Cells

The formation of tight junctions between adjacent Sertoli cells results in a partioning of the seminiferous epithelium into two compartments (reviewed in Griswold et al., 1988). The basal compartment is in contact with the vascular supply and contains spermatogonia and preleptotene spermatocytes, while the adluminal compartment contains most of the meiotic germ cells and all the spermatids. The secretory products of Sertoli cells directed to the adluminal compartment create a unique serum-free environment in which spermatogenesis and meiosis can occur (Griswold et al., 1988). Because of the tight barrier between adjacent Sertoli cells, luminally secreted proteins must functionally replace some serum proteins which have restricted access to the adluminal compartment of the seminiferous epithelium. Furthermore, Sertoli cells secrete a number of other proteins which are important for the maintenance of spermatogenesis. This repertoire of secreted proteins includes binding and transport proteins that are responsible for the movement of nutrients, vitamins, hormones, and waste products to and from the developing germ cells (Sylvester, 1993). As more information is gathered, it is likely that the long list of proteins provided by Griswold (1993) will grow and some of them will even be reclassified. The most abundant proteins secreted by the Sertoli cell were first detected in the medium of cultured cells by the use of polyacrylamide gel electrophoresis followed by fluorography (Kissinger et al., 1982). Many secreted polypeptides can be detected by this technique, but only transferrin, ceruloplasmin, and sulfated glycoproteins 1 and 2 (SGP-1 and SGP2) have been characterized or identified (Skinner and Griswold, 1980, 1983; Skinner et af., 1984; Sylvester et al., 1984). Transferrin, ceruloplasmin, and SGP-1 and SGP-2 comprise more than 80% of the total mass of

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proteins secreted by cultured Sertoli cells, and SGP-2 alone represents as much as 50 percent (Griswold et al., 1988). Anti-Miillerian hormone and androgen binding protein (ABP) are in low abundance and cannot be detected by these methods without selectively concentrating the protein by immunoprecipitation (Griswold, 1993). The glycoproteins secreted by Sertoli cells can be classified into several groups based on their known biochemical properties (Griswold, 1993). The transport or bioprotective proteins are secreted in relatively high abundance and include metal ion transport proteins such as transferrin and ceruloplasmin, and SGP-I and -2. The function of secreted SGP-I is unknown but SGP-2 may either bind and transport lipids or be involved in immunosuppression. Another important category of proteins secreted by Sertoli cells consists of the proteases and protease inhibitors, which may be important in tissue remodeling processes that occur during spermiation and in movement of preleptotene spermatocytes into the adluminal compartment. Sertoli cells also synthesize and secrete some of the glycoproteins which form the basement membrane underlying these cells. Finally, Sertoli cells secrete glycoproteins that function as growth factors or paracrine factors. The complete spectrum of growth factors made by Sertoli cells is still undefined but includes such proteins as Mullerianinhibiting substance and inhibin. This last class of regulatory glycoproteins is in low abundance yet still carries out its biochemical role (Griswold, 1993). In the sections to follow we examine recent data obtained in our laboratory concerning testicular SGP-I, SGP-2, and transferrin; we discuss the potential roles of these glycoproteins in the testis in relation to our data and that of others.

1. Secreted SGP-1 SGP- I is biochemically and functionally different from SGP-2. SGP- 1 is a monomer of 70 kDa with isoelectric points in the range of 4.0 to 4.8 (Griswold et al., 1988). SGP-I shares sequence similarity with human prosaposin, the precursor of four heat-stable saposins. Prosaposin is coded for on human chromosome 10 (Fujibayashi et al., 1985) and is encoded on a messenger RNA of 2740 nucleotides that gives rise to a 65-kDa glycosylated precursor. In addition to generating the four saposins localized within secondary lysosomes (see Section 11,C,2), prosaposin also exists as an unprocessed 70-kDa protein which is secreted by several human tissues. The highest concentration of this secreted form is in testis, seminal plasma, cerebral gray matter, and human milk (O’Brien et al., 1988). Collard et af.(1988) have cloned and sequenced the secreted form of prosaposin (SGP-1) from rat Sertoli cells. This form was found to be

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a 70-kDa glycoprotein containing a 3 1-residue, proline-rich, amino acid sequence that was not present in the lysosomal human prosaposin. The 3 1-residue, proline-rich stretch is similar to those found in secretory proteins of salivary glands (O’Brien et d . , 1988). Recently, it has been demonstrated that Sertoli cells synthesize two forms of SGP-1. A 64-kDa form is targeted to the secondary lysosomes, where it is further processed to the smaller saposins (15 kDa) (Igdoura and Morales, 1994). In addition, Sertoli cells also produce a 70-kDa form that is secreted into the adluminal compartment of the seminiferous epithelium, where it binds to the plasma membrane of the late spermatids (steps 16-19) (Sylvester et al., 1989). In some cells, lysosomal enzymes, including arylsulfatases, are secreted into the extracellular space. It is possible therefore that the secreted form of SGP-1 may act as a coating protein protecting the sperm membrane from lysosomal enzymes by virtue of its glycolipid binding sites (Collard et al., 1988). In fact, sulfoglycerolipids(seminolipids)are the most abundant component of the spermatid membrane (Lingwood et al., 1981).Arylsulfatase A, which is secreted by the seminal vesicles, has also been shown to desulfate the seminolipids of mammalian spermatozoa (Gadella et al., 1991). Desulfated seminolipids have potent membrane fusogenic properties that are important during acrosome reaction. Thus, the binding of SGP-1 to the plasma membrane of late spermatids may be important for preventing early acrosome reaction by lysosomal enzymes that may be secreted into the lumen of the seminiferous tubules by Sertoli cells. 2. Secreted SGP-2

SGP-2 is the major protein secreted by rat Sertoli cells. It is composed of two disulfide-linkedsubunits of 34 and 47 kDa. Pulse-chase experiments have shown that this protein is synthesized as a cotranslationallyglycosylated 64-kDa precursor molecule that is modified to a negatively charged 73-kDa form by sialylation and sulfation of carbohydrate moieties; this takes place before intracellular cleavage to the mature 34- and 47-kDa subunits (Collard and Griswold, 1987; Tsuruta et al., 1990). A plasmid cDNA library has been constructed from immunopurified mRNA, and the recombinant clone containing the entire coding sequence of SGP-2 has been isolated from the library and analyzed. The derived SGP-2 sequence has a molecular weight of 51,379 and contains six potential N-glycosylation sites. Proteolytic processing sites for the preprotein were determined by amino terminal sequencing of the isolated SGP-2 subunits. Pro-SGP-2was converted to the mature protein by proteolytic cleavage at the amino acid Arg-226 (Collard and Griswold, 1987). Recently the complete amino acid sequence of two serum glycoproteins

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known as SP-40,40 in the human and apolipoprotein J (apo J) in the human and mouse, showed an overall identity with SGP-2 of 77%. The biochemical properties of these two proteins were similar to those of SGP2 since they consisted of two nonidentical, disulfide-linked subunits which were extensively glycosylated and sulfated, and showed isoelectric points ranging between 4.5 and 5.4 (de Silva et al., 1990b). SP-40,40 was first identified as a component of the SC5b-9 membrane attack complex of complement 4,5 and was shown to have an inhibitory capacity on C5b-6-initiated complement hemolysis. In consequence, it is likely that in the serum this protein combines with nascent C5b-7, preventing membrane insertion of the complement and cell lysis (O’Bryan et al., 1990). However, the role of SGP-2 in the testis in this context is questionable since none of the complement components has been shown to be present in the seminiferous tubular fluid. Apo J is a protein associated with high-density lipoproteins (HDL) in human plasma. HDL together with low-density lipoproteins transport 80% of the plasma cholesterol in humans. HDL cholesterol is inversely correlated with the risk of premature atherosclerosis. The antiatherogenic nature of HDL is attributed, in part, to its possible role in the transport of cholesterol from peripheral tissues, which are unable to catabolize excess cholesterol, to the liver for clearance, a process referred to as reverse cholesterol transport. Analysis of the primary structure of apo J predicts the existence of amphiphilic helices which may account for the association of apo J with lipoproteins and with heparin-binding motifs in both of its subunits (de Silva et al., 1990a; Smith et al., 1990). Thus, SGP-2 may be involved in the transport of lipids from the Sertoli cell to germ cells in the adluminal compartment. It has also been shown that SGP-2 is homologous to a TRPM-2 protein found in the regressing prostate (LCger et al., 1987) and to a protein designated as glycoprotein I11 found in the secretory granules of chromaffin cells of the adrenal medulla (Palmer and Christie, 1990). SGP-2 was first shown to be synthesized by Sertoli cells of the rat testis and to bind to the plasma membrane of late spermatids by Sylvester et al. (1991). Studies using Sertoli cells in culture in a dual chamber system and in vivo suggested that SGP-2 is secreted apically (Danahey et al., 1986). In situ hybridization of testicular sections and Northern blotting analysis of mRNAs also demonstrated that Sertoli cells transcribe SGP2 mRNA (Morales et al., 1987a). Recently, we have proposed a model for the binding of SGP-2 to sperm in the rat testis and its subsequent release in the rete testis and efferent ducts. The basic elements of this model include (1) synthesis and secretion of SGP-2 by Sertoli cells into the lumen of seminiferous tubules, where it binds to late spermatids; (2) binding of SGP-2 to a specific germ cell

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receptor; (3) release of SGP-2 after lipid transfer and its endocytosis by rete epithelial cells and nonciliated cells of the efferent ducts (Fig. 10) (Hermo et af., 1991a; and Sections V,A,3 and V,B,2).

111. Germ Cells Few studies have been done on the secretion and endocytosis of proteins by germ cells. However, there is evidence that these cells exhibit a degree of secretory and endocytic activity. This section reviews some known aspects of these functions.

A. Secretion There is little doubt that the Sertoli cell in vivo interacts with germ cells by direct contact or through a paracrine mechanism mediated by secretory proteins. Some of these interactions have been examined in v i m in a multichamber cell culture (Onoda and Djakiew, 1990, 1991; Djakiew and Onoda, 1993). For example, the influence of round spermatids and pachytene spermatocyte proteins on the total Sertoli cell protein secreted bidirectionally was studied by Onoda and Djakiew (1990, 1991) and Onoda ef al. (1991). These authors found that total Sertoli cell secretory protein was increased 133% above control levels by round spermatid proteins and 119% above control levels by pachytene spermatocyte proteins (Onoda and Djakiew, 1991). Interesticgly, this stimulation occurred only when the germ cell protein was added in the upper reservoir (which is equivalent to the adluminal compartment) but not when it was added to the basal reservoir. These experiments suggest that a receptor-mediated stimulation may occur specifically on the apicolateral membrane of the Sertoli cell, but this has yet to be demonstrated. More recently, a 29-kDa, round, spermatid-derived protein (RSP-29) was purified by gel-filtration chromatography (Onoda and Djakiew, 1993). Using a polyclonal antibody against RSP-29, these authors showed that total protein and transferrin secretion from Sertoli cells were significantly reduced following incubation with the RSP-29 round spermatid protein, while preimmune serum treatment of round spermatid proteins revealed no significant reduction of either biological marker (Onoda and Djakiew, 1993). Over the years, there has been an extensive amount of work on the structure and function of the Golgi apparatus of early spermatids (steps 5-8) and its role in formation of the acrosomic system (Susi et af., 1971;

RETE TESTIS

EFFERENT DUCT

FIG. 10 A diagram illustrating the endocytosis of testicular SGP-2 by epithelial cells of the rete testis and efferent ducts and secretion of epididymal SGP-2 by principal cells. Testicular SGP-2 (represented as open circles) is secreted by Sertoli cells and binds to the surface of late spermatids. During transit through the rete testis and efferent ducts, it detaches from the sperm and is endocytosed by the cuboidal rete epithelial cells and nonciliated cells of the efferent ducts. Receptors (Y) for testicular SGP-2 presumably exist on the apical plasma membrane of these cells. Following its receptor-mediated uptake by invaginations of the cell surface, the receptor-ligand complex appears in endosomes (E), multivesicular bodies (MVB), and secondary lysosomes (L).In the endosome, one may postulate that the receptor and ligand dissociate from one another, with the receptor being recycled to the apical plasma membrane, while the ligand is destined for degradation in secondary lysosomes. In the epididymis (except for the proximal area of the initial segment), the epithelial principal cells appear to be involved in the secretion of an epididymal SGP-2 (represented as solid black circles). The epididymal SGP-2 is found in 150-300-nm vesicles of the principal cell next to the Golgi apparatus as well as in the apical region of the cell; these are considered the secretory vesicles (sv). Following its secretion, the epididymal SGP-2 binds to the plasma membrane of the sperm, especially in the area of its head and the principal piece of its tail. (Reproduced from Hermo er a / . , 1991a.)

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Hermo et al., 1980; Clermont et al., 1981, 1990). In late spermatids (steps 9-17), radioactive substances are incorporated by these cells (Tang et al., 1982). However, despite numerous electron microscope studies, the secretory vesicle for the transport of proteins to the cell surface has yet to be identified. B. Endocytosis

Preliminary evidence also indicates that adluminal germ cells have the capacity to perform endocytosis. Intraluminal injection of NF and other tracers demonstrates their incorporation into the endocytic apparatus of spermatids (Figs. 1 1 , 12), as was also shown by Segretain et al. (1992). Internalization of these markers also occurs in spermatocytes (Segretain et al., 1992). In fact, acid phosphatase activity in spermatids was detected in membrane-bounded secondary lysosomes (Fig. 13), which are similar in appearance to those labeled by tracers. Petrie and Morales (1992) showed that both spermatocytes and spermatids possess high-affinity transferrin binding sites, complementing the finding of transferrin receptor mRNA expression in these cells (Roberts and Griswold, 1990; Petrie and Morales, 1992). Furthermore, by in uiuo radioautography, it was determined that following binding of [ '2SI]transferrin,

FIG. 1 1 Pale multivesicular body (asterisk) in the cytoplasm of a step 7 spermatid, IS min after injection of native femtin into the lumen of the rete testis. The tracer (arrowheads) appears in the matrix ofthis body. Small vesicles (arrows) enveloping this body are unlabeled. x 47,000.

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FIG. 12 Dense multivesicular body (asterisk)and lysosomal element (L) in a step 7 spermatid labeled with native ferritin 1 hr after its injection into the lumen of the rete testis. ER, endoplasmic reticulum. x 47,000.

FIG. 13 Numerous cytidine monophosphatase-positive secondary lysosomes (L) of a step 7 spermatid are present next to the chromatoid body (CB) consisting of a finely granulofilamentous material (asterisks) and associated vesicular profiles (arrowheads). x 47,000.

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transferrin was internalized within endosomes of round spermatids and thereafter recycled back to the cell surface (Petrie and Morales, 1992). Iron is a nutritional requirement for all cells as a constituent or cofactor of enzymes involved in such processes as respiration and nucleic acid synthesis (Morgan, 1981). In the testis, iron must be in constant supply since germ cells, which have a functional requirement for iron, are continuously replaced in the seminiferous epithelium. However, Sertoli cell tight junctions prevent the diffusion of macromolecules from the systemic circulation. Adluminal germ cells (spermatocytes and spermatids) are thus prevented from obtaining their iron requirements directly from serum transferrin (sTf). Therefore there must exist an iron supply route that bypasses the tight junctions to supply differentiating adluminal germ cells with iron. sTf has been shown to undergo receptor-mediated endocytosis at the basal pole of Sertoli cells and to recycle back to the interstitial space (Fig. 9) (Morales and Clermont, 1986). In the process, iron must dissociate from sTf and become associated, by means of an undetermined mechanism, with testicular transferrin (tTf) synthesized by Sertoli cells (Fig. 9). The diferric tTf has been shown to be secreted by Sertoli cells into the adluminal compartment, where it binds to transferrin receptors on the surface of adluminal germ cells and is endocytosed (Fig. 9) (Petrie and Morales, 1992). In this way iron would be delivered to adluminal germ cells.

IV. Intermediate [Terminall Region of the Seminiferous Tubule

The intermediate region of the seminiferous tubule is where the seminiferous tubule proper ends and the rete testis begins. This region has been described in detail by Hermo and Dworkin (1988). Tall, columnar epithelial cells referred to as transitional cells line this region of the tubule. From the basement membrane these cells extend upward and show a characteristic bending and orientation toward the lumen of the rete testis. In this way they form a potential plug, presumably preventing the backflow of spermatozoa and other substances into the seminiferous tubules. Germ cells are sparse in this region. Transitional cells clearly differ morphologically from Sertoli cells, yet while they share some features in common with these cells, they also share features in common with the rete epithelial cells. Thus they appear to be in a state of transition between these two distinct cell types. Tracers introduced into the lumen of this region are taken up within a welldeveloped endocytic apparatus in these cells. These cells are involved in both fluid-phase and adsorptive endocytosis (Hermo and Dworkin, 1988).

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They also possess an endocytic apparatus in the basal region which appears to act independently of that in the apical and supranuclear region of the cell. Studies showing the uptake of specific proteins as well as a secretory role for these cells have not been done.

V. Structure and Function of the Rete Testis and Efferent Ducts The epithelial cells lining the rete testis and efferent ducts of the adult rat have distinct morphological features and functions. In the following sections, we examine some of these features and the role of these cells in the endocytosis of SGP-2, SGP-1, and transferrin. SGP-2 is one of the major proteins secreted by Sertoli and epididymal cells (Sylvester et a!., 1991). Biochemical analysis of the testicular and epididymal forms of SGP-2 yields proteins of similar molecular weight, but shows differences in glycosylation, suggesting that the two forms of SGP-2 occur because of tissue-specific, post-translational modifications (Sylvester et al., 1991). Studies in dual chamber Sertoli culture systems suggest that SGP-2 is secreted apically and that it binds preferentially to spermatozoa (Danahey et al., 1986). Interestingly, the detergent-extracted SGP-2 protein recovered from washed testicular sperm is of a higher molecular weight than that recovered from washed epididymal sperm. However, no modification of the high-molecular-weight testicular form by epididymal cells or fluids can be detected in incubation media (Sylvester et al., 1991). Since SGP-2 has been shown to be a species homolog of apolipoprotein-J (de Silva er al., 1990a), it may be, like apolipoprotein-J, involved in lipid transport. Testicular SGP-2, transferrin, and testicular SGP-1 are all found in high concentration in the lumen of the seminiferous epithelium and rete testis and then decrease significantly in concentration in the caput epididymidis (Sylvester er al., 1989).Evidence for the endocytosis of these proteins and of various nonspecific tracers by epithelial cells lining the rete testis and efferent ducts and for the secretion of epididymal SGP-2 is presented in the next section.

A. Rete Epithelial Cells

1. Fluid-Phase and Adsorptive Endocytosis The anastomotic labyrinth channel referred to as the rete testis is lined by low, cuboidal, epithelial cells which possess few cisternae of rough endoplasmic reticulum and a relatively small Golgi apparatus (Dym, 1976;

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Morales et al., 1984). The lateral plasma membranes of adjacent rete cells are highly interdigitated and often show small vesicular blebs (Fig. 14). The apical plasma membrane displays short microvilli as well as small invaginations or pits which may or may not be coated (Fig. 14). In the cytoplasm are small uncoated vesicles and endosomes as well as occasional pale and dense MVBs and secondary lysosomes (Fig. 14); the latter have been shown to be acid phosphatase-positive (Morales et al., 1984). These membranous elements comprise the endocytic apparatus of the cell.

FIG. 14 Electron micrograph showing extensive interdigitations of the lateral plasma membranes (LPM) of adjacent epithelial cells of the rete testis. Beneath the apical cell surface which shows short microvilli (open arrows) are several small vesicular elements (arrowheads) which also appear in other regions of the cytoplasm. Vesicles are also seen in continuity with the lateral plasma membrane (arrows). Scanty irregular elongated profiles of the endoplasmic reticulum (ER) are visible. Secondary lysosomes (L) are also evident. Several invaginations or pits (curved arrows) of the basal plasma membrane are apparent. m, mitochondrion; Lu, lumen; BM, basement membrane. ~ 4 3 , 4 0 0

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Electron-dense tracers, such as NF, HRP-G, and Alb-G (fluid-phase markers), and CF and Con A-F (adsorptive markers) show a rapid but progressive incorporation by the endocytic apparatus of the rete epithelium (Morales and Hermo, 1983; Morales et a / . , 1984). At 2 min after injection into the lumen of the rete, the tracers appear in pits of the apical cell surface and by 5 min in small uncoated vesicles and endosomes. At 15 and 30 min, the tracers are found in the matrix of pale and dense MVBs respectively. At 1 hr and later intervals, secondary lysosomes become labeled (Figs. 15, 16). These results suggest that the rete epithelial cells are capable of endocytosing proteins from the luminal fluid and then degrading them in secondary lysosomes. Since it is documented that a dramatic decrease in protein concentration of the seminiferous tubular fluid occurs as it passes through the rete (Hinton and Keefer, 1983), it is probable that a significant proportion of the proteins lost are endocytosed by the rete epithelial cells and degraded by lysosomes. Internalized adsorptive tracers are also observed to be carried by small uncoated vesicles to the lateral plasma membrane of the rete epithelial

FIG. 15 Electron micrograph of a rete epithelial cell I hr after injection of horseradish peroxidase bound to colloidal gold. The tracer (arrowheads) is found in the lumen (Lu),pits of the apical cell surface and subsurface vesicles (arrows), a multivesicular body (asterisk), and in secondary lysosome (L). ER, endoplasmic reticulum. X 45,990.

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FIG. 16 Diagram of receptor-mediated endocytosis of SGP-2 (on left) and receptor-mediated endocytosis of transfemn (on right) in the rete epithelial cells. SGP-2(open circle) presumably binds to its receptor (Y) and is internalized into the endocytic apparatus consisting of pits (P), vesicles (V), endosomes (E), multivesicular bodies (MVB), and secondary lysosomes (L).Tubules emanating from the endosome serve to carry the receptor back to the apical surface. Transferrin carrying iron (A) binds to its receptor (T) and is internalized within pits, vesicles, and endosomes. In the latter, iron dissociates from transferrin, which with its receptor is recycled back to the apical cell surface via tubules (AT) emanating from endosomes.

cell (Morales et al., 1984). At 30 and 60 min after injection, vesicles containing tracer are seen in the cytoplasm either close to or connected to the lateral or basal plasma membrane; these tracers are also found in the lateral intercellular space as well as in the lamina lucida of the basement membrane. Thus the capability also exists for these cells to take up proteins from the lumen and deliver them by transcytosis to the underlying connective tissue space by which they can reach the circulation. However, such proteins have yet to be characterized. 2. Endocytosis of SGP-1 and SGP-2 by Rete Epithelial Cells LM and EM immunolocalization results demonstrate that testicular forms of SGP-1 and SGP-2, synthesized by Sertoli cells, bind to the surface of late spermatids in the seminiferous tubules (Sylvester et al., 1984, 1991; Gnswold et al., 1988). In the rete testis, LM immunolocalization of SGP2 (Hermo et al., 1991a) and SGP-1 (Hermo et al., 1992b) reveals a dense reaction product over the apical and supranuclear regions of the rete epithelial cells (Fig. 17). EM imrnunogold labeling localizes SGP-1 and SGP-2 over the various compartments of the endocytic apparatus of these

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FIG. 17 Photomicrograph of the cuboidal epithelial cells of the rete testis immunostained with the anti-SGP-2 antibody. The immunoperoxidase reaction product appears as a dense band (arrows) in the apical region of these cells; the basal region and nucleus (n) are unreactive. Lu. lumen. x 760.

cells (Fig. 18). Because spermatozoain the lumen of the rete are unlabeled, we conclude that SGP-1 and SGP-2 detach from spermatozoa during transit through the rete testis and consequently are internalized within the endocytic apparatus of the rete epithelial cells (Fig. 10). It is possible that SGP-1 and SGP-2 are internalized by way of a receptor-mediated process. Their final destination appears to be secondary lysosomes in the rete epithelium, where they are presumably degraded. No evidence of a transcytotic route for these proteins has been observed.

3. Endocytosis of Transferrin In the case of transferrin, which is synthesized by Sertoli cells (Skinner et al., 1984; Sylvester and Griswold, 1984), we observe that the rete epithelial cells are also involved in its endocytosis (Morales and Hermo, 1986), most probably by a receptor-mediated process. At the earliest time intervals (2 and 5 min) after injection of diferric transferrin into the lumen of the rete testis, the tracer appears in coated and uncoated pits of the apical cell surface and then in small subsurface uncoated vesicles and early endosomes (Fig. 16). At later intervals (15 and 30 min), the tracer appears in late endosomes as well as pale MVBs. In addition, tubular elements connected to or closely associated with endosomes and pale MVBs, and isolated tubules seen in close proximity to the apical plasma membrane become labeled (Fig. 16). At 60 and 90 min, the majority of

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FIG. 18 Electron micrograph of a cuboidal epithelial cell of the rete testis immunolabeled with anti-SGP-2 antibody. Gold particles representing SGP-2 antigenic sites are found in an endosome (E), multivesicular body (asterisk), and secondary lysosomes (L). Lu, lumen; N, nucleus. x 16,800.

endosomes and pale MVBs are no longer labeled. At no time is transferrin seen in secondary lysosomes. These results suggest that after the dissociation of iron from transferrin within endosomes and pale MVBs, transferrin is recycled back to the lumen via tubules emanating from these structures (Fig. 16). Thus we postulate that the rete cells are involved in the recovery of surplus iron originating from the seminiferous epithelium (Morales and Hermo, 1986). Morphological evidence for secretion of proteins by the rete epithelial

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cells is clearly lacking. Only small, empty-looking vesicles are apparent in the Golgi region, but a role for these in secretion has yet to be determined. 6 . Epithelial Nonciliated Cells of the Efferent Ducts

1. Fluid-Phase and Adsorptive Endocytosis The efferent ducts are lined predominantly by epithelial nonciliated cells (Fig. 19) and occasional ciliated cells. The columnar nonciliated cells contain a loosely arranged network of cisternae of rough endoplasmic reticulum (Fig. 20) and a large Golgi apparatus possessing a well-developed trans Golgi network (Fig. 19 and Rambourg et al., 1987). However, there is a conspicuous absence of typical dense core secretory granules, and the secretory vehicle of these cells has not been identified. The nonciliated cells have been shown to be involved in uptake of nonspecific substances from the lumen (reviewed by Robaire and Hermo, 1988). Confirming this function, we have shown a highly developed endocytic apparatus in these cells (Fig. 19) which includes tubular coated pits, an abundance of apical tubules, endosomes, MVBs (Fig. 20), and numerous acid phosphatase-positive secondary lysosomes (Hermo and Morales, 1984; Hermo et al., 1988a). Our time course studies, following injection of fluid-phase or adsorptive tracers into the lumen of these ducts, show a progressive labeling of the endocytic apparatus such that by 1 hr secondary lysosomes begin to be labeled (Fig. 21). Thus the nonciliated cells, like the rete epithelial cells, appear to be actively involved in the endocytosis of substances from the seminiferous fluid. However, no evidence of transcytosis is observed in the nonciliated cells (Hermo and Morales, 1984; Hermo et al., 1988a). The reason for such effective endocytic cells at this level of the reproductive duct system is clearly still a mystery, as are the various proteins and substances which are endocytosed. However, the lack of myoid cells in the connective tissue areas underlying the rete testis may necessitate a means for the exit of sperm from this region. This may be accomplished by the rapid uptake of water by the nonciliated cells, leading to a negative pressure or suction effect to move sperm out of the rete testis. Interestingly, it has been documented that the less abundant epithelial ciliated cells of the efferent ducts are also involved in fluid phase and adsorptive endocytosis, but that they possess a much less elaborate endocytic apparatus (Hermo et al., 1985). Early LM immunocytochemical studies suggested that androgen binding protein is taken up by the epithelial cells lining the efferent ducts (Pelliniemi etal., 1981;Attramadal et al., 1981).This was demonstrated recently

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at the electron microscope level in the ram by Veeramachaneni and Amann (1991), who also provided evidence for the endocytosis of transferrin and SGP-2 by these cells. LM immunolocalization of inhibin in the epithelium of the ovine rete testis and efferent ducts (Veeramachaneni er al., 1989) is also indicative of an endocytotic function by these cells.

2. Endocytosis of SGP-2 by Nonciliated Cells of the Efferent Ducts Immunoperoxidase localization of SGP-2 reveals a distinct reaction over the apical and supranuclear regions of the nonciliated cells (Fig. 22 inset and Hermo et al., 1991a). This localization corresponds in the EM to an immunogold labeling of tubular coated pits, apical tubules, endosomes, MVBs, and secondary lysosomes (Fig. 22). Since spermatozoa in the lumen are unlabeled, we deduce that testicular SGP-2 detaches from spermatozoa in the duct and is subsequently endocytosed by the nonciliated cells; its presence in secondary lysosomes suggests that it is degraded there (Fig. 10) (Hermo et al., 1991a).

3. SGP-1 in Nonciliated Cells of the Efferent Ducts Unlike SGP-2, SGP-1 has been shown to follow two different targeting routes in Sertoli cells: a lysosomal route, presumably occurring from the Golgi apparatus to secondary lysosomes (Igdoura and Morales, 1993) and a secretory route, ending in the binding of SGP-I to the tails of late spermatids (Sylvester el al., 1989). The secretory form of SGP-1 is found in high concentration within the lumen of the rete testis but decreases significantly in the caput epididymidis (Sylvester er al., 1989). In the efferent ducts, EM immunocytochemistry reveals that SGP-1 is localized in all compartments of the endocytic apparatus of nonciliated cells, including secondary lysosomes (Fig. 23 and Hermo et al., 1992b). Spermatozoa in the lumen of these ducts are unlabeled and remain so throughout the rest of the epididymis. We suggest therefore that the fate of Sertoli-derived SGP-1 is its endocytosis by the rete epithelial cells and nonciliated cells of the efferent ducts (Hermo et al., 1992b). In addition to an endocytic-derived source of SGP-1, we have evidence that efferent duct cells synthesize their own SGP- 1 . Evidence for this FIG. 19 Electron micrograph of a portion of the epithelium of the efferent ducts. The columnar nonciliated epithelial cells possess numerous large secondary lysosomes (L) of various intensities in their supranuclear region, while their apical region contains vesicular and tubular elements (arrows). Lipid droplets (Lip) appear in the basal region. N, nuclei: Mv, microvilli; G , Golgi apparatus. X 6160.

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FIG. 20 Electron micrograph of the apical region of adjacent nonciliated epithelial cells of the efferent ducts. Tubular coated pits (arrowheads) are continuous with the apical plasma membrane. Numerous tubules (arrows) seen as straight, curved, or wavy elements occupy this region. Referred to as apical tubules, they appear to be filled with a moderately dense material and are seen in cross section as circular elements with a content. Small and large membranous elements referred to as early and late endosomes (E) are also present. The endoplasmic reticulum (ER) is not plentiful and appears in frontal view as an anastomotic sheet (asterisks). m, mitochondria: Mv, microvilli; L, secondary lysosome. x 22,890.

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FIG. 21 Diagrammatic representation of the endocytic process in the nonciliated cell of the efferent ducts. Tubular coated pits invaginate from the apical plasma membrane, pinch off, and undergo constriction accompanied by the gradual loss of the coat to form apical tubules. The average time required for this process is 5 min. Apical tubules fuse to form endosomes; 30% of apical tubules recycle back to the apical plasma membrane, the rest partake in the transformation of endosomes to multivesicular bodies to secondary lysosomes. The average time required for an apical tubule to fuse with an endosome is 2 min. Recycling of apical tubules requires an average turnover time of 30 min. (Reproduced from Herrno et al., 1988a.)

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FIG. 22 Electron micrograph of a nonciliated cell of the efferent ducts irnmunolabeled with anti-SGP-2 antibody. Gold particles are present in tubular coated pits (arrows), endosomes (E),multivesicular bodies (asterisks), and secondary lysosomes (L).Mv, microvilli. x 32,000. Inset. Light micrograph of an efferent duct immunostained with anti-SGP-2 antibody. The dense immunoperoxidase reaction product (arrows) is seen in the apical and supranuclear regions of the nonciliated epithelial cells. Lu, lumen. x 200.

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:i TCP AT

-E 1VB

L

FIG. 23 Diagram of the events taking place in the nonciliated cell of the efferent ducts from a control, untreated animal. The testicular Sertoli-derived SGP- I (t-SGP-I represented as open squares) is endocytosed, possibly via a receptor-mediated process, and appears within the endocytic apparatus, which includes tubular coated pits (TCP). apical tubules (AT), endosomes (E), multivesicular bodies (MVB), and secondary lysosomes (L). It is assumed that a receptor (T) may dissociate from SGP-I and be recycled to the apical cell surface by means of tubules. An efferent duct form of SGP-I (represented as dark triangles) appears to be synthesized and targeted from the Golgi apparatus (G) via small vesicles to the late endocytic apparatus (MVB and L), where it is presumably processed to saposins (dark circles). A secretory form of SGP-I (dark square) may also be synthesized by these cells and ferried by small vesicles to the apical cell surface. N . nucleus. (Reproduced from Igdoura er a / . , 1993 with permission of Wiley-Liss, a division of John Wiley & Sons, Inc.)

comes from the localization of SGP-1 mRNA in these cells and the EM immunolocalization of SGP- 1 within smaller vesicles in the vicinity of the Golgi apparatus, dense MVBs, and secondary lysosomes of these cells (Hermo et al., 1992b). On this basis we postulate that an endogenous lysosomal form of SGP- 1 is synthesized by the nonciliated cells and trans-

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ported by small vesicles from the Golgi apparatus to dense MVBs and secondary lysosomes (Fig. 23). In order to test this hypothesis, a combination of various experiments were designed. One was ligation of the efferent ducts near the rete testis to restrict the entry of testicular fluids and, therefore, of Sertoli-derived SGP-1 into the lumen of these ducts. As early as 4 hr after ligation, there was a marked decrease in the number of gold particles representing SGP1 antigenic sites in the endocytic vesicles, endosomes, and pale MVBs (early endocytic apparatus) compared with control untreated animals; at 14 and 24 hr, little to no immunolabeling was found in these early endocytic organelles. These results demonstrate that the sole source of SGP-1 for the early endocytic apparatus of nonciliated efferent duct cells is testicular (Igdoura ef al., 1993). On the other hand, anti-SGP-1 labeling of the late endocytic apparatus (dense MVBs and secondary lysosomes) was not significantly decreased compared with control untreated animals, even at 24 hr after ligation. This finding suggests either a slow turnover of testicular SGP-1 within the late endocytic apparatus or the presence of an endogenous lysosomal form of SGP-1 synthesized by the nonciliated cells and possibly targeted to the late endocytic apparatus via Golgi-derived vesicles. Since the presence of mRNA transcripts for SGP-1 was found in the efferent duct cells together with small anti-SGP-l-labeled vesicles or clusters in the Golgi region, and in close proximity to dense MVBs and secondary lysosomes, it is postulated that small vesicles serve to target SGP-1 or SGP-l-derived proteins from the Golgi apparatus to the late endocytic apparatus (Igdoura et a)., 1993). This hypothesis was tested by using groups of animals treated with tunicamycin and sacrificed at different time intervals. Tunicamycin, a potent inhibitor of protein glycosylation, interferes with the formation of dolichol-diphospho-N-acetylglucosamine and blocks the glycosylation of asparagine residues in glycoproteins (Elbein, 1981; Kuo and Lampen, 1974). Since SGP-1 is extensively N-glycosylated (Collard ef al., 1988), tunicamycin was used as a potential inhibitor of its targeting to secondary lysosomes. At a late time interval (24 hr), SGP-1 immunolabeling of the late endocytic apparatus decreased significantly compared with untreated control animals and animals with their efferent ducts ligated. The decrease in labeling observed in the late endocytic compartment is interpreted as a blockage of the SGP-1 lysosomal pathway from the Golgi apparatus. Pulse experiments using [3SS]cysteinewere aimed at defining the sequence of events from the initial products of translation to the final fully processed mature forms of SGP-1 peptides in efferent ducts in uiuo. SGP1 forms were immunoprecipitated from homogenized efferent ducts at different intervals post radiolabeling. The results of this experiment re-

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vealed that the first form of SGP-1 biosynthesized by the efferent ducts is a 55-kDa protein which appears to be post-translationally modified to a 65-kDa product. The 65-kDa protein, which precedes the 70-kDa protein, is then proteolytically processed to 15-kDa mature saposins (Igdoura et al., 1993). As discussed earlier, the 65-kDa form of SGP-1 was isolated from Sertoli cell lysosomes and identified as the saposin precursor (Igdoura and Morales, 1993). On this basis we suggest that the 65-kDa protein in the efferent duct cells is the lysosomal form of SGP- I (Igdoura et al., 1993). The extensive sequence similarity between the rat 70-kDa SGP-I and human prosaposin (O’Brien et al., 1988) suggests that both proteins are homologous. While it is known that a 70-kDa SGP-1 is secreted by Sertoli cells (Griswold et al., 1988),our study showed a similar form in the efferent duct cells. The results, however, could not confirm whether the 70-kDa form was secreted or retained in an intracellular compartment (Fig. 23). However, Hineno et al. (1991) demonstrated that various secretory fluids contain prosaposin and suggested a possible extracellular function for SGP- 1 /prosaposin.

4. Quantitative Studies on the Endocytic Apparatus of Nonciliated Cells After a single injection of C F (an adsorptive tracer) into the lumen of the rete testis, an EM kinetic analysis was performed on the labeling indices of tubular coated pits and apical tubules at numerous time intervals ranging from 0.5 to 120 min after injection (Hermo et al., 1988a). The results from this study indicated that the endocytic process begins with tubular coated pits, followed by their transformation into apical tubules (Fig. 21). It was further shown that two classes of apical tubules exist. Those in one class fuse with each other to form endosomes, while those in a second class detach from endosomes to recycle back to the apical plasma membrane. Calculations were that it takes 5 min for tubular-coated pits to transform into apical tubules, 2 min for one class of apical tubules to fuse together to form endosomes, and 30 min for a second class of apical tubules to recycle back to the apical cell surface (Fig. 21). It was further computed that about 30% of the apical tubules recycle from endosomes, leaving 70% to go on to completion of the endocytic process. Following labeling of endosomes, the labeling sequence proceeded in this way: pale MVBs + dense MVBs + secondary lysosomes (Fig. 21) (Hermo et al., 1988a). Since numerous morphological images of transition states between endosomes and pale MVBs, between pale and dense MVBs, and between dense MVBs and secondary lysosomes were observed, it was suggested that these endocytic compartments transform into one another. This was

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given further credibility due to the apparent lack of small labeled vesicles which could act as shuttle elements between the different compartments (Hermo er al., 1988a).

VI. Epididymis: Cell Types and Functions The major epithelial cell type lining the epididymis is the principal cell, a cell recognized to be involved in secretion and endocytosis (reviewed in Hamilton, 1975;Cooper, 1986;Robaire and Hermo, 1988).Interestingly, principal cells of the different regions of the epididymis, that is, initial segment, intermediate zone, caput, corpus, and cauda (Fig. 24), show distinctive morphological and functional features. This also appears to be the case for the highly endocytic epithelial clear cells. In this section we first consider the general complexity of epididymal secretion and then provide an integrative analysis of the morphological and functional features of principal and clear cells in different regions of the epididymis. Initial Segment proximal

'rent Ducts

Cauda

FIG. 24 Diagram of different regions of the epididyrnis of the adult rat. Outlined are the proximal, middle, and distal areas of the initial segment; intermediate zone; and proximal and distal areas of the caput epididyrnidis and corpus and cauda epididymidis. (Reproduced from Hermo et al., 1991a.)

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Our emphasis is on the secretion and endocytosis of specific proteins that we have studied. A. Secretion by Principal Cells of the Epididyrnis

Over the past 25 years the epididymis has been shown to be involved in the secretion of glycoproteins into the lumen. In the early 1970%suggestive evidence for protein secretion arose from a series of studies in which the electrophoretic pattern of protein bands from the epididymal fluid was shown to differ not only from plasma but also among different epididymal regions (Barker and Amann, 1971; Amann et al., 1973;Jones, 1974; Koskimies and Kormano, 1975; Dacheux and Voglmayr, 1983). Neutra and Leblond (1966) presented the first L M radioautographic evidence that labeled galactose was taken up by the epididymal principal cells. Initially, silver grains were present over the Golgi apparatus and subsequently over the apex of the cell and lumen. This was substantiated in later studies by others (Kopecny and Peck, 1977). In the case of labeled amino acids, epididymal principal cells first showed silver grains over the rough endoplasmic reticulum (ER) and later over the Golgi apparatus, apical cell surface, and lumen (Flickinger, 1979, 1981, 1983, 1985). Flickinger (1981) showed in mouse principal cells that it takes about 2 hr for the completion of events involved in protein synthesis and secretion, and that synthesis and transport of proteins were faster in the caput and corpus epididymidis than in the cauda region. The synthesis and secretion of various glycoproteins have been shown by LM immunocytochemistry to vary in distribution within the epididymal epithelium. Examples of this variation are given below. In many cases, the secreted proteins become associated with spermatozoa in specific regions, suggesting that they may be involved in sperm maturation. Indirect immunofluorescence of a sperm maturation antigen number four (SMA 4), a determinant on the surface of mouse sperm tails, revealed its localization in epithelial principal cells to a specific segment of the distal caput and proximal corpus; a variable reaction in staining of individual epithelial cells in this segment was also noted (Vernon et al., 1982). Secretion by principal cells in the same region was also found in the rabbit and hamster epididymis using antisera raised against epididymal-specific proteins purified by isoelectric focusing (Moore, 1980) and in the rat epididymis using antisera against a 37-kDa epididymal sialoprotein (Faye et al., 1980), a 33-kDa acidic epididymal glycoprotein (Lea et al., 1978), and a specific epididymal protein (SEP) (Kohane et al., 1980). Other examples of proteins secreted by the epididymis in the same or different regions include the acrosome stabilizing factor (Thomas et al., 1984), bovine for-

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ward motility protein (Brandt et al., 1978), and secretory proteins of the human epididymis (Tezon et al., 1985). Recently, Rankin et al. (1992) have characterized and immunolocalized several mouse epididymal proteins (MEP) which are secreted by principal cells of the epididymis. One of these, MEP 7, secreted by principal cells of the distal caput, corpus, and cauda, is a 29-kDa glycoprotein. It has properties similar to the proteins AEG (Lea et al., 1978),CP 27 (Flickinger et al., 1988), D and E (Brooks and Higgins, 1980),PES (Fournier-Delpech et al., 1973), protein IV (Jones et al., 1980) and a 32-kDa protein (Wong and Tsang, 1982). In fact, these glycoproteins may be the same because their electrophoretic mobility and isoelectric points are identical and because their immunolocalization in the epididymis is similar (Rankin et d., 1992). Indeed, the nucleotide sequence of mRNA and the amino acid sequence of MEP 7 compared with AEG and proteins D and E, indicating that MEP 7 is the mouse homolog of rat AEG and proteins D and E (Rankin et al., 1992). MEP 10 is immunolocalized within principal cells at the junction of the distal caput and corpus. MEP 10, an 18-kDa polypeptide has a molecular mass and amino acid sequence (Rankin et al., 1992) similar to proteins B and C isolated from rat epididymis by Brooks and Higgins (1980).Proteins B and C are members of the a2pglobulin superfamily (Brooks, 1987) and are identical to two novel retinoic acid binding proteins-epididymal binding proteins (EBP)l and 2-which are present in rat caudal fluid (Newcomer and Ong, 1990). MEP 10 has also been shown to bind retinoic acid (Rankin et al., 1992). MEP 9 is a 25-kDa glycoprotein immunolocalized within all principal cells of the distal caput and within only some principal cells of the corpus and cauda epididymidis (Rankin et al., 1992;Vierula et al., 1992). Proteins identified with a molecular mass similar to MEP 9 are a 24-kDa protein reported to be secreted by the mid- and distal caput epididymidis of the mouse (Jimenez et al., 1990), a 26-kDa protein found in the cauda region of the mouse epididymis (Murphy and Carroll, 1987), and a rat 26-kDa sperm binding protein present in caudal fluid and on caudal sperm but not in the caput epididymidis (Olson et al., 1987). All of these proteins differ in their distribution and biochemical properties from MEP 9 (Rankin et al., 1992). However, a recent study pointing to a similarity in the immunolocalization of MEP 9 with the Yo subunit of glutathione S-transferase (Veri et al., 1993), suggests that MEP 9 is the mouse homolog of this subunit of glutathione S-transferase. Moore et al. (1990) described the synthesis and secretion of an androgenregulated 18-kDa component of rat epididymal a-lactalbumin-like complex to principal cells of the proximal caput which they found similar to a protein reported by Brooks et al. (1986). This immunolocalization fits that

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reported by Byers et al. (1984) for a-lactalbumin. Cornwall et al. (1992) showed by in situ hybridization cystatin-related epididymal-specific (CRES) mRNA within principal cells of the proximal caput epididymidis. A specific 135-kDa protein from porcine cauda fluid was localized in epithelial cells from the initial segment to the corpus epididymidis (Okamura et al., 1992). In situ hybridization revealed that proenkephalin mRNA is expressed at abundant levels in the principal cells of the initial segment (Garrett et al., 1990). The initial segment and proximal caput regions of the epididymis also contain cellular retinol-binding protein (Porter et al., 1985; Kato et al., 1985). The fate of some proteins secreted in the proximal epididymis appears to be their reabsorption in the distal epididymis: for example, Faye et al. (1980) noted that a 37-kDa SP protein, secreted by the epithelial cells of the caput and corpus epididymidis, is reabsorbed by epithelial cells, possibly clear cells, in the cauda region. Flickinger et al. (1988) indicated that a 27-kDa glycoprotein (CP 27) secreted by the distal caput-proximal corpus epididymidis is reabsorbed in the distal corpus and cauda epididymidis by clear cells (see Section V1,C). All of these studies as well as others clearly indicate that principal cells are involved in the secretion of a large variety of proteins which are differentially secreted along the length of the epididymis. A key question that arises is why such a regional variation of protein secretion has evolved. Presumably each successive segment of the epididymis must fulfill some essential and cumulative role in the maturation of the sperm. The key to understanding the maturational process of the sperm no doubt lies in resolving the role of each secretory protein and the identification of the regulatory factors involved in its secretion.

6 . Secretion and Endocytosis by Principal Cells of Different Epididymal Regions

1. Initial Segment: Structure and Functions of Principal Cells Principal cells of the initial segment of the epididymis are tall columnar cells characterized by an extensive secretory apparatus and a less distinguished endocytic one. The endocytic apparatus of these cells is characterized by coated pits and vesicles and few endosomes, pale and dense MVBs, and acid phosphatase-positive secondary lysosomes (Figs. 25,261. These structures gradually become labeled in a temporal and sequential manner when tracers such as N F or CF are introduced into the lumen of the duct (Figs. 27, 28). These cells are thus involved in fluid-phase and adsorptive endocytosis. Early EM studies noted the uptake of Thorotrast

FIG. 25 Electron micrograph of the apical region of an epithelial principal cell of the initial segment of the epididymis. Coated pits (arrows) of the apical cell surface are evident, as are subsurface coated vesicles. Smooth-surfaced vesicles (150-300-nm diameter) without any apparent content are evident in this region and correspond to the secretory vesicles (sv) of the cell. Cisternae of the sparsely granulated endoplasmic reticulum (asterisks) are plentiful and appear as large, irregularly shaped, dilated, membranous elements containing a fine flocculent material; they may come in close proximity to the apical cell surface. Numerous bundles of filaments (f) are present. Small vesicles (v) are also present. Lu, lumen. x 43,100.

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FIG. 26 Electron micrograph of the supranuclear region of adjacent principal cells of the initial segment of the epididymis. Stacks of Golgi saccules (S) are evident and associated on both cis and trans faces with the sparsely granulated endoplasmic reticulum. The latter (asterisks) is dilated, irregular in appearance, and contains a fine flocculent material. There are pale and dense multivesicular bodies (MVB) but few secondary lysosomes (L) in this cell. x 15,300.

FIG. 27 Electron micrograph of the apical region of a principal cell of the epididymal initial segment 2 hr after injection of native ferritin into the lumen. Noticeably labeled are dense multivesicular bodies (MVB) and an endosome (E) showing a tubular extension (arrowheads). A few vesicular and tubular elements near the surface are weakly labeled (arrows). The 150-300 nm empty-looking, smooth-surfaced vesicles referred to as secretory vesicles (sv) are unlabeled. Lu, lumen; asterisks, sparsely granulated endoplasmic reticulum. x 41,040.

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FIG. 28 Electron micrograph of the Golgi region of a principal cell of the epididymal initial segment 2 hr after injection of native ferritin into the lumen. Secondary lysosomes (L) are well labeled but there is a complete absence of labeling of the 150-300-nm empty-looking, smooth-surfaced, secretory vesicles (sv). Small vesicles (arrowheads) are plentiful in association with and at a distance from the Golgi stacks of saccules (S) but they are always unlabeled. Asterisks, sparsely granulated endoplasmic reticulum. x 36,650.

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by these cells (Hoffer er al., 1973). LM immunocytochemical studies first suggested that androgen binding protein was taken up by these cells (Pelliniemi et af., 1981; Attramadal er af., 1981), which was later verified in the ram by EM immunocytochemistry (Veeramachaneni and Amann, 1991). LM immunocytochemistry has also provided evidence for the endocytosis of inhibin in the proximal part of the excurrent duct system of the ram (Veeramachaneni et af., 1989). The secretory apparatus of these cells is characterized by two distinct types of endoplasmic reticulum and an elaborate Golgi apparatus (Figs. 25,26). In the basal region, flattened cisternae of rough ER are arranged in parallel rows, while in the apical and supranuclear regions of the cell, numerous large, dilated, irregular profiles of ER are present which show on their outer surface only an occasional ribosome; their lumen contains a uniform, finely filamentous material (Figs. 25,26). They are referred to as the sparsely granulated endoplasmic reticulum (sgER; Hoffer er al., 1973; Flickinger, 1979).The Golgi apparatus is extensive, filling the supranuclear region and showing on both its cis and trans faces a close association with the sgER (Fig. 26 and Hermo et al., 1991b). From the Golgi region, the sgER extends upward to the apical region of the cell, where it is in close proximity to the apical plasma membrane, although not in continuity with it (Fig. 25 and Hoffer et al., 1973; Hermo et al., 1991b). Hoffer et al. (1973) speculated that secretory proteins formed in the sgER might be released directly into the lumen, thus bypassing the Golgi apparatus. The radioautographic study of Flickinger (1979), however, did not support this idea. Injection of radioactive isotopes showed silver grains initially over the sgER and then in succession over the Golgi apparatus, apical cell surface, and lumen (Flickinger, 1979, 1981). Although it has been shown that the synthesis and secretion of proteins involve the ER and Golgi apparatus (Flickinger, 1979,1981),the transport carrier from the Golgi apparatus to the apical cell surface has not yet been identified. However it was recently shown that on the trans face of the Golgi apparatus, several CMPase-positive trans Golgi networks are present (Fig. 29) (Hermo et al., 1991b). In frontal views, they appear as an elaborate anastomotic tubular network containing pale, dilated areas (Fig. 30). These areas are similar in appearance to smooth-surfaced, electronlucent vesicles averaging 150-300 nm in diameter seen in proximity to the Golgi apparatus (Fig. 28) as well as in the apical region of the cell (Figs. 25,27). Experiments involving the injection of tracers into the lumen consistently reveal that these vesicles are unlabeled (Figs. 27,28). Based on these observations and findings to be described later, these vesicles can be tentatively classified as the secretory vesicles of the cell (Hermo et al., 1991b). A close relationship between the trans Golgi networks and the sgER is often observed in these cells (Fig. 30) but the rationale for this association is unclear.

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FIG. 29 Electron micrograph of stacks of Golgi saccules (S) of a principal cell of the epididyma1 initial segment showing cytidine monophosphatase activity. The reaction is present in the trans Golgi network (arrows) on the trans face of the Golgi stacks, and in secondary lysosomes (arrowheads). x 28,080.

Secretion of SGP-2 In an LM immunocytochemical analysis using an anti-SGP-2 antibody, a distinct difference in staining was obtained over principal cells between the proximal and distal regions of the initial segment (Hermo et al., 1991a). Although ultrastructurally the principal cells of these regions are identical in terms of their morphological appearance, those of the proximal region were unreactive while those of the distal region were distinctly reactive. The reaction was seen throughout the cytoplasm of every principal cell in the distal region.

Q.

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FIG. 30 A diagram of a stack of Golgi saccules of a principal cell of the initial segment as seen in cross section (top). The compact (CZ) and noncompact (NCZ) zones are illustrated. Overlying the cis aspect of the stack are several cisternae of sparsely granulated endoplasmic reticulum (ER), which show the occasional bud-like projection (arrow) and clusters of small 80-nm vesicles (v). The stack is formed of the cis element which is osmiophilic, the first saccule; saccules 2-4, which are strongly NADPase-positive and which together with the first saccule form the so-called wells (W), which contain a few small vesicles; saccules 5-7 and the eighth element, which are TPPase-positive; and several CMPase-positive trans Golgi networks (TGN). One of the trans Golgi networks shows a “peeling-off” configuration; the other is separated from the stack. Cisternae of the sparsely granulated endoplasmic reticulum are often interposed between the eighth element and the peeling-off trans Golgi network. These ER cisternae are also found between adjacent trans Golgi networks. The threedimensional appearance of the seventh saccule, eighth element and underlying trans Golgi networks and their close relationship with cisternae of sparsely granulated ER is illustrated

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Immunogold labeling at the EM level revealed SGP-2 predominantly over the 150-300-nm electron-lucent vesicles present in the Golgi and apical regions of principal cells of the distal initial segment, with some label over ER and Golgi saccules (Hermo et al., 1991a). There was no labeling of the endocytic apparatus. These results therefore give further credibility to the notion that the electron-lucent vesicles are secretory in nature.

b. Secretion of Immobilin LM immunoperoxidase localization of immobilin revealed a differential staining of the initial segment, allowing its subdivision into a proximal, middle, and distal region (Hermo et al., 1992a). In the proximal region the staining of principal cells was negligible (Fig. 31). In the middle region, a checkerboard-like staining was apparent, with some cells being reactive while others were unreactive (Fig. 32). In the distal region, many of the principal cells were intensely reactive (Fig. 33). At the EM level, immunogold labeling of immobilin revealed the secretory apparatus, including the 150-300-nm vesicles, of the reactive principal cells to be immunolabeled (Hermo et al., 1992a). Gold particles were also found over small patches of a flocculent material seen sparingly in the lumen of the duct of this region. 2. Intermediate Zone: Structure and Function of Principal Cells The intermediate zone of the epididymis is an area situated between the initial segment and proximal caput epididymidis (Fig. 24). It is referred to as such because grossly the epididymal tubules of this region are intermediate in size between those of the initial segment, which are small in diameter, and those of the proximal caput, which are larger. There are no studies on principal cells of this region; however, it is clear that these cells possess morphological features as seen in the EM that differ dramatically from those of the other regions. Indeed, the Golgi apparatus and endoplasmic reticulum both are structurally different from that seen in principal cells of the initial segment or caput epididymidis. A key feature of these cells is the presence of extremely large vacuoles in the apical

at the bottom. Note the pale, dilated regions (asterisks) along the trans Golgi network. Such regions are electron-lucent in the electron microscope and presumably are liberated as electron-lucent vesicles from the trans Golgi networks as the latter break down and fragment, leaving behind residual trans Golgi networks (RTGN) and free tubules (T). The electronlucent vesicles (150-300 nm, stars) are considered as secretory vesicles. (Reproduced from Hermo e r a / . , 1991b.)

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FIGS. 31-33 Photomicrographs of the proximal (Fig. 32), middle (Fig. 33), and distal (Fig. 34) regions of the epididymal initial segment immunostained with anti-immobilin antibody. Principal cells (P) of the proximal region are weakly stained, while in the middle region some principal cells are intensely stained (arrowheads) while others are weakly reactive (P). In the distal region, principal cells are moderately or intensely stained while a few are weakly reactive (arrows). There is a progressive increase in staining of material in the lumen (asterisks) associated with spermatozoa. IT. intertubular space. X 225.

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region (diameters of 2 pm and greater). These vacuoles (usually 1-3 per cell) contain patches of an electron-dense material, membranous profiles, and small vesicles (Hermo et al., 1992~).The function of these vacuoles in endocytosis or secretion is still unknown. Principal cells of this region are immunoreactive for anti-SGP-2 and anti-immobilin antibody. The reaction with SGP-2 appears moderately intense and uniformly distributed throughout the apical and supranuclear regions of the cell. In the case of immobilin, the reaction was intense throughout the cell. These cells thus appear to be involved in the secretion 1991a, 1992a). Aside from these of SGP-2 and immobilin (Hermo et d., observations, however, this zone of the epididymis has not been examined systematically but included as part of the initial segment. The role that principal cells of this zone play in sperm maturation is still unknown.

3. Caput, Corpus, and Cauda Epididymidis-Structure and Function of Principal Cells a. Secretion and Endocytosis by Principal Cells of the Epididymis Principal cells lining these different regions of the epididymis share many morphological features in common and yet also present their own distinct characteristics. The Golgi apparatus is elaborate (Fig. 34) and consists of many stacks of saccules (Fig. 35). Because these cells become progressively shorter distally along the epididymis, the Golgi occupies a progressively larger area of the supranuclear region of the cell and even extends into the apical region (Fig. 34). Numerous electron-lucent, 150-300-nm vesicles are present in the Golgi region as well as in the apex of the cell (Figs. 35,36). The rough endoplasmic reticulum is plentiful and arranged in parallel arrays in the basal region while in the remainder of the cell it consists of loosely distributed, flattened, irregularly shaped cisternae (Figs. 34-36). No sparsely granulated ER is evident in principal cells throughout these regions of the epididymis. The endocytic apparatus consists of coated pits and coated and uncoated vesicles (100 nm), endosomes, pale and dense MVBs, and acid phosphatase-positive secondary lysosomes (Figs. 36,37). Some of these structures have been shown to become labeled when nonspecific tracers are introduced into the lumen of the duct (Moore and Bedford, 1979; Friend, 1969). At the LM level, principal cells of the caput have been reported to be involved in the endocytosis of ABP (Pelliniemi et al., 1981; Attramadal et al., 1981). This has been verified ultrastructurally in the rat (Gerard et al., 1988) and ram (Veeramachaneni and Amann, 1991). a-2macroglobulin and transferrin have also been shown to be endocytosed in a receptor-mediated manner by rat caput principal cells (Djakiew et al., 1984, 1985, 1986). The pathway taken up by these proteins involved

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FIG. 34 Electron micrograph of epithelial principal cells of the distal caput epididymidis. The Golgi apparatus is elaborate and composed of many stacks of saccules (s) distributed in the apical and supranuclear regions of the cell. Numerous small vesicles (arrowheads)

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FIG. 35 Electron micrograph of the Golgi region of a principal cell of the caput epididymidis. Several stacks of saccules (S) are present in association with numerous smooth-surfaced, empty-looking 150-300-nmsecretory vesicles (sv). Numerous small vesicles (arrows) also appear in proximity to the stacks. ER, rough endoplasmic reticulum. x 24,450.

as well as a few pale multivesicular bodies (asterisks) are seen beneath the apical cell surface. Few dense secondary lysosomes (L) are present in this cell. The base of the cell contains stacked cisternae of rough endoplasmic reticulum (arrows) and several lipid droplets (Lip). Mitochondria (m) are plentiful throughout the cytoplasm. N , nucleus: Mv, microvilli; My, myoid cells. x 6200.

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FIG. 36 Electron micrograph of the apical region of a principal cell of the caput epididymidis. Coated pits (cp) of the apical cell surface are visible as well as subsurface coated vesicles (cv). An endosome (E) and pale multivesicular bodies (asterisks) are present, each showing plaques of a fuzzy material (arrowheads) on their delimiting membrane. Smooth-surfaced secretory vesicles (sv, 150-300-nm diameter) are plentiful. Also evident are small (50 nm) uncoated and coated vesicles (arrows). Several elongated flattened cisternae of rough endoplasmic reticulum (ER) are present. f, filaments; Mv, microvilli. x 31,810.

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FIG. 37 Electron micrographs of cytidine monophosphatase-positive secondary lysosomes (L) in a principal cell of the caput epididymidis. S. Golgi stack; ER, endoplasmic reticulum. X 28,665.

coated pits and vesicles, endosomes, and MVBs at early time intervals. At later intervals, a-2-macroglobulin was found in secondary lysosomes, while transferrin was reported to be recycled back into the lumen. Djakiew et al. (1986) also showed that the net amount of transferrin taken up by receptor-mediated endocytosis was equivalent in the caput and corpus, despite its decreasing amounts in luminal fluids from the caput to the cauda epididymidis. Secondary lysosomes of principal cells from distinct epididymal regions have specific morphological features (Robaire and Hermo, 1988). Acid phosphatase-positive, these organelles in the caput often present a pale, stained matrix containing electron-dense spherical or doughnut-shaped masses, small vesicles, and membranous profiles. In the corpus and cauda these bodies contain a homogeneous electron-dense material in which membranous and vesicular profiles are embedded (Robaire and Hermo, 1988).The heterogeneous nature of these lysosomal elements in the different regions of the epididymis has also been shown immunocytochemically by Suarez-Quian et al. (1992), but the meaning of this is unclear. Principal cells of all these regions also contain numerous small, uncoated vesicles (50-60 nm) in the supranuclear and apical regions of the cell as well as small, 50-60-nm coated vesicles, the functions of which are unknown. Few tubular elements are seen in the apical region. In our studies of endocytosis in principal cells, nonspecific tracers (NF and CF) injected into the lumen of the cauda epididymidis were seen at

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early intervals in coated pits and vesicles, endosomes, and pale MVBs (Fig. 38) and at later intervals in dense MVBs and secondary lysosomes. No labeling was present in the 150-300-nm electron-lucent vesicles present in the Golgi or apical region of the cell (Fig. 38).

6 . Secretion of SGP-2 LM and EM immunocytochemical analysis of SGP-2 revealed a pattern of staining for principal cells indicative of SGP2 synthesis and secretion (Hermo et al., 1991a). In the caput epididymidis, principal cells show a checkerboard-like immunoperoxidase staining pattern such that in a given tubular section cells are either intensely, moderately, or weakly reactive or unreactive (Fig. 39). In the case of the reactive cells, the entire cytoplasm is always stained (Fig. 40). In the corpus and cauda epididymidis, a similar but weaker pattern of staining is found. Spermatozoa in the lumen of these regions are highly reactive (Fig. 39). Northern blotting analysis supports these findings since a single 2.1-kb band, corresponding to the mRNA of SGP-2 (Morales et al., 1989), is found in the epididymis (Hermo et al., 1991a). Two-dimensional Western blotting analysis and two-dimensional gel electrophoresis revealed that SGP-2 was secreted primarily by the caput epididymidis, where it bound to sperm in the lumen (Mattmueller and Hinton, 1991). Based on our immunocytochemical results, the events in the secretion and endocytosis of SGP-2 in the testis and epididymis can be summarized as follows (see Fig. 10). Testicular SGP-2 secreted by Sertoli cells binds to the plasma membrane of late spermatids in the lumen. During transit through the rete testis and efferent ducts, testicular SGP-2 is lost from the sperm surface. This loss coincides with the endocytosis of testicular SGP-2 by the rete and efferent duct epithelium and its degradation in the secondary lysosomes of these cells. The uptake of testicular SGP-2 by the rete testis and efferent ducts is given further credibility by the fact that the principal cells and spermatozoa of the proximal initial segment do not contain SGP-2. Excluding the proximal initial segment, the principal cells of the epididymis secrete an epididymal form of SGP-2 which binds to the plasma membrane of spermatozoa in the lumen. As discussed earlier, the purpose of SGP-2 may be either to transport lipids from principal cells to the plasma membrane of spermatozoa or to solubilize lipids within the plasma membrane of spermatozoa.

c. Secretion of Immobilin LM immunostaining with an anti-immobilin antibody revealed that principal cells of the proximal caput are also involved in the synthesis and secretion of immobilin (Hermo et al., 1992a).

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FIG. 38 Electron micrograph of the apical region of a principal cell 30 min after the injection of cationic ferritin into the lumen. Femtin particles are present on microvilli (Mv) in coated pits and small subsurface vesicles (arrows), tubular elements (arrowheads), and pale multivesicular bodies (asterisks). Smooth-surfaced secretory vesicles (sv) are unlabeled. ER, endoplasmic reticulum; m, mitochondria. x 35,300.

In this region all principal cells are equally and intensely reactive. Immunostaining further down the duct becomes progressively less intense and terminates before the cauda epididymidis.

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FIGS. 39 and 40 Photomicrographs of tubules of the caput epididymidis immunostained with anti-SGP-2antibody. Note checkerboard-like staining pattern of the epithelium where principal cells (P) are intensely (arrows), moderately (arrowheads), or weakly immunoreactive. A reaction also appears in association with spermatozoa in the lumen (asterisk).IT, intertubular space. Fig. 39, x 225, Fig. 40, x 540.

At the EM level, immunogold labeling of immobilin is observed over the basal cisternae of rough ER, the Golgi apparatus, and the Golgi-related electron-lucent 150-300-nm vesicles (Fig. 41), which are also present in the apical region of the cell. The presence of antigenic sites for immobilin and SGP-2 (as seen earlier) but the absence of tracers (NF and CF) in these vesicles gives strength to the notion that the 150-300-nm, electronlucent vesicles represent the secretory vesicles of this cell.

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FIG. 41 Electron micrograph of the Golgi region of a principal cell of the caput epididymidis immunolabeled with anti-SGP-2 antibody. Gold particles appear over the Golgi saccules (S) and associated 150-300-nm smooth-surfaced secretory vesicles (sv) on the trans face. x 34,200.

Immobilin is also found in the lumen over a fine flocculent material which progressively increases in amount from the caput to the cauda epididymidis (Fig. 42). It is especially prominent in the lumen of the cauda, where spermatozoa are stored, and probably functions in immobilizing spermatozoa by virtue of its viscoelastic properties (Usselman and Cone, 1983; Usselman et al., 1985).

d . Synthesis of SGP-I LM immunoperoxidase staining of SGP-1 revealed an intense reaction over small bodies of various shapes and sizes in the supranuclear region of principal cells of all epididymal regions except the cauda epididymidis (Hermo et al., 1992b). In the EM this staining corresponded to a strong immunogold labeling of secondary lysosomes. No labeling was noted over coated pits and vesicles, endosomes and pale MVBs, and the lumen and its contents, thus eliminating the possibility that SGP-1 could be endocytosed from the lumen to ultimately end up in secondary lysosomes. Similar observations were made for the epithelial clear cells. Messenger RNA encoding SGP-1 was detected by Northern blotting analysis in all regions of the epididymis except the cauda (Hermo et al., 1992b). Coincident with mRNA expression, immunogold labeling of SGP1 , in addition to being present in secondary lysosomes, was associated with the Golgi apparatus and was found in close proximity to secondary lysosomes. Based on these observations and the lack of evidence for the

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FIG. 42 Electron micrograph of the lumen of the cauda epididymidis immunolabeled with anti-immobilin antibody. Gold particles (arrows) are abundant and present over a fine flocculent material in the lumen. The tails of spermatozoa are not labeled; a distinct pale area or halo (H)is seen around each. Outer dense fibers (ODF), axoneme (A), and mitochondria1 sheath (m) of the tail are indicated. x 27,200.

secretion of SGP-1 into the lumen, it is suggested that SGP-1 is synthesized by principal cells and then ferried to secondary lysosomes by small vesicles derived from the Golgi apparatus (see Fig. 43). Because SGP-1 has recently been shown to have substantial sequence similarity to prosaposin, it may

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FIG. 43

Diagrammatic representation of a principal cell of the epididymis illustrating the synthesis and trafficking of SGP-I. Presumably via small Golgi-derived vesicles, SGP-1 is channeled to multivesicular bodies and secondary lysosomes, where it was shown to be immunolocalized. No labeling was seen in coated pits, vesicles, or endosomes, indicating that SGP-I was not being internalized from the lumen. The 150-300-nrn, smooth-surfaced secretory vesicles were unlabeled. CP, coated pits; V, vesicles, E, endosome; MVB, multivesicular body; L, lysosornes; sv, secretory vesicles. (Reproduced from Hermo er a / . , 1992b with permission of Wiley-Liss, a division of John Wiley & Sons, Inc.)

be speculated that SGP-1 is instrumental in the degradation of membrane glycolipids present within secondary lysosomes of principal cells.

4. Studies on Principal Cells during Postnatal Development Electron microscope examination of animals at early postnatal day 21 revealed that principal cells of the entire epididymis appeared structurally undifferentiated. A full complement of secretory and endocytic organelles

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was only seen by day 39 (Hermo et al., 1992c), a time coinciding with high levels of androgen (Scheer and Robaire, 1980). It was of interest therefore to correlate this structural-hormonal relationship with the expression of anti-SGP-2 and anti-immobilin, two functional markers of postnatal development. Immunolocalization of SGP-2 was examined during postnatal development in animals ranging in age from day 7 to 56 (Hermo er af., 1994a). In the LM, principal cells of the initial segment showed an intense reaction for SGP-2 beginning at day 39, correlating well with high androgen levels noted at this age (Scheer and Robaire, 1980). However, principal cells of the caput revealed an intense checkerboard-like staining pattern for SGP2 only by day 49, while principal cells of the corpus and proximal cauda epididymidis showed an adult staining pattern only by day 56, at a time when spermatozoa appear in the lumen (Robaire and Hermo, 1988). The distal cauda on the other hand, revealed its adult-like staining pattern at day 49, which is well before the arrival of spermatozoa in the lumen and after androgen levels reach peak values at day 42 (Hermo et al., 1994a). Ligation of the efferent ducts of 15-day-old animals subsequently sacrificed at later intervals, that is, 49 and 64 days after birth, revealed that expression of the SGP-2 protein was not affected in the distal initial segment and caput (Hermo et al., 1994a). Thus luminal factors emanating from the testis do not seem to play an important role in SGP-2 expression in these regions. On the other hand, expression of SGP-2 in the corpus and proximal cauda was absent and thus dramatically affected, suggesting that testicular factors did play a role in SGP-2 expression in these regions (Hermo et al., 1994a). Similar findings have recently been reported for SGP-2 mRNA levels in adult animals (Cyr and Robaire, 1992). Postnatal immunolocalization of anti-immolibin antibody revealed that maximal reaction in principal cells was reached in the initial segment and caput by day 39 (Hermo et al., 1994b). Examination of 15-day-old ligated animals revealed a strong reaction in the initial segment but little reaction in principal cells of the caput, even at postnatal day 64, suggesting that luminally-derived testicular factors were important in the expression of this protein in the caput region. It thus appears that for principal cells, different factors are involved in the expression of different proteins along the epididymis and that even in the case of a given protein, different factors appear to play a role in its expression within the various epididymal regions. C. Roles of Epithelial Clear Cells in Endocytosis

Clear cells of the epididymis are present in the caput, corpus, and cauda but not in the initial segment or intermediate zone of the epididymis.

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These cells possess an elaborate endocytic apparatus (Fig. 44). Their apical region is filled with numerous small, medium, and large-sized endosomes and occasional MVBs (Fig. 44). Coated pits are present, as are large, irregular, often branching microvilli (Fig. 45). Secondary lysosomes shown to be acid phosphatase-positive (Hermo et al., 1988b)fill the supranuclear region of this cell (Fig. 44). Injection of tracers into the lumen of the cauda epididymidis reveal labeling at early intervals in the numerous apically located endosomes of various sizes and at later intervals in MVBs and secondary lysosomes of the clear cells (Fig. 4 3 , thus demonstrating that these cells are involved in the uptake of substances from the lumen (Hermo et al., 1988b). Endocytosis as the primary role for these cells has been substantiated by the finding that endocytic activity is much greater in clear cells than in principal cells (Moore and Bedford, 1979). Radioautographic detection of labeled amino acids has also revealed that a large proportion of proteins synthesized by clear cells are transferred to MVBs (Dadoune et al., 1985). One particular endocytic role for the clear cell appears to be the uptake of membranous and small particles (35 nm) which emanate from disrupting cytoplasmic droplets of spermatozoa in the cauda epididymidis (Fig. 46 and Hermo et al., 1988b). Membranous profiles and the 35-nm particles, resembling elements found in the droplet, were seen throughout the endocytic compartments of the clear cells in the cauda epididymidis.

1. Endocytosis of Immobilin We have shown by L M immunocytochemistry that immobilin is endocytosed by clear cells only in the distal cauda epididymidis (Fig. 47) (Hermo et al., 1992a). At the EM level, immunogold labeling representing immobilin antigenic sites is present in endosomes, MVBs, and secondary lysosomes of these cells (Fig. 48). However, the observation that a significant amount of immobilin is not endocytosed by the clear cells but remains in the lumen of the cauda epididymidis (Fig. 42) suggests that the clear cells of this region function in maintaining a steady state of immobilin within the lumen (Hermo et al., 1992a). Several other proteins have been shown by immunocytochemistry to be endocytosed by clear cells along the epididymis. MEP 7 is endocytosed by clear cells of the distal corpus and cauda region, and MEP 10 by clear cells of the distal corpus (Rankin et al., 1992). CP 27 is taken up by clear cells of the distal corpus and cauda (Flickinger et af., 1988), and acidic epididymal glycoprotein appears to be taken up by clear cells of the cauda (Lea et al., 1978). These observations point to a regional selectivity in the endocytosis of proteins by clear cells. Evidence for secretion of proteins by these cells has not been documented.

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FIG. 45 Electron micrograph of the apical region of a clear cell of the caput epididymidis 1 hr after injection of native femtin into the lumen. The tracer is present in small vesicles (v), endosomes (E), and dense secondary lysosomes (L).Lu, lumen. ~ 4 2 , 0 0 0 .

2. Studies on Clear Cells during Postnatal Development A recent postnatal study revealed that clear cells of the cauda epididymidis become structurally differentiated by day 39 (Hermo et al., 1992~).At this age their endocytic apparatus was highly developed and comparable FIG. 44 Electron micrograph of an epithelial clear cell of the cauda epididymidis. The supranuclear region is filled with large secondary lysosomes (L). Endosomes (E) and small vesicles (arrowhead) are seen in the apical region. G , Golgi apparatus; N , nucleus. The principal cell (far right) shows stacks of Golgi saccules (S) and secondary lysosomes (L). x 7400.

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FIG. 46 A diagram illustrating the position of the cytoplasmic droplet of spermatozoa in the testis and different regions of the epididymis. The cytoplasmic droplet is a small bulge of cytoplasm that is retained by spermatozoa as they are released into the lumen of the seminiferous tubule. The bulk of the late spermatid’s cytoplasm, referred to as the residual body, detaches and is phagocytosed by the Sertoli cells lining the seminiferous epithelium. In the testis and ductuli efferentes, the droplet surrounds the neck region of the tail, while in the caput epididymidis, the droplet appears near the junction of the middle and principal piece of the tail. The droplet shows a lateral displacement in the corpus epididymidis, suggesting that it is being shed, while in the cauda, most spermatozoa are devoid of droplets. While intact droplets are not conspicuous, their contents are found free in the lumen as well as within the endocytic organelles of the epithelial clear cells. This suggests that once droplets are released from spermatozoa, they break up in the lumen, liberating their contents, which appear to be endocytosed selectively by clear cells presumably to be degraded within their numerous secondary lysosomes. (Reproduced from Hermo er al., 1988b.)

to that of adult animals. The presence of numerous degenerating germ cells in the lumen at this time was thought to be a factor contributing to their differentiation. Clear cells of the caput and corpus, however, become differentiated only by day 49 (Hermo et al., 1992~). Using anti-immobilin antibody on rats at different postnatal ages, a

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FIG. 47 Light micrograph of tubules of the distal cauda epididymidis immunostained with anti-immobilin antibody. An immunoperoxidase reaction is present in association with stereocilia. Only clear cells (C) of the epithelium are intensely reactive. IT, intertubular space; Lu, lumen. x300.

weak to moderate reaction was present over clear cells of the distal cauda at day 39, at an age when small amounts of immobilin were present in the lumen of this region (Hermo et af., 1994b). However, by day 49, clear cells of this region became intensely reactive. This corresponded to the age when the lumen of the distal cauda was filled with immobilin. Examination of animals whose efferent ducts were ligated at day 15 and then sacrificed at postnatal day 49 revealed that clear cells of the distal cauda were only weakly stained with the anti-immobilin antibody at a time when little immobilin was seen in the lumen. However by day 64, these cells became intensely reactive at a time when large amounts of immobilin were seen in the lumen (Hermo et al., 1994b). Because degenerating germ cells are absent from the lumen under duct ligation, immobilin may be one factor involved in clear cell differentiation. It also appears that the clear cells are able to respond to increasing concentrations of immobilin. The delay in reactivity of clear cells could be explained by the fact that in ligated animals, immobilin is only secreted by the principal cells of the initial segment but not those of the caput, which normally occurs. Thus the accumulation of immobilin in the lumen of the distal cauda would be expected to take much longer. In any case, the delayed but intense reaction of clear cells in ligated animals indicates that the structural and functional differentiation of clear cells is not influenced by luminally-derived testicular factors (Hermo et al., 1994b).

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FIG. 48 Electron micrograph of the apical region of a clear cell of the distal cauda epididymidis immunolabeled with anti-immobilin antibody. Gold particles are seen in the lumen (Lu). In the clear cell, small vesicles (v), endosornes (E), and secondary lysosomes (L) are immunolabeled. x 22.930.

D. Epithelial Narrow Cells

Narrow cells of the epididymis are found only in the initial segment and intermediate zone. These goblet-like cells in the LM present a large, dilated, apical region but only a narrow, attenuated, basal region (Robaire and Hermo, 1988). In fact, the nucleus occupies the apical half of the cell. These cells are characterized in the EM by the presence of numerous small, spherical, or C-shaped vesicles (Robaire and Hermo, 1988), which clearly become labeled with tracers such as CF and NF, as do the few endosomes, MVBs, and secondary lysosomes in the cytoplasm (L. Hermo, unpublished). Endocytosis of specific proteins has not been demonstrated.

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Cisternae of ER are few in number but the Golgi apparatus is fairly well developed (Robaire and Hermo, 1988). There is no evidence as yet for secretion by these cells. Recently, these cells in the middle region of the initial segment have been shown to be immunoreactive for the anti-Yf subunit of glutathione S-transferase P (Veri et al., 1993), while in the proximal initial segment when they were reactive for its Yo subunit (Veri et al., 1994). In this region, therefore, these cells may be involved in protecting the epididymal epithelium from electrophilic attack. Aside from these observations, little is known about the function of narrow cells. E. Epithelial Basal Cells

Basal cells are small dome-shaped cells which do not reach the lumen and which are present throughout the entire epididymis and vas deferens (Robaire and Hermo, 1988). These cells possess an endocytic apparatus with coated pits seen on their basal plasma membrane, and few endosomes, MVBs, and secondary lysosomes. The Golgi apparatus is well developed in some cells but ER cisternae are not plentiful. A role in secretion or endocytosis for these cells has not as yet been determined. Recently we have shown that basal cells are immunoreactive for the anti-Yf subunit of glutathione S-transferase P (Veri et al., 1993). These cells were especially reactive in the region of the corpus and proximal cauda epididymidis. As seen in the LM, EM, and confocal microscope, the intensely stained basal cells showed extensive processes that covered a large part of the base of the epididymal tubule. The intense reactivity of these cells for this Pi glutathione S-transferase subunit, coupled with the discovery of their processes and overall organization around the base of the tubule, led us to speculate that these cells may be involved in protecting the epididymal epithelium from electrophilic attack (Veri et al., 1993).

VII. Vas Deferens: Secretion and Endocytosis by Epithelial Principal Cells

As in the case of the epididymis, the vas deferens shows regional differences in epithelial principal cell structure and can be divided into a proximal, middle, and terminal region (Hamilton and Cooper, 1978). In each region, principal cells display their own unique structural features. These cells have been shown to be involved in the uptake of sugars and their incorporation into substances that are secreted into the lumen (Bennett

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et al., 1974). They are equipped with a secretory apparatus endowed with numerous cisternae of rough endoplasmic reticulum and an elaborate Golgi apparatus, which like that of principal cells, shows numerous 150300-nm electron-lucent secretory-like vesicles which do not become labeled when tracers are introduced into the lumen (Hermo and De Melo, 1987). In the terminal region, discrete groups of epithelial cells have been implicated in the phagocytosis of spermatozoa (Cooper and Hamilton, 1977). Principal cells of the vas deferens contain coated pits and vesicles, endosomes, MVBs, and secondary lysosomes which become labeled temporally in a sequential manner when tracers are injected into the lumen (Friend and Farquhar, 1967; Hermo and De Melo, 1987). Involved in fluidphase and adsorptive endocytosis, these cells thus appear to be involved in getting rid of proteins and other substances present in the lumen. Such substances upon being endocytosed are degraded within secondary lysosomes of the cell (Hermo and De Melo, 1987). Adsorptive tracers such as CF and Con A-ferritin were also seen in small vesicles adjacent to the lateral plasma membrane as well as within the lateral intercellular space at late (15,30 min) but not early ( 5 , 10 min) time intervals (Hermo and De Melo, 1987), suggesting transcytosis from the lumen to the lateral intercellular space. Such an event also appears to take place in the rete testis. It is still not known which substances are being transcytosed because appropriate experiments have not been done.

VIII. Modification of the Sperm Membrane during Epididymal Transit

It is well documented that the plasma membrane of mammalian spermatozoa acquires a negative surface charge during epididymal maturation (Bedford, 1963, 1979; Cooper and Bedford, 1971; Moore, 1979; Holt, 1980; Yanagimachi, 1988). The acquisition of negative surface charge, as spermatozoa migrate from the caput to the cauda epididymidis, coincides with an increase in the amount of lectin binding sites (Nicholson et al., 1977; Olson and Danzo, 1981; Hamilton and Gould, 1982; Hamilton et a / . , 1986). Holt (1980) found that the amount of colloidal iron hydroxide (CIH) bound by cauda epididymal spermatozoa of the ram was 4-fold higher than that bound by caput spermatozoa. Based on the fact that the CIH-binding capacity of cauda epididymal spermatozoa was almost totally abolished by neuraminidase treatment, Holt (1980) concluded that the increase in the net negative surface charge on spermatozoa during epididyma1 transit is attributable to the acquisition of sialic acid groups on the sperm surface. Furthermore, since Ficoll washing of epididymal sperm

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did not affect the capacity for CIH binding, he concluded that the sialic acid carbohydrate groups were attached to proteins which were integral components of the plasma membrane. In an attempt to identify sperm membrane proteins which acquire sugar residues during epididymal transit, Olson and Hamilton (1978) made use of galactose oxidase and sodium metaperi~date-[~H]sodium borohydride techniques in order to radioactively label sperm plasma membrane glycoproteins possessing terminal galactose and sialic acid residues, respectively. They found that cauda epididymal spermatozoa of the rat possess a 37-kDa glycoprotein which was labeled by both of these techniques, whereas no such protein was detected on caput epididymal spermatozoa. This study prompted a further investigation by Brown et al. (1983), using an identical technique, to compare changes in plasma membrane glycoproteins of rat testicular sperm and of cauda epididymal spermatozoa. They found that unique to the plasma membrane of cauda epididymal spermatozoa were 1 3 3 , 32-, 47-, 84-, and 150-kDa glycoproteins, of which the 32-kDa protein was the most conspicuous. Another group of researchers in a series of studies (Olson and Danzo, 1981; Olson et al., 1987) demonstrated, by lactoperoxidase-catalyzed radio-iodination of the rat epididymal spermatozoon surface, that a 26-kDa glycoprotein appears on the plasma membrane of cauda sperm but is not found on caput sperm. Immunohistochemistry with monospecific antibodies obtained against this protein showed that this antigen was not detectable on caput sperm but first appeared on sperm from the proximal corpus epididymidis. A recent in v i m study showed that the incorporation of [14C]fucose,after rat spermatozoa from five representative regions of the epididymis were incubated with GDP-[14C]fucose,was highest in plasma membranes of sperm obtained from the distal caput (Tulsiani et al., 1993). Most conspicuously, the fucose label was incorporated into an 86-kDa plasma membrane protein. Thus it is established that the sperm plasma membrane is modified by the addition or alteration (i.e., glycosylation) of glycoproteins during epididymal maturation, but the outstanding question remains of how this is accomplished. One possibility for how the plasma membrane of epididymal spermatozoa acquires new glycoproteins is that the proteins could be secreted by the epididymal epithelium and subsequently bind to the sperm surface. Precedents regarding this possibility are many. For example, acidic epididymal glycoprotein (Lea et al., 1978), forward motility protein (Brandt er al., 1978; Acott and Hoskins, 1978), acrosome stabilizing factor protein (Thomas et al., 1984), His proteins (Rifkin and Olson, 19851, SGP-2 (Sylvester et al., 1984; Hermo et al., 1991a), and androgen-dependent proteins (Jones et al., 1980; Brown et al., 1983; Brooks et al., 1986; Brooks, 1987; Moore er al., 1990) have all been shown to be secreted by

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various regions of the epididymal epithelium and then become associated with the sperm surface. In fact, Klinefelter and Hamilton (1985) showed by perifusion organ culture of proximal and distal rat caput epididymal polypeptides are synthetubules that at least five ~-[~~S]methionine-labeled sized and secreted by these cultured tubules and become associated with luminal sperm. However, in all the above cases, the mechanisms of association (e.g., receptor mediated) of these secreted proteins with the sperm membrane are unknown; it is not known whether any of these proteins are integral components of the membrane. Furthermore, it does not appear that epididymal-secreted proteins account for all of the new glycoproteins acquired by the sperm membrane during epididymal transit. Another possibility for sperm membrane modification during epididymal transit, which has received little consideration, is by extrinsic glycosylation of preexisting membrane proteins. This possibility assumes that there would be glycosyl transferases secreted into the epididymal lumen by either the seminiferous or epididymal epithelium. In considering this hypothesis, Hamilton (1980) measured the enzymatic activity of N-acetylglucosamine galactosyl transferase in fluids from rat rete testis and epididymis. By cannulation of the rete and exudation of epididymal fluid, Hamilton (1980) found that the specific enzyme activity of galactosyl transferase in the luminal fluid was significantly higher’(>Sfold) in the rete than in the caput epididymidis; the enzyme activity per unit volume of the fluid then decreased abruptly in the distal cauda. This decrease in galactosyl transferase activity led Hamilton (1980) to suggest that this enzyme has a relationship to spermatozoon maturation. An equally important interpretation was that this enzyme, which was shown to be kinetically more analogous to milk galactosyl transferase than to serum galactosyl transferase, must be synthesized and secreted by the testis. Tulsiani et al., (1993) measured the total activities of sialyl transferase, fucosyl transferase, galactosyl transferase, and N-acetyl transferase in the supernatant and sperm pellet suspension of epididymal fluid obtained from five representative regions of the epididymis. Although a small proportion of glycosyl transferase activities sedimented with spermatozoa, the largest proportion was found in a soluble form. On measuring the sperm-associated glycosyl transferase activities per lo6 spermatozoa, they found that only sialyl transferase and fucosyl transferase activities showed a gradual reduction or “maturational-dependent change” as the spermatozoa moved from the caput to the cauda region of the epididymis. The higher levels of the two enzymes found on caput spermatozoa led Tulsiani et al. (1993) to suggest that a transient binding of these enzymes to endogenous sugar acceptor molecules on the sperm surface occurs, followed by their release in the distal part of the epididymis, after sialylation and fucosylation of the acceptor molecules. However, as acknowledged by

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these investigators, additional proof for an extrinsic mechanism of glycosylation of sperm surface components lies in the detection of endogenous nucleotide sugar donors. Furthermore, an immunolocalization of these glycosyl transferases onto the sperm membranes is imperative because the exact location of these enzymes cannot be precisely determined by biochemical means. Another question which remains to be answered, and which was raised indirectly by Hamilton’s (1980) study, is the initial source of the glycosyl transferases. Are the glycosylases secreted by the seminiferous or epididymal epithelium or are they intrinsic to the sperm? An additional consideration of epididymal sperm plasma membrane protein modification which we would like to propose is one of intrinsic glycosylation by which the sperm itself has this capability. For this to work, several requirements must be met. First, epididymal spermatozoa would have to have a compartment in which to store functionally active glycosyl transferases since new proteins are no longer synthesized at this stage of maturation. Second, the proteins or substrates (i.e., endogenous acceptors) to be glycosylated would also have to be in plentiful supply. Third, if the glycosyl transferases were to be stored in a membrane compartment, as they are in the saccules of the Golgi apparatus, a functionally active nucleotide sugar transporter system would have to be present. Finally, there would have to be a mechanism for the transport of newly glycosylated acceptors to and into the plasma membrane of epididymal sperm. In a recent study (Oko et al., 1993) we have been able to provide evidence for the first three requirements and indirect evidence for the fourth. In the case of the first requirement, we have been able to show in situ that the saccular elements of the cytoplasmic droplet of caput rat spermatozoa (Figs. 49,50) are strongly immunolabeled with antisialyl and galactosyl transferase antibodies; no immunolabeling was found elsewhere on the spermatozoa. This localization was confirmed biochemically on isolated saccular elements by the demonstration of a significant enrichment in the specific activity of these two enzymes in this fraction (Oko et al., 1993). As for requirements two and three, endogenous glycosylation performed on the isolated saccular elements of the droplet, using both uridine diph~sphate-[~H]galactose and cytidine m~nophosphate-[~H]sialic acid as glycosyl donors, showed convincingly that the labeled sugars were transferred into several endogenous proteins ranging in molecular masses from 10 to 145 kDa (Oko ef af., 1993). Since the incubations were done in the absence of detergents and membrane perturbants, nucleotide sugar transport through the saccular membrane most probably is operating. Circumstantial evidence which is consistent with transport of glycoconjugates from the saccular elements to the adjacent plasma membrane arose

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FIG. 49 Electron micrograph of cytoplasmic droplets of the spermatozoa within the lumen (Lu) of the efferent ducts. The droplet contains many flattened saccular elements (S),which appear randomly distributed with respect to each other but localized to one pole. The edges of the saccules often come in close association with each other (arrows) as well as with the plasma membrane (arrowheads). At times they are loosely stacked (curved arrows). In frontal view they appear as pale discs (asterisks). Several small vesicular profiles are also evident (small arrows). T. tail of spermatozoon. x 28,160.

from our lectin labeling in situ. We found that both Ricinus communis agglutinin (RCA I) and Helix pomatia lectin (HPL) labeling revealed high concentrations of the cognate sugar ligands (i.e., galactose and N-acetylgalactosamine) over the saccular elements of the cytoplasmic droplet and adjacent plasma membrane (Oko et al., 1993). Furthermore, ultrastructural observations revealed spot associations of saccule membranes with each other as well as with the adjacent plasma membranes (Figs. 49,50). These membrane associations were previously identified by Friend and Heuser (1981) as fusion intermediates and may signify a site of exchange. However, proof for such a unique transport system will ultimately depend upon the ability to follow the newly derived glycoconjugates from the saccular elements to the associated plasma membrane of epididymal spermatozoa. in situ.

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FIG. 50 Electron micrograph of a portion of a cytoplasmic droplet. The saccular elements (S) are flattened, usually curved membranous elements and often contain a uniform, moderately dense material that fills the lumen. Their edges often come in close association with each other (arrows) but other points of close association are visible (arrowheads). Some saccules are loosely stacked together (open arrows). x 74.000.

It has been emphasized recently that a decrease in glycosyl transferase activity, as spermatozoa transit from caput to cauda epididymidis, most probably reflects a maturation-dependent change (Tulsiani er al., 1993). This is because most sperm surface glycoconjugates are modified relatively early in epididymal transit (Hammerstedt and Parks, 1987; Eddy, 1988; Orgebin-Crist, 1987; Yanagimachi, 1988). In our study (Oko er al., 1993) we have shown biochemically and immunocytochemically that both sialyl transferase and galactosyl transferase in the saccular elements of the cytoplasmic droplet undergo a maturation-dependent decrease as spermatozoa transit from the caput to cauda epididymidis. This drop in activity complements the findings of Bernal et al. (1980), who demonstrated that caput but not cauda spermatozoa can replace disialo-fetuin as an acceptor of sialic acid in sialyl transferase assays. It also complements the data of other investigators who found that the amount of sialic acid bound to the

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sperm membrane increases rapidly to a maximum in the corpus epididymidis (Prasad et al., 1973; Laporte et al., 1975). In summary then, we have considered three potential ways by which the sperm membrane can acquire new, integral glycoconjugates during epididymal transit. They are (1) by epididymal epithelium secretions, (2) by extrinsic glycosylation, and (3) by intrinsic glycosylation. The validity of each requires consideration and remains open for investigation.

IX. Concluding Remarks

It is clear that the epithelial cells of the entire male reproductive duct system, from the testis to the vas deferens, contribute to a proper milieu for sperm maturation through two distinct activities: secretion and endocytosis. We have provided examples of these activities by following the origin and fate of SGP-1, SGP-2, and immobilin in the excurrent duct system. These proteins typify the regional variations that exist for the secretion and endocytosis of proteins along the reproductive duct. The reasons for such regional variations in secretion and endocytosis of different proteins will ultimately lie in the genetic regulatory factors for each protein, the type of association of each protein with the spermatozoa, if any, and the functional contributions that each protein plays in the final maturation of spermatozoa. As is evident from this review, these areas of endeavor are still in their infancy. An important concept raised in this review is that the spermatozoon itself may contribute to its own maturation (i.e., glycosylation) providing that the appropriate conditions of its external milieu are met by the secretory and endocytic activities along the excurrent reproductive duct system. Acknowledgments This work was funded by Medical Research Council of Canada grants to L. Hermo, R. Oko, and C. R. Morales and by an Natural Sciences and Engineering Research Council grant to R. Oko. Dr. Carlos R. Morales is a fellow from Fonds De La Recherche En Sante Du Quebec. The technical assistance of Jeannie Mui and Matilda Cheung is gratefully appreciated. The secretarial assistance of Ann Silkauskas is gratefully appreciated.

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Segretain, D., Egloff, M., Gerard, N., Pineau, C., and JCgou, B. (1992). Cell Tissue Res. 268347 1-478. Sertoli, E. (1865). Morgagni 7, 31-40. Skinner, M. K., and Griswold, M. D. (1980). J. Biol. Chem. 255, 9523-9535. Skinner, M. K., and Griswold, M. D. (1983). Biol. Reprod. 28, 1225-1229. Skinner, M. K., Cosand, L. W., and Griswold, M. D. (1984). Biochem. J . 218, 313-320. Smith, E. P., Dickson, B. A., and Chernausek, S. D. (1990). Endocrinology (Balfimore) 127, 2744-275 1. Soares Pessoa, J. F., and David-Ferreira, J. F. (1980). Biol. Cell. 39, 301-304. Suarez-Quian, C. A., Jelesoff, N., and Byers, S. W. (1992). Anat. Rec. 232, 85-96. Susi, F. R., Leblond, C. P., and Clermont, Y. (1971). Am. J . Anat. 130, 251-278. Sylvester, S. R. (1993). I n “The Sertoli Cell” (L. D. Russell and M. D. Griswold, eds.), pp. 201-216. Cache River Press, Clearwater, FL. Sylvester, S. R., and Griswold, M. D. (1984). Biol. Reprod. 31, 195-203. Sylvester, S. R., Skinner, M. K., and Griswold, M. D. (1984). Biol. Reprod. 31, 1087-1 101. Sylvester, S. R., Morales, C. R., Oko, R., and Griswold, M. D. (1989). Biol. Reprod. 41, 94 1 -948. Sylvester, S. R., Morales, C. R., Oko, R., and Griswold, M. D. (1991). Biol. Reprod. 45, 195-207. Tamaru, T., Fujubayashi, S., Brown, W. R., and Wenger, D. A. (1986). Histochemistry 86, 195-200. Tang, X . M., Lalli, M. F., and Clermont, Y. (1982). A m . J . Anat. 163, 283-294. Tezon, J. R., Ramella, E., Cameo, M. S., Vazquez, M. H., and Blaquier. J . A. (1985). Biol. Reprod. 32, 591-597. Thitry, G., and Rambourg, A. (1976). Biol. Cell 26, 103-106. Thomas, T. S., Reynolds, A. B., and Oliphant, G. (1984). Biol. Reprod. 30, 693-705. Tsuruta, J. K., Wong, K., Fritz, I. B., and Griswold, M. D. (1990). Biochem. J . 268,571-578. Tulsiani, D. R. P., Skudlarek, M. D., Holland, M. K., and Orgebin-Crist, M.-C. (1993). Biol. Reprod. 48, 417-428. Usselman, M. C., and Cone, R. A. (1983). Biol. Reprod. 29, 1241-1253. Usselman, M. C., Cone, R. A., and Rossignol, D. P. (1985). J . Androl. 6, 315-320. Veeramachaneni, D. N. R., and Amann, R. P. (1991). J . Androl. 12, 288-294. Veeramachaneni, D. N. R., Schanbacher, B. D., and Amann, R. P. (1989). Biol. Reprod. 41, 499-503. Veri, J. P., Hermo, L., and Robaire, B. (1993). J . Androl. 14, 23-44. Veri, J . P., Herrno, L., and Robaire, B. (1994). J . Androl. (accepted for publication). Vernon, R. B., Muller, C. H., Herr, J. C., Feuchter, F. A., and Eddy, E. M. (1982). Biol. Reprod. 26, 523-535. Vierula, M. E., Araki, Y., Rankin, T. L., Tulsiani, D. R. P., and Orgebin-Crist, M.-C. (1992). Biol. Reprod. 47, 844-856. Wong, P. Y. D., and Tsang, A. Y. F. (1982). Biol. Reprod. 27, 1239-1246. Yanagimachi, R. (1988). I n “The Physiology of Reproduction” (E. Knobil and J. Neill, eds.), pp. 135-185. Raven Press, New York. Ziemer, M. A., Swain, W. F., Rutter, W. J., Clements, S., Ann, D. K., and Carlson, D. M. (1984). J. Biol. Chem. 259, 10475-10480.

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Evolution of Mammalian Sex Chromosomes and Sex-Determining Genes Jennifer A. Marshall Graves and Jamie W. Foster' Department of Genetics and Human Variation, LaTrobe University, Melbourne, Victoria 3083, Australia

1. Introduction Mammals have a chromosomal sex-determining system in which XX individuals are female and XY male. There are two unique and interesting features of X and Y chromosomes. First, and most obviously, the sex chromosomes control sex. We know that the Y chromosome determines the male phenotype, and we would like to know how. Second, the X and Y chromosomes have become very different from each other in size and gene content. These genetic differences have far-reaching consequences in the behavior of the sex chromosome pair at male meiosis, and in setting up gene dosage differences between the sexes, which are compensated for by a chromosome-wide X-inactivation system. In this chapter, we explore the organization and function of mammalian X and Y chromosomes. Information has come from genetic differences among individuals and among species. We have emphasized the latter. Such comparisons enable us to deduce the process by which the X and Y chromosomes differentiated from each other, and took on their functions in sex determination and dosage compensation.

II. Organization and Evolution of Sex Chromosomes

Mammals all have an almost identical gene content, and all have genomes of about 3 x lo6 kb. The very diverse karyotypes result from the arrangements of this genome into a few large chromosomes, or many small ones.

' Present address: Department of Genetics, University of Cambridge, Cambridge, U K . Inrrrnurionrrl Reuieu, of Cyrology, Vol. I54

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Copyright D I 9 4 by Academic Press. Inc. All rights of reproduction in any form reserved.

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Since mammals are diploid organisms, each chromosome is present in duplicate. At meiosis, the two members of each of these homologous pairs recognize each other, synapse and recombine, then are distributed singly into gametes. The sex chromosomes present an exception to these rules. At mitosis in male-derived cells, the X and Y are distinguishable by their different sizes and morphologies. The X is remarkably conserved in size among different species, amounting to about 5% of haploid length, and containing about this proportion of genes (estimated at 2500-5000). The Y is invariably smaller and heterochromatic, being composed largely of repeated sequences, and containing few active genes. The genetic difference between the X and Y chromosomes is obvious at male meiosis, when the X and Y undergo pairing and recombination over only a tiny homologous region. Of the thousands of mammalian species, by far the most closely studied have been humans and mice. With the advent of the human genome project (National Center for Human Genome Research, 1990),increasingly detailed gene maps and physical maps of the human sex chromosomes are becoming available, and it makes sense to regard humans as the type species for comparisons. We therefore describe the human X and Y in some detail before drawing comparisons with other closely and distantly related mammals. A. Organization of Human Sex Chromosomes

Humans have 22 pairs of autosomes, a medium-sized X, and a small, heterochromatic Y (Fig. I). The X is divided by the centromere into a long arm (Xq)and a short arm (Xp), which can be subdivided by G-banding. In the recent flurry of gene mapping, 93 genes and numerous other unique DNA sequences have been located on the human X (Schlessinger et al., 1993). The positions of a few of these genes, referred to in this chapter, are depicted in Fig. I , and their names listed in Table I. The identified genes seem to code for a rather average mix of housekeeping functions (such as the ubiquitously expressed G6PD and P G K ) , and specialized functions (such as the color pigment gene RCP and the blood-clotting factors F8 and F9). The Y chromosome, making up about 2% of haploid length, has its centromere near one end. It stains differentially with DNA-binding fluorochromes, revealing large stretches of noncoding heterochromatin, concentrated in the distal portion of the long arm (Yq) (Cooke, 1976). N o Ylinked genes were ever identified by the classic methods of observing the inheritance patterns of variant traits, but several functions in male fertility

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S RY RPS4Y Z FY AMGY ADMLY STSP

DMD OTC MAOA

HYA

UBE 1 AR

RPS4X PGKl XlST

GLA

HPRT F9 F8 GGPD RCP

SOX3

FIG. 1 Human sex chromosomes. The X and Y are different in size and gene content, but share a small pseudoautosomal region (shown shaded) as well as a number of active and inactive genes outside this region (listed on the right). Other genes discussed in this chapter that are unique to the X are listed on the left. The names of the genes are given in Table I.

have been assigned to regions of the Y from the phenotypic effects of deletions (Bardoni et al., 1991). A male-specific transplantation antigen, HYA, has been ascribed to a gene on the Y. More recently, several expressed sequences have been revealed by molecular techniques (Goodfellow and Weissenbach, 1992; Wolff et al., 1992). Some of these have been discovered by positional cloning, and identified by their possession of a potential coding sequence (e.g., a zinc finger gene ZFY; Page et al., 1987);others have been discovered unexpectedly with probes to sequences elsewhere on the genome (e.g., RPS4Y, which codes for a ribosomal

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TABLE I Human X and Y Genes Discussed

Locus symbol“

Gene name

MIC2 CSF2RA S TS AMG ADML ZFXIZFY DMD OTC MAOA UBEl

Antigen detected by monoclonal antibody I2E7 Colony-stimulating factor 2 receptor Steroid sulfatase Amelogenin Adhesion molecule (Kallman syndrome) Zinc finger protein Dystrophin (Duchenne muscular dystrophy) Ornithine transcarbamylase Monoamine oxidase A Ubiquitin-activating enzyme 1

AR RPS4 PGKI XIS T

Androgen receptor (testicular feminization) Ribosomal protein subunit 4 Phosphoglycerate kinase I X (inactive)-specific transcript

GLA HPRT SR Y

Galactosidase alpha Hypoxanthine phosphoribosyl transferase Sex region, Y chromosome

SOX3

SR Y-like HMG box-containing

HYA F9 F8 RCP G6PD

Histocompatibility antigen on Y Blood coagulation factor 9 (hemophilia B) Blood coagulation factor 8 (hemophilia A) Red cone pigment (color blindness) Glucose-6-phosphate dehydrogenase

Comments PAR PAR Pseudogene on Y Active genes on X, Y Pseudogene on Y Active genes on X, Y

Active genes on X, Y in other mammals Active genes on X, Y Putative X inactivation center

Testis-determining factor Closest relative (ancestor?) of SRY Male specific

~

~~

Human gene nomenclature as recommended by McAlpine e t a / .(1993)is used throughout this chapter except where mouse genes (notated in lower case) are specifically discussed. a

protein, was discovered because of its sequence similarity with a gene, RPS4X, on the X ; Fisher et al., 1990). No cytological homology is apparent between the X and Y, but at meiosis the X and Y do pair at the tips of their short arms, ensuring regular segregation (Chandley et al., 1984). Burgoyne (1982) predicted that a region of genetic homology shared by the X and Y would be found, containing genes which undergo recombination like autosomal genes. Indeed, such “pseudoautosomal” inheritance was observed for the MZC2 gene mapping to the tips of the human X and Y (Goodfellow et al., 1986). More recently, a growth factor receptor gene CSF2RA, and several other functional genes (Gough et al., 1990; Ellison et al., 1992; Scheibel et al., 1993; Yi et al., 1993; Schlessinger et al., 1993) have been demonstrated to

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lie within this pseudoautosomal region (PAR). A sharp pseudoautosomal boundary has been demonstrated, above which sequences on X and Y are identical, and below which they diverge rapidly (Ellis et al., 1989). The dosage of genes within the PAR does not differ between males and females, so that it is not surprising to find that genes in this region are not subject to X-chromosome inactivation. There is now some evidence for a second, and smaller, PAR shared between the tips of the X and Y long arms. A region of about 500 kb is found to be homologous between distal Xq and Yq, and to undergo recombination (Freije et al., 1992). There is also considerable homology between the X and Y outside the PAR. Several regions of the X and Y share repeated sequences, or even considerable stretches of unique sequence (Cooke and Brown, 1984; Page et al., 1984; Geldwerth et al., 1985). More significantly, many-perhaps all-of the few functioning genes characterized on the Y have related sequences on the X. Moreover, probes to some X-linked genes (e.g., the steroid sulfatase gene S T S ) detect related nonfunctional sequences (pseudogenes) on the Y (reviewed in Graves and Schmidt, 1992; Wolff et al., 1992). Is this homology between the human X and Y the result of recent shuffling of sequences by translocation or retroposition? Or is it a relic of ancient homology between an ancestral X and Y? If this is so, is gene order preserved? How and why was X-Y homology lost? Comparisons of gene content of the X and Y chromosomes of other mammalian species can help to answer these questions.

6. Variation of Mammalian Sex Chromosomes Comparisons between closely related species can provide information about recent changes in genome organization and function, and comparisons between the most distantly related mammalian groups can inform us about ancient evolutionary events. Both have been extremely valuable in following the changes in mammalian sex chromosome organization and function. There are three major groups of mammals. Figure 2 shows that eutherian (placental) mammals diverged about 130 million years ago from metatherians (marsupials), and their common therian ancestor diverged about 170 million years ago from prototherians (the egg-laying monotremes) (Hope et al., 1990). Within Eutheria, the extremes are orders (such as primates and rodents) that diverged 80 million years ago, and species (such as man and the other primates) that diverged only a few million years ago. Within the marsupials there are similar divisions. The sex chromosomes of a number of eutherian mammals were exam-

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JENNIFER A. MARSHALL GRAVES AND JAMIE W. FOSTER

MONOTREMES MARSUPIALS

EUTHERIANS

0 50 100

150

200 MYr

/

FIG. 2 Phylogeny of mammals, showing the divergence of monotremes (subclass Prototheria), marsupials (infraclass Metatheria), and eutherians.

ined in the 1960s. The X was found to constitute about 5% of the genome, regardless of the sizes of the autosomes. This remarkable conservation of size, combined with the observation that G6PD and PGK were sex linked in a variety of mammals, led Ohno (1967)to predict that the mammalian X was absolutely conserved in gene content, perhaps because of its involvement in a chromosome-wide X inactivation system. Much more detailed mapping (O’Brien and Graves, 1991),made possible by the advent of somatic cell genetic and molecular mapping methods, has confirmed “Ohno’s law.” Detailed mapping studies in the mouse showed no exceptions to conservation among 53 loci which mapped to the X in both mouse and man, suggesting that the gene content of the X has not changed in the 80 million years since these species diverged.

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However, this conservation breaks down for at least one human pseudoautosomal locus, CSF2RA, which is found to be autosomal in mouse (Disteche et al., 1992). Although the gene content of the X seems hardly to have changed, differences in X-chromosome morphology and gene order indicate that internal rearrangements have occurred. At least five separate segments can be aligned between gene maps of the human and mouse X (Searle et al., 1989), and small homologous segments may contain numerous internal rearrangements (Lava1 and Boyd, 1993). However, the mouse may be atypical, for human and mink X chromosomes have a similar gene order, and even bear a cytological similarity (Zhdanova et al., 1988). There are few data concerning gene position in the sex chromosomes of other species. Is the mammalian Y chromosome also conserved in gene content? The Y is rather variable in size between species, but this may merely reflect its different heterochromatin content. Limited gene mapping suggests at least that the eutherian Y is monophyletic, for the human Y-borne genes, ZFY and HYA, detect male-specific homologs among a number of eutherian species. However, there are an increasing number of exceptions. The human RPS4 gene detects an X, but no Y-linked homolog in mouse (Fisher et al., 1990), whereas a sequence, Ubel, coding for a ubiquitin-activating enzyme, has active copies on both X and Y in mouse, but only an X-linked copyinman(Kayetal., 1991;Mitchelletal., 1991). Steroidsulfatase, STS, has an active copy on the Y in mouse (Keitges et al., 1985), but there is only an STS pseudogene on the human Y (Yen et al., 1988). Comparisons of base sequence and map position can reveal whether regions shared between the X and Y chromosomes resulted from recent transposition, or are vestiges of ancient homology. Some unique sequences shared by the X and Y, and processed pseudogenes, clearly arose from recent transposition to the Y from the X or autosomes (Page et al., 1984; Wolff et al., 1992). However, comparing the positions of genes on the X and Y chromosomes of closely related species reveals that all the functional genes and some pseudogenes on the Y were originally partners to X-linked genes. This is most evident in studies of closely related species. The great apes have X chromosomes which appear to be cytologically and genetically identical to the human X. However, the different centromere position in the human and gorilla Y implies that the Y has undergone at least one rearrangement in the primate lineage. Because of the similarity in base sequence between homologous genes in closely related species, human gene probes can be used to map the positions of human X-Y shared genes in other great apes. Several have been shown to lie in the same order on the gorilla X and Y , but are inverted in the human X and Y (Yen et al., 1988; Wolff et al., 1992; Schempp and Toder, 1993). Thus the

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original configuration of genes was probably the same in the X and Y, strongly suggesting that the two sex chromosomes were once homologous, at least within the human Xp region. If the X and Y originally shared a much larger region of homology than the tiny PAR of humans, it might be expected that the gene content of the PAR will vary among species. Primate X chromosomes share a similar early replicating distal Xp, which may indicate the absence of X inactivation in a similarly located PAR (Schempp et al., 1989). However, detailed comparisons of DNA sequences around the pseudoautosomal boundary provide evidence that its demarcation and position have undergone change (Ellis et al., 1990). Of particular interest is the finding that STS in prosimians shows no gene dosage difference between males and females, suggesting that in these species STS is located within the PAR, and that the rearrangement of the Y may have disrupted this relationship and led to the inactivation of the human Y-borne STS allele (Schempp and Toder, 1993). In contrast, there are active STS alleles on the mouse X and Y which recombine at meiosis (Soriano et al., 1987). Few other data are available, but this evidence of variation is consistent with the hypothesis that the PAR may represent a rather variable relic of a much larger region of homology shared by the X and Y of an ancestral eutherian. If the X and Y chromosomes were originally homologous, how did they become so different? The absence of variation in the size and known gene content of the eutherian sex chromosomes originally suggested that a single major rearrangement may have removed most active genes from the Y, possibly adding them to the X (Lyon, 1974). Alternatively, the Y may have been gradually degraded by progressive inactivation and deletion (Lucchesi, 1978). To evaluate these hypotheses, it is an advantage to compare the sex chromosomes of the most distantly related groups of mammals, in order to find variants that will permit us to reconstruct the genome of a long-extinct common ancestor, and to deduce ancient rearrangements. Indeed, marsupials and monotremes provide the variation in sex chromosome size, pairing relationships, and gene content that is lacking among eutherians (Fig. 3). Marsupials are a diverse group of about 250 species, yet they present an extraordinarily conserved karyotype, relating to a 2n = 14 configuration which was undoubtedly ancestral (Rofe and Hayman, 1985). The basic X chromosome is smaller (3% of haploid length) than the 5% of the standard eutherian X, suggesting that the gene content may be correspondingly smaller. The Y chromosome of many marsupial species is minute, and there is no cytological evidence that the marsupial Y undergoes homologous pairing with the X (Sharp, 1982). Initial family studies showed that the three genes, G6PD, PGK, and GLA, which are sex linked in eutherian mammals, were all sex linked in

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MAMMALIAN SEX CHROMOSOMES AND SEX-DETERMINING GENES

X

Y

X

Y

U MONOTREMES

X

Y

HUMAN MARSUPIALS

EUTHERIANS

FIG. 3 Sex chromosomes of the three major mammal groups. The large human X (about 5% haploid genome) is almost completely differentiated from the smaller heterochromatic

Y. except over the pseudoautosomal region (shown shaded). over which they pair and recombine. The basic marsupial X is smaller (3%) and completely differentiated from the Y. The larger monotreme X and Y are observed to pair at meiosis over the large hatched region (Xp and Yq).

a variety of marsupials (reviewed in Graves et al., 1993b). Somatic cell genetic studies using isozyme variants confirmed these assignments in several Australian marsupial groups, and added HPRT to the list of markers conserved on the X in all therian mammals. More recently, Southern blotting analysis of DNA from these cell hybrids, using cDNA probes to detect the marsupial homologs of highly conserved human X-linked genes, has added a number of other human X markers to the marsupial X chromosome. Hybridization of these probes in situ to chromosomes of the tammar wallaby (Mucropus eugenii), a species now used as a genetic model, has confirmed their assignment to the X, and provided a rough map of the X in this species. All these human genes map to the long arm and pericentric region of the human X, suggesting that this region is genetically equivalent to the marsupial X and represents a conserved region (amounting to about 3% of the genome) which was probably on the X chromosome in an ancestral therian mammal (Graves and Watson, 1991). However, further gene mapping showed that Ohno’s law breaks down for a number of genes located on the short arm of the human X. Surprisingly, human Xp genes, such as OTC and DMD, were found to be autosoma1 in marsupials (Graves et al., 1993b), lying in two clusters which are similar in dasyurid and macropodid marsupials. Since these groups represent marsupial orders which last shared a common ancestor about 50 million years ago, this finding suggests that human Xp genes were

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present on autosomes at least in a marsupial ancestor. There are two alternative explanations; either the original mammalian X included the human Xp region, which was subsequently lost from the marsupial X, or the original X lacked this region, and it was subsequently added to the eutherian X (Graves and Watson, 1991). To distinguish between these two hypotheses, it is necessary to look at the third mammal group, which is distantly related to both eutherians and marsupials-the monotremes. The three extant species of the egg-laying monotreme mammals, the platypus and two closely related echidna species, have almost identical karyotypes of 23 (27) autosome pairs (Wrigley and Graves, 1988a). The X and Y chromosomes (defined by comparisons of male and female karyotypes) are large (about 6 and 4% of the genome), and pair over about a third of the X chromosome length. Echidna species have an X,XlX2X2 female/X,X,Y male sex chromosome system (in which the X I is equivalent to the platypus X, Watson et al., 1992) produced by translocation of the Y with an autosome. All monotreme species share the added peculiarity that, at male meiosis, the X and Y form a translocation chain with several unpartnered elements (Murtagh, 1977). Comparing the gene content of monotreme sex chromosomes with those of therian mammals (eutherians and marsupials) has not been easy. Classic gene mapping has been impossible because the animals do not breed in captivity. Limited data have been wrung from the few stable somatic cell hybrids, but gene localization has largely relied on in situ hybridization, using human cDNA probes to highly conserved genes (Graves et al., 1993b). These studies show that the human Xq markers that map to the marsupial X are also present on the monotreme X, confirming that this region has been conserved on the X for at least the 170 million years since monotremes diverged from therian mammals. However, the human Xp genes that are autosomal in marsupials map within two similar clusters on platypus autosomes (Watson et al., 1991). Since marsupials and monotremes diverged independently from eutherians, this implies that the human Xp region was originally autosomal, and has been added to the X relatively recently in eutherian evolution (Graves and Watson, 1991). Since this region of the human X contains several genes shared with the Y, it must have been added to both sex chromosomes. This is unlikely to have happened independently, and may have occurred at a stage at which the original X and Y were partly homologous. Addition to one member of the pair was shortly followed by recombination within this ancient PAR, adding it to the other member of the pair (Fig. 4). The addition of this region to both X and Y, making a greatly enlarged PAR, accounts for the patchy homology between the eutherian Xp and Y, which is now seen to be the relic of autosomal homology. However, some of the homology between the eutherian X and Y is

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MAMMALIAN SEX CHROMOSOMES AND SEX-DETERMINING GENES

X

Y

Original sex chromosomes

X

Y

Addition of autosomal region t o X

X

Y

Transfer of autosomal region to Y by recombination

X

Y

Autosomal region

on both sex chromosomes enlarges PAR

FIG. 4 Diagrams to show possible addition of autosomal segment to the ancestral eutherian chromosomes. The autosomal region X (white) and Y (black) is added to the original pseudoautosomal region (stippled) of the X. then transferred to the Y by recombination within the PAR (horizontal lines). The X and Y which have this addition may now pair over the length of the enlarged PAR. The original addition could equally well have been to the Y. and transfer via recombination to the X.

clearly more ancient, since there is at least one X-Y shared gene in the conserved region defined by inclusion on the marsupial X. Sequences corresponding to the ubiquitin binding enzyme gene Ubel are shared by the mouse X and Y, and similar sequences have also been cloned from the marsupial X and Y (Mitchell et al., 1992). This suggests that even the smaller ancestral sex chromosomes were at one stage at least partially homologous. The cloning of UBEl from the marsupial, as well as the mouse Y , also implies that eutherian and marsupial Y chromosomes are monophyletic, and this is confirmed by the observation of a sequence on the marsupial Y that is similar to the human and mouse SRY genes (see Section VI). What is the ultimate origin of mammalian sex chromosomes? To answer this question, we should make even wider comparisons, between the sex chromosomes of mammals and those of other vertebrate groups. A survey of sex determination in birds, reptiles, fish, and amphibians reveals a bewildering variety of chromosomal mechanisms, as well as systems in which an environmental factor, such as nest temperature, determine the sex of the hatchlings (Deeming and Ferguson, 1988). Some reptiles share with mammals an XX/XY system of male heterogamety, whereas others, like birds, have a ZW/ZZ system, in which the female is the heterogametic

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sex. No other vertebrate group shows dosage compensation. There are many variants of these systems, and also many species, particularly among fish and amphibians, which do not have cytologically distinct sex chromosomes at all. The dimorphisms between Z and W chromosomes show striking parallels to those that distinguish mammalian X and Y chromosomes (Singh and Majumdar, 1993). It is attractive to consider that there might be some genetic homology between the sex chromosomes and sex-determining systems of different vertebrates. The pathway of sex determination may be basically the same, involving homologous genes which perform homologous duties in mammals, birds, and snakes. However, it seems that mammals do not share any recognizable vestige of an ancestral sex chromosome pair with other vertebrates. Of six human X-linked genes mapped in birds, five ( P G K , G6PD, HPRT, DMD, Z F X ) are located on autosomes (Dominguez-Steglich et al., 1990), and only OTC (which, as we have seen, is a recent addition to the mammalian X ) is located on the chicken Z and W (Dominguez-Steglich and Schmid, 1993). The only two genes assigned to the bird Z chromosome are autosomal in mammals (Baverstock et al., 1982; Morizot et ul., 1987). Thus it follows that the mammalian XY pair and the bird ZW pair must have independent evolutionary origins, as proposed by Ohno (1967). It follows that, in a common ancestor to birds and mammals that existed 200 million years ago, the XY and ZW chromosomes must both have been represented by homologous autosomal pairs. Perhaps the specialization of different autosomes into sex chromosomes in the two lineages means that two quite different genes have taken on a sex-determining function in birds and mammals. C. Evolution of Mammalian Sex Chromosomes

Given the independent origins of sex-determining systems in mammals and birds, we cannot follow the process of mammalian X-Y differentiation directly by comparing sex chromosomes of other vertebrates. However, we can trace changes within groups that seem to be analogous to stages we infer for mammalian X and Y chromosomes. The first step in the differentiation of the X and Y may parallel those vertebrate species which have no cytologically distinct sex chromosomes, but which show a regular segregation of sexes among the progeny, revealing that the homomorphic chromosomes must contain different alleles of a sex-determining gene(s) (Ohno, 1967; Bull, 1983). The next step may be represented by amphibians that have no morphologically distinct sex chromosomes, but in which the two members of an autosomal pair display

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differences in replication timing (Schempp and Toder, 1993), which may correlate with differential gene activity. A series of intermediate steps of cytological differentiation may be seen among snake species, which show a gradation in W chromosome size and heterochromatin content, from virtual homomorphy in some primitive snakes to extreme differentiation between Z and W in more advanced species (Ohno, 1967; Jones and Singh, 1985). These may represent stages in a progressive loss of active genes from the W chromosome, and may parallel the stages in the evolution of the mammalian Y chromosome. Comparisons of gene arrangements in the sex chromosomes of distantly related mammals may be used to define regions of the eutherian sex chromosomes in terms of their evolutionary origins (Graves and Schmidt, 1992). Figure 5 depicts the origins of different regions of the human X and Y. The human Xq and pericentric region, shared by the X of all mammals, represents a conserved region of the X (XCR) which constituted the ancestral X. Most of the human Xp region is on the X only in eutheri-

X

Y PAR

PAR YDR

XDR

PAR

- Pseudoautosornal

region

XDR - X differential region XCR - X - conserved region XAR - X - recently added region YDR - Y differential region (conserved YCR and added YAR not resolved) FIG. 5 Diagrams for possible evolutionary origins of human sex chromosomes. The X and Y differential regions (XDR and YDR) are each composed of a conserved region (representing the ancient proto-sex chromosomes) and a recently added region (representing ancient autosomal regions added to the X and Y in eutherian evolution). The XCR (X-conserved regions) and XAR (X recently added region) were resolved by comparative mapping, but there are insufficient gene locations on the Y to resolve YCR and YAR.

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ans, and represents a recently added region (XRA). Although not so obvious because of the few genes available for comparisons, the differential region of the Y is probably also composed of an ancient conserved region (YCR), represented by SRY and UBEl Y , and an added region (YRA) revealed by the presence of genes shared with the XRA region. Does the pseudoautosomal region represent merely the vestige of this recently added autosomal region? Or was it part of the original X and Y, whose critical function in pairing and recombination ensured its conservation? The lack of conservation of the mouse and human PAR suggests that the gene content of the PAR is not important to its function. However, we await mapping of human or mouse PAR genes in marsupials and monotremes to determine whether the PAR was a part of the conserved or the added region of eutherian sex chromosomes. A picture of mammalian sex chromosome evolution is now emerging (Fig. 6). Comparative mapping of mammalian sex chromosomes shows that the heteromorphic X and Y chromosome of humans and other eutherians can be traced back to a smaller pair of chromosomes which were at least partly homologous. Comparisons with the sex chromosomes of other vertebrates imply that the X and Y must have ultimately evolved from a homologous pair of autosomes that existed 200 million years ago. Homology was lost, probably in steps, as the Y chromosome was progressively deleted and inactivated. The X was conserved in gene content, but not in gene order. Addition of a large autosomal region to the eutherian sex chromosomes occurred between 130 and 80 million years ago. The region added to the Y, like the original Y, became subject to deletion, and the region added to the X, to inactivation. There has been much speculation on the forces which drive this progressive degradation of the Y. It is generally agreed that the first step is the genetic isolation of a region of the Y, perhaps by rearrangement with respect to the X, which suppresses recombination and permits divergence. Such a rearrangement may be selected for if it protects a group of sexdetermining genes from disruption (Maynard Smith, 1978). There is little

FIG. 6 Schematic diagrams for possible evolution of mammalian sex chromosomes. In a mammalian ancestor, the proto-sex chromosomes were essentially homologous and paired (horizontal hatching), differing only in alleles of a sex-determining gene(s). Differentiation of the X and Y occurred by progressive loss and heterochromatinization of the Y (black). In the monotremes, this primitive situation prevails, except for the addition of autosomal region C. This process continued in the therian lineage, leading to complete differentiation of the X and Y in the marsupials. In the eutherian lineage, at least two autosomal regions, A and B, were translocated to an ancestral PAR, and the process of Y degradationcontinued, producing almost completely differentiated X and Y , which retain homology only over part of one added autosome.

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evidence for a cluster of sex-determining genes in extant mammals (Graves and Schmidt, 1992) (see Section IV), but it is possible that, originally, mammalian sex determination may have been controlled by quite different gene(s) on the proto-X and Y chromosomes. Also, there is evidence that rearrangements have occurred relatively recently. For instance, in the primate lineage, at least one inversion has changed gene order between the XRA and YRA (Wolff et al., 1992; Schempp and Toder, 1993) within the region, which was added long after the mammalian sex chromosomes were defined. There are several mechanisms which may account for the loss of active genes from the isolated regions of the Y. A severalfold higher than average mutation rate for genes confined to the Y is expected from the numbers of premeiotic divisions of germ cells (Miyata et af., 1987); this would at least provide added variation. The preferential deletion and inactivation of genes on the Y has been ascribed either to accidental and progressive loss of the class of Y chromosomes with the fewest changes (“Muller’s ratchet”), or to a hitchhiking effect, in which a favorable mutation may be selected along with any detrimental changes on the same Y (Rice, 1987; Maynard Smith, 1978; Charlesworth, 1991). The loss of active alleles from the Y sets up 2 : 1 dosage differences between female and male. These differences are compensated for by a system of X inactivation, in which one or other X becomes genetically inactive leaving both sexes with a single active X (Section 111). It is obvious that the unique functions of the sex chromosomes in dosage compensation, as well as sex determination, have had a major role in shaping the differences between the X and Y chromosomes. We now examine the mechanisms of these functions and ask how they evolved.

111. Dosage Compensation and X Chromosome Inactivation Although female mammals have two X chromosomes and males only one, the amount of product of X-linked genes is found to be the same in the two sexes. Dosage compensation is achieved by inactivation of one or another X chromosome in females (Lyon, 1961). X inactivation is a property of an entire X chromosome, or most of it, and is a remarkable example of genetic control on a grand scale. It involves several cytological and molecular changes, and may represent a complex, multistep control mechanism. Because X inactivation is a chromosome-wide system, X-autosome

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rearrangements may alter chromosome activity. The conservation of the X has been attributed to selection against disruption of X inactivation (Ohno, 1967). The progressive loss of active alleles from the Y has been accompanied by the spread of X inactivation, but it is not clear which change drives which. An understanding of the organization and function of mammalian X and Y chromosomes will therefore require an understanding of the mechanism of X inactivation, and of how-and why-it evolved.

A. Expression of X-Linked Genes

The hypothesis that only a single X is active in the somatic cells of female mammals was put forward to explain the mosaic phenotype of females heterozygous for alleles of a sex-linked coat color gene, as well as the equality of expression of X-linked genes in males andfemales. Lyon (1961) suggested that one or another X chromosome, chosen at random, became genetically inert early in embryogenesis. This state is stable and somatically heritable, giving rise to clones of tissue in which either the maternal or paternal X is active. An extension of this hypothesis, stating that, in individuals with aberrant sex chromosome constitutions, all but a single X are inactive, explains the atypically benign phenotypes of sex chromosome aneuploids. The major tenets of Lyon’s hypothesis have been amply confirmed for a variety of human or mouse X-linked genes (Gartler et al., 1992), including PGK, G6PD, and GLA in the conserved region (XCR), as well as OTC and MAOA in the recently added region (XRA) of the eutherian X. What makes X inactivation uniquely interesting is that it seems to be a whole chromosome phenomenon, in the sense that all the alleles on the maternal or paternal X are expressed or repressed coordinately. Patterns of expression of genes on fragments of the X, separated by X-autosome translocations, suggest that X inactivation is controlled from a single inactivation center (XIC) located near PGK in the XCR. Inactivation spreads from this center, even into translocated autosomal regions (Russell, 1963). The genes which are represented on both the X and Y chromosome present a special case in which dosage compensation would not be expected to be necessary. Genes within the PAR are represented by two equivalent copies in both sexes, and would therefore have no need for compensation. In fact, MIC2 and other genes in the human PAR are not inactivated, and Sts in the mouse PAR is active from both X chromosomes (Ellison et al., 1992; Scheibel et al., 1993; Keitges er al., 1985; Gartler et al., 1992). X-Y shared genes outside the PAR (ZFXIZFY, RPS4XIY) also escape X inactivation in humans (Schneider-Gadicke et al., 1989; Fisher

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et al., 1990), and, since these genes are interspersed with inactivated genes, it is obvious that, if there is a signal emanating from the inactivation center, it is able to skip genes that do not respond to it. The X-Y shared genes (Zfxly, Ubelxly) in mice, however, are subject to inactivation (Adler et al., 1991; Ashworth et al., 1991). It has been suggested that the Y-linked alleles of these genes have evolved different, male-specific functions in the mouse, but not in man, and that the difference in dosage of active genes might explain why XO mice are much less severely affected than XO humans. Genes which are shared by the human X and Y chromosome, and are not subject to inactivation, are normally present in two active copies. If X-Y shared gene(s) in the mouse have different functions, and the X-linked copy is subject to inactivation, XY, XX, and XO mice would all have a single dose of active X-linked gene product (Ashworth et af., 1991). A deficiency of an X-Y shared gene has been suggested to be responsible for the short stature of human XO (Turners) individuals lacking one X or part of it, and RPS4XlY, which codes for a ribosomal protein, was proposed to be the missing growthcontrolling gene (Fisher et al., 1990). However, the critical region has been mapped to the PAR (Ogata et al., 1992b), which may be autosomal in the mouse, explaining the absence of growth retardation in XO mice. The onset of X inactivation in the embryo and its reversal in female germ cells have been closely studied. Sensitive enzyme microassays and the polymerase chain reaction (PCR) have been used to detect activity of paternal or maternal alleles in single embryos or embryonic tissues (Monk, 1992). These have shown that inactivation occurs at different times in embryonic and extraembryonic tissues, and is qualitatively different. In the earliest differentiating extraembryonic lineages, cytogenetic and enzyme studies show that inactivation is paternal rather than random, and may be less stable (Takagi and Sasaki, 1975; Migeon et al., 1986). Random inactivation occurs some days later in the inner cell mass which forms the embryo. Recently, a transgenic mouse has been constructed in which a bacterial P-galactosidase gene inserted into the X chromosome is subject to X inactivation. The cell-localized product of this transgene can be monitored by a staining reaction, so it has been possible to see very directly the activity of each X chromosome in individual cells of a heterozygous embryo at different stages of development. This “blue mouse” system has confirmed the difference in timing and randomness between extraembryonic and embryonic lineages, and provided a wealth of detail about the process (Tan et al., 1993). The inactive X must be reactivated at some stage of egg formation, since both alleles can be inherited in an active state. Expression of both alleles in the same cell has been observed to occur at female meiosis

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(Monk, 1992). In male meiosis, curiously, the X and Y have both been observed to become heterochromatic and genetically inactive (Lyfschitz and Lindsley, 1972), but it is not clear how comparable this process is with somatic X-chromosome inactivation.

6. Cytological Changes and Molecular Mechanisms Several cytological changes accompany the genetic inactivation of the X in humans, mice, and other eutherian mammals, and might provide some clues to the mechanism of inactivation. Most striking is the appearance of the inactive X as a highly organized heterochromatic “sex chromatin body” on the periphery of interphase nuclei (Gartler et af., 1992). The inactive X is also found to replicate later in the DNA synthesis phase than its active counterpart, a difference which is apparent at the level of individual genes (Schmidt and Migeon, 1990). It was always assumed that inactivation was due to transcriptional repression, but the absence of RNA transcript of an inactive allele was not demonstrated experimentally until 15 years after Lyon’s discovery (Graves and Gartler, 1986). Transcriptional repression has now been confirmed for many inactivated genes using the PCR to detect allele-specific transcripts (Adler et al., 1991; Ashworth et al., 1991; Gartler et al., 1992). Reactivation of alleles on the inactive human and mouse X by inhibitors of DNA methylation (Mohandas et al., 1981; Graves, 1982) suggested that methylation of cytosine residues was involved, and the development of methods for mapping the positions of methylated sites has now provided detailed correlations of activity with changes in methylation patterns. The 5’ region of the inactive mouse Pgk gene, including the promotor, was found to be heavily methylated, and footprinting methods showed that it was wrapped around nucleosomes, whereas the promotor of the active allele is bound to several proteins which are thought to be transcription factors (Pfeiffer and Riggs, 1991). Inactivation and DNA methylation occur close in time in the early mouse embryo, and may process from the inactivation center (Grant et al., 1993), although there was some suggestion that inactivation might precede methylation (Lock ef al., 1987). Methylation evidently interferes with the transformation of an inactive, but not an active allele via purified DNA; this difference in transformation efficiency is not evident for the active and inactive alleles of extraembryonic cells (Gartler et al., 1992). Genetic inactivation by DNA methylation is an attractive hypothesis

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because enzymatic maintenance of methylation patterns offers an explanation of the stability and heritability of repression. However, there is some evidence that DNA methylation cannot be the only molecular difference between the active and inactive X, and may not even be the primary difference. Reactivation is induced at high frequencies in somatic cell hybrids and species hybrids, but not in normal, or even transformed cells. This suggests that inactivation must be at least a two-step process, involving heterochromatinization as well as methylation (Gartler et ul., 1985). A more elaborate multistep regulation (Riggs, 1990) may involve differential timing of DNA synthesis on the active and inactive X. There is a very general relationship between early replication and transcriptional activity, and differential timing of replication domains could act as a heritable control of gene activity (Goldman, 1988). The recent demonstration that one X chromosome contains little acetylated histone H4 (Jeppeson and Turner, 1993) confirms the involvement of protein modification at some level in X chromosome inactivation. The recent cloning of a candidate gene for the X inactivation center offers to tell us much about the molecular biology of X inactivation. A sequence, XZST, has been cloned from the control region of the human X and its counterpart, Xist, from the equivalent region of the mouse X (Borsani et al., 1991; Brockdorff et al., 1991; Brown et al., 1991). The XZSTgene has the unique property of being transcribed from the inactive, but not the active X. This gene produces a large transcript in both species, but it remains in the nucleus and is evidently not translated (Brockdorff et a/., 1992; Brown et al., 1992). The timing of XZSTexpression is suggestive, for it makes its appearance in the mouse embryo just before X inactivation is manifest (Kay et al., 1993). It is first expressed from the paternal X, but later from both X chromosomes, corresponding to the onset of random X inactivation. Significantly, the XZST gene is expressed during male meiosis, just before the inactivation of the X (McCarrey and Dilworth, 1992; Richler et al., 1992; Salido et al., 1992). Just how the XZST transcript might have the effect of coordinately inactivating thousands of genes in cis is a matter for intense speculation. Perhaps the RNA transcript itself is involved in a localized change of configuration of the X which expresses it. XZST product may control spreading by cooperatively binding DNA, or may be constrained within the sex chromatin body. This is one of the most active areas of research on mammalian sex chromosomes, since understanding the action of the XZST gene may clarify the most mysterious aspects of X inactivation: the large-scale coordinate control and spreading effect.

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C. Comparative Studies and the Evolution of X Inactivation

As well as molecular studies of X inactivation in humans and mice, a comparison of X chromosome behavior among other species, especially the distantly related marsupials and monotremes, offers an alternative approach to unravelling the complex molecular mechanism of X inactivation, and deducing how X inactivation has accompanied-or shaped-sex chromosome evolution. X inactivation, as observed by mosaic expression of X-linked genes or inferred from asynchronous DNA replication, occurs in all the eutherian species studied. In marsupials, too, one X chromosome in female-derived cells was observed to replicate late. However, this late-replicating X was subsequently found always to be paternally derived, and genetic studies showed that only the maternal alleles of the X-linked genes G6PD and PGK were expressed in blood cells. Paternal X inactivation has been found to be general for all marsupials (VandeBerg et al., 1987; Cooper et al., 1993). X inactivation in marsupials is also incomplete and tissue specific. Alleles on the paternal, as well as the maternal X were found to be expressed in cultured fibroblasts and in other body tissues. The paternal allele was inactive, or partially or fully expressed, depending on the locus and the species. This gave rise to the idea that X inactivation in marsupials might be a patchwork of independent, locus-specific controls (VandeBerg et al., 1987). However, the patterns of activity seem to be related to gene position, suggesting that inactivation could be controlled by variable spreading from an inactivation center (Graves and Dawson, 1987). Marsupial X inactivation has also been found to be much less stable than inactivation of the human or mouse X (Migeon et al., 1989). Thus X inactivation in marsupials differs from that in eutherians in being paternal, incomplete, less stable, and tissue specific. Which system better represents the ancestral X inactivation system? Cooper (1971) suggested that paternal inactivation represents the primitive condition, a proposition supported by the subsequent demonstration that X inactivation in eutherian extraembryonic membranes is also paternal. There is some evidence that at least some genes may escape X inactivation in eutherian extraembryonic tissues (Migeon et al., 1986). That incomplete and tissuespecific inactivation may also reflect an ancestral X-inactivation system is also suggested by the finding of replication asynchrony (the only clue to genetic inactivity) in a part of the monotreme X in blood lymphocytes, but not fibroblasts (Wrigley and Graves, 1988b). If marsupial X inactivation reflects a more flexible ancestral system, it

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will be important to investigate its molecular mechanism. There is little evidence that DNA methylation is involved in marsupial X inactivation. The few molecular studies of cloned marsupial genes have failed to find evidence of differential DNA methylation in the 5' region of marsupial G6PD or PGK (Kaslow and Migeon, 1987; Cooper et al., 1993), although there are differences within the gene that are comparable to internal sites in human and mouse genes which are unrelated to gene activity. Nor is chromatin condensation consistently associated with X inactivation in marsupials or monotremes, being apparently absent in adult cells (McKay et al., 1987), but very obvious in embryonic tissues (Johnson and Robinson, 1987). The only cytological change consistently associated with X inactivation in all mammals is therefore delayed DNA replication. Perhaps this means that X inactivation was originally a simpler system, relying on replication asynchrony , and that the evolution of the random, hyperstable eutherian inactivation system involved the addition of other levels of control, including DNA methylation. Paternal X inactivation in marsupials was the first demonstration of genomic imprinting in mammals in which the activity of a gene depends on its parental source. Genomic imprinting in man and mouse has since been implicated in abnormal development of embryos which had received both nuclei, both copies of a chromosome region, or even of a single gene, from individuals of the same sex. Certain human genetic diseases and specific mouse phenotypes are associated with the inactivation of a normal allele inherited from the male, or in other cases the female, parent (Hall, 1990; Tilghman, 1992). Several imprinted regions have been identified on mouse autosomes (Cattanach and Kirk, 1985), and four imprinted autosomal mouse and human genes have now been cloned. There are parallels between X inactivation and genomic imprinting (Grant et al., 1993). Both are confined to mammals and both result from transcriptional repression. An association with DNA methylation has been established at least for transgenes, which are commonly imprinted. Imprinted genes replicate late, as does the inactive X. Perhaps X inactivation was originally instituted as maternal imprinting of the XZST locus; alternatively, perhaps imprinted autosomal loci were originally part of the X inactivation system. Comparative mapping of these imprinted genes in distantly related mammals may enable us to deduce this relationship. D. X Chromosome Inactivation and Sex Chromosome Evolution

How has X chromosome inactivation constrained-or accelerated-sex chromosome evolution in mammals? Ohno (1967) originally proposed that

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the gene content of the mammalian X is absolutely conserved because a change in gene arrangement would disrupt the chromosome-wide X chromosome inactivation system. This hypothesis is supported by the invariance of gene content among the X chromosomes of eutherian mammals. The only exceptions are the absence from the mouse X of genes which are located in the human PAR, and would therefore have no need for dosage compensation (Section 11). However, marsupials and monotremes defy Ohno’s law, since all the markers within the eutherian XRA are autosomal in both these groups. Does this breakdown of conservation contradict the hypothesis that X inactivation protects the X from rearrangement? Not necessarily. The XRA was evidently added to the ancestral eutherian Y as well as X, so is most likely to have occurred in an ancient pseudoautosomal region, homologous between the partially differentiated (and presumably only partially inactivated) proto-X and Y (Fig. 4). The conservation of the XCR within the monotreme X is harder to understand, since at least part of the monotreme XCR is paired with the Y and shows no sign of inactivation (Watson et al., 1990). There is little doubt that the differentiation of the X and Y and the spread of inactivation occurred together, but which came first? The simplest hypothesis is that the progressive degradation of the Y produced dosage inequity for genes which had lost their partners. There was therefore selection for the recruitment of these newly unpartnered genes into the X-inactivation system, causing inactivation to spread along the X, keeping pace with the progressive differentiation of the X from the Y (Fig. 6). Comparisons of X inactivation and sex chromosome evolution between species reveals several stages in this process. X inactivation was evidently a property of the original XCR, since the system is common to marsupials and perhaps even monotremes. This region contains the XZSTlocus, which may have been the site of the most primitive X inactivation in the partially differentiated proto-X. Conversely, the XRA, having been originally autosomal, was originally paired and not inactivated. Its addition to the eutherian sex chromosomes, and the subsequent degradation of the YRA, was evidently accompanied by X inactivation, since most of the loci within the XRA are inactive. There has been no spread of inactivation into the PAR, which has no need for dosage compensation. X-Y-shared genes outside the PAR present a muddled picture, but it is particularly instructive to examine these apparent breakdowns in the relationship between gene dosage and X inactivation, since they may represent intermediate stages in the evolution of X inactivation. Of genes with active relatives on the Y, human ZFX, AMGX, and RPS4X are not subject to inactivation, whereas mouse Zfx and Ubelx are apparently fully

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inactivated (Adler et al., 1991; Ashworth et al., 1991). Conversely, human STS and ADMLX, which are represented only by inactive pseudogenes on the Y, are expressed (though not equally) from both X chromosomes (Shapiro et al., 1979; Migeon et al., 1982; del Castillo et al., 1992). There is some suggestion that Sts is partially inactivated in mouse, although it lies in the PAR and has a fully active allele on the Y (Jones et al., 1989). Part of the explanation for these exceptional genes may lie in the divergence of function of X- and Y-linked alleles, as can be seen in the different patterns of expression of mouse Zfx and Zfy (Mardon et al., 1990; Schneider-Gadicke et al., 1989). The absence of a strict correlation between gene dosage and X inactivation must mean that dosage differences are not critical for many or most genes, a conclusion obvious from the normal phenotype of heterozygotes for null alleles at very many loci. Deletion of the Y allele and incorporation into the X inactivation system may, therefore, be events that can be separated in time. We have presented only the generally accepted hypothesis that X inactivation is selected for by dosage differences that arise as active alleles are lost from the Y. However, the observation that some X-Y-shared genes are subject to inactivation might suggest that X inactivation could precede, and even cause, Y degradation. Perhaps the abnormal spread of inactivation into a region on the X might set up dosage differences and select for the loss or inactivation of the corresponding region of the Y (Graves and Schmidt, 1992). These evolutionary arguments both rest on the usual assumption that X inactivation is primarily a dosage compensation mechanism. How true is this? How true was it 200 million years ago? E. X Inactivation-Dosage Sex Determination?

Compensation or

Dosage compensation for X-linked genes was first described for Drosophila, and with the formulation of the X-inactivation hypothesis, the concept was extended to mammals. Equalization of the expression of X-linked genes in mammals would seem to make functional sense, since it is obvious that extra copies, or, particularly, missing copies, of autosomal regions are poorly tolerated in mammals. However, dosage compensation is unknown in other vertebrates, even those with highly differentiated sex chromosomes (Chandra and Nanjundiah, 1993; Baverstock et al., 1982). Did mammalian X inactivation evolve for some other reason? Chandra has made the point that gene dosage differences, wherever and however they occur in the animal kingdom, are invariably associated

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with sex determination. In Drosophilu and Caenorhubdiris, dosage compensation forms an integral part of the very well-characterized sexdetermining systems (Hodgkin, 1990). In scale insects (in which genomic imprinting was first described), gene dosage is modified by inactivation of sex chromosomes or of an entire haploid set (always paternal), or sex is determined by haplodiploidy (i.e., the presence or absence of the paternal genome). In birds, dosage of genes on the Z chromosomes affects sexual phenotype (Thorne and Sheldon, 1993). It may be that genomic imprinting and/or dosage differences underlie all these sex-determination systems (Chandra and Nanjundiah, 1993). Chandra (1985b) pointed out that a sexdetermining gene(s) shared by the X and Y and subject to X inactivation would have two active alleles in males and a single active allele in females. Could X dosage be involved in mammalian sex determination after all? This might seem unlikely, given the unequivocally male phenotype of XXY and the female phenotype of XO humans and mice, which would appear to establish that the Y uniquely determines maleness, and that the dosage of X chromosomes is immaterial in mammals (Section V). However, the phenotypes of X-aneuploid individuals do not really test the X dosage hypothesis, because all but a single X are inactivated. The phenotypic effects of two active X chromosomes is impossible to observe directly in diploid animals since a 2X-active state is lethal, at least in mouse embryos (Takagi and Abe, 1990). However, duplications of parts of the active human X are viable (Schmidt et a/., 1991). The observations of human XY females with duplications of parts of Xp suggest that dosage of a gene(s) in this region can override the male-determining effect of the Y (Scherer et al., 1989). The first X(dup)Y females described had duplications in the region Xp21.1-p22.3, which contains the ZFX gene: however, additional cases have excluded ZFY (Ogata et a/., 1992a), and a candidate Y-suppressing gene has now been isolated from a minimal duplicated region (Am et a/., 1994). The recent observation of a stillborn female child who was triploid with an XXXY set chromosome constitution (Maaswinkel-Mooij et a/., 1992) also suggests that an aberrant dosage of active X-linked genes might modify sex determination. In marsupials, there is an obvious X dosage effect on some aspects of sexual differentiation. XXY animals have testes, but instead of a scrotum, they possess a pouch with functioning mammary glands. XO animals have no testis, but a scrotum may replace the mammary glands (see Section V ) . This suggests that X dosage controls some aspects of sexual phenotype. A gene which controls a scrotum or mammary gland switch (Sharman et al., 1990) must therefore lie on the marsupial X. This gene is likely to be conserved in eutherians, but because it must map within the XCR, it is distinct from the human Y-suppressing gene, which maps within the XRA. If the action of this gene depends on dosage, the incomplete and tissue-

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specific X inactivation of marsupials may be seen, not as a primitive and rather sloppy dosage compensation system, but as part of a complex system which regulates sexual differentiation. An alternative hypothesis is that the function of this switch gene is regulated, not by dosage, but by imprinting (Cooper, 1993). A paternally derived X, normally present only in females, may determine pouch and mammary glands, and suppress scrotum formation. The cloning and characterization of a testis-determining gene from the mammalian Y chromosome (Section VI) would seem to undermine the hypothesis that X inactivation is directly involved with sex determination. However, the data we have reviewed above might suggest that, even if dosage of X-linked genes is now irrelevant to mammalian sex determination, X-chromosome inactivation may have originally evolved as part of the sex-determining system in an ancestral mammal.

IV. Gonadal Differentiation and Sexual Dimorphisms

In all mammals, there are, besides the obvious differences in gonads and germ cells, many other differences between males and females imposed by their reproductive roles; genitalia and reproductive tracts, mammary gland development and function. In addition, there are a number of sometimes quite spectacular secondary sexual dimorphisms; differences in body size and hair distribution, antlers and horns; pitch of the human voice, breast development, perhaps differences in brain anatomy and function which underlie differences in behavior. These sexual dimorphisms must be the products of very many genes in many developmental pathways. Are they all under some common genetic control? A. Gonadal Differentiation and Hormone Function

A common step in a complex sex differentiation pathway could be controlled by a single sex-determining gene. A decade of brilliant physiological experiments gave reality to this single-switch theory when it was discovered that the first detectable difference between male and female development was the differentiation of the embryonic gonad into either a testis or an ovary. The decision to form or not form a testis was then shown to determine all other sexual characteristics (Fig. 7). This decision is made comparatively late in development. Male and female embryos follow a common developmental pathway up to 10.5 days

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FIG. 7 Schematic diagrams to show the differentiation of the testis from the undifferentiated gonad occurring as the result of TDF action. The fetal testis then secretes testosterone and MIS, which results in a male phenotype. In the absence of TDF, the undifferentiated gonad becomes an ovary, and in the absence of testicular hormones, a female phenotype results.

in mouse, or 6 weeks in human. By this stage, an undifferentiated gonad has developed as a “genital ridge,” indistinguishable between male and female. The ducts which will develop into male (Wolffian) and female (Miillerian) reproductive tracts are both present. Testis transplantation experiments and hormone injection showed that testicular hormones have an overriding effect, causing regression of the Miillerian ducts and completely masculinizing a female fetus (Jost, 1970). Experiments in which the embryonic testis is surgically removed, or the action of testicular hormones blocked, show that in the absence of testicular hormones, development proceeds along a female pathway. Development of the ovary in female embryos is later than testis de-

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velopment in the male, suggesting that it is the failure to develop testis, rather than the presence of an ovary, which determines female structures. These experiments show that once the undifferentiated gonad develops into a testis, it secretes hormones which determine all other male-specific somatic sex characteristics (Wilson et al., 1981).There are two hormones produced by two cell types within the testis. Testosterone (produced by the Leydig cells) promotes the development of the Wolffian duct into the male reproductive tract; and its more potent (but short-lived) derivative, dihydroxytestosterone, promotes development of male genitalia. A second hormone, the Miillerian inhibiting substance (MIS, alternatively named anti-Miillerian hormone, AMH), produced by Sertoli cells, acts to inhibit the development of the Miillerian duct into the female internal ducts. In the absence of testosterone, the Wolffian ducts degenerate, and female genitalia result. In the absence of MIS, the Miillerian ducts develop into the Fallopian tubes, oviducts, uterus, and part of the vagina. Because female development results from the failure at any step of the hormoneinduced pathway, it has been suggested that female development passively follows a “default pathway.” For instance, the X-linked condition, “testicular feminization” (androgen insensitivity), results from a mutation in the X-borne AR gene which codes for the androgen receptor, and converts XY embryos into phenotypic females. Although testis determination is accepted as the critical first step in the mammalian sex-determining pathway, there is some evidence of sexual dimorphism at much earlier stages. When mouse embryos of particular strains were separated into early and late developing classes at stages between morula and neurula, the early-developing class was mostly XY, and the late-developing class XX. Analysis of this growth difference in XY, XX, and XO mice ascribed growth-promoting properties to the Y and growth-retarding properties to the paternally derived X. These genes were expressed at different stages (Burgoyne, 1992). This growth difference between XX and XY mouse embryos has been directly demonstrated by PCR sexing (Zwingman et af., 1993). Differences in the rate of gonad growth are also apparent early in XX and XY fetuses, and are ascribed to factors on the Y chromosome (Hunt and Mittwoch, 1987), which might also account for different testis size among adult males. It is not clear whether an early growth advantage is a necessary step in testis determination (as proposed by Mittwoch, 1988), or whether it reflects independent genetic pathways unrelated to sexual differentiation; however, there are interesting parallels with the relationship of growth and gonad differentiation in other mammals, and even other vertebrates.

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B. Gonadal Differentiation and Independently Controlled Sexual Dimorphisms in Marsupials Marsupial young are born at an immature stage of development, and complete their development attached to a teat, usually in an external pouch. Gonad size, position, and histology are indistinguishable in newborn male and female pouch young (Tyndale-Biscoe and Renfree, 1987), as are the Wolffian and Mullerian duct systems. N o testicular hormones are detected at birth. Gonadal differentiation occurs fairly rapidly after birth in the tammar wallaby. The germ cells migrate and begin to proliferate in the gonad about 12 hr after birth, and after 2 days, MIS production becomes detectable in developing testes. After 3 days, the first signs of testis differentiation can be seen in males as an aggregation of Sertoli cells to form testis cords. N o ovarian differentiation is observed in female embryos until a few days later. As in eutherians, the differentiation of the Mullerian or Wolffian ducts and their derivatives is under the control of testicular hormones (Shaw et al., 1988). Removal of the testis from male pouch young, or transplantation into a developing female shows that testicular hormones control the masculinization of the Wolffian ducts and the development of the penis (Tyndale-Biscoe and Hinds, 1989). However, not all sexual dimorphisms are under the control of testicular hormones in marsupials. 0 er al. (1988)described several consistent differences in morphology between male and female newborns. In the male wallaby embryo, at birth and even as early as 5 days before birth, scrotal bulges appear anterior to the phallus, which are never seen in female embryos. Conversely, the female embryo has mammary anlagen flanked by two mesodermal ridges which later form the pouch. Sexual dimorphism is also apparent at this stage in the gubernaculum and processus vaginalis. All these dimorphisms are therefore established before gonadal differentiation and testicular hormone production, and their differentiation must therefore be independent of gonad function (Renfree and Short, 1988). Treatment of pouch young with exogenous hormones confirms that some sexually dimorphic characters in marsupials develop independently of the gonadal hormones (Shaw et af., 1988). Treatment of female pouch young with testosterone stimulated the Wolffian duct system, but had no effect on the pouch or mammary glands, nor did it elicit scrotum formation. Conversely, treatment of male pouch young with estradiol had no effect on scrotal development and did not induce pouch and mammary gland formation in male embryos. Removal of testes from male pouch young prevented the masculinization of the internal reproductive tract but had no effect on the developing scrotum, and grafting testes into female embryos,

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while masculinizing the internal reproductive tract, had no effect on the development of the pouch and mammary glands (Tyndale-Biscoe and Hinds, 1989). These experiments clearly indicate that the development of scrotum, pouch, and mammary glands does not depend on testicular hormones. Instead, the development of these sexual dimorphisms appears to be under primary genetic control, which is independent of gonadal differentiation (Renfree and Short, 1988; Shaw ef al., 1990).

C. Sex Determination in Other Vertebrates: A Role for Differential Growth? We may also gain a deeper understanding of mammalian sex determination by studying the process in other higher vertebrates, particularly those which display female heterogamety (a ZZ ma1e:ZW female sex chromosome system), or homogamety and environmental sex determination. Ovary determination in ZW female birds and snakes has many parallels to testis determination in XY male mammals (Ohno, 1967; Bull, 1983). Development of a single ovary occurs in female embryos before testis development in male embryos. Removal of ovaries from female avian embryos induces them to develop along a male pathway; indeed, removal of an ovary even from an adult bird causes the second gonad (which remains undifferentiated) to develop into a testis. In species with female heterogamety, female development is advanced, and if there is any interruption to this pathway, the male (default) pathway is followed. Environmental sex determination seems, superficially, to be a completely different system, yet growth differences may be a common denominator. In some species of fish, removal of the male induces the largest female to change sex. In many reptiles, temperature is the factor that determines whether eggs will develop as males or females. In some species, the higher temperature is male determining and in others female determining, but in all of these systems, the sex determined by the higher temperature develops from the more advanced embryos. For instance, a higher incubation temperature induces male development in alligators (Deeming and Ferguson, 1988). The temperature-sensitive period lasts a week, during which time a testis is differentiated. Eggs incubated at this temperature have larger and more developed embryos than eggs incubated at a lower temperature, and testis development occurs some days before ovary development. In turtles, however, a higher temperature is female determining; embryos incubated at the female-producing temperature are larger and more advanced, and ovary development precedes testis devel-

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opment. This has led Mittwoch (1988) to propose that growth advantage plays an integral part in all sex-determining systems. D. Testis Determination in Mammals

The first and critical step in somatic sex determination in eutherian mammals is the development of testes. This is followed by the secretion of hormones from this organ, inducing a cascade of hormone-induced changes. Testis determination and hormone production is also a necessary first step for most (though not all) aspects of sexual dimorphism in marsupials. In order to understand the genetic control of sex determination, we must understand how testis formation is induced. The undifferentiated gonad is composed of four cell types, each of which is diverted into different fates in the ovaries of female embryos or the testes in males (Burgoyne ef al., 1988). Primordial germ cells become eggs or sperm. Supporting cells develop into follicles which surround the egg, or Sertoli cells which form testis cords. Steroid cells become estrogensecreting theca cells in the ovary, or testosterone-secreting Leydig cells in the testis. Even connective tissue cells form a much more complex network in the testis than in the ovary. Of the four cell types in the undifferentiated testis, which is the critical tissue that is the target for the testis-determining step? We can eliminate the germ cells immediately, for they have a separate embryonic origin (the yolk sac) and migrate into the gonad. That their development follows an independent pathway is demonstrated by the observation that mutations which preclude germ cell development or migration in male mice have no effect on external phenotype. The somatic testis tissue that mediates the Y chromosome-controlled step was determined by observing mice developing from embryos constructed by amalgamating XX and XY blastomeres. In these XX:XY chimeras, the only tissue that was exclusively made up of XY cells was the supporting cells of the undifferentiated gonad (Burgoyne, 1992). Thus the lineage which gives rise to Sertoli cells in the testis must be the lineage in which the testis-determining switch acts. This result also shows that the testis-determining switch is cell autonomous; these results support classic work on XX cattle embryos, which develop into partly masculinized freemartins under the influence of factors provided by a male twin via the shared circulation (Jost, 1970). The absence of testicular tissue in freemartins indicates the absence of a diffusible testis-determining factor, and suggests that the first and subsequent steps of testis development are under cell-autonomous control.

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The commitment of the other cell lineages to their male fates does not depend on the Y chromosome, so must somehow be triggered by the Sertoli cells. How this is accomplished is quite unknown. One of the first products produced by the Sertoli cells is MIS, the hormone that inhibits the development of Miillerian (female) ducts. However, MIS has no effect on subsequent testis differentiation, as shown by the phenotype of boys who lack this hormone, yet possess developed (though undescended) testes, as well as persistent Mullerian ducts. The same interpretation is suggested by the lack of any testicular development in XX mice transgenic for MIS (Burgoyne, 1992). Testis determination is likely to be typical of other pathways of organogenesis, and it is even possible that the testis-determining factor may belong to a family of genes which have similar functions in controlling differentiation of other organs. As such, it presents an ideal opportunity to study a developmental pathway which, although it has a dramatic phenotypic effect, is not essential for survival (Goodfellow and Darling, 1988). Mutations which interrupt the pathway are therefore viable, and many human conditions of aberrant sexual differentiation are known and have been well studied. Most of what we know of mammalian sex determination comes from this study of sex chromosome variants and mutants in sex-determining genes, which alter the sex of the individual. Valuable information has also come from a second source of variation-that found among distantly related species. The comparative approach has been of particular value in the search for the testis-determining factor, and the evaluation of candidate genes.

V. The Search for the Testis-Determining Factor

All the information is consistent with the idea that a single testis-determining event controls the subsequent gonad differentiation and development of sexual dimorphisms. This single event has been long attributed to the action of a single testis-determining factor (TDF in humans, Tdy in mouse). Many attempts have been made over three decades to identify this crucial gene and to study its functioning. Molecular methods of gene mapping, and a positional cloning approach in which an unknown function can be localized to a small chromosome region and isolated, have provided new tools for the search for the TDF. The progressive narrowing down of the sex-determining region, then the isolation and identification of the mammalian testis-determining gene, has been one of the most dramatic success stories of modern genetics.

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A. Influence of the Y Chromosome on Sex Determination and Differentiation

A great advantage in the search for the testis-determining factor is that we know, from the phenotypes of humans and mice with abnormal numbers of X and Y chromosomes, that the TDF must logically be borne on the Y chromosome. Humans with a single X chromosome and lacking a Y (XO) developed as females with Turner syndrome (Ford et al., 1959), whereas individuals with two X chromosomes in addition to the Y (XXY) developed as males with Klinefelter syndrome (Jacobs and Strong, 1959). Individuals with three, four, or even five X chromosomes also developed into males if a Y chromosome was present, but into females if no Y was present. Similarly, XO mice are female and XXY mice are male (Welshons and Russell, 1959; Cattanach, 1961; Russell and Chu, 1961), and the same holds for other eutherian mammals studied. These data implicate the Y chromosome in somatic sex determination, and suggest that the numbers of X chromosomes present have no influence on sex. This was a surprising finding at the time, since the phenotypes of Drosophila with abnormal numbers of sex chromosomes showed that sex in fruit flies is determined by the ratio of X to autosomal sets. Does the Y chromosome directly determine sexual dimorphisms other than testis development? The unequivocally male o r female development of most X aneuploids implies that development of secondary sexual characteristics depends on testis determination, and is therefore indirectly Y determined. However, as we have discussed in Section IV, there are some sexual dimorphisms that appear to be independently determined by the Y chromosome. At least some of the accelerated growth of male preimplantation embryos can be attributed to genes on the Y (Burgoyne, 1992). Growthpromoting properties have also been ascribed to the human Y by comparing the heights of boys with different numbers of Y chromosomes (Ratcliffe et al., 1991). A possible role of the H Y A gene in regulating growth has been suggested (Heslop et al., 1989), and, since it is expressed in the heterogametic sex in a variety of vertebrates, this could make sense of the general correlation between the more advanced growth of the heterogametic sex. The ribosomal subunit gene RPS4, and the zinc finger gene ZFY, with its transcription factor structure, are also obvious candidates for a growth-promoting role, as is the pseudoautosomal growth factor (Ogata et al., 1992b). There is good evidence that the Y chromosome also directs aspects of germ cell development (Burgoyne, 1992). It has been demonstrated that, although XX as well as XY cells may form prospermatogonia in chimeras, spermatocytes in adult testes are exclusively XY. This has been ascribed

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to inhibition of spermatic development by the activity of two copies of the X chromosome in chimeras and XX sex-reversed mice; however, a complete block at metaphase I of meiosis is also observed in XO/XY mosaics. Since XO male mice carrying the deleted Sxr region show an identical block, there must be a spermatogenesis factor (named Spy) which maps on the short arm of the mouse Y near the testis-determining factor. Since the spermatogenesis factor Spy maps in the same region of the short arm of the mouse Y, near to Hya and Zfy, it was thought that either of these two genes might prove to be Spy. However, a gene, U b e l y , has recently been identified in this region which may be a better candidate for the Spy gene (Mitchell et al., 1991; Kay et al., 1991). This gene codes for ubiquitin-activating enzyme 1, and has a homolog U b e l x on the X, as discussed in Section 11. It appears to be homologous to a gene identified on the human X by its action in complementing a yeast cell cycle mutant; however, there is no Y-borne homolog in man or other primates. Mouse U b e l y probe identifies X- and Y-borne homologs in all other eutherian mammals, as well as marsupials (Mitchell et al., 19921, suggesting that the Y-borne copy fulfills some important function. The loss of UBEl Y from the Y in primates has evidently been recent. Deletions of the proximal region of the long arm of the human Y are also associated with azoospermia (Bardoni e f al., 1991), suggesting that a spermatogenesis-controlling gene lies on the human Y chromosome. However, this gene is unlikely to be the homolog of the mouse Ubel Y gene, since this probe detects no signal on the human Y. Nor is its map position consistent with either HYA or ZFY, and its identity remains unclear. Deletion analysis of the mouse Y also provides evidence for other, less well defined, factors on the long arm of the Y, which are required for normal sperm differentiation (Burgoyne, 1993). Partial deletions of the long arm increase the frequency of abnormal sperm, suggesting that multiple copies of a gene, distributed along the long arm of the Y, are required for normal sperm head development. The X chromosome also contains factors which affect embryo growth and gonad size, as well as factors which are required for germ cell development (Burgoyne, 1992). There are no oogonia in the gonads of human XO (Turner) females, and in the absence of follicles, no estrogen or progesterone is produced, and an undifferentiated “streak” gonad results. Although XO mice are fertile, the oogonia degenerate rapidly. The degeneration of germ cells in XO humans and mice suggests that two copies of at least one gene on the X are required in the germ line. The sterility of XXY individuals may be accounted for by the detrimental effect of a second (paternal?) X on the developing germ cells. Thus both sex chromosomes have effects on sexual dimorphisms in

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eutherian mammals that are independent of testis determination. However, these effects on growth and germ cell differentiation are usually regarded as peripheral to determination of sex. Although it is still possible that it is the growth-promoting properties of the Y chromosome which indirectly control the development of the gonad into a testis (Mittwoch, 19881, it seems more likely that testis determination is induced directly by TDF on the Y chromosome, and that somatic sex differentiation is largely controlled by testicular hormones. In marsupials, the X and Y both have striking effects on sexual dimorphisms, such that we must consider to what extent sex is determined by the possession of a Y-borne testis-determining factor. While it is clear that the possession of a testis or ovary correlates precisely with the presence or absence of a Y chromosome, this is not the case for other sexual dimorphisms. The several XXY animals that have been described are all intersexes, having undescended testes and male genitalia, but instead of a scrotum they have a pouch with functioning mammary glands, and the short gubernaculum and cremastor muscle typical of females (Sharman et al., 1990). The phenotype of XO animals is variable, but generally the reverse; there is no testis, but a scrotum often replaces the pouch and mammary glands (Hughes et al., 1993). While the phenotypes of some animals are more complex mixtures of the above (some of these may be mosaics), it is fairly clear that scrotum development is independent of testis determination. Taken with the observation (0 et al., 1988) that scrota1 bulges are evident in marsupial embryos considerably before testis determination has occurred, this must mean that testis determination and production of testicular hormones does not control all other male characteristics in marsupials. Determination of scrotum in males, or pouch and mammary glands in females, appears to be under independent genetic control, perhaps mediated by the dosage or parental origin of a scrotum-determining gene(s) on the X chromosome (Sharman et al., 1990; Cooper, 1993). However, it is clear that in marsupials, as in eutherians, the Y determines testis and presumably carries a TDF gene. Are there genes on other chromosomes that control o r modify sex determination? We have already reviewed evidence (Section 111) that human sex determination can be modified by a gene on the X. Perhaps an X-linked suppressor of Y function may also explain examples of variant sex determination in other species. Horses which are XY or XXY have phenotypes ranging from normal fertile female to highly masculinized intersex; XY sex reversal may follow an X-linked pattern of inheritance (Kent et al., 1986). In many natural populations of two lemming species, XY females are present which have a rearranged X. XX*Y animals may be male or female, according to whether X* is inactivated at high o r low

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frequency. This X* has a different banding pattern, which suggests a complex inversion and deletion (Fredga, 1988). It includes at least some Y-derived material, detected by expression of the HY antigen by X*Y females. Evidently this X* contains a factor which suppresses the action of the Y chromosome. It bears an unusual ZFX variant (Lau et ul., 19921, and it is exciting to speculate that it may contain a duplication or variant of the Y-suppressing gene identified on human Xp (Section 111). There are also a number of autosomal mutants which will reverse sex, apparently by preventing the normal action of the testis-determining factor. In humans, XY females and intersexes with campomelic dysplasia have been found with rearrangements to chromosome 17, in a region with some homology to the T locus on mouse chromosome 1 1 (Tommerup et al., 1993). In mice, there are at least three mutants which map to autosomes. Mutants of these genes, with seemingly different functions, all exert a dominant sex-reversing effect (Eicher, 1988). For instance, Tus lies within the T locus, mutations of which affect development of the skeleton. The genes defined by these mutants were also proposed to be responsible for the mismatch of Mus domesticus type Y chromosomes, which did not determine male phenotype in particular mouse strains. Eicher (1988) suggested that these genes act in a coordinated fashion in a testis or ovarydetermining pathway. However, an alternative explanation is that all these mutations have a nonspecific effect on sex determination, perhaps by retarding growth (Burgoyne, 1992). These mutants resulted from small deletions, and may act to produce a timing mismatch between T D F and its target tissue. These X-linked and autosomal sex-reversing mutants are unlikely to define other genes involved in the primary testis-determining step, but may well define upstream or downstream steps in a sex-determining pathway. All the evidence points to a paramount role for a TDF gene on the Y chromosome in testis determination. B. Regional Localization of the TDF

A gene mapping approach has been taken to pinpoint the T D F on the Y chromosome, and to evaluate candidate sex-determining genes. Deletion mapping depended on the occurrence of variant humans and mice possessing only a part of the Y chromosome (Fig. 8). Initially, the sexual phenotype of rare humans with cytologically detectable structural abnormalities of the Y chromosome (gross deletions or translocations, ring Y chromosomes or isochromosomes) enabled a rough localization (Davis, 1981). From the sex of these patients, it could be concluded that removal of all or part of the short arm of the Y produced a phenotypic female,

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whereas removal of the long arm did not: thus, the TDF must lie on the short arm of the human Y (Goodfellow and Darling, 1988). More precise localization of the T D F on the human Y chromosome was provided by a study of patients whose sex was discordant with their sex chromosome complements. So-called XX human males were described who had an apparently normal XX female complement; conversely, human females were described who had an apparently normal Y chromosome. Ferguson-Smith ( 1966) suggested that these variants were generated by illegitimate recombination between the tip of the X and Y chromosomes, outside the pseudoautosomal region to which recombination is normally restricted. As a result, a part of the differentiated region of the human Y was exchanged for a region of the X (de la Chapelle et al., 1984; Disteche et a l . , 1986; Ferguson-Smith and Affara, 1988: Levilliers et al., 1989). In XX males, therefore, the tip of Yp replaces the tip of Xp; conversely, in XY females the tip of Xp replaces the tip of the Yp. These exchanges are invisible cytologically, but probing with DNA markers specific for Yp can detect the addition of small Y regions in XX males and the deletion of Y regions in XY females (Fig. 8). Indeed, Y-specific sequences were later detected in the DNA of several XX males. The next step was to use these rearrangements to construct a molecular map of DNA markers on the human Y chromosome. Using sequences specific to Yp as probes, the extent of deletions or additions of Y material could be mapped, the markers ordered, and the regions present in XX males or absent in XY females delineated (Vergnaud et al., 1986). Using this approach, TDF was mapped to the distal region of Yp (Disteche et al., 1986; Ferguson-Smith et al., 1987; Muller et al., 1986; Page et al., 1988). In a recent tour deforce, a detailed deletion map of the euchromatic portion of the human Y, and a map of overlapping fragments cloned into yeast artificial chromosomes (YACs) were prepared (Foote et al., 1992; Vollrath er al., 1992). A long-range restriction map of the sex-determining region, from the MIC2 gene within the pseudoautosomal region to the most distal breakpoint in XX males, was then constructed using pulsed field electrophoresis to separate large genomic fragments (Goodfellow and Darling, 1988). Several groups then set out to clone and search this entire region and locate the TDF gene. Since nothing at all was known of the size, structure, or function of the TDF gene, a search was initiated for a sequence which contained an open reading frame (ORF), and recognizable signal sequences. In addition, it seemed likely that the T D F gene would be highly conserved among mammals with an XX fema1e:XY male sex chromosome mechanism. Several groups refined the position of the TDF on the human Y chromosome and screened the sequences for conserved coding sequences. In particular, Peter Goodfellow’s group in London

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worked from the pseudoautosomal boundary toward the centromere, and David Page's group in Boston worked up from a deletion breakpoint toward the pseudoautosomal region. Concurrently, a search for the mouse testis-determining factor Tdy was undertaken, using similar mapping procedures to localize the gene to the small, short, arm of the Y. A great deal of information has been provided by a sex-linked mouse mutation Sxr (sex reversed) which converts XXsx' mice into phenotypic (but sterile) males. This mutation was found to result from the duplication of about 5000 kb of the normal Yp onto the tip of Yq adjacent to the pseudoautosomal region, and its transfer to the tip of the Xq by X-Y recombination within the pseudoautosomal region (Cattanach, 1975; Evans et al., 1982; Singh and Jones, 1982). The male phenotype of XXsx' mice implied that Tdy lay within this small translocated region of the Y. Again, identification and mapping of Y-specific DNA probes provided a detailed map of this sex-determining region of the mouse Y chromosome (Mitchell and Bishop, 1991). C. The Rise and Fall of Candidate TDF Genes

The search for the testis-determining gene over the past two decades involved a huge amount of work on what turned out to be false trails. However, the pursuit of each presented surprises, and enriched our understanding of sex chromosome organization, function, and evolution. Initially, the approach to identifying the TDF gene was to identify protein products expressed in males but not females, then to map the gene producing this product. The first sex-specific product was identified as a weak transplantation antigen raised by injecting female mice with spleen cells from males of the same inbred strain. The antisera were later found to cross-react with cells from males, but not females of a range of mammalian species, including humans, suggesting that it was encoded by a gene (HYA in man and Hya in mouse) born on the mammalian Y. Wachtel et al. (1975b) proposed that the HY antigen was the product of the testis-determining gene. The antisera also cross-reacted with cells from avian, reptile, and amphibian species. In species in which the male is the heterogametic sex, the male-derived cells cross-react, but in species in which the female is the heterogametic sex, it was female cells which cross-reacted. The hypothesis had to be modified (Wachtel et al., 1975a) to state that H Y A directs the indifferent gonad to develop toward whichever mature gonad (testis or ovary) typifies the heterogametic sex. At this stage HYA was the only function ascribed to the Y chromosome, so it was widely supposed to be coded for by the sex-determining gene.

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However, this became less likely when HYA was found to be ubiquitously expressed, even in preimplantation embryos, and its expression did not correlate in any way with testis determination. The function of H Y A is still not known, but Heslop et al. (1989) have proposed that it acts as a sexspecific growth regulatory factor, and may be involved in the differences in growth rate between early male and female embryos in mammals, and perhaps in other vertebrates (Section IV). Despite intense interest in this gene, it took almost a decade to provide genetic evidence that distinguished it from the TDF. The expression of the HY antigen by XXsxr male mice was consistent with the identity of HYA and the TDF. However, mice lacking HYA expression were found to develop testes, and mice derived from the Srx mutant, which had part of the added Y material deleted, were found to be male although they lacked HYA (McLaren et al., 1984, 1988). The TDF and HYA were later mapped to different locations on the short arm of the mouse Y chromosome (McLaren et al., 1988; Roberts er al., 1988). Thus HYA and the TDF were shown to be separable genetically in mice. In humans, even fairly crude mapping of the Y was sufficient to remove HYA from consideration as the human TDF, for the expression of the human HY antigen was concordant with the retention of the long arm of the human Y chromosome, whereas maleness seemed to depend on the retention of the short arm (Simpson et al., 1987). Since H Y A was still the only active gene ascribed to the Y by the early 1980s, it had little competition as a candidate sex-determining gene. However, the possibility that sex could be determined by noncoding sequences was raised by Singh and co-workers. They found that a minor satellite DNA sequence, Bkm, cloned from repetitive DNA of the banded krait (an Indian snake), was remarkably conserved, at least among vertebrates. They detected sex-specific bands in DNA in mice as well as birds and reptiles (Singh et al., 1980; Jones and Singh, 1981, 1985). This sequence detected male-specific bands localized on the Y in mice, but femalespecific bands localized on the W chromosome of snakes and birds. The simple GATA repetitive nature of this ubiquitous Bkm sequence was obviously incompatible with a coding function. How could it possibly determine sex? Chandra (1985a) suggested that sex could be determined by a sequence which did not code for a product, but which acted as a sink for high-affinity binding of a controlling (female producing?) substance, which could be the product of a gene on any chromosome. The observation that Bkm sequences were concentrated in the small Y-derived sex-determining region translocated to the X in XXsxrmale mice supported this contention. However, Bkm sequences were eventually eliminated because the male mice with the deleted Srx region lacked most or all of these Bkm sequences, although they must have had TDF. The

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involvement of Bkm in human sex determination was always in doubt because Bkm sequences are not concentrated on the human Y. Thus, although the location of Bkm sequences on vertebrate heteromorphic sex elements still elicits suggestions of a functional role (Singh and Majumdar, 1993), it is unlikely to play a role in sex determination. More recently, accurate gene mapping, followed by positional cloning and characterization have been used to pinpoint the male-determining function on the Y chromosome. This approach, as well as eliminating both of the original candidate sex-determining genes, was used to narrow down the sex-determining region of the Y chromosome which must bear the TDF. Page et al. (1987) used this technique to define a small sex-determining region which was present in an XX male with the terminal 280 kb ofthe Yp, and absent in a female with a complex Y:22 translocation who appeared to have a single 140-kb deletion from the Y. Lining up the region of the Y present in the XX male and absent in the XY female left a 140-kb region which, logically, had to contain the TDF gene (Fig. 8). This entire 140-kb region was subcloned and searched for coding sequences highly conserved against a nonconserved background. Fragments within it were used to probe Southern blots containing DNA from males and females of a number of species (“Noah’s Ark blots”). A single probe detected a highly conserved sequence with an open reading frame. This sequence appeared to satisfy the requirements of TDF very adequately. It was the only probe within the defined region to detect a malespecific sequence in human DNA. It detected male-specific bands also in several species of eutherian mammals. The largest open reading frame (1.2 kb) defined within it coded for a predicted protein with 13 polypeptide “fingers,” held together by zinc bridges. This zinc finger protein was reminiscent of known transcription factors, such as the frog TFZZZA, and, like them, was presumed to fulfill a DNA binding function. The gene was termed ZFY, for zinc finger, Y chromosome. One surprising feature of this ZFY gene was that the probe also detected a band shared by both male and female DNA in all mammals tested. Dosage differences suggested that this second band represented an Xlinked copy of a closely related gene, a hypothesis which was confirmed by subsequent cloning, sequencing, and mapping of the human sequence ZFX (Schneider-Gadicke ef al., 1989). The ZFXgene mapped to the short arm of the X chromosome at position Xp12-13 (Muller and Schempp, 1989; Page ef al., 1990a), well outside the pseudoautosomal region. The ZFX gene was found to have a structure similar to that of ZFY, and was also predicted to code for a protein with multiple zinc finger domains, differing from ZFY product only in 10 of 393 amino acid positions. The genes were subsequently found to be 97% homologous within the DNA

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FIG. 8 Deletion map of the short arm of the human Y chromosome, used to deduce the position of the TDF in the normal Y. So-called XX males have part of the Y , which must contain the TDF. One XX male had a piece of the Y, including ZFY (Page et a / . , 1987). but others had smaller regions of the Y which lack this gene. All include SRY (Palmer et al., 1990). XY females lack the part of the Y containing the TDF. The complex deletion in the XY female on the right misled researchers into thinking that only ZFY was lacking (Page et al., 1987). but the female was later found to have a second deletion containing S R Y (Page et a/., 1990b).

binding domains (Palmer et al., 1990), and are calculated to have diverged about 100 million years ago. In mice, an X-borne Zfx and duplicated Zfyl and Zfy2 genes on the Y were described (Mardon et al., 1990). Page suggested four ways in which the products of ZFY and ZFX could interact to determine sex. They could have completely different functions or act antagonistically on the same target, or their products could interact to produce male-specific protein. A particularly interesting proposal was that the X- and Y-linked copies might operate as Chandra (1985b) had suggested, determining male phenotype by means of dosage of active

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genes. In this hypothesis, a male would have an active ZFY plus an active ZFX, whereas a female would have only a single active copy of ZFX on the active X chromosome. The first indication that ZFY was not likely, after all, to be the sexdetermining gene came from gene mapping studies in marsupials. For the ZFY gene to be a credible candidate TDF gene, it should detect malespecific homologous sequences on the minute marsupial Y chromosome, since the Y is testis determining in marsupials. In addition, mapping a ZFX homolog in marsupials would provide a stringent test of the hypothesis that ZFX dosage differences are involved in sex determination via X chromosome inactivation, because the region of the human Xp which contains ZFX lies within the recently added XRA region, which is autosoma1 in marsupials and not inactivated (see Section 111). Southern blotting analysis showed that the marsupial genome contained sequences highly homologous to human ZFY. However, marsupial Noah’s Ark blots showed that these ZFY homologs were not male specific, so they could not lie on the Y chromosome (Sinclair et al., 1988). Southern blotting analysis of rodent-marsupial cell hybrids excluded ZFY homologs from the X. The gene was mapped to autosomal locations in marsupials, and, significantly, these lay within the clusters containing other human Xp genes. Subsequently, ZFY has been mapped to two similar autosomal gene clusters in monotremes (Watson et al., 1993), suggesting that this gene was autosomal in a common mammalian ancestor, and was part of the XRA region translocated to the eutherian X between 80 and 150 million years ago. This would require marsupials and monotremes (and, indeed, the ancestral mammal) to use some other gene as the testis-determining switch. The conclusions were either that ZFY took on a sex-determining role in eutherians relatively recently, or that ZFY is not the TDF. Independent evidence that ZFY does not function as the testis-determining gene in eutherian mammals was obtained by studying its time and tissue of expression. Unexpectedly, human ZFY and ZFX were both found to be expressed ubiquitously, a curious property for a gene whose effect must logically be felt in the genital ridge immediately prior to testis development. In mouse, Zfx was found to be expressed ubiquitously, but Zfy1 and Zfy-2 were testis specific, and Zfy-1 was expressed in fetal testis (Mardon et al., 1990; Nagamine et al., 1990), which is concordant with its suggested function as the TDF.However, further studies with a strain of mutant mice which lack germ cells (Koopman et al., 1989) showed that Zfy expression was confined to germ-line, rather than somatic cells of the testis. Since we know that the target for testis determination is the Sertoli cell lineage, and that germ cells follow an independent developmental pathway, this expression profile is incompatible with a role in testis determination.

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The possibility that human ZFX and ZFY determine sex by means of differential gene dosage brought about by X-chromosome inactivation had to be abandoned when it was subsequently shown that the human ZFX was not subject to inactivation (Schneider-Gadicke et af., 1989), so that both males and females would have two active copies of the gene. A sexdetermining function for the Zfy genes in mice was also made unlikely by the construction of a strain of XY female mice derived by introducing a retroviral vector into XY embryonal stem cells, then introducing these cells into chimeric mice. Normal structure and expression of both Zfy genes in these mice confirmed that sex reversal did not result from the disruption of the Zfy gene (Gubbay et af., 1990a). The definitive exclusion of human ZFYfrom a role in sex determination was made by more detailed deletion mapping of the sex-determining region of the human Y chromosome. DNA from a number of XX males was tested, using a probe derived from the Y-specific sequences at the pseudoautosomal boundary. Among these patients were four who had pseudoautosomal boundary sequences, but lacked ZFY. Reasoning that the acquisition of regions of the Y could hardly be coincidental to their male phenotype, Palmer et al. (1989) concluded that ZFY was not included in the region of the Y that determines maleness. They then used the information from these patients to redefine a region, immediately adjacent to the PAR, in which the TDF must lie. It was in this region that the TDF was finally discovered. VI. Identification and Characterization of the Mammalian Testis-Determining Factor SRY

The four XX males studied by Palmer et al. (1989) had received less than 60 kb of the Y chromosome-unique region, exchanged with the tip of the X. The search for the TDF gene shifted to this region. A. Cloning and Characterization of the SRY Gene

The entire 60-kb region had already been cloned (Ellis et al., 1989), and these clones were now used to map the breakpoints very accurately in the XX males (Palmer et al., 1989; Sinclair et al., 1990). This allowed the region which must contain the TDF to be narrowed down to a 35-kb region immediately proximal to the pseudoautosomal boundary (Fig. 8). This 35-kb sex-determining region was then subcloned and sequenced, and meticulously screened for sequences which fulfilled the requirements expected of the testis-determining factor. The TDF should be unique to

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JENNIFER A. MARSHALL GRAVES AND JAMIE W. FOSTER

the Y chromosome and present in all the XX males with Y-derived sequences. It should be highly conserved and male specific in other mammals with an XX/XY sex chromosome system, and it should also be present within the minimal sex-determining region in XXsxrmice. It should contain an open reading frame and recognizable promotor and translation signals, and be expressed in fetal gonads. This search was made difficult by the presence of many highly repetitive sequences in the region. However, a detailed map of unique sequences could be constructed, and each of these could be tested. Several of these detected male-specific fragments in human DNA. However, only one, a 2. I-kb Hind11 fragment, also detected male-specific fragments in a range of other eutherian species, as well as a number of weaker bands shared by males and females. The probe detected homologous sequences in XXsxr males, as well as in normal male mice. This sequence, given the name SR Y (for sex region, Y chromosome), was put forward as the best candidate for the testis-determining factor (Sinclair et al., 1990). Curiously, corroborating evidence came from more detailed studies of the original XY:22 translocation female on whom the original identification of ZFY as the sex-determining gene had depended (Page et al., 1987). DNA from this patient was now found to lack, not only the ZFY region, but also the region in which SRY was identified (Page et al., 1990b). Thus the SRY sequence satisfied at least two of the criteria for the TDF gene. It was present on, and specific to, the smallest region of the human and mouse Y chromosomes known to be testis determining. It was absent in an XY female who lacked only small regions of the Y. It detected homologous male-specific sequences in other mammals. The sequence was then further characterized, in the expectation that it contained the human TDF gene. Sequencing the fragment revealed two open reading frames, the larger of which coded for a putative polypeptide of 223 amino acids. A search of databases showed that this product had some homology to several DNA-binding proteins, in a conserved motif common to hUBF (human upstream binding factor), NHP6 (non-histone protein 6) and HMG I (highmobility group protein I ) , as well as part of the yeast mating casette (Mc) protein. Homology was limited to a conserved motif of 80 amino acids, termed the HMG box. Cloning and sequencing the SRY genes of other eutherian mammals showed that they had homology only within the small HMG box region (Sinclair et af., 1990). The human SRY probe was used to clone homologous sequences from mice, and the corresponding malespecific mouse Sry gene was isolated, as well as other SRY-related sequences shared by males and females. The SRY gene therefore seemed to belong to a family of genes which shared the HMG box (Gubbay et al., 1990b).

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The SR Y gene structure is still incompletely known. Northern blotting analysis of human testis RNA revealed a 1. I-kb SRY message, but it has not been possible to determine the size of the embryonic SRY transcript. It has proved difficult to obtain complete cDNA clones from human or mouse testis libraries, but comparisons between genomic and available cDNA sequences show that at least the coding region comprises a single exon. In an attempt to resolve the transcriptional unit of the human SR Y gene, Su and Lau (1993) have recently transfected mouse L cells with a cosmid containing the genomic sequence, and made cDNA from RNA expressed by these cells. The transcripts were the expected 1.1 kb, and primer extension revealed two putative start sites 5’ of the initiating AUG codon. However, it is not clear whether either of these putative promotor sequences is active in vivo. The human transcript has also been studied in normal adult testis (Clepet et al., 1993). This analysis reveals multiple potential sites of transcript initiation consistant with a major transcript of 1.1 kb and a minor transcript >400 bp longer. Identification of the transcriptional unit of the mouse Sry has been even more problematical. Mouse testis cDNAs were found to be remarkably rearranged, and it was later shown that the testis transcript is a circular RNA molecule (Cape1 er al., 1993). Given the significance of the observation that ZFY was autosomal in marsupials, it was a question of some special interest as to whether a gene homologous to SR Y was located on the Y chromosome in marsupials, in which the Y is at least testis (if not entirely male) determining. A considerable effort has been put into identifying and characterizing SR Y homologous sequences in marsupials. This proved to be not a straightforward task, for at low stringency, the human SRY gene identified a number of homologous sequences. Most of these were shared by males and females and were assumed to be the autosomal homologs of the HMG box gene family described by Gubbay er al. (1990b). However, one band seemed to be male specific in the tammar wallaby, and this was eventually cloned from a size-selected subgenomic library and shown to detect male-specific sequences in two marsupial species (Foster et al., 1992). Shortly thereafter, a clone was identified in a testis cDNA library from the distantly-related, striped-face dunnart, which also detected a male-specific band in this species. Sequencing of the marsupial Y-borne SRY homologs revealed an open reading frame from which a sequence of 208 amino acids could be predicted. This protein contained a 79-amino acid HMG box motif with homology to those of eutherian SR Ygenes. As for eutherian mammals, homology is restricted to the HMG box motif. The recovery of an SRY homologous cDNA from a testis cDNA library provides some evidence that this gene

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is expressed in the testis. The gene was mapped to the Y chromosome, and is therefore a good candidate for the marsupial testis-determining factor. When the putative SRY protein products of a human and mouse are compared with two marsupial species (Fig. 9), the overall amino acid similarity within the HMG domain is about 80% (including conservative substitutions). As for eutherian mammals, there is no recognizable homology outside the box. Many amino acids were identified that remain identical across all SR Y genes; since these have remained unchanged for 80-150 million years, it is likely that these positions are important in SRY DNAbinding activity or specificity.

6 . Demonstration That SRY Is Sex Determining in Man and Mouse

Mutation analysis of human XY females directly implicates the SR Y gene in testis determination. Two XY females with apparently intact Y chromosomes were found to have independent SR Y mutations within the HMG box. One had a point mutation not shared by her father or brother which would substitute isoleucine for methionine (Berta et al., 1990), and the second a 4-base deletion near the 3’ end of the HMG box not shared with her father which would cause a frame shift (Jager et al., 1990). Since this time, several de n o w point mutations, all within the HMG box, have been identified among XY females (Hawkins et al., 1992; Goodfellow et al., 1993). The amino acid positions within the human HMG box identified by mutation analysis as critical for the gene to function are those conserved among humans, rabbits, mice, and marsupials. Other XY females examined in these studies appeared to have a normal HMG box sequence, or have an alteration shared with the father. This suggests that mutations elsewhere in the SRY gene, or in other genes, may affect SR Y function. Reports of a number of XY female horses among the progeny of a single stallion (Kent et al., 1986) may have a similar basis in a conditional SRY mutation. The cloning of the homologous gene, Sry, in mice (Gubbay et al., 1990b) also allowed analysis of mutant mice with altered sexual phenotype. The Sry gene was found to be located on the short arm of the mouse Y chromosome, near ZfL-l,ZfY-2, and Ubely, and is present in male XXsxr mice, on the smallest region of the Y known to be testis determining. The Sry sequence was also shown to have been deleted in a strain of XY female mice with normal ’2 (Gubbay et al., 1990a; Lovell-Badge and Robertson, 1990). Studies of the expression of the human and the mouse SRY gene also

Human SRY Mouse Sry S. mac Y sox 3

RVKRPMNAFMVWSRGQRRKMALENPKMHNSEISKRLGADWKLLTDAEKRPFIDEAKRLRAVHMKEYPDYKYRPRRKTK

FIG. 9 Sequences translated from the HMG boxes of SRY and SOX3 genes of human, mouse, and marsupial. The SOX3 genes code for identical product in this region, whereas the SRY gene products have diverged considerably from each other and from their putative ancestor, SOX3. Below, the SRY sequences have each been compared to the consensus SOX3 sequence. Amino acids identical with the SOX3 sequence are represented as closed squares, similar amino acids as open squares, and dissimilar amino acids as gaps.

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JENNIFER A. MARSHALL GRAVES AND JAMIE W. FOSTER

support the claim that it acts as the testis-determining factor. SRY transcripts of 1 . 1 kb were detected by Northern blotting analysis of human testis RNA, but not RNA from ovary or other tissues (Sinclair et al., 1990). Detailed studies of Sry expression during mouse development were undertaken using the extremely sensitive reverse transcription (RT)-PCR technique, in which DNA transcripts of expressed RNA (reverse transcripts) are amplified by the polymerase chain reaction. Transcripts from Sry were first detected in embryos at 10.5 days, and were present up to 12.5 days, after which transcription ceased (Koopman era/., 1990). In situ hybridization showed that the expression was localized in the developing genital ridge, and was apparent only at 11.5 days. The 2 day window of Sry expression in the developing mouse genital ridge coincides exactly with the first visible signs of testis differentiation in the mouse. However, the correlation between Sry expression and testis differentiation may not be absolute. Recent studies of Sry expression using RT-PCR in very early mouse blastocysts (Zwingman et al., 1993) have detected transcripts as early as the 2-cell stage. The mouse and human SRY gene is also expressed in adult testis, but is localized in the germ cells (Koopman et a / . , 1990) and, at least in the mouse, is circular and probably nonfunctional (Cape1 et al., 1993). Whether the early blastomere transcripts are functional is not yet known. The ultimate test of the function of the Sry gene was the production of sex-reversed XX male mice transgenic for the Sry gene. Koopman et al. (1991) injected fertilized mouse eggs with a 14-kb mouse genomic fragment containing Sry. Of these, six eggs developed into XX embryos containing the transgene, and two of these were found to have testes when they were examined at 14 days. Among embryos allowed to develop to term, five were transgenic for Sry, and one of these was chromosomally XX but had a male phenotype. This mouse (the famous “Randy”) had well-developed testicles, male reproductive ducts with complete regression of the Miillerian ducts, male genitalia, normal hormone levels, and male mating behavior. Germ cells were lacking, because of the presence of two X chromosomes, but, phenotypically, this XX Sry transgenic was a normal male. Thus all the information required for testis determination must be present within this 14 kb, a conclusion which confirms that SRY is equivalent to the TDF. C. Mechanism of SRY Function

The mechanism by which the SR Y gene acts is now under intense study. The HMG box it contains undoubtedly codes for a DNA-binding domain, suggesting that SR Y controls the action of other genes by binding to DNA.

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We are only now acquiring direct information on what sequences it binds to, and how it might activate or repress target genes. Much of what we currently understand about SRY function relies on analogy with other genes which share the highly conserved HMG box. The human upstream binding factor binds to specific sequences in the enhancer region of the ribosomal RNA genes, interacting with a second factor to promote transcription. The insulin response element binding protein (IRE-ABP) seems to mediate the activation of genes after insulin treatment. Two closely related HMG box-containing transcription factors, LEFl and T C F l , are involved in T-cell-specific gene expression and differentiation, binding to specific sequences within enhancer elements of receptor genes. Although these three genes are highly homologous within the HMG box, they bind to different DNA sequences. By analogy with these genes, the SRY gene was predicted to encode DNA binding proteins which would recognize related target sequences, and act as transcriptional activators. Two studies have borrowed the DNA binding sites from other closely related genes to confirm that the SRY gene codes for a sequence-specific DNA binding protein. Fusion protein translated from the cloned normal SRY was able to bind to the core sequence of the enhancer region recognized by TCFl, whereas the protein from several mutant SR Y sequences failed to bind to this sequence (Harley et al., 1992). Similarly, the HMG box translated from the mouse Sry was shown to bind a related enhancer element in a sequence-specific manner (Nasrin et al., 1991). A core sequence of A/, A/, CAAG was identified by analogy to other proteins, but recent experiments using recombinant SR Y identify an optimal SR Y binding site of A/, A/, CAATG (Harley et ul., 1994). The SRY protein, as well as other HMG box factors, fits into the minor groove of the DNA helix, and bends the DNA (Giese et al., 1992). This could have the effect of bringing distant regions of the Y chromosome, or protein factors bound to these regions, into close contact. The HMG box of SRY product also recognizes four-way junction DNAs, regardless of sequence (Ferrari et al., 1993). What genes does SRY regulate as the next step in the sex-determining pathway? Genes whose activities are regulated by SR Y might be identified by their possession of high-affinity binding sites for SRY. Haqq et al. (1992) identified HMG box binding sites within the promotors of two malespecific genes, coding for p450 aromatase and the Mullerian inhibiting substance. However, the significance of these observations is not clear, because such sites have low affinity for SRY and appear frequently in the genome. Although MIS is one of the first identifiable products of the developing testis, it would be surprising if it were induced directly by SRY.

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More recently, Cohen et al. (1994) observed that the SRY protein binds the same DNA sequence as another HMG box-containing gene, IRE-ABP. This sequence motif appears in the promotor of agenefra-1 (a transcription factor related to the oncogene fos), which is expressed both after insulin treatment and during spermatogenesis. They demonstrated that the purified SRY protein binds strongly to sequences in the fra-1 promotor, and enhances fra-1 transcription. If these in uitro binding studies reflect the control in uiuo, SRY may function in a cell-specific way by activating a more general transcription factor. The assumption has been made that SRY is a transcriptional activator because the dominant male-determining action of TDF suggests that it acts to turn on other testis-differentiating genes. Alternatively, as Jost (1970) originally proposed, TDF could act by inhibiting one or more ovarydetermininggenes. Or it could have an even less direct action; for example, by repressing a repressor of testis-differentiating genes (Fig. 10). The action of SR Y as an activator of testis-differentiating genes is consis-

MALE normal XY

SR?

FEMALE normal XX

t

-*-TESTIS-~

-1

no testis

TDG

b

MALE

normal XY

FEMALE normal XX

sRy\

FIG. 10 Two possible modes of action of the SRY gene in determining testis differentiation. (a) Direct action: SRY activates testis-differentiatinggenes directly in the male and has no action in the female. (b) Indirect action: SRY represses inhibitor(s) of testis-differentiating gene(s) (ITD) in the male and leaves these genes active to inhibit the testis in the female. Much more complex circuitry can readily be proposed.

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tent with its similarity to other transcriptional activators. However, the only conserved region known is a DNA binding domain, which could equally well belong to a transcriptional repressor. The poor homology between SRY genes from different species (Foster et al., 1992) might be more consistent with a repressor role, since sequence would be expected to be less constrained in evolution (Graves et al., 1993a). A repressor action by SRY would also make it easier to understand the occasional observation of XX males who apparently lack any Y-derived sequences (McElreavey et al., 1993), whose phenotype could easily be explained by mutations in the target ovary-determining or testis-repressing genes. This hypothesis also provides an explanation for the baffling observation that mouse ovaries form testicular tissue when grafted into the kidney capsule of castrated male or even female hosts (Taketo-Hositani and SinclairThompson, 1987). It is clear that SRY acts to determine testis. However, it is not yet known what its targets are, or even whether SRY exerts positive or negative control. To date, the hope that the identification of the testisdetermining factor will provide a direct entrance into the sex-determining pathway have not been realized. Not only is it proving difficult to identify genes which are the targets for SRY control, but there is also the suspicion that SRY may have a very indirect action. If SRY is, for instance, a repressor of a repressor of an activator of a testis-differentiating gene, then we are still a long way from the sex determining pathway itself. We may yet find that it is quicker to identify testis-differentiating genes from scratch, by cloning autosomal sex-reversing genes, than it is to identify genes which possess SRY binding sequences. Perhaps comparisons of the organization, sequence, and expression of SRY and related genes in a range of mammals will provide clues to the function of SR Y in sex determination, as well as the evolution of this function.

D. Evolution of SRY Organization and Function

Is SRY the universal male-determining gene in all mammals? All vertebrates? Even all animals? At what point in animal evolution did SRY become male-determining? SR Y is shared by the Y chromosomes of eutherian and marsupial mammals (Foster et al., 1992). This observation, together with the finding that UBEl is located on the marsupial, as well as the mouse Y (Mitchell et al., 19921, suggests that therian Y chromosomes have a common origin and probably a common function in sex determination. However, in many vertebrates the female is the heterogametic sex. In others, sex determina-

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tion is under environmental control. Is SR Y involved in sex determination in birds and alligators, maybe even in Drosophila? Comparative studies have shown that bird Z and W chromosomes bear no genetic relationship to the mammalian X and Y (see Section II), suggesting that the XY and ZW chromosome pairs evolved from different homologous pairs in a common ancestor. It seems unlikely, therefore, that these groups share a common sex-determining gene. Certainly, the well-characterized genes involved in sex determination in Drosophila bear no homology to mammalian SRY. We conclude, therefore, that SRY assumed a sex-determining function in the last 300 million years since mammals diverged from birds and reptiles. Comparisons of SRY-related sequences may reveal the origin of the mammalian sex-determining gene, and perhaps provide clues to its function. There are many SR Y-related sequences. Even preliminary characterization of SRY produced evidence that this gene is a member of a large family which shares at least the HMG domain. When human SRY was hybridized to Southern blots containing DNA from a range of other eutherian and marsupial species, the probe detected, as well as a single malespecific fragment, bands that were shared between males and females (Sinclair er al., 1990; Gubbay et al., 1990b; Foster er al., 1992). These shared bands must have derived from the X chromosome or autosomes. Four of these SRY-related sequences were isolated from an 8.5-day mouse embryo cDNA library using the human SRY probe (Gubbay er al., 1990b). None were identical to the Y-borne Sry gene, but all shared strong homology within the HMG box. Southern analysis of DNA from males and females suggested that they were autosomal, and they were initially named a l , a2, a3, a4; subsequently they were renamed Sox I , 2, 3, and 4 (for Sry-like HMG box-containing genes). An ever-growing number of SOX genes has been detected in the human genome, as well as other vertebrates and even Drosophila (Denny e l al., 1992; Griffiths, 1991). It seems, therefore, that the SRY gene is a member of a family of related sequences, which could be involved in regulation of other developmental pathways in all animals. How did SRY come to lie on the therian Y and acquire its sexdetermining function? One possibility is that SR Y was derived by insertion into the Y of a DNA copy of a transcript from one of the autosomal SOX genes. Su and Lau (1993) noted that cloned human SRY cDNAs they derived from in uitro transcripts had poly (AT) tracts, and suggested that this is consistent with reverse transcription and insertion. The lack of introns is consistent with such a retoposon origin for SRY, but, as this seems to be a general feature of the SOX gene family, there is little evidence that the SR Y gene was derived by retroposition. An alternative hypothesis is that SRY evolved from a SOX-like gene

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which originally had alleles on the undifferentiated proto-X and Y of an ancestral mammal. This hypothesis is strongly supported by the observation that the SOX gene most similar to SRY lies on the X chromosome. The discovery was a by-product of the original attempts to clone marsupial SRY, for one of the male-female shared fragments, revealed by probing male and female marsupial DNA with human SRY, showed clear malefemale dosage differences, typical of a gene on the marsupial X chromosome (Foster et al., 1992). This gene was assigned to the marsupial X by Southern blotting analysis of cell hybrids and mapped to the X near F9 by in situ hybridization (Foster and Graves, 1994). Sequencing this marsupial X-linked SRY-like gene showed that it had a typical SOX gene structure, and was, in fact, completely homologous at the protein level to the mouse Sox3 cloned and characterized previously (Gubbay et al., 1990b). The human homolog of this SOX3 gene has now been cloned too, and mapped to the long arm of the human X near F9 (Stevanovic et af., 1993). The position of SOX3 on the human and mouse X, as well as its inclusion in the marsupial X , places it within the original conserved XCR. Thus it seems likely that, after all, there is a conserved SRY-like gene on the mammalian. Has this SOX3 gene any function in sex determination? Perhaps Chandra’s dosage hypothesis, which initially provided a plausible explanation for the roles of ZFX and ZFY in sex determination, may be once again exhumed and applied to the SOX3ISRY gene pair. Related Sox genes are expressed in neuronal cells of male and female embryos suggesting a general role in development of the central nervous system. The male phenotype of a hemophiliac patient with a deletion of a region of the X which includes the SOX3 gene (Stevanovic et al., 1993)is inconsistent with a role for human SOX3 in sex determination. Of all the SOX genes, SOX3 is the most closely related to SRY (Griffiths 1991). Comparisons of SRY and the SOX3 gene among mouse, human, and marsupial species shows that the Y-borne SRY gene has evolved rapidly, whereas the SOX3 sequence has undergone few changes (Fig. 9). Whereas the SRY genes show only 65% base sequence homology between marsupial and mouse, the SOX3 gene retains 99% homology over these species. The SRY genes of man, mouse, and marsupial are more similar to the mouse Sox3 gene than they are to each other (Graves et al., 1993a). The SRY gene shows rapid sequence divergence, even among primates (Whitfield et al., 1993) and rodents (Tucker and Lundrigan, 1993). Thus it now seems likely that SOX3 and SRY were originally alleles on the undifferentiated mammalian proto-X and -Y chromosomes. We have proposed (Foster and Graves, 1994) that this ancestral gene was not involved in sex determination, but was active in gonad development and

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neuronal differentiation. The evolution of its function in controlling testis determination may have preceded, and initiated, X-Y divergence in mammals. Alternatively, the evolution of a sex-determining function may have followed, and resulted from, X-Y divergence. If this is so, the original mammalian X and/or Y must have contained another, more primitive sexdetermining gene, perhaps one that operated by differential dosage. As the X and Y chromosome differentiated by progressive loss and inactivation of genes on the Y, the SOX3 alleles borne on the X and Y were excluded from recombination, and diverged in sequence and function. Thus SOX3 evolved into the testis-determining SRY (Fig. 1 1 ) . Rapid divergence and evolution of novel function may be a general feature of the few active sequences which have survived the degradation of the Y chromosome, as discussed in Section 111. Certainly the Y-borne ZFY has evolved more rapidly in primates than the X-borne ZFX (Shimmin et al., 1993; Pamilo and Bianchi, 1993), and at least in mice, its expression has become tissue specific. Similarly, Ubelx is ubiquitously expressed in mouse, whereas Ubely is testis specific and may have evolved a malespecific function as a spermatogenesis factor (Mitchell et al., 1991). These changes have occurred independently, at least for genes (UBElXI Y and SOX3ISRY) in the original XCR and genes (ZFXIZFY) in the recently added XRA, and we suggest that the evolution of the sex-determining SR Y gene has paralleled the evolution of the other X-Y shared genes (Fig. 11). The consistent difference between the rate of evolution of X- and Yborne sequences may simply reflect the increased number of spermatogonial divisions suffered by the Y (Miyata et al., 1987). Alternatively, there may be selection for variant SRY (or ZFY or Ubely) sequences which, being all involved in sex determination or male fertility, could impose reproductive isolation and accelerate speciation.

VII. Conclusions

All genetic analysis depends on a comparison of variants. The investigation of the genetics of mammalian sex determination has used comparisons between wild-type and mutant organisms,and among mammals of different species. In this chapter, we have emphasized how genetic comparisons among widely divergent mammals can provide information about the evolution and function of highly conserved genetic pathways. Mammalian sex chromosomes are clearly related by descent. The X chromosome shares a region (XCR) conserved between even the most

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MAMMALIAN SEX CHROMOSOMES AND SEX-DETERMINING GENES

a ZNFl

,

ZNF2 ,

- -

-

ZNFn

- -

-

UBEll

- - -

sox11

ZNF

4

zx b

ZFY

5

UBE2

,

UBE3 ,

UBElX

UBElY

C

SOX1 ,

SOX2

, SOX3 ,

A

/\

SOX3

FIG. 11 Divergence of sequence and function of genes on the sex chromosomes. Each gene was represented by alleles on the initially homologous proto-X and -Y. The X-borne and Yborne alleles then diverged as X-Y recombination was suppressed and the sex chromosomes differentiated. The X-borne gene maintained its original function in both sexes, while the Y-borne partner became free to assume a male-specific function. (a) The large zinc finger gene family had at least one representative (ZNF)which was originally autosomal, but was transferred to the X and Y in eutherians. X- and Y-borne alleles diverged, giving rise to the conserved, ubiquitously expressed ZFX,while in the mouse at least, the Zfygene assumed a testis-specific expression and male-specific function. (b) The ubiquitin-binding enzyme family had a representative (UBEI) on the proto-X and -Y. X- and Y-borne alleles diverged and Ubely assumed a testis-specific expression and male-specific function in spermatogenesis. (c) The HMG-box containing the SOX gene family had one representative on the protoX and -Y. X- and Y-borne alleles diverged and SRY assumed a male-specific function as the testis-determining factor.

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distantly related mammals-eutherians, marsupials, and monotremes. The Y chromosome of eutherians and marsupials shares at least two genes, SRY and UBEZ. The mammalian X and Y chromosomes undoubtedly evolved from a pair of chromosomes which were homologous except for a small sex-determining region or a single locus. The small pseudoautosomal region of present-day mammals, as well as several X-Y shared genes outside the PAR, appear to be relics of this homology. Comparative studies have demonstrated that the X and Y were differentiated in stages. The progressive degradation of the Y was accompanied by the spread of X-chromosome inactivation; which change drove which is not clear. There is good evidence for at least one major addition of an autosomal region (XRA) to the original mammalian X and Y, and of the subsequent differentiation and inactivation of this recently added region. Classic work on sex determination identifies a positive male-determining function of the mammalian Y chromosome. In embryos possessing a Y, the undifferentiated gonad is transformed into a testis, and the production of testicular hormones induces most of the sexual dimorphisms apparent in humans and other eutherian mammals. In marsupial mammals, too, the Y chromosome has a testis-determining function, but some male characters are determined independently of testicular hormones. The identification and characterization of the Y-borne testis-determining gene, which initiates the male-determining pathway, is expected to clarify the steps involved in the development, not only of testis, but of all organ systems. Gene mapping has been of great importance in assessing the qualifications of rival candidate sex-determining genes. The finding that the HY antigen mapped to a different location on the mouse and the human Y than did the testis-determining factor eliminated this gene from consideration. Likewise, the finding that Bkm sequences were not concentrated on the human Y, and were separable from TDF in sex-reversed mice, showed that these sequences were probably not involved in sex determination. The location of ZFY within the sex-determining region, defined by deletion mapping of the human Y, initially suggested that this gene was the TDF. However, the finding that ZFY is autosomal in marsupials was the first indication that ZFY could not be a universal mammalian testis-determining factor. The subsequent redefinition of the sex-determining region of the human Y led to the identification of the SR Y gene. SRY has all the characteristics expected of the TDF. It is located on the Y in eutherian and marsupial mammals, expressed in the genital ridge of the undifferentiated embryo, and promotes male development in XX transgenic mice. SRY evidently controls the activity of other genes in the testis-determining pathway, but the upstream and downstream steps of this pathway have yet to be defined.

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It is obvious that the myriad differences between males and females must result from differential expression of a very large number of genes in a complex sex-determining pathway. SR Y may control an initial testisdifferentiating or ovary-determining step of this pathway, but steps upstream as well as downstream may be controlled by genes shared by males and females. Hopes that the isolation of the testis-determining gene would immediately unravel this developmental pathway have not, so far, been realized. It may be that the action of the SRY gene in controlling one or more of the steps in testis differentiation is very indirect. SRY is a member of a large gene family, whose members share the HMG box coding for a DNA binding domain. The finding that one of these genes, SOX3, lies in the conserved XCR suggests that SOX3 and SRY were originally allelic, but, like several other X-Y shared genes, diverged in sequence and function during early mammalian development. Originally involved in gonad and neural development in both sexes, S R Y may have taken over a sex-determining role from a more primitive sexdetermining gene, perhaps one which still determines sex in other vertebrates by means of dosage differences. A common origin of vertebrate sex chromosomes appears to be unlikely, since no genes are shared between the ancestral mammalian X and the bird Z . This does not require that the molecular mechanisms of sex determination and sexual differentiation be different in mammals and other vertebrates. The sex-determining pathway may well be common to all vertebrates, but different genes may have taken on a master switch role in different lineages. This might have occurred several times, when a duplicate gene, or an allelic variant evolved a novel control function over steps in the sexual differentiation pathway, as has evidently occurred in the evolution of SRY. Once upstream and downstream genes in the mammalian sex-determining pathway are identified, it will be possible to screen for them in other vertebrates and discover common steps. Some of the steps in mammalian sex determination may have evolved independently. Indeed, comparisons of the phenotype of sex chromosome aneuploids in eutherians and marsupials reveal the operation of an independent genetic pathway for determining some phenotypic differences between the sexes. The finding that scrotum and mammary gland development is a function, not of the presence or absence of a Y chromosome, but of the dosage of X chromosomes, suggests that the scrotum and mammary gland evolved later than did sex differences shared with other vertebrates. Originally they may have been determined by an independent genetic pathway, but have come under hormonal control (and therefore, indirectly, under SR Y control) in eutherians. In all these investigations, comparisons between normal and mutant sex chromosomes, and among men, mice, and marsupials have provided

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a rich source of information on how mammalian sex chromosomes and sex-determining genes evolved, and how they function. Acknowledgment The authors wish to thank JoAnne La Rose for her patient preparation of the rapidly evolving diagrams.

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Organization of Replication Units and DNA Replication in Mammalian Cells as Studied by DNA Fiber Radioautography Natalia A. Liapunova Institute of Human Genetics, Medical Genetics Research Center, Russian Academy of Medical Science, Moscow 115478, Russia

1. Introduction

The process of DNA replication in eukaryotic chromosomes has been studied intensively over the past 30 years. Data have been obtained on the organization of DNA replication at the levels of mitotic chromosomes, DNA molecules (replicon characteristics), and molecular events in the replication fork. Despite the abundant information obtained at all three levels, until now the knowledge of eukaryotic chromosomal DNA replication has remained fragmentary. Jacob and Brenner (1963) proposed a replicon model for bacterial chromosome replication. The replicon was defined as a genetic element (DNA molecule) which replicates as a whole using a unique origin, fixed at a membrane, in which replication is initiated and regulated. The elongation of a replicon was thought to occur by replication fork movement (Cairns, 1963). At the same time, it was shown that, unlike prokaryotic chromosomes, large DNA molecules of eukaryotic chromosomes are replicated in multiple sections (Taylor, 1960, 1963). Following the terminology of Jacob and Brenner, Taylor (1963) proposed a definition for the eukaryotic replicon as a stretch of DNA molecule which replicates as whole from a single origin. In succeeding years, the molecular mechanisms for replication of bacterial, mitochondrial, viral, and chromosomal DNA of lower eukaryotes (yeasts) were studied and reviewed in detail. Progress in establishing the molecular mechanisms of DNA replication in chromosomes of higher eukaryotes was noticeably slower. Nevertheless, important information on molecular mechanisms of DNA replication in chromosomes of higher eukaryotes was obtained by using SV 40 and polyoma virus chromosomes lnrernorional Review of Cvfologv. Val. 154

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as relatively simple but appropriate models for mammalian replicons (DePamphilis et al., 1983). Chromosomes of these viruses have nucleosomal structure and, except for initiation, all the stages of DNA replication appear to be carried out by the host cell. In viral chromosomes, replication is initiated at a genetically defined unique site. In contrast, multiple sites for initiation of replication in mammalian chromosomes are represented by “many preferred DNA sequences rather than by a single required sequence. Preferred sites can be rearranged, deleted, or inserted without impairing DNA replication” (DePamphilis et al., 1983). This is the main difference between the concept of replicons of eukaryotes and that of prokaryotes. Many investigators believed that application of the term “replicon” to eukaryotes was not correct (Painter, 1976) and they used other terms, such as replicating regions (Cairns, 1966), replication sections (Huberman and Riggs, 1968), units of DNA replication (Taylor and Miner, 19681, replicating units (McFarlane and Callan, 1973; Callan, 1976) or replication units (Housman and Huberman, 1975; Hand, 1975a,b, 1977; Wilson, 1975; Hand and German, 1977; Hand et al., 1983). Keeping in mind the aforesaid and following other authors (Edenberg and Huberman, 1975; Kapp and Painter, 1982b; Ockey, 1982; Sawada et al., 1982; Jagiello et al., 1983; Taylor, 1984), we use here the short and convenient term “replicon” together with the term “replication unit.” A complete description of replication of genomic DNA should include a full characterization of replicons, such as their sizes and number, the rate of elongation, and the method of termination. It should also include known regularities of temporal and spatial organization of chromosome duplication and the parameters of the period of DNA synthesis (S phase) in the cell cycle. More or less satisfactory descriptions of genomic DNA replication have been reported for only a few eukaryotic organisms with a relatively simple genome and a small amount of DNA. These are the yeast Saccharomyces cerevisiae (Petes et a!. , 1974; Petes and Williamson, 1975; Rivin and Fangman, 1980; Fangman et d., 1983) and the flowering plant Arabidopsis rhaliana (Van’t Hof et al., 1978). A general idea of the organization of mammalian genomic DNA for replication in time and space is lacking. Data on mammalian DNA replicons were obtained by a variety of techniques. A number of hydrodynamic methods have been successfully used to estimate the rates of replication fork movement and the sizes of replicative intermediates both in normal cells and in cells where DNA synthesis was affected by various agents (Taylor, 1968; Povirk and Painter, 1976; Laughlin and Taylor, 1979; Kapp and Painter, 1979, 1981). The adequacy of these methods for the study of replicon sizes is rather questionable, but the matter is not discussed in this chapter.

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A great body of information on eukaryotic replicons, including mammalian ones, was obtained by DNA fiber radioautography (DFR). These data confirm the concept of “clusters of small replication units” for mammalian DNA replication. By a cluster is meant a tandem arrangement of 10 to 100 replicons about 30 pm long that replicate synchronously (Huberman and Riggs, 1968). This concept is accepted by many researchers. Its main postulates are entered in monographs and textbooks (Alberts et al., 1983; Lime-de-Faria, 1983) and are used intensively for interpretation of new experimental data on DNA replication in mammalian cells (Vogelstein ef ul., 1980; Heintz et nl., 1983; Murata-Collins and Clark, 1987; Clark et al., 1987; Lug0 ef d., 1989; Handeli ef d., 1989; Vaughn et ul., 1990). At the same time, data in favor of another concept may be advanced. These data were obtained by improved DFR method. According to this concept, replication of large DNA molecules in mammalian chromosomes occurs mainly via large replicons (30-600 p m ) . DNA synthesis in most of these replicons takes from one third to one half of the S phase. Doubling of DNA molecules in individual chromosomal bands occurs via single replicons or simultaneous functioning two to four large replicons rather than by clusters of small replicons (Liapunova, 1985). In order to elucidate the source of disagreement between the two concepts, this chapter focuses on a critical view of radioautographic analysis of replicating DNA molecules. Thereafter the characteristics of replicons in mammalian chromosomes, their sizes, the replication fork rate (RFR), the methods of replicon termination, and the question of the existence of a replication fork barrier (RFB) are discussed on the basis of data obtained mainly by DFR. At the beginning of this chapter, the current concepts of chromosome replication are briefly reviewed. At the end, the replicon model for DNA replication in mammalian chromosomes is proposed.

II. Organization of Mammalian Chromosomes for Replication Haploid genomes of the majority of mammals contain about 90 cm of linear DNA molecules. In human cells this DNA is packed into 23 chromosomes. Each chromosome has a DNA molecule 2 to 6 cm long. The universal characteristic of chromosome structure in higher organisms is their subdivision into chromatin bands of contrasting properties. With light microscopy it is possible to visualize up to 2000 separate G (Giemsa-positive), R (reverse or G-negative), and C (constitutive heterochromatin) bands per haploid set of human chromosomes at midprophase of mitosis (Yunis,

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1981). This number may be increased to 3000 by specific electron microscopic techniques (Bak et al., 1981). It means that on average an elementary prophase band contains a segment of DNA molecule about 300450 p m long; the segment can reach more than 1000 p m in large bands. Replication of chromosomal DNA proceeds during the S phase of the cell cycle. The S phase lasts 6 to 10 hr in various somatic tissue cells of warm-blooded animals, with the exception of early embryo cells. DNA synthesis, as evidenced by labeled thymidine incorporation, starts simultaneously at numerous sites along the chromosomes, and different regions of chromosomes are replicated at specific times in the S phase (Taylor, 1960). The most clear-cut results on the pattern and timing of chromosome replication have been obtained with the thymidine analog 5bromodeoxyuridine (BUdR). The regions of chromosomes which incorporated BUdR at a defined period of the S phase could be detected at the following mitotic phase either by the fluorochromes Hoechst 33258 or acridine orange (Latt, 1973; Dutrillaux et al., 1973; Stubblefield, 1973, by fluorochrome-photolysis-Giemsa staining (Perry and Wolff, 19741, or by antibody to BUdR (Vogel et al., 1986). The resulting pattern was called “dynamic replication banding” (Drouin and Richer, 1989). A correlation between replication banding and structural R- or G-bandings has been established for metaphase chromosomes (Grzeschik et al., 1975; Zakharov and Egolina, 1976; Lau and Arrighi, 1980) as well as for high-resolution banded (at the 1000-1200 bands level), human prometaphase chromosomes (Meer et al., 1981; Camargo and Cervenka, 1982; Drouin and Richer, 1989; Drouin et al., 1990; Lemieux er al., 1990). It has been found that DNA in all R-bands replicates within the first half of the S phase, in G-bands within the second half of the S phase, and in C-bands within the last third of the S phase. DNA with the same name bands starts replication more or less simultaneously. It was shown that in human lymphocyte chromosomes, the pattern of replicative R-bands was complete even after application of a 15-min BUdR pulse at the beginning of the S phase (Schmidt, 1980). The term “chromosomal replicon” was proposed for an individual band, which replicates as a whole at the chromosomal level (Lau and Arrighi, 1981; Lug0 et al., 1989). The relation between the time of replication and transcription of mammalian chromosomal bands has also been studied. The first attempts to link the structural R- and G-bands with transcriptional functions were reported by Yunis et al. (1977) and Yunis and Tsai (1978). The authors performed in situ hybridization of the G-banded human chromosomes with the labeled probes of cytoplasmic polyadenylated mRNA, polysomal mRNA, and heterogeneous nuclear RNA. It has been found that most of the hybridizing grains were localized in Giemsa lightly stained R-bands.

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The conclusion has been drawn that the bulk of the transcribing genes and middle repetitive transcribing sequences are replicated early in the S phase. In good agreement with this were data on replication of DNA from a number of transcribed genes early in the S phase (Suzuki and Okada, 1975; Balazs and Schildkraut, 1976; Nakazawa and Klevecz, 1976; Chang and Baserga, 1977). Weintraub and Groudine (1976) have shown that DNA of transcribed genes in chicken erythrocyte nuclei was preferentially digested with DNase I. It was also shown that DNA of ribosomal genes has the highest sensitivity to DNase I among other DNAs (Weintraub, 1975). The mammalian DNA replicating at different time intervals during the S phase was tested by DNase I digestion. It was found that DNA replicating early in the S phase was digested effectively with DNase I. Digestion of the newly synthesized DNA with DNase I decreased sharply during the S phase and DNA replicating late in the S phase was resistant to DNase I (Liapunova and Khaitova, 1979; Ono and Okada, 1981). Contrary to this, Holmquist et al. (1982) have found no differences in sensitivity to pancreatic DNase I of chromatin alternately labeled in the early or late S phase. Later Goldman et al. (1984) using blot hybridization of the early or late replicating DNA with a recombinant DNA probe of specific genes demonstrated that housekeeping genes, which are active in all cells, replicate early in the S phase, whereas most of the tissue-specific genes which were not expressed in the cells tested were replicating late in the S phase. This means that housekeeping genes are localized in R-bands, whereas most of the tissue-specific genes are localized in G-bands (Holmquist , 1987). The established relation between DNA transcription and the time of its replication during the S phase allows one to conclude that all characteristics of the early S replicons may be attributed to the actively transcribed DNA, whereas those of late S replicons may be attributed to DNA of nontranscribed genes or to DNA that is devoid of true genes.

111. DNA Fiber Radioautography as a Method for Replicon Analysis A. Basic Principles

The method of DNA fiber radioautography was proposed by Cairns (1963) and first used to study replication of bacterial circular DNA molecules. Later it was used in investigations of DNA replication in mammalian cells (Cairns, 1966; Huberman and Riggs, 1966, 1968). According to the

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modified Cairns method, after incubation with tritium-labeled thymidine (trTh), the cells are lysed on the surface of microscope slides coated with gelatin or albumin (Lark et al., 1971; Huberman and Tsai. 1973). The large chromosomal DNA molecules are spread linearly along a slide. After being washed and dried, the preparations are coated with radiationsensitive liquid emulsion, exposed in darkness for 6-9 months, and then developed. After a short (15-60 min), simple, pulsed label, short labeled fragments are seen in radioautographs. Sometimes they are arranged in linear sequences (Fig. 1). Other labeling protocols were also used. In pulsechase experiments, an excess of nonradioactive thymidine was added to the medium before cell lysis, or the labeled medium was replaced by an unlabeled one. In double-pulse experiments, incubation of cells with trTh of high specific activity (hot pulse) was followed by incubation with trTh of low specific activity (warm pulse). In both cases there was a decreasing density of label on one or both ends of the hot-labeled fragments, indicating the direction of replication fork movement (Fig. 2). Decreasing label density on both ends of hot-labeled fragments indicates that replication proceeds bidirectionally from a single initiation site (Huberman and Riggs, 1968; Huberman and Tsai. 1973; Hand, 1975a,b; Callan, 1976). The decreased label density on only one end of the hot track was interpreted as evidence of a one-way replication process (Lark et al., 1971; Hand and Tamm, 1973; Huberman and Tsai, 1973; Hand, 1975a,b). In experiments with a double-pulse label, bidirectional replication units with a gap at the center of the hot track and without it were observed. The first units represent prepulse replicons in which initiation sites (origins) were activated before the provision of label. The second ones represent postpulse replicons, in which origins were activated simultaneously or shortly after the provision of label (Fig. 2). Usually, preincubation with 5-fluorodeoxyuridine (FUdR) was used to reduce the intracellular thymidine pool and to block cells at the beginning of the S phase (Huberman and Riggs, 1968). That is why the authors proposed that most if not all initiation sites lie at the center of simplepulse-labeled fragments. This enabled them to propose a method for measurement of the sizes of replication units as the distances between the centers of adjacent labeled fragments arranged in linear, tandem segments, the so-called “center-to-center’’ (C-C) method (Huberman and Riggs, 1968). By the middle of the 1970s, when my colleagues and I started our research in this field, a concept of replicon organization in eukaryotic chromosomal DNA had been established (Huberman and Riggs, 1968; Callan, 1973, 1974, 1976; Prescott and Kuempel, 1973; Edenberg and Huberman, 1975; Painter, 1976; Hand, 1978, 1979). According to this concept, DNA of eukaryotic chromosomes is synthesized via replicons

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FIG. 1 DNA fiber radioautogram from human fibroblasts. Simple pulse label (30 min). Short labeled fragments are sometimes arranged in tandem. Bar = 100 pm.

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FIG. 2 DNA fiber radioautogram from mouse L cells. DNA was labeled for 30 min with trTh of high specific activity and then for 30 min with trTh of low specific activity. Arrows indicate prepulse (a) and postpulse (b) units. More than one labeled DNA molecule lies under the linear radioautographic track indicated by the arrow (c). Bar = 100 pm. (Reprinted from Hand, 1975b, with permission of the author and the Histochemical Society Inc.)

arranged in clusters (tandems, series). Each cluster consists of 10-15 or more (up to 100) replicons (Stubblefield, 1974). The sizes of most of the replicons range from 15 to 100 pm, with a mean of about 30-50 pm. Replicons in the cluster appear to initiate replication synchronously (Hand, 1975a). In most cases, replication of DNA molecules within one replicon

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is presumed to be bidirectional. Termination occurs when the two growing chains meet. In warm-blooded animal and human cells, the average rate of DNA chain elongation reaches 0.6-0.8 pm/min (Kapp and Painter, 1982a).According to these results, the time needed to complete replication in a cluster of replicons is less than 1 hr. On this basis in most investigations of mammalian cells that used the DFA method, the duration of cell incubation with trTh as a rule did not exceed 1 hr.

B. Critical Remarks The advantage of the DFA method is the possibility of direct microscopic analysis of DNA fiber fragments that incorporated the radioactive label in the known regime in the course of replication. At the same time interpretation of these data is somewhat subjective since one cannot discern the DNA molecules themselves behind the radioautographic tracks. Since the silver grains in photoemulsion are 0.5- 1 p m in diameter and the diameter of DNA molecules is 2 nm, a number of side-by-side molecules may occur in linearly arranged tracks of silver grains (Fig. 3). This problem has been discussed in a number of publications (Ananiev et ul., 1977; Yurov and Liapunova, 1976, 1977; Taylor and Hozier, 1976; Taylor, 1977; Hand, 1978). Here I emphasize that if there is more than one labeled molecule in a bundle of side-by-side DNA molecules, the short, labeled fragments will fall on the same line in the radioautogram and may be interpreted as a cluster. This is diagrammed in Fig. 4. One can see that the results of measurement of C-C distances between adjacent labeled fragments in tandem depend on the fragment sizes. Namely, the shorter the labeled fragments, the higher the possibility of finding them lying separately along a straight line, and the shorter the measured C-C distances between the adjacent fragments will be (compare Figs. 4a and 4b). The results published by Wilson (1975) illustrate this assumption. She studied parameters of replicons from four species of amphibians with different DNA content per haploid genome. After 30 min of incubation with trTh, the sizes of replicons of all the species studied ranged from 10 to 25 pm. However, after 10 min of incubation, the average size of replicons decreased to 8 pm and after a longer incubation (over 45 rnin), increased to more than 45 pm. Wilson did not analyze the origin of these differences. The questions then arise: What is the size of the replicons? Why are different results obtained for the same cells under the same conditions with different labeling protocols? To test the methods for measurement of replicons sizes, Yurov (1979b) determined replicon sizes in human and Chinese hamster cells after incubation of cells with trTh (simple-pulse label) for 4, 10, 30, and 60 min. He

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FIG. 3 DNA fibers from MCF-7 human cells. Bundles of DNA molecules and the single molecules between them can be seen. The preparation was obtained as for DNA fiber radioautography (Lark rf a / . . 1971) with some modification. One drop of cell suspension was mixed with a drop of lysing solution (SDS-EDTA-Tris) on the surface of a microscope slide. Released DNA molecules were spread along the slide. After being rinsed with 45% acetic acid (30 min at room temperature), the preparation was stained with propidium ( I pg/ml PBS). The photomicrograph was made using a Leitz UV-photomicroscope, with a l O 0 X objective. Bar = 10 p m . (Courtesy of N. V. Tomilin.)

found that the average sizes of the labeled DNA fragments in human cells ranged from 3.1 ? 0.2 pm to 37.2 2 1.6 pm in proportion to the duration of the labeling pulse, and those of Chinese hamster cells ranged from 4.2 2 0.2 pm to 51.0 + 2.4 pm. The replicon sizes determined as C-C distances in linear tandems of labeled fragments ranged in these cases from 5.5 k 0.3 pm to 45.5 k 2.0 pm in human cells, and from 7.8 k 0.3 prn to 64.7 & 2.6 pm in Chinese hamster cells. In Fig. 5 the data from several papers are combined (Wilson, 1975; Yurov, 1979b; Sawada et af., 1982; Jagiello et af., 1983). One can see that a well-defined linear function exists independently of the organisms studied (cold-blooded or warm-blooded animals); the larger the average size of the labeled fragments, the larger the average size of replicons measured. It will be recalled that in the majority of studies the replicon sizes in mammalian cells were measured after 30-60 min (simple pulse) or 30 + 30 min (double pulse) of labeling. The sizes of the majority of the labeled

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FIG. 4 Scheme illustrating the appearance of clusters of the labeled fragments in DNA fiber radioautographs when different labeling protocols are used. Four DNA molecules (1-4) lie side by side, thus forming the bundle. Each molecule has one (numbers 2 and 4) or two (numbers 1 and 3) labeled fragments with the origin of replication in the center (arrows). The resulting linear radioautographic tracks are shown within frames ( 5 ) . (a) Short simple pulse label. (b) The same as (a) but the pulse is two times longer. (c) Short doublepulse label. Three (RI-R3, b) or four (R,-R.,. a, c ) replicons can be measured as center-tocenter distances in clusters. Note that the mean sizes of replicons in (b) are larger in comparison with those in (a) and (c). Solid thick lines indicate high grain density tracks, dotted lines indicate low grain density tracks. (Reprinted with some modifications from Liapunova, 1985, with permission of the author and "Naukova Dumka.")

fragments under these conditions ranged from 20 to 40 pm and the resulting replicon sizes measured as C-C on adjacent tracks were about 30-50 pm on average. If tandems of labeled fragments do occur as a result of side-by-side adhesion of several fragmentary labeled DNA molecules, it is reasonable to suggest that the probability of tandems of labeled fragments appearing in preparations will be lower the smaller the fraction of labeled DNA

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measured as center-to-center distances in linear tandems of radioautographic tracks. I , human fibroblasts; 2, Chinese hamster cells (Yurov, 1979b); 3, Bufo cognutus; 4. Tritirus uiridescens (Wilson. 1975); 5, mouse spermatocytes (Jagiello er a / . , 1983); 6. L5178Y mouse cells (Sawada et ul., 1982). Numbers of measurements for different points were abscissa, 100 (1 and 2). 400 (3 and 4). 2535 and 1576 (5); ordinate, 100 (1 and 2), 60-300 (3 and 4). 621 and 503 ( 5 ) . Data for ( 6 ) were obtained by measurements of track length and replicon sizes in published radioautograms. The number of measurements were, respectively, 13, 16, 17 (abscissa) and 9 , 8 , and 6 (ordinate). (Reprinted with some modifications from Liapunova, 1985, with permission of the author and “Naukova Dumka.”)

molecules in lysate. Published micrographs (Callan, 1973) support this assumption (Fig. 6). These radioautograms were obtained from neurula of Triturus vulgaris. A typical example of a tandem or cluster of labeled fragments is shown in Fig. 6a. Figure 6b shows a fragments arranged in pairs which represent the result of the movement of two replication forks in single replicons, and these replicons do not form clusters. The absence of clusters in Fig. 6b is the result of dilution of labeled cell suspension with an unlabeled one just before lysis. Callan used a dilution of labeled cells to visualize tracks belonging to single, labeled cells. Callan also pays attention to the bidirectional movement of replication forks in virtually all replicons but he does not discuss the disappearance of clusters. It is clear that if replicons are grouped on one or several DNA molecules in a bundle, after dilution with unlabeled molecules they will show up in radioautograms as distinct, true clusters. Figure 7 illustrates this assumption. The pictures which were observed in experiments correspond to variant “b” of Fig. 7. Thus this is an additional argument against cluster organization of small replicons. As pointed out in the preceding section, individual bands of chromo-

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FIG. 6 DNA fiber radioautograms from Trifirrirs vulgaris neurula labeled at 18°C for (a) I hr or (b) 2 hr. (a) The labeled fragments are arranged in a linear tandem (cluster). (b) Pairs of labeled fragments represent bidirectional, single replicons. One can see that all replicons but two (arrows) belong to different DNA molecules. In (b). the labeled cells were massively diluted with unlabeled ones just before cell lysis. Bar = 100 pm. (Reprinted from Callan, 1973. with permission of the author and Plenum Publishing Corp.)

somes replicate as a whole and include DNA molecules up to 1000 p m long and more. It was postulated that one band corresponds to one cluster of replicons (Stubblefield, 1974). This means that 10 and more (up t o 100) replicons can be visualized in clusters. However, as a rule, no more than 3-5 linearly arranged labeled fragments are usually observed. In pulsechase and double-pulse experiments, different authors did not observe more than 2, rarely 3-4 replicons lying in a line (Hand, 1975a, 1977; Richter and Hand, 1979a; Takeuchi et al., 1982; Zannis-Hadjopoulos er al., 1980; Hand et al., 1983; Dahle et al., 1979; Ockey, 1979). It has been suggested that in the course of preparative procedures, DNA was sheared into comparatively small pieces. However, even in the earlier reports (Huberman and Riggs, 1968), it was demonstrated that after long (12 hr and more) incubation with labeled thymidine, one can observe a large number of DNA fragments up to 500-800 p m long. In our experiments, using the method of Lark et al. (1971), we have also shown that after long labeling the sizes of the majority of the labeled DNA molecules ranged from 200 to 700 p m and the maximum size of unbroken molecules reached more than 2000 p m (Yurov and Liapunova, 1976). Misinterpretation in

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6

I -FIG.7 A scheme illustrating the effect of dilution of the labeled cells with unlabeled ones before cell lysis on the resulting fiber radioautographs. (A) Among six (1-6) side-by-side adhering DNA molecules, two (numbers 2 and 5 ) contain clusters of three replicons each. (B) Each of six DNA molecules of a bundle contains only one functioning replicon. It can be assumed that the smaller the portion of labeled DNA molecules among all DNA molecules of lysate. the lower the probability that more than one labeled molecule will occur in a bundle. Consequently, if cell suspension is not diluted. in both cases, a cluster of irregular tracks will be seen on the radioautographs (shown within frames A. 7 and B. 7). In the case of dilution of cell suspension with unlabeled cells, in (A) two separate clusters are seen (A, 8), but in ( B ) six single replicons are lying independent of each other (B, 8).

replicon analysis which might result from side-by-side adhesion of labeled DNA molecules may be minimized by using the modified DFR method. C. DFR after Long Double-Pulse Labeling

Double-pulse labeling was used in a number of DFR studies on mammalian cells. Following a common practice based on the concept of clusters of small replication units, the overall duration of the label in most of them did not exceed 1 hr (Hand, 1975a,b; Hand and German, 1975; Dahle et af., 1979;Zannis-Hadjopoulus et al., 1980;Hand et al., 1983). A representative example of DFR of double-pulse (30 + 30 min)-labeled mouse DNA is shown in Fig. 2 . This radioautogram allows at least two conclusions: ( 1 ) Practically all well-defined single replication units belong to different DNA molecules; they do not lie along one line. ( 2 ) One hour is not enough time for completing replication in the majority of replicons. The same can be seen in Plate 111 in the paper by Huberman and Riggs (1968) and Fig. 1 in the paper by Hand and German (1977).

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275

Assuming that the maximum C-C distance is 200 ,urn and the mean oneway RFR is about 40 p m per hour, it was supposed that 3 hr of labeling was enough for completing replication in almost all replicons within clusters (Yurov 1977a; Yurov and Liapunova. 1977). According to the labeling protocol used by these authors, incubation of cells for 30 or 60 min with trTh of high specific activity (hot pulse) was followed by a 150- or 120min warm pulse (Fig. 8). The selected labeling regime allowed one to expect the formation of both informative and noninformative tracks (Fig. 9). Informative variants are as follows: ( I ) continually labeled DNA molecules, in which heavily labeled regions are connected by lightly labeled ones; these pictures should correspond to clusters of replicons with postpulse initiation (Fig. 9b); (2) labeled molecules similar to the preceding variant but having an unlabeled region at the center of the heavily labeled one; they should correspond to clusters of replicons with prepulse initiation (Fig. 9c, d). The noninformative variants should be the continually lightly labeled molecules, indicating that their replication coincided with the warm pulse (Fig. 9f), or the tandems of hot-labeled fragments formed when the replicons in cluster had finished functioning during the hot pulse ( Fig. 9e). In the case of informative variants, the authors expected to observe on the order of 5 to 10 (variant “b”) or 10 to 20 (variant “c”) hot tracks on the fragments of molecules about 200 to 400 p m long (Yurov and Liapunova, 1977).The replication patterns observed in radioautograms obtained in different experiments proved to be somewhat unexpected (Fig. 10). Three hours of double-pulse label incorporation produced informative labeled DNA molecules which corresponded to the schemes presented in Fig. 9. But there were no more than 2-3 initiation sites on DNA molecules 200-1000 pm long. The micrograph of DNA molecules 1360 p m in length is shown in Fig. 1Od. This picture is especially informative because it shows two separated daughter DNA molecules with the same replication pattern. Three replication units are clearly visible. Sequences consisting of 2 or 3 replicons which account for 10-15% of all molecules are useful analysis. Another 10-15% make up the single replication unit (Fig. l l a ; see also Figs. 2 and 15). More often, fragments useful for analysis were produced by one replication fork (Fig. I 1 b). These are either the halves of symmetric replicons that are sometimes torn apart or widely spaced, or they are actually replicons originated by one replication fork. Other examples of micrographs of DNA fiber radioautograms after long double-pulse labeling have been presented in a number of publications (Hand, 1977; Hand and German, 1977; Yurov and Liapunova, 1977; Yurov, 1977a,b, 1978, 1979a,b, 1980; Liapunova and Khaitova, 1977; Liapunova and Dulatova, 1989). The micrograph and scheme (Fig. 12)

FIG. 8 DNA fiber radioautogram from human diploid fibroblasts labeled with a 30-min hot pulse followed by a 150-min warm pulse. Bar = 100 pm.

277

MAMMALIAN DNA REPLICATION

a I

b......

+...........

I c.....-l-.........-

I I

?2

03

I

I

+.......+. I

1-

-...-

I

. . . I-.. . . . .

-

I

I

94

..........+.I ..... I I -.....

-

1 I I I I 1 1 eI I I I f . . . . . . . . t . . . . . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . .I. . . . . . . . . . . . . .

d..-

-

I

I

-..

I...

-

I

I

-

l

I

FIG. 9 Scheme illustrating the expected tracks in DNA fiber radioautographs after long double-pulse labeling assuming the simultaneous initiation of replicons in a cluster. DNA molecule (a) has four origins of replication (0,-04). Three replicons (R,, R2. R,) can be measured as center-to-center distances. (b, c , d) are the informative variants, (e) and (f) the noninformative ones. (b), postpulse initiation; (c, d), prepulse initiation; (e), replication is completed during the hot pulse: (f), replication took place during warm pulse.

illustrate how false clusters of labeled fragments might have arisen in the case of a simple-pulse experiment. Thus informative labeled tracks observed in experiments with long double-pulse label incorporation were not consistent with the concept of “clusters of small replicons.” At the same time, the radioautograms provided material for a sufficiently conclusive analysis of the sizes of replication units as well as of RFR and RFB in individual replicons.

IV. Sizes of Replication Units Figure 13 shows three types of labeled fragments which may be used for estimation of replication unit sizes in radioautographs from cells exposed to double-pulse labeling. Type A and A‘ fragments (Fig. 13 a-f) represent DNA molecules on which two or more initiation sites became effective for replication more or less simultaneously. Type A’ fragments (Fig. 13 d-f) appear when the overall time of the label incorporation is not enough for replication forks from the adjacent replicons to complete functioning. They are more commonly observed when the duration of double-pulse labeling is less than 3 hr. In this case, one cannot be sure that the replicons belong to the same DNA molecule, and false measurements may be made. Following a common practice, C-C distances on type A and A’ fragments are measured as the sizes of replicons. According to the definition of a replicon, it would be more proper to measure the distances between

FIG. 10 DNA fiber radioautograms from (c. d) human embryo hepatocytes and (a. b. c) fibroblasts. Double pulse label: 30 rnin hot pulse followed by I50 rnin warm pulse (a-d) or 60 rnin hot pulse followed by 240 rnin warm pulse (e). Bars = 100 pm. Arrows indicate the initiation points. (c and d reprinted from Yurov and Liapunova, 1977. with permission of the authors and Springer Verlag.)

MAMMALIAN DNA REPLICATION

279

FIG. 1 1 DNA fiber I'ddiOaUtOgrdmS from human fibroblasts, double-pulse label (30 + 150 min). (a) A microscopic field showing several single bidirectional replicons. The initiation points are indicated by arrows. (Reprinted from Yurov and Liapunova, 1977. with permission

of the authors and Springer Verlag.) (b) Several examples of labeled fragments produced by one replication fork.

280

NATALIA A. LIAPUNOVA

FIG. 12 Radioautogram (a) and scheme (b) illustrating the descent of “false clusters.” Human fibroblast cells were double pulse labeled (30 and 150 min). The hot-labeled fragments indicated by arrowheads lie along a straight line. In the absence of a subsequent warm pulse they would have been considered as clusters. The numbers (b) represent center-to-center distances in micrometers (the replicon sizes). In fact, these fragments belong to different molecules which can be traced by the lightly labeled tracks (a). Bar = 100 pm.

28 1

MAMMALIAN DNA REPLICATION

TYPeA

. . . . . . . . .t- . . . . . . . . . . . . . . . . . . . . . . a ......-I--............. 7 ........ b ......- +--. ..........-3 -. .... c -t 7 .... .... .... ....f *...- t- -... ....-.... ... .- r-. . . . . . . . -3 -. ...

Type A'

t................

e f

Type

................. -+g -.... .........f. . h

.............r........-................... .-f

d

TYPeC ................

7 i j

FIG. 13 Schematic representation of the labeled DNA fragments (types A. B. and C) suitable for measurement of replicon sizes after double-pulse label. See description in the text.

termination sites, but these sites cannot always be identified unambigously on the labeled tracks. Type B fragments correspond to prepulse and postpulse single replicons (see Figs. 2, I la, 15). The origin of initiation may be located in the center of the replicon (Fig. 13 g, h) as well as slightly off center (Fig. 13i). Among type B fragments, the entire replicon size can be estimated if elongation is completed during the time of cell incubation with a label. The sizes of replicons will be underestimated if one or both replication forks have not finished functioning. Type C fragments (Fig. 13j) correspond to a unidirectional track. Assuming that these are the halves of the replicon ( r ) , the entire replicon size will be (2r + x) pm, where x is the length of the DNA segment that replicated before the label was added (Yurov and Liapunova, 1977). Data on replicon sizes in mammalian cells obtained in experiments with double-pulse labels are scarce (Table I). In order to present the results in comparable form here, in a number of cases the mean values are calculated on the basis of the published histograms and micrographs of DFA. Analysis of Table I allows the following conclusions: (1) The average sizes of all three types of distances measured fall within 50-300 pm, with the limits ranging from 20 to 670 pm. There is some increase in average val-

TABLE I Sizes of Replication Units in Mammalian Cells

Duration of labeling (hours): Type of cell

Sizes of fragments (pm), mean

Total

Hotandwarm

Human, HeLa

0.5

Chinese hamster, V-79 cells

0.8

Human fibroblasts

1

+ 0.25) (0.4 + 0.4) (0.5 + 0.5)

Human fibroblasts

1

(0.5

+ 0.5)

70 2 3d

Mouse L-929 cells

I

(0.5

+ 0.5)

70

(0.25

Type A

Type B

1

(0.5

+ 0.5)

S.E."

Type

c

References

47

?

2.5

Ockey (1982)

75

?

9.0

Dahle ei

?

6'

48

?

ti/. (

1979)

Hand ( 1977)

90 ? 3 (30-250)h'

Hand and German ( 1977) 78

?

4'

(50-103)

Mouse L5178Y cells

5

31 -t I ' (29-4 I )

Hand (l975b) Sawada ('I

2

ti/.

(1982)

( 15- 100)

+ 0.5)

110 ? 12'.'

Chinese hamster. V-79 pur 1 cells

1

(0.5

Human lymphocytes

2

( 1 + 1)

Human lymphocytes

2

(1

+

1)

91 k 5 (20-250)"

Human lymphocytes

2

(1

71 2 4 d

2

(1

+ +

1)

Human fibroblasts Human hepatocytes

3

(0.5

77

1)

+ 2.5)

Zannis-Hadjopoulos f't

128 ? 7 (40-280)

5

ti/. (

1980)

Yurov (1978)

2

Hand (1977) Hand and German (1977) 187 5 21' ( I 10-370)

73 5 2 (30-140)

N . A. Liapunova and Yu. B. Yurov (unpublished)

176 2 6 (80-320)

115 5 4 (40-220)

Yurov and Liapunova (1977)

+

2)

116 2 6 (40-280)

Human hepatocytes

(1

Human fibroblasts

(0.5 + 2.5) a n d ( 1 + 2)

Human fibroblasts

(0.5

160 2 6 (80-320)

105 2 3 (40-220)

Yurov and Liapunova ( 1977)

199 2 8 (80-440)

91 2 3

Yurov (1977b)

+ 2.5)

Human fibroblasts

N

+

Human fibroblasts

(0.5

Chinese hamster. BI IFAF, clone 237

(0.5 + 2.5) a n d ( 1 + 2)

Human fibroblasts

(1

Human fibroblasts

"

(1

+ +

2.5)

107 2 3 (40-240)

Yurov (1978)

115 2 3 (40-220)

Yurov (1978) Yurov (1979b)

114 2 3 (20-250)'

4) 5)

( 30-200)

157 2 23' (50-330)

127 ? 2 (40-280)

Yurov (1977a)

144

N . A . Liapunova and and Yu. B . Yurov (unpublished)

5

(I

304 2 33L 10-670)

( 6O-3OO)

321 2 15 (70-670)

154 2 6 (30-350)

2

Liapunova ( 1989)

The numbers of measurements were 100 and more in all but indicated cases.

* The ranges are shown in parentheses according to published histograms. ' Fourteen out of 394 units ranged from 250 to 460 p m .

Median and standard error. Tracks of type A and type A' (see Fig. 13) were measured. Twelve fragments of type B and 22 of type C were measured in the published radioautogram (see Fig. 2 in this chapter) Geometric mean and standard error. " Four out of 200 units ranged from 250 to 420 p m . ' n = 11.

Thirty-three out of 300 units ranged from 250 to 530 y n .

' n = 20. 'n

=

76.

284

NATALIA A. LIAPUNOVA

ues with an increase in total labeling time. This may be explained thus: (1) As the duration of the pulse increases, more replicons finish elongation (see Section VI) and reach their terminal sizes. As a result, the upper limits of the sizes measured increase, whereas the lower limits remain unchanged and the average values increase too. (2) The distances between initiation sites in the type A labeling pattern fall in the range of 20 to 300 pm, with a mean of about 50-150 pm. (3) In the case of the type B labeling pattern, the average sizes of replicons range from 160 to 320 pm. The replicons with a maximum length of up to 670 pm were measured after 5-6 hr of labeling. (4) The sizes of type C labeled fragments range from 30 to 350 pm, with a mean of about 100-150 pm. This closely corresponds to the halves of type B replicating units. On the basis of various methods, for HeLa cells (Cairns, 1966; Painter et al., 1966), Chinese hamster cells (Taylor, 1968), and murine lymphoma L5178Y cells (Lehmann and Ormerod, 1972; Jolley and Ormerod, 19741, it was calculated that DNA is synthesized on replication units several hundreds microns in length. The appearance of the paper by Huberman and Riggs (1968), “On the mechanism of DNA replication in mammalian chromosomes,” was perceived as a sensation and stimulated activity in the study of replication units in eukaryotic cells. The mere fact of an agreement between the results obtained and the results of Huberman and Riggs was taken as evidence of their correctness. All the earlier mentioned reports on the large replicons were explained by the fusion of small replicons in clusters (Taylor, 1968; Taylor and Miner, 1968; Painter, 1976; Edenberg and Huberman, 1975). There was a tendency to explain the large sizes of replicons from cells exposed to the long double-pulse label as a result either of conditions of cell cultivation or of some peculiarities of cell proliferation (Hand, 1978). However, these explanations are not valid. Yurov and Liapunova (1977) have performed control experiments that contradict this explanation. Along with double-pulse-label experiments, they measured the sizes of replicons as C-C distances between adjacent labeled fragments in DNA fiber radioautographs from human cells exposed to 30 or 60 min of simplepulse label. These results did not differ from those of other researchers, which are summarized in the reviews cited (Edenberg and Huberman, 1975; Hand, 1978, 1979). The sizes of replication units were demonstrated to remain the same throughout the S phase. In human fibroblasts, after 3 hr of double-pulse incubation of cells with trTh in the early, middle, and late S phase, the sizes of type C fragments were in the range of 30 to 150 pm, with a mean of about 82, 104, and 99 pm, respectively (Khaitova, 1980). Replication unit sizes remain the same in normal human cells of different origin: embryo and skin fibroblasts, brain and heart cells, hepatocytes,

MAMMALIAN DNA REPLICATION

285

and lymphocytes (Yurov, 1977b; Yurov and Liapunova, 1977); as well as in the cells obtained from patients or fetuses with trisomy 21 (Down syndrome), trisomy 7, karyotypes 45, X (Turner syndrome), 47, XXY (Kleinefelter syndrome), 47, XXX, 49, XXXXX; and in the cells from patients with xeroderma pigmentosum (De Sanctis-Cacchione syndrome) (Yurov, 1978). Reviewing this section, a general conclusion can be made that in mammalian somatic cells, DNA replication occurs via replicons whose sizes vary widely. The sizes of the majority of replicons are within the limits of 50 to 300 pm. Maximum replicon sizes reach 600 pm and more. The existence of small replicons (less than 20-40 pm) cannot be ruled out (for example, see Fig. 4 in Yurov, 1979b), but they are rarely observed. It should be noted here that the probability for the appearance of small replicons increases when FUdR is used to synchronize cell growth and to decrease the endogenous thymidine pool. It was shown that with increasing duration of FUdR block (lo-’ M ) , there is a linear increase in incorporation of trTh added to the culture medium 4 min before cell fixation (Kurek and Taylor, 1977). To interpret these observations, the authors proposed the existence of potential sites for replicon initiation. When replication is blocked by FUdR, these potential sites become activated by binding the protein initiation complexes, but initiation does not occur until thymidine is supplied (Taylor, 1978). When the FUdR block is removed, most of these initiation sites become the starting points for replication fork movement. Using the DFA method, Taylor and Hozier (1976) presented evidence for the existence of groups of four micron replication units in Chinese hamster ovary (CHO) cells released from FUdR block. Later potential initiation sites located at a distance of 12 kb (4 pm) were reported for Xenopus DNA molecules (Watanabe and Taylor, 1980; Taylor and Watanabe, 1981). The existence of replication origins located at a distance of 12 kb has been demonstrated on a fragment of DNA molecule adjacent to the gene for dihydrofolate reductase (DHFR) in Chinese hamster cells (Handeli et al., 1989). In the same work, active origins were not detected on a 60-kb (20 pm) DNA fragment located within the region of the adenine phosphoribosyl transferase gene (APRT). Two proposals can be made in this connection. First, the method developed and used by the authors does not rule out that in addition to functioning origins, weak (potential) origins exist and their activation occurs either with lower probability or under special conditions, or at specific stages of development. Second, potential and active origins may be unevenly distributed along DNA molecules in different regions of a genome. Our general conclusions differ from the generally accepted concept that replicon sizes in mammalian cells lie in the range of 15-100 pm, with a

286

NATALIA A. LIAPUNOVA

mean of about 30-50 pm. In the preceding sections I tried to show that the cause of disagreement lies in the details of using DNA fiber radioautography to measure the sizes of replication units.

V. R a t e of Replication Fork Movement

The replication fork rate can be measured as the grain track length on DNA fiber radioautograms from cells pulse labeled for a definite time interval. In the case of simple pulse labeling, the length of fragments measured will be heterogeneous. Only some of them produced by one replication fork (halves of prepulse replicons) allow one to estimate the actual RFR. The other fragments will be either the postpulse units originated by two divergent replication forks, or will result from fusion of the neighboring replicating units as well as from occasional overlapping of the labeled fragments belonging to different DNA molecules. These questions have been discussed in some papers and reviews (Callan, 1973, 1976; McFarlane and Callan, 1973). These difficulties are resolved by making a large number of measurements and by determining the RFR at least in two lines of experiments that differ in the duration of the labeling (Callan, 1973, 1976). In experiments with a double-pulse label, hot-labeled fragments have been measured in which DNA replication occurred unidirectionally during the whole time of cell incubations with trTh of high specific activity. On one side these fragments are flanked by unlabeled fragments which replicated before the cells were subjected to the label and on the other side by the slightly labeled track (Huberman and Riggs, 1968;Hand, 1975b; Hand and German, 1975; Yurov and Liapunova, 1977).The measurement of hot-track lengths on radioautograms is successful when there is a welldefined border between the high and low grain density tracks. Reproducible results were obtained by this method for human and animal cells in cultures (Hand, 1975b, 1977; Hand and German, 1975, 1977; Yurov and Liapunova, 1977; Liapunova and Khaitova, 1977; Yurov, 1977a,b, 1978, 1979a,b, 1980; Dahle et al., 1979). Apart from DFR, a number of hydrodynamic methods [CsCl equilibrium density gradient, alkaline sucrose gradient, BUdR - 313 nm photolysis] have been used to estimate RFR in DNA of mammalian cells (Painter and Schaefer, 1969; Povirk and Painter, 1976; Kapp and Painter, 1978, 1979, 1981; Kapp et al., 1979a; Richter and Hand, 1979b). Some aspects of RFR in DNA from mammalian cells have been reviewed in detail (Kapp and Painter, 1982a). This allows us to focus on a limited number of problems. Various techniques used to measure the RFR in mammalian cells gave

MAMMALIAN DNA REPLICATION

287

comparable results. RFR was studied in human cells of different origin: normal diploid cells (skin and embryo fibroblasts, lymphocytes, fetal brain, heart cells, hepatocytes), in cells from donors with genetic diseases (xeroderma pigmentosum, ataxia-telangiectasia, Down syndrome, Fanconi anemia) and transformed cells (HeLa, basal cell nevus, basal cell carcinoma, etc.) (for references, see Kapp and Painter, 1982a, Table I). The mean RFR ranged mainly from 0.5 to 0.8 pm/min. The average RFR for human cell lines is about 0.6 pmlmin (Kapp and Painter, 1982a). Disagreements arose only when RFRs in Bloom syndrome (BS) cells were studied. Some authors reported slower than normal RFR in the BS cells (Hand and German, 1975; Kapp, 1982)whereas Ockey (1979) did not find differences. Studying the question in detail, Ockey (1979) demonstrated that the decreased RFR in BS cells is a result of the altered capacity of BS cells to adapt to in vitro conditions. Rodent cells have higher RFRs than those of humans. The average values are as follows: mouse cells, about 0.7 p d m i n ; Chinese hamster cells, about 0.8 pmlmin; kangaroo rat (Dipodornis ordii), 1.0 pm/min (Kapp and Painter, 1982a); Microtirs ugrestis, 0.87 p d r n i n ; and rat-like hamster (Tsherskia triton),0.83 pmlmin (Liapunova and Khaitova, 1977). Higher RFRs were reported for shrew cells (Sorex ciraneus), 0.87 and I . 13 pm/min (Ockey and Saffhill, 1976: Ockey, 19781, whereas in monkey CV-1 cells, the RFR is relatively low, 0.4-0.6 pm/min (Richter and Hand, I979a). Simultaneously with the relative constancy of the mean RFR for the given animal and human cell types, there is considerable RFR variability among individual replicons within the same cell line. This heterogeneity is significant even when the RFR is studied in double-pulse-labeled DNA (Figs. 14, 15) (see also Housman and Huberman, 1975, Fig. 3a). In human cells the RFRs range from 0.2 to 1.2 pm/min (Yurov and Liapunova, 1977; Yurov, 1979a, 1980), and in Chinese hamster cells from 0.3 to 1.5 pm/min (Yurov, 1977a; Yurov and Liapunova, 1977). These more than 5-fold differences overlap the error of measurement of the lengths of labeled fragments, which does not exceed 15%. A number of examples of prepulse single replicons (type B labeled fragments, Fig. 13) obtained from one micropreparation are shown in Fig. 15 a-e. Three observations have attracted attention: (1) the considerable variability in the rate of replication fork movement among different replicons; (2) the similarity of RFR between the halves of a replicon; (3) the constancy of the RFR within the given replicon during the time of replicon functioning. The third observation is evident from the fact that the lightly labeled regions which were formed during 2 hr of labeling in all the cases are approximately two times longer than the heavily labeled regions formed during I hr of labeling (Fig. 15 a-e). Figure 15f shows a replicon in which,

MAMMALIAN DNA REPLICATION

289

according to the labeling regime (30 + 150 rnin), the length of the heavily and lightly labeled regions in both halves is about 20 and 100 pm. It means that during the first 30 min as well as subsequent 150 min the DNA chain replicated at the rate of about 40 pm/hr. In the replicon shown in Fig. 15g the DNA chain replicated at a rate about 20 pm/hr. Additional experimental data confirming this observation can be found in the paper by Yurov (1979a). What is the cause of different RFRs in different replicons? This question still remains to be solved. A difference in RFR in DNA molecules which replicated at the early, middle, or late S phase is one of the assumptions. Attempts to study the RFR in successive time intervals of the synchronous S phase have been reported in a number of papers (Kapp and Painter, 1982a). The conclusions are conflicting. In some works a 2-3-fold increase in the average RFR at the late S phase was observed compared with that reported for the early S phase (Painter and Schaefer, 1971: Housman and Huberrnan, 1975; Ockey, 1982). There were also data on the absence of any differences (Huberman and Riggs, 1968; Ockey, 1978; Kapp and Painter, 1979; Richter and Hand, 1979b; Probst et al., 1980) and on the lower rate in the mid S phase (Kapp and Painter, 1979). The differences reported depend neither on the objects (CHO, HeLa, human diploid fibroblasts, shrew, monkey CVl, mouse ascites cells), nor the experimental techniques used for RFR measurement (DFR, equilibrium density gradient, BUdR-3 13 nm photolysis), nor the synchronization methods (mitotic selection, FUdR for 12 hr, double thymidine block, S phase induction by serum). Our studies in the past decade have contributed to resolving the question. We studied the rate of replication fork movement in DNA replicating at different intervals of synchronous S phase in diploid cultures of postnatal and embryonic human fibroblasts (Liapunova and Khaitova, 1980, 1982; Khaitova et al., 1980). To avoid artifacts which might have resulted from the action of synchronizing agents, we used the property of diploid cells to parasynchronously pass the first S phase after reseeding of stationary culture into fresh medium. The cells were grown in a dense monolayer for 10-12 days without changing the medium and then replated into bottles with fresh medium. The halves of the coverslips (9 x 18 mm) were placed at the bottom of the culture bottles. Twelve to 14 hr after reseeding, when the DNA synthesis began, the cells were incubated at hourly intervals

FIG. 14 DNA fiber radioautograms from human fibroblasts. Double-pulse label (30 and

I50 min). Three microscopic fields (a-c) from one slide are shown. The mean rate of replication fork movement is about 24 prn/hr (a), 40-60 prn/hr (b), or 70-80 pm/hr (c). Bar = 100 wm.

290

NATALIA A. LIAPUNOVA

FIG. 15 DNA fiber radioautograms from human diploid fibroblasts. Double-pulse label: 60 min and 120 min (a-e) or 30 and 150 min (f, g). Arrows indicate the initiation points. (a-f are reprinted from Yurov and Liapunova. 1977, with permission of authors and Springer Verlag.)

with trTh for 15 rnin. The cells on coverslips were fixed and washed with cold 5% trichloroacetic acid and their radioactivity was measured in a scintillation radiometer. The same cells were exposed later to radioautography. The percentage of labeled cells, mitotic indices, and the fraction of cells among all the labeled cells that incorporated label at the early or

291

MAMMALIAN DNA REPLICATION

late S phase were analyzed. At different times during the S phase, the cells were incubated in a pair of bottles with trTh of high and low specific activity. DNA fiber radioautograms were prepared and RFRs were measured (Fig. 16). The data obtained show that the average RFR increases throughout the S phase. This increase is determined by a progressive decrease in the fraction of replicons with comparatively low fork rates and by an increase in the fraction of replicons with high fork rates toward the end of the S phase (Khaitova et d., 1980; Liapunova and Khaitova, 1982). Parallel analysis of DNA fiber radioautograms and the pattern of label in interphase nuclei reveals a positive correlation between the increase in the fraction of replicons with high fork rates and the fraction of cells

t 0

d

10 20 30 40 50 0 20 40 60 80 100 Length (pm)

FIG. 16 Frequency distribution of the sizes of the labeled DNA fragments from human skin fibroblasts after incorporation of hot label for 30 (a, c. e) or 60 (b, d, f ) min in double-pulse experiment. The cells were labeled beginning from 18 hr (a, b), 20 hr (e. d). or 22 hr (e. f ) after reseeding. The left-shaded parts of the histograms include the fragments growing with RFR of 30 gm/hr and less. The right-shaded parts are 50 pmihr and more. At 18, 20. and 22 hr of subcultivation, the fraction of the former decreases and reaches 43, 19. and 7% (means for two variants). whereas the fraction of the latter increases and reaches 4, 15, and 4396, respectively. The average overall RFR increases and reaches approximately 30 (a, b). 40 (c. d ) , and 50 (e, f ) wm/hr. (Reprinted from Khaitova et a/., 1980. with permission of the authors and Publishing House Nauka.)

292

NATALIA A. LIAPUNOVA

in which replication proceeds mainly in the condensed heterochromatin. The authors suggested that the replicons produced by the forks with high rates (50 pm/hr and more) belong mainly to genetically inert DNA of heterochromatin replicating at the late S phase (Khaitova ef al., 1980). Yurov (1980) studied the RFR in single human cells and found significant (up to 3-5-fold) differences in the fork movement rates. This is additional evidence that the RFR heterogeneity might result from variation in RFRs among replicons belonging to different chromatin fractions. It may be proposed that very slow RFRs (0.2-0.3 pm/min) in human cells may characterize, for example, DNA replication in clusters of ribosomal genes which possess very high transcriptional activity and replicate very early in the S phase (Balazs and Schildkraut, 1976). The highest RFR (0.8- 1.2 pm/min in human cells) may characterize DNA replication in genetically inert constitutive heterochromatin. RFRs in DNA of R and G chromosomal bands may range from 0.3 to 0.8 pm/min, according to the amount and activity of housekeeping or tissue-specific genes involved (Holmquist, 1987). Further investigation and new experimental approaches should verify this speculation. VI. Termination of Replicons

When a number of replicons initiate simultaneously on the same DNA molecule, termination occurs when two forks from adjacent replicons meet. This mechanism of termination is beyond question. Within the scope of the hypothesis for the clusters of small replicons, it is the main if not the sole mechanism for termination. Discussion has been under way for many years about the existence of special sites for replicon termination on chromosomal DNA molecules. In this chapter we use the term “replication fork barrier” proposed by Brewer and Fangman (1988) for yeast chromosomes. A theoretical ground for doubting the existence of fixed RFBs was put forward by Blumental et al. (1973): if RFBs existed, chromosomal rearrangements such as inversions, translocations, or deletions might create DNA segments without origins between two adjacent RFBs. Such segments would not be replicated and would prevent daughter chromosomes from separating in mitosis. The significance of these doubts is reduced, however, when it is considered that the interval between potential initiation sites on DNA molecules is as small as 12 kb (4 pm) (Taylor and Hozier, 1976; Taylor, 1977, 1984; Watanabe and Taylor, 1980; Taylor and Watanabe, 1981). Since DNA of R-, G-, and C-bands along the chromosome replicates at different time intervals in the S phase, the suggestion can be made that RFBs should exist at least at the boundaries of the bands. Nevertheless no evidence for the existence of RFBs in amphibian, mammalian, and

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avian DNA has been found in DFR experiments with short time labeling (Callan, 1973; McFarlane and Callan, 1973; Hand, 1975a). However, evidence for the existence of termination sites was obtained upon analysis of the labeled tracks after 3 hr of double-pulse labeling (Yurov and Liapunova, 1977). Keeping in mind that RFR is more or less constant during the time of replicon functioning, the authors supposed that the fragment would not be terminated if its length ( r ) corresponded both to an RFR ( u ) , which can be measured as the length of the hot track, and the duration of trTh incorporation ( t ) , that is, r = ut. When Y < ut, the fragment may be considered terminated. It was found that in human hepatocytes about 60% of the labeled fragments produced by one replication fork (halves of type B fragments, see Fig. 13 h, i) were terminated. Later, Liapunova (1989) analyzed more than 1500 fragments (type C and halves of type B, Fig. 13) in different experiments with human cells where the duration of the double-pulse label varied from 2 to 6 hr. The results can be summarized as follows: ( I ) The fraction of terminated fragments increased with an increase in the labeling time: about 25% of fragments analyzed were terminated after 2 hr of labeling, about 40% after 3 hr, about 60% after 5 hr, and up to 80% after 6 hr of labeling. ( 2 ) The fraction of terminated fragments increased with an increase in RFR under the same labeling time (Fig. 17). These data indicate the existence of RFBs on DNA molecules from human chromosomes. Circumstantial evidence of the existence of RFBs can be ruled out by a mechanism of sister chromatid exchange (SCE) formation. Painter (1980) proposed a replication model of SCE. According to this model, the sites of SCE formation are associated with junctions between completely replicated replicon clusters (or replicons) and replicon clusters (or replicons) which have not yet completed replication. The delayed fusion of replicons caused by some agents may enhance the probability of SCE. Ikushima (1990) reported very efficient induction of SCE by luminol, a strong inhibitor of poly(ADP4bose) synthetase, the enzyme involved in DNA repair and recombination. He observed a bimodal pattern of SCE induction during synchronous S phase in Chinese hamster V79 cells. He suggested that two peaks of luminol-induced SCEs might correspond to early/mid and mid/late chromosomal replicon fusions and might have resulted from misligation of newly synthesized daughter DNA strands with the parental strands in the adjacent replicons. Using the premature chromosome condensation-sister chromatid differentiation (PCC-SCD) technique and induction of SCE with cyclophosphamide, a group of authors (Lug0 et a / . , 1989) demonstrated that SCEs in CHO cells occurred at a boundary between chromosomal replicons. Although the molecular nature of the RFB remains unknown, all the data mentioned here assume the existence of RFBs on DNA molecules of mammalian chromosomes.

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~

+.-a

a

m

-b A ...........J c

o--+

10

20

d

30 40 50 60 70 Replication fork rate (prn/h)

FIG. 17 Termination of the type C labeled fragments (see Fig. 13) with different replication fork rates. Human embryo fibroblasts were double pulse-labeled for 6 hr ( 1 hr hot pulse followed by 5 hr warm pulse) beginning at (a) 15 and 18 hr after subcultivation. (b) 21 and 24 hr, (c) 27 and 30 hr. and (d) 33 and 36 hr. The replication fork was considered to be terminated when B < 6A (see scheme at the bottom of right corner) and not terminated when B = 6A. In each variant (a-d) 200 fragments were analyzed.

New methods for investigation of the functional sites for eukaryotic chromosome replication have been developed. As an example, we mention two-dimensional agarose gel electrophoresis, which allows one to analyze the restriction fragments containing replication forks (Brewer and Fangman, 1988) and scanning of sequenced genomic regions to detect the functional origins and terminals (Handeli ef al., 1989). We hope that the invention of such methods will provide us in the near future with a knowledge of replication fork barriers on DNA from mammalian chromosomes.

VII. Replicon Model for DNA Replication in Mammalian Chromosomes

A. Experimental Prerequisites The total properties of replication units and the order of chromosome replication briefly reviewed in this chapter, permit us to propose a model for DNA replication in mammalian chromosomes. The model covers the

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events during the S phase in mammalian late embryo and adult somatic tissues. We do not discuss replication of genomic DNA in early embryogenesis because in this case a radically different organization of the process of DNA doubling cannot be ruled out (Benbow, 1985; Benbow et a / . , 1985). The following data on mammalian genome replication are used as the basis for the model: 1. Replication of chromosomal DNA occurs via a multitude of bidirectional replication units (replicons). Replicon sizes vary over a wide range, from 20 to 600 pm, with the size of the majority of replicons about 50300 p m . Replication of DNA molecules corresponding to elementary chromosomal bands (chromosomal replicons) is accomplished by single replication units or simultaneously functioning groups of two to four replicons. 2. The rate of replication fork movement in different replicons varies in the range of 12 pm/hr to 80 pm/hr. The mean RFR is about 40 pmlhr in human and monkey cells and about 50 pm/hr in some rodent and other mammalian species studied. As the cell progresses through the S phase, the average RFR increases. This increase is accounted for by a decrease in the fraction of replicons produced by the forks with low rates and an increase in the fraction of replicons produced by the forks with high rates. Within the given replicon, the RFR does not change significantly over time. 3. Within the given replicon, the time of DNA elongation varies from 30 min (for example, if the replicon size is 50 p m and one-way RFR is 50 pm/hr) up to 6-8 hr (if the replicon size is 500 p m and RFR is 3040 pm/hr). The majority of the middle-sized replicons should be under replication for 3 to 4 hr. 4. Along a chromosome there are sites (barriers) of termination for replication fork movement. They are located at the boundaries of elementary chromosomal bands (chromosomal replicons). Within a group of simultaneously functioning replicons, termination takes place when two forks from adjacent replicons meet. 5 . The fraction of DNA in R-bands which contains constantly active (housekeeping) genes replicates at the beginning of the S phase. DNA in G-bands which contains repressed tissue-specific genes replicates later. Genetically inert DNA of heterochromatin (C-bands) replicates late in the S phase. 6. Finally, the model is based on a statement that initiation acts are grouped in discrete time intervals during the S phase. In many investigations performed on synchronous mammalian cell cultures, the existence of three peaks of DNA synthesis intensity has been demonstrated by measuring radioactivity after a temporal pulse of labeled thymidine incorporation (Klevecz and Kapp, 1973; Klevecz etal., 1975; Kapp and Painter,

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1977, 1982b; Kapp et al., 1979b; Dulatova and Liapunova, 1985; Liapunova et al., 1989). Sometimes, on the left flank of the curve, a small fourth peak appears (Kapp et al., 1979b, Fig. 2 a,d; Kapp and Painter 1982b, Fig. 1; Klevecz et al., 1975, Fig. 4b; Liapunova et al., 1989, Fig. 3). It is possible that the peak reflects the very early replication of a small portion of DNA containing more intensively transcribing housekeeping genes. Bloch et al. (1981) studied the rate of DNA synthesis in Ehrlich ascites tumor cells by flow cytometric analysis of DNA amount in cells and by analysis of the radioactivity of pulse-labeled, sorted cells. Three main peaks were shown during the S phase in which approximately 15,60, and 35% of the total amount of DNA was replicated. Synchronous initiations of clusters of replicons followed by periods of low initiation frequency have been proposed by Klevecz and Kapp (1973) to explain peaks in trTh incorporation. Kapp and Painter (1982b) using X-ray irradiation of diploid human cells at different time intervals during synchronous S phase obtained experimental evidence for this proposal.

To study the distribution of initiation acts during the S phase, Liapunova et al. (1989) used the antibiotic bleomycin. It was known that bleomycin inhibits DNA synthesis in mammalian cells exhibiting radiomimetic properties (Cramer and Painter, 1981; Cohen and Simpson, 1982; Jaspers et al., 1982). Studies by alkali density-gradient centrifugation showed that bleomycin, like X-rays, inhibits replicon initiation (Noda, 1988). It was demonstrated by DFR that bleomycin does not affect elongation of functioning replicons (Lavrushina, 1989). Four discrete time intervals have been revealed in the course of synchronous 10-hr S phases in human embryo lung cells in which bleomycin dramatically inhibited incorporation of trTh (Liapunova el al., 1989). This finding supports the conclusion about grouped initiation of replicon portions during the S phase in mammalian cells.

B. Description Figure 18 represents DNA replication at the level of DNA molecules and Fig. 19 that at the level of mitotic chromosomes. Replication of mammalian genomes can be described as follows. At an appropriate time of the cell cycle when the products of specific genes induce cell transition from G I into the S phase (Huberman, 1991), replicons of R-bands initiate. In the cells synchronized with methotrexate this process takes no more than 15 min (Schmidt, 1980). During the succeeding 3-4 hr, elongation and gradual termination of the first fraction of replicons (S,) takes place. By 3-4 hr of the S phase (the border between S Iand S2subphases), replicons of G-bands initiate. It is possible that this process is accompanied by

297

MAMMALIAN DNA REPLICATION G-band

RFB

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RFB

I

GI1

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;

B

RFB 11 .p12

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5 k-

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.....

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__I

....

-

.....-I

.....!-. .... ..__I

.......I

FIG. 18 A model of replication of a large DNA molecule in a mammalian chromosome. (A) A DNA molecule forming three bands (G, R, C) is shown in successive subphases (GI, S,-S3, Gz) during interphase. (B) and (C) More detailed schemes of replicons on a DNA molecule from one band. There are four steps when one replicon functions (B,1-4) or five

steps when two replicons function (C, 1-5). Solid thin lines indicate unlabeled DNA molecules before (single line) or after (double line) replication; solid thick lines indicate labeled fragments of a DNA molecule of high grain density, dotted lines indicate that of low grain density. RFB, replication fork barrier. Arrows indicate the initiation points (origins).

transition of the replication complex from replicons of R-bands which have completed replication into the origins of replicons of G-bands (Holmquist et al., 1982; Holmquist, 1987). By 6-7 hr of the S phase, the DNA of the C-band initiates (Fig. 19). The scheme (Fig. 18) shows all the typical patterns of radioautographic tracks that originate from double-pulse-labeled DNA molecules. Moreover, one can easily trace the origin of type A, B, and C fragments (Fig. 13), and can also understand the high frequency of occurrence of the type C fragments (Fig. 18b, lines 3 and 4; Fig. 18c, lines 3, 4, and 5). It will

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S-phase (hours) 0

1

2

3

4

5

6

7

8

9

-

SAT NOR -c CENC’

I I

G d

\

,R

f

I

1

I I

I 1 1

I

1

FIG. 19 The model of mammalian chromosome replication. Horizontal lines indicate the number of replicons by which the chromosomal bands (R.G , C) are replicated and the time of replicon operation. SAT, satellite; NOR, nucleolar organizer region: CEN, centromere; S , . Sz. S3. subphases of S phase.

be recalled that on average an individual chromosomal band contains a DNA molecule 300-450 p m in length. Variability in the sizes of bands (Yunis, 1981) suggests that the length of DNA molecule in the smallest bands should not exceed 100 p m and that in large bands it will be more than 1000 pm. Replication in small bands is likely to occur via single replicons. In the bands of more than medium size, two and more replicons may function. It is apparent that unidirectional fragments which are either the halves of single bidirectional replicons (Fig. 18b, lines 3 and 4) or the flanking fragments of replicon groups (Fig. 18c, lines 4 and 5 ) may be located at a distance of several hundred micrometers and will not be identified along with the other labeled fragments as belonging to the same DNA molecule. The scheme (Fig. 19) shows that subphase S, is shorter than S , , and S, shorter than S,. This assumption is based on the fact that the average sizes of replication units do not change significantly during early, middle,

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and late S phase (Khaitova, 1980) whereas the average RFR values increase in replicons functioning during s,,S2, and s,. The model allows us to understand the appearance of three peaks of intensity of DNA synthesis during the S phase. At each time interval during the S phase, the rate of labeled thymidine incorporation into cells is determined by the number of functioning replicons and the rates of replication fork movement in replicons. According to the scheme (Fig. 19), the bursts in trTh incorporation will occur at the time intervals between 0 and 1, 4 and 5 , and 7 and 8 hrs. It was shown that in human diploid cells, R- and G-bands accounted for approximately 48 and 41% of the overall chromosomal length (Yunis et al., 1977; Yunis and Tsai, 1978). This suggests that the DNA content of R- and G-bands in these cells is approximately equal and the number of replicons should be also equal. Since the average RFR in S, is lower than that in S,, the first maximum should be lower than the second one. The third maximum represents replication of approximately 10% of the human genomic DNA, and, although the average RFR in S, is high, it may be expressed more weakly than the S, maximum. These predictions emerging from the model are in agreement with the reported data. Although in experiments it is difficult to reproduce the patterns of the curves, the following general tendency is observed in studies of diploid human cells: the middle peak is the strongest, whereas the third peak is the weakest and appears sometimes as a shoulder on the right flank of the curve (Kapp and Painter, 1982b; Dulatova and Liapunova, 1985; Liapunova et al., 1989). A decrease in S phase duration is accompanied by an increase in transmission of component distributions and a decrease in the probability of the appearance of separate peaks in the resultant curve of DNA synthesis intensity. Indeed, the intensity of DNA synthesis in cell lines where the length of the S phase does not exceed 7 hr is described by a curve with the central positive excess. It is characteristic for transformed human cells and for a number of rodent cells (Kapp et al., 1979b; Dulatova and Liapunova, 1985). The model allows for partial overlapping of replicons functioning in S , , S,, and S, (Fig. 19). This may be manifested in heterogeneity of RFR in replicons from single cells (Yurov, 1980). C. Some Consequences

The model advanced here allows us to make some proposals on the organization of DNA replication in mammalian chromosomes. The major thrust of this chapter is directed toward establishing the proposition that in mammalian cells replication of chromosomal DNA occurs via replication

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units whose sizes fall mainly in the range of 50-300pm. It is obvious that the overall number of replication units required for doubling of all chromosomal DNA molecules depends on their size. Taking an average replicon size as 200 pm, 9000 replicons will be required to accomplish replication of a diploid set of chromosomes containing about 180 cm of linear DNA molecules. According to a rough estimate, about 3000 replicons should function simultaneously in each of three subphases (Sl, S,, and S,) during the S phase of the cell cycle. According to an alternative concept based on cluster organization of small (on average 30 pm) replicons, DNA synthesis in diploid cells is accomplished by no less than 60,000 replicons. This means that in each subphase of the S phase, on the order of 20,000 origins for replication should be activated. Attempts to evaluate the number of functioning replication units in mammalian cells were undertaken in only a few studies. Laughlin and Taylor (1979) reported that in Chinese hamster ovary cells, about 2000 origins functioned at the beginning of the S phase. This number of operative origins per cell increased to about 6000 within 15 hr if cells were blocked with FUdR. The results of this investigation support the model proposed here. At the same time, we need new experimental approaches which will enable us to determine either the number of active origins, or the number of molecules of protein-initiator of origins, or the number of replicative protein complexes functioning simultaneously during the S phase in the cell. Another consequence emerges from the established data on different rates of replication fork movement in replicons at different time intervals during the S phase. The model suggests that in actively transcribed genes, DNA synthesis proceeds in early S phase and, consequently, is accomplished by replication forks with relatively low rates. This fact may be of importance from an evolutionary point of view, since the lower the rate of polymerase reaction the less the probability of errors of replication occurring. On the other hand, the high rates of replication suggest the high probability of replicative errors. Since DNA of heterochromatin replicates in late S phase, its replication proceeds at a high rates. It is likely to be one of the reasons for the high variability in nucleotide composition of DNA in heterochromatin and its heterogeneity, even in the related species. The model imposes definite requirements on the levels of regulation of DNA replication during the cell cycle. These include transition from GI into S phase and activation of origins in three groups of replicons functioning in s,,s,, and S3at definite time intervals. It is apparent that chromatin conformation within R-, G- and C-chromosomal bands should play an important role at this level of regulation. The model does not require special mechanisms for replicon regulation at the level of individual clus-

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ters. Such a level is inevitably implied in discussion of eukaryotic DNA replication in clusters of small replicons. Since DNA synthesis in such clusters is completed within 1 hr of 6-10 hr of the S phase, continuous DNA replication in mammalian cells may take place only if replication occurs successively in 6-10 cluster groups. At present there are rapid advances in studies of the mechanisms of regulation of genomic DNA replication in mammalian cells (Huberman, 1991; Hamlin, 1991). Advances in the isolation and study of functions of proteins-the products of oncogenes and antioncogenes-have had a stimulating influence on these investigations. Knowledge of the organization of chromosomal DNA replication at the level of replicons may be of use in determining lines of investigations and will keep scientists from erroneous searches.

VIII. Conclusion It is impossible to discuss and analyze critically all the variety of data on replicons from mammalian cells within the framework of one chapter. Therefore, the readers will not find the answers to many questions connected with the concept of large replicons and organization of mammalian DNA for replication which I tried to substantiate here. The main purpose of this chapter is to encourage a critical approach to analysis of replication units functioning during DNA replication in warm-blooded animals. More than 10 years ago Painter (1980) noted that “Yurov (1979b) has recently questioned the existence of [replicon] clusters; however, his limited data are subject to other interpretation.” Nevertheless, up to now, nobody has suggested another interpretation. I hope that this chapter will stimulate new studies on DNA replication in mammalian chromosomes using the improved DFR method. Together with other new methods (see, for example, Brewer and Fangman, 1988; Handeli et af., 1989; Burhans et af., 1990; Dijkwel et al., 1991) this will provide an insight into one of the main problems of mammalian somatic cell genetics-the organization of genomic DNA for replication.

Acknowledgments I would like to thank Dr. Nina B. Varshaver and Dr. Yuri F. Bogdanov for critical reading of the manuscript and for helpful discussion and advice. I thank also A. Mirzabekova, E. Gromova, and V. Victorov for their help in preparation of the manuscript.

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proteins: searching for the principles of eukaryotic chromosome replication. In "New Approaches in Eukaryotic DNA Replication" (A. M. de Recondo. ed.), pp. 203-233. Plenum. New York. Dijkwel, P. A.. Vaughn. J. P., and Hamlin, J. L. (1991). Mapping of replication initiation sites in mammalian genomes by two-dimensional gel analysis: stabilization and enrichment of replication intermediates by isolation on the nuclear matrix. Mol. Cell Biol. 11, 3850-3859, Drouin. R., and Richer, C.-L. (1989). High resolution R-banding at the 1250-band level. 11. Schematic representation and nomenclature of human RBG-banded chromosomes. Genome 32,425-439. Drouin, R.,Lemieux, N., and Richer, C.-L. (1990). Analysis of DNA replication during Sphase by means of dynamic chromosome banding at high resolution. Chromosoma 99, 273-280. Dulatova. Sh. N., and Liapunova, N. A. (1985). Methylnitronitrosoguanidineeffect on DNA synthesis in human and animal cells. Inhibition of DNA synthesis in synchronous and asynchronous cell cultures. Mol. Genet. Mikrohiol. Viriisol. 9,23-27. Dutrillaux. B., Laurent. C., Couturier. J.. and Lejeune, J. (1973). Coloration des chromosomes humains par I'acridine orange apres traitement par le 5-bromodeoxyuridine. C . R . Hebd. SPances Acad. Sci., Ser. D 216, 3179-3180. Edenberg, H. J., and Huberman, J. A. (1975). Eukaryotic chromosome replication. Annrc. Rev. Genet. 9,245-284. Fangman, W. L.,Hice, R. H., and Chlebowicz-Sledziewska. E. (1983). ARS replication during the yeast S . Cell (Cambridge, Mass.) 32, 831-838. Goldman, M. A., Holmquist, G. P . , Gray, M . C., Caston, L. A., and Nag, A. (1984). Replication timing of genes and middle repetitive sequences. Science 224, 686-692. Grzeschik. K-H., Kim, M. A., and Johannsmann, R. (1975). Late replicating bandsofhuman chromosomes demonstrated by fluorochrome and Giemsa staining. Humangenetik 29, 41-59. Hamlin. J . L. (1991). Regulation of DNA replication in mammalian chromosomes. In "Perspectives on Cellular Regulation from Bacteria to Cancer" ( J . Campisi et a / . , eds.), pp. 297-315. Wiley-Liss, New York. Hand, R. (1975a). Regulation of DNA replication on subchromoso- ma1 units of mammalian cells. J . Cell Bid. 64, 89-97. Hand, R. (1975b). Deoxiribonucleic acid fiber autoradiography as a technique for studying the replication of the mammalian chromosome. J . Hisfoehem. Cytoclzem. 23, 475-481. Hand, R. (1977). Human DNA replication: Fiber autoradiographic analysis of diploid cells from normal adults and from Fanconi's anemia and ataxia telangiectasia. Hum. Genet. 37, 55-64. Hand, R. (1978). Eucaryotic DNA: Organization of the genome for replication. Cell (Cumbridge, Mass.) 15, 317-325. Hand. R. (1979). Eukaryotic DNA replication and its regulation. In "Cell Biology: A Comprehensive Treatise" (D. M. Prescott and L. Goldstein. eds.). Vol. 2, pp. 389-437. Academic Press, New York. Hand, R., and German, J. (1975). A retarded rate of DNA chain growth in Bloom's syndrome. Proc. Natl. Acud. Sci. U.S.A . 12, 758-762. Hand, R.. and German, J. (1977). Bloom's syndrome: DNA replication in cultured fibroblasts and lymphocytes. Hum. Genet. 38, 297-306. Hand. R., and Tamm, J. (1973). DNA replication: Direction and rate of chain growth in mammalian cells. J . Cell Biol. 58, 410-418. Hand, R., Eilen, E., and Basilico, C. (1983). A mutation that blocks initiation of DNA synthesis in hamster cells. In "New Approaches in Eukaryotic DNA Replication" (A. M. de Recondo. ed.), pp. 265-275. Plenum, New York.

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Instability of the Homogeneous State as the Source of Localization, Epigenesis, Differentiation, and Morphogenesis Yoram Schiffmann Department of Applied Mathematics and Theoretical Physics, University of Cambridge, Cambridge CB3 9EW, United Kingdom

1. Introduction

Some biologists believe that there is no genetic program for development and often they accordingly believe that natural selection is of no great importance in development. The majority of biologists, however, do believe that there is a genetic program for development, which leaves open the question of where in the DNA this genetic program is located and how it operates. The key question is the genetic source of spatially differential, coherent, behavior, for example, gene expression. I claim here that in order to identify this genetic source of spatial nonuniformity, it is of crucial importance to invoke the principle of the instability of the homogeneous state. I suggest that this principle is realized in the genes for enzymes of the household intermediary metabolism. These genes constitute a program which is chemically a highly improbable one, and which can arise only through a combination of both natural selection and self-organization. It is transmitted from generation to generation and it is this transmittance that is the only element of preformation in development. This transmitted genetic potential for development has still to be unfolded. This unfolding occurs through the activation of catabolism, which results in an efficient energy metabolism. The conditions for this efficiency are also the conditions for the instability of the homogeneous state in a (CAMP, ATP) reaction-diffusion Turing system. The emergence of a spatially differential quantitative difference in metabolism as a source of a qualitative difference in a cell’s fate was first advanced by Child (1941, and earlier works) and was met with great resistance. The unfolding of genetic potential through the activation of glycogen or lipid metabolism is triggered by a homogeneous signal, and Inremotional Review. of C.vto1og.v. Vol. 154

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a structure emerges spontaneously for each developmental episode without a requirement for an external, spatially localized cue. This spontaneous generation of asymmetry, epigenesis, not only conforms to experimental reality, but is the only way for robust development to occur. To benefit from spontaneous self-organization via the agency of the Turing-Child metabolic system, all other biochemical processes have to be coupled to this particular metabolic field. This coupling also resides in molecular structure, and therefore the elusive biological coherence is also reduced to the linear sequence of nucleotides in the DNA. The reluctance of biologists to envisage spatially autonomous development without preexistent, external, spatially localized causes arises from the fact that a de nouo spontaneous emergence of a spatial structure seems to fly in the face of not only common sense and intuition but also of celebrated physical laws: the second law of thermodynamics, which deals with the universal drive to molecular chaos, and Curie’s symmetry principle. However, the possibility of obtaining an autonomous emergence of spatially differentiated, nonphosphorylative ATP hydrolysis, phosphorylative ATP hydrolysis (spatially differential ATP synthesis will secure spatially differential phosphorylation by all types of kinase), and a reduction potential, is precisely what is needed to correlate all molecular biological processes in space and time. These three fields will simultaneously affect proteins as diverse as channels, pumps, enzymes, and transcription factors, and alter their activity in a correlated manner. This theory of spontaneous symmetry breaking in the (CAMP,ATP) Turing system solves major biological enigmas, such as how a homogeneous egg in a homogeneous environment can begin to differentiate “by itself,” or the “paradox” with respect to Drosophilu, one major model used here, of how structure can emerge when both the receptor and ligand are homogeneous. I argue that for all four maternal systems in Drosophifu and in general for other organisms, it is the metabolic field that is responsible for the localization of cytoplasmic morphogenetic determinants. This localization is also helped by the localization of the cytoskeleton and of electric potential, which are in turn affected by the same Turing-Child metabolic field. I emphasize that, corresponding to the vision of Turing and Child, it is the spatially differential chemistry that determines all other spatially differential properties, such as tissue deformation, stiffness, viscoelasticity and electric potential, and not the other way round, as suggested in some recent theories. In this article I focus mainly on the origin of order and localization in development, but many of the problems considered are fundamental in other areas of biology. Thus the origin of electrical potential differences is central to electrobiology, and the origin of the localized assembly of the cytoskeleton permeates most of biology. The problem of the Cambrian

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explosion in biological diversity, also considered in this article, is the holy grail of evolutionary biology and paleontology (Gould, 19911, Since the solution to all these problems is based on the principle of the instability of the homogeneous state realized in the particular universal bioenergetics that natural selection has carved, it is appropriate that I introduce this principle in an intuitive way according to my results (Schiffmann, 1991). Let a = [CAMP] and h = [ATP] and also let f(a,h) and g(a,h) represent the rate of production of cAMP and ATP, respectively. I have showed (Schiffmann, 1990, 1991) that upon the application of a nonlocalized extracellular signal that removes the substrate inhibition from adenylate cyclase, the following conditions hold: {f, > 0, g , > 0,fh < 0, gh < O}, where&, = af/da, etc., the partial derivatives are evaluated at the homogeneous steady state,and Dh > D , , that is, ATP diffuses faster than CAMP. cAMP is designated the activator since if its concentration increases above its homogeneous level, the rate of production of both cAMP and ATP increases (f, > 0, g, > 0). Similarly, ATP is designated the inhibitor since if its concentration rises above its homogeneous level, the rate of production of both cAMP and ATP decreases (A, < 0, gh < 0). I proceed to show that these five inequalities are the (Turing) conditions for the instability of the homogeneous state. Consider Fig. 1 , which is an adaptation of the graphic illustration introduced by J. Maynard Smith (cited in Newman and Comper, 1990). Figure 1A represents a small, random, local fluctuation of LI above its homogeneous steady state (represented by a horizontal line for both chemicals). Because f, > 0 (autocatalysis), a rises further, and because g, > 0, h (gray line) also rises, as shown in Fig. IB. Although the peak of h is centered at the same point as that of a , it is a broader peak because D,, > D,.This faster diffusion of ATP (the inhibitor) into the surroundings of the peak contributes to the decrease in the inhibition of cAMP (the activator) and ATP production in the peak region. Thus, the resulting relative presence of CAMP and relative absence of ATP in the peak region, together with the fact that cAMP increases the production of both cAMP and ATP whereas ATP inhibits the production of both, show that in the peak region the production of cAMP and ATP will increase even further and the two peaks of a and h will become even sharper, as shown in the transition from Fig. 1B to Fig. IC. The situation on each side of the peak is the opposite of the situation on the peak. Here we have the relative presence of ATP and relative absence of CAMP. The replacement of the predominance of the activator by the predominance of the inhibitor will lead to a decrease in the concentration of both cAMP and ATP since there is relatively more inhibitor and less activator present. This is seen in Fig. IC, where a trough develops on either side of the initial peak. Consider also, for example, the points marked by the arrows in Fig.

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A

FIG. 1 An intuitive demonstration of the principle of the instability of the homogeneous state. A standing wave is developed spontaneously from a random fluctuation. The black line represents the activator (CAMP)concentration, and the gray line represents the inhibitor (ATP) concentration. See text for discussion.

1 B. Because a decreases from the peak more steeply than h, it has reached the homogeneous steady state at these points, whereas h is still above its homogeneous steady-state level. Therefore at this point cAMP does not contribute to the rate of production of either cAMP or ATP, but ATP will contribute to inhibition of the production of both, and their net destruction will contribute to the formation of troughs (Fig. 1C). The process described in Fig. 1 also explains the general phenomenon of lateral inhibition (Schiffmann, 1991), for example, in experiments on hydra where a second head arises only beyond a minimum distance from the first head; this is explained by the inhibitory regions surrounding the center of activation (as in Fig. 1); only beyond these regions can additional centers of activation arise, as shown in Fig. 1 . The result is a standing wave (Fig. 1D). The natural chemical wavelength depends not only on the values of the reaction and diffusion parameters, but also on the size, with more peaks, troughs, and nodes fitting into a larger system; these intuitive results not only correspond to the rigorous mathematics, but also to Child’s results on metabolic patterns (Schiffmann, 1991). The number of peaks, the chemical wavelength, and other quantities of interest are derived from formulas that depend only on fa, g , , f h , g h ,

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D,, Dh (Turing, 1952). By plotting the experimentally determined rates, f a n d g , as functions of a and h (four curves), and measuring the slope of the tangent to the curve in each case, the sensitivities&, g o , f h , and g , can be determined experimentally and then used in these formulas to predict the observed number of peaks and other quantities of interest. Figure 1 also presages another important conclusion of the rigorous mathematics, which I found useful (Schiffmann, 1991): colocalization. We indeed see from the figure that the peaks of cAMP correspond to those of ATP, as do the troughs and the shapes of the two curves in general. This colocalization correlates in space the activation of kinase by cAMP and the availability of the phosphoryl group donated by ATP, and results in an effective phosphorylation field. The figure also demonstrates the spontaneous nature of the symmetry-breaking instability which is set off merely by a random local fluctuation around the homogeneous state. No prelocalization is required.

II. Antithesis between Preformation and Epigenesis

The debate between, on the one hand, proponents of preformation or preexistence of the embryo, or of prelocalization within o r without the egg, and on the other hand, proponents of epigenesis; that is, gradual differentiation from an amorphous beginning, is essentially synonymous with the history of embryology (Needham, 1959; Bowler, 1971 ; essays in Horder et al., 1986; Moore, 1987). (See Fig. 2 for a visual contrast of the two notions.) The formulation in the nineteenth century of the CarnotClausius second law of thermodynamics, the essence of which is the natural tendency toward disorganization, disorder, and increase in entropy, has made epigenesis even more difficult to conceive. The founders of thermodynamics indeed excluded living phenomena from that law. Thus Sir William Thomson (Lord Kelvin) (Guye, 1942; Leff and Rex, 1990) restricted his formulation of the second law to inanimate matter; he did not believe in the “materialistic hypothesis of life” and thought that “the real phenomena of life infinitely transcend human science. Similarly, Helmholtz exempted living phenomena from the restrictions of the second law and he imagined an entity like Maxwell’s demon (Guye, 1942; Monod, 1972; Harold, 1986; Leff and Rex, 1990) that can recognize and manipulate individual molecules and so lead to a decrease in entropy. With the solution of the problem of epigenesis suggested in Schiffmann (1991) and this chapter, I can now say that Helmholtz’s “life principle” was essentially correct since the faculty of recognition of the demon is the essence of the property of the enzymes and receptors recognizing their substrates, (allosteric) effectors and ligands, thus fulfilling the Turing ”

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FIG. 2 Preformation versus epigenesis. The original system in the center of the figure has circular symmetry. In the transition denoted by preformation, the asymmetry (localization) of the effect, indicated by the differentially shaded circles, is already preexistent in the cause, indicated by localized small solid circles, which may represent a localized ligand acting on a uniformly distributed transmembrane receptor. This intuitive situation, corresponding to Curie’s principle, is believed to apply in, for example, the generation of dorsoventral polarity in Drosophiln: the figure then represents a cross section of a blastoderm embryo (e.g.. as in Steward and Govind, 1993). In the transition denoted by epigenesis. the cause has the original circular symmetry but nevertheless the effect is asymmetric. This situation, in which the effect has less symmetry than the cause, is called spontaneous symmetry breaking.

conditions [equation (2) in Schiffmann, 1991; see also the set of inequalities in the introduction considered in relation to Fig. I]. Furthermore, since the recognition and binding properties of the proteins depend on their structure, which is in turn determined by their DNA, the often-raised question as to whether development can be reduced to molecular genetics

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(Monod, 1972; Lewin, 1984; Goodwin, 1985; Harold, 1986; Maynard Smith, 1986; Davies, 1987; Sheldrake, 1989; Casti, 1991; Garcia-Bellido, 1993) is to be answered in the affirmative, and the trend against reduction to molecular genetics (Weiss, 1968, 1969, 1973; Elsasser, 1975; Thorn, 1975; Stent, 1978, 1982; Goodwin, 1985; Oyama, 1985; Harold, 1986; Nagl, 1986; Davies, 1987; Gordon and Brodland, 1987; Sheldrake, 1989; Newman and Comper, 1990; Nijhout, 1990; Casti, 1991; Bentil and Murray, 1993; Lewin, 1993; Wilkins, 1993) is not justified. Many statements in the literature are incorrect. Some examples are: The noxious impact of molecular biology on embryology came about because the tenet that the gene is a one-dimensional description of the primary structure of a particular protein molecule was turned, willy-nilly, into the doctrine that the genome is a one-dimensional description of the whole animal. In particular, it came to be believed that the genome embodies, not merely a protein catalog, but also a genetic program for development, from zygote to adult. (Stent, 1985) There is no genetic program for development, no program that guides the system through its morphogenetic transitions. (B. Goodwin, in Lewin, 1993) The standard idea, that DNA “programs” the fertilized egg (and the cells that arise from it) to undergo a sequence of changes, is simply incorrect. (Newman, 1988)

Opinions such as those of Goodwin and Newman that development is not dictated by the DNA but by the dynamics (equivalently, the instability, self-organization), stem from the lack of appreciation that the dynamics of the positional information field is dictated by a highly specific DNA sequence. This lack of appreciation is clearly seen, for example, in the following statements: Among his [Turing] key ideas is the spontaneous formation of patterns arising from instabilities of the homogeneous state. . . . The specific mechanism that he proposes did not stand up to detailed experimental scrutiny, and has not found much favor among mainstream biologists, especially in an age that is strongly oriented towards the molecular view of biology and the DNA code. . . . There is a growing body of evidence that the development of biological form must involve dynamic, as well as molecular, processes. (Stewart, 1993)

It is also seen in: “The main difficulty in accepting development as a self-organizing process is that we do not have a simple description of heritability and self-replication for such a system” (Nijhout, 19901, and in Newman’s “nonprogrammatic model” characterized by “[a] pattern [which] is not codified as information in any chronological or material variable prior to its emergence” (Newman and Leonard, 1983) even though he considers, in particular, a reaction-diffusion field. I also note that since the field of positional information is dictated by the genes (since the

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conditions for the instability of the homogeneous state are dictated by the genes), and since this field in turn acts on the genes, the whole of development is inscribed in the genes. Despite the tendency to believe that there is no genetic program for development, it is probably true that a large majority of biologists do believe that “perhaps the greatest challenge for modern embryologists is to explain in molecular terms how the simple, one-dimensional array of genetic information in DNA can be transformed into the complex, three-dimensional organization of the adult” (Watson er al., 1987). This chapter and my earlier work (Schiffmann, 1991) can answer this challenge. Instead of development being determined by the linear sequence of the DNA nucleotides, many authors suggest electrical fields or mechanical and viscoelastic fields; for popular reviews of these and other morphogenetic fields, see Sheldrake (1989) and Casti (1991). Thus for example, calcium-regulated strain fields were suggested (Goodwin and Trainor, 1985; Colli, 1993). The Oster-Murray mechanical approach to biological pattern formation (Bentil and Murray, 1993) also belongs to this alternative (nonchemical, nongenetic) approach, as well as P. Ortoleva’s and coworkers’ nongenetic, electrical self-organization models (Ortoleva er al., 1982; Ortoleva, 1984). Gordon and Brodland (1987) suggested another mechanical, nongenetic theory that does not invoke gradients of morphogens but advances the notion of a cytoskeletal apparatus called the “cell state splitter” that can create mechanical instability, and which is composed of ubiquitous building materials; this ubiquity leads the authors to question the very existence of a genetic program for development. Interestingly, Waddington did not even expect the universality of the morphogenetic field: “Only if the forces are always the same or of very few kinds as they are in gravitational or electromagnetic fields . . . would the field concept be a unifying paradigm; and we know that none of these conditions is fulfilled” (Sheldrake, 1989). In fact, it is the vision and hope, with respect to development, of the founders of molecular biology, that are correct. The working hypothesis, which was expressed in the following statements, has now been shown to be true (Schiffmann, 1991): “The point of faith is: make the polypeptide sequences at the right time and in the right amounts, and the organization will take care of itself” (Lederberg, 1966), and “In the end only the shape-recognizing and stereospecific binding properties of proteins will provide the key to these phenomena [morphogenetic field, macroscopic morphogenesis, epigenesis]” (Monod, 1972). It should be remarked that Monod tended to believe that his basic vision is realized by surface interactions between cells (Monod, 1972), an approach nowadays championed by G. Edelman in his theory of topobiology which Casti (1991) refers to as the “last chance to include embryological development” in the molecular biology vision. However, a theory of morphogenesis

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based on differential distribution of cell adhesion molecules still has to explain the origin of the succession of spatially nonuniform patterns of adhesion molecules. I noted in an earlier work (Schiffmann, 1991) that the morphogenetic movement involved in neurulation has the same shape and symmetry as the metabolic patterns found by Child in neurulation. I further note here that the spatial patterns of cell adhesion molecules involved in neurulation also have the same shape and symmetry as the metabolic patterns (Takeichi, 1987). It makes sense to postulate that in general the positional information embodied in the Child-Turing metabolic field is responsible for the spatially differential expression of the adhesion molecules, just as it is responsible for spatially differential gene expression in general. It is the recognition capacity of the enzymes involved in bioenergetics (Schiffmann, 1991) that is of essence in the struggle against entropy increase. This struggle is the essence of life as recognized by another founder of thermodynamics, Boltzmann, as well as by Schrodinger, who was influenced by Boltzmann. The enigma about the opposing evolution of biological systems and physical systems is discussed at length in Guye (1942). One solution advanced there is based on an entropy-reducing fluctuation when the number of molecules is small so that the laws of large numbers do not apply. However, as Schrodinger (cited in Schiffmann, 1991) has emphasized, systems that contain only a few particles should have random, nonreproducible and unpredictable behavior-not a robust base for development and life. Note that even in the middle of this century, great physicists, always under the spell of the awesome second law, have seriously considered the possibility that living systems are exempt from the second law; for example, Bridgman (1943) states “that the tendency of living organisms is to organize their surroundings, that is, to produce ‘order’ where formerly there was disorder. Life then appears in some way to oppose the otherwise universal drive to disorder. . . . Does it mean that living organisms may violate the second law of thermodynamics?” Brillouin (1949) notes: “The evolution of species, as well as the evolution of individuals . . . has been progressing from the simplest to the most complex structures . . . and appears almost as a contradiction [to] the second principle. . . . It is hard to reconcile these two opposite directions of evolution. . . . Is there not, in living organisms, some power that prevents the action of the second principle?” The Boltzmann idea that the phenomenon of life-the creation of islands of order (negentropy) amidst increasing disorder in their surroundings-is characterized as an ongoing struggle against the natural tendency toward disorganization and increase in entropy, as dictated by the second law of thermodynamics, was adopted by other thinkers on the nature of life, for example, Katchalsky (1976) and Bergson (191 l), who writes: “Life is an effort to remount the decline that matter descends.”

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Both ontogeny and phylogeny are characterized by a progression from the homogeneous to the heterogeneous, and by ever-increasing heterogeneity (Gould, 1977). Biologists have always recognized this biological trend of increasing complexity. Various principles were conceived whose essence is an increase in complexity and which are antithetical to the second law, for example, von Baer’s law of development from the homogeneous to the heterogeneous in ontogeny (Gould, 19771, but they could not conceive the mechanisms underlying these principles. In considering the ever-increasing complexity and heterogeneity, the most difficult part to imagine was the beginning of the process, namely, how an originally homogeneous system in a homogeneous environment became heterogeneous in the first place: “An original homogeneity is equally unthinkable, for out of a system all whose parts are absolutely alike, by no imaginable process could any heterogeneity ever be evolved” (Jenkinson, 1909). This is echoed by Huxley and De Beer (1934): “It is logically almost impossible to conceive how a non-polarised fragment of living matter can acquire polarity by self-differentiation . . . it seemed impossible to understand epigenesis on mechanistic lines.” Similarly, Child (1941) states: “The conception of developmental pattern as independent of environment seems to demand a teleological principle.” Wheeler states: “The pronounced ‘epigenecist’ . . . must gird himself to perform Herculean labors in explaining how the complex heterogeneity of the adult organism can arise.” (cited in Horder er nl., 1986). The first and most penetrating logical analysis of the origin of localization and differentiation was given by Driesch (1929). He starts by recalling his own famous experiments disproving that qualitatively unequal nuclear division involving the disintegration of a morphogenetic structure in the nucleus, as suggested by Weismann, could be the source of specification of the blastomeres. According to Driesch, Weisman advanced such a preformation type of theory because he thought that “an epigenetic theory would lead right beyond natural science.” Next, Driesch considers whether the cytoplasm can be the origin of localization. One major reason why he rejects this possibility is that “there exists in every sort of egg an earliest stage, in which all parts of its protoplasm are equal. . . .” He then turns to a third source of localization, the environment of the egg (or the organ). This too cannot be the source of localization since “no exterior formative stimuli are responsible for the intimate details of animal organization . . . this morphogenetic independence in animals is due to their comparatively far-reaching functional independence of those external agents which have any sort of direction” and “in our harmonious systems no localizing stimulus comes from without.” So he can conclude that neither the nucleus nor the cytoplasm nor the environment can be the source of localization. He then takes a crucial step and introduces the idea of vitalistic entelechy precisely because of the impossibility to ac-

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count, “for all future time,” for the typical localization of every morphogenetic effect in the differentiation of a harmonious equipotential system by the discovery of a single external cause. From a leading embryologist, whose experiments and theories are largely responsible for our modern understanding of the constant transmission of the original chromosomal endowment to all somatic cells of an organism, that genes are not spread among cells or lost o r acquired, and that it is the position of the cell that determines its fate, such a conversion is most astounding and dramatic. However, in order to justify his nonmaterial entelechy as the ordering principle, he goes to first principles in physics. In particular, he invokes the second law of thermodynamics, namely, “that a something that is homogeneous cannot become heterogeneous ‘by itself’”; “for the sufficient reason of happening would be wanting in a system which was uniform throughout, wanting at least so far as the system was uniform.” Clearly, spontaneous instability of the homogeneous state is not envisaged by him. A closer analysis of his formulation of the second law makes it clear that he equates (without saying so explicitly) the second law with Curie’s principle (that a spatial dissymmetry is needed for a physical process to occur and that this dissymmetry cannot be found in the effect if it is not preexistent in the cause) which was prominent in the physics of the time. Some of his statements in Curie’s spirit are: “nothing can happen without diversities, and that the originating of diversities demands pre-existing diversities”; and “the effect with regard to its manifoldness must have its equivalent in the manifoldness of the cause; or, in other words, that the effect cannot be more manifold than the cause. If, therefore, there seems to be an increase in manifoldness during an event, this increase must be accounted for within the cause. In short: A system, in the course of becoming, is unable to increase its manifoldness by itself. The modern notion of symmetry breaking in nonlinear systems does not respect the Curie principle, a principle we indeed now know is not always valid. Thus we see that his absolute confidence in the “impossibility for all future times” of explaining epigenesis within physics lies in his mistake in equating a principle which always holds (the second law) with a principle which holds often but not always (Curie’s principle). Driesch emphasizes in many places that the essence of differentiation and morphogenesis is epigenesis-the production of new, not preexisting, manifoldness and heterogeneity. It is precisely for this production that he created the concept of entelechy. So supernatural entelechy has precisely the same function as the principle of the instability of the homogeneous state (Schiffmann, 1991). Woodger (1929, 1930) also rejects the three sources of preformation considered by Driesch: Weismannian nuclear preformation because the nucleus divides equally; cytoplasmic preformation because “as develop-

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ment proceeds more and more cellular parts are elaborated, and if these are all to be referred back to the egg-cytoplasm the latter will become, it seems, intolerably overcrowded.” In addition, localization is not favored in liquid or semiliquid substances and differences in different regions of the cytoplasm in certain eggs can be centrifuged away without disturbing the development. He then concludes: “When nucleus and cytoplasm fail it is the custom to turn to the environment of the egg but this again has proved of little avail. In birds and mammals the environment is probably remarkably uniform and can therefore hardly be invoked to account for the progressive increase in organization during development.” Child (1941) deals with the origin of embryonic pattern in Chapter 16 and considers as supernatural both the Roux-Weismann theory of qualitative unequal nuclear division and Driesch’s concept of development which is independent of spatial nonuniformity in the environment. Yet he had seen time and again the emergence of metabolic patterns in homogeneous systems with homogeneous environments. He also notes, for example, that the homogeneous oocyte of annelids develops patterns in a homogeneous fluid, and for another type of annelid, groups of eight identical cells autonomously turn into one oocyte and seven accessory cell groups. Child also notes that “since the more highly developed follicles usually surround the oocyte uniformly, they apparently do not provide an environmental differential.” Child is compelled to admit that the question of how asymmetry originates is unanswered. Yet despite the experimental evidence before him of pure epigenesis, he still cannot imagine the spontaneous generation of polarity and asymmetry and he finds refuge in a preformation type of theory; namely, that there are spatial environmental cues donated by the previous generation: “The view commonly held at present is that, whatever the nature of embryonic pattern, it does not originate autonomously in the egg but originates in relation to its intraorganismic environment that is, to factors in the relations of the oocyte to the parent organism.” Accordingly he sets out to search for the parental environmental spatial nonuniformity that provides the positional information. He recognizes that the oocyte environment is often a mechanically mixed and agitated fluid due to ovarian contractions, peristaltic contraction, and other movements of the animal. In those cases where environmental differentials seem to determine the pattern, the result is very variable. He also observes that, “in other forms of development polarity is determined by various external differentials, and polarity determined by one differential may be obliterated and a new polarity determined by another in many animals.” Indeed, we can now interpret such an external differential as only favoring a particular fluctuation around the homogeneous state of the oocyte or of the germ cluster, but a Turing system is still needed to amplify even this

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favored fluctuation. I also note that a robust development of a biological pattern cannot depend on a particular fluctuation since the environmental differential that favors a particular fluctuation is often fragile, changeable, and transient. Huxley and De Beer (1934), who adopt in general the metabolic gradient theory of Child emphasize in their introduction that embryological development is rigorously epigenetic, and are also aware, for the eggs of amphibians and sea-urchins, for example, that the exact location of the anteriorposterior or dorsoventral axis is very labile and can be overriden by a variety of external localized factors. Yet on the other hand, they cannot bring themselves to dispense with the requirement for external localization. Thus their claim that “certain external factors set up quantitative differentials in the egg and embryo, as a result of which qualitative differences of structure ultimately ensue” and that “external differentials may serve to release the capacity of the egg to develop polarity,” is based on the mistaken notion that a favored fluctuation is an absolute necessity, whereas we now know that it is not. It is because the Curie principle’s line of thought, that asymmetry of the effect (the morphogenetic localization in the oocyte or in the germ line) is already preexistent in the cause (the external differential), corresponds to our intuition and common sense, while the notion of spontaneous breaking of symmetry does not, that the search for external localization (a sort of preformation) continues, as it did in Child’s day, and it in fact pervades all of present-day biology. It is again to the credit of Driesch that at the beginning of the century he had already courageously discerned and concluded that, “morphogenesis, we have learned, is ‘epigenesis’ . . . manifoldness in space is produced where no manifoldness was” (Driesch, 1929). This, “the problem of morphogenetic localization . . . the autonomy of life,” is his main “proof” of vitalism. However, modern experimental biology like Child’s work does not envisage either spontaneous breaking of symmetry or vitalism. It is still assumed-as we saw in Child (1941) and Huxley and De Beer (1934)-that a localized spatial cue is provided by the parent organism. For example: “Thus, it is conceivable that the animal-vegetal polarity originates from the asymmetric intercellular contacts between the oocyte and the surrounding ovary cells, by a process of imprinting stable structural features into the plasma membrane and/or the cortex of the oocyte” (Zivkovic and Dohmen, 1989); see also Wall (1990) and Fig. 2.7 therein for the concept of a cortical map. Similarly, “The animal-vegetal polarity of the egg may not be formed de n o w during oogenesis but may be transferred from one generation to the next by means of cytoplasmic continuity through the germ cells” (Nieuwkoop et al., 1985). Typical of the search for an external differential is the leading example of Drosophila. St Johnston and Nusslein-Volhard (1992) consider

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that “positional information is transmitted from one generation to the next” and that “for an inductive process to be spatially controlled, either the inducer or the competence to respond must be localized.” For the terminal and dorsoventral systems, the origin of polarity is suggested to reside in a localization in the somatic follicle cells ( =the environment), leading to localized induction in the oocyte (Nusslein-Volhard, 1991).The origin of the localization of the morphogenetic mRNA for the anterior and posterior systems is not explained. In later sections 1 suggest that for all four maternal systems in Drosophilu, localization (positional information) will emerge in the germ line with receptor and extracellular ligand both remaining homogeneous, and that the previous generation does not necessarily have to contribute positional information (spatial cue, localized ligand), as is normally assumed.

111. Improbability of the Turing Couple and of Biological Coherence

We can conclude from the preceding section that despite experimental evidence for pure epigenesis, few biologists faced this fact like Driesch or Woodger did. The majority were, and are, inclined to develop various theories in which an element of spatial preformation is always maintained. To account within the sciences for epigenesis, a new principle of nature was needed (Prigogine, 1980),a principle counter to the spirit of the second law of thermodynamics. This new principle, sometimes referred to as the local reversal of the second law, would not constitute a violation of the second law, but would be an additional law and would constitute “the law of life” (Samuel, 1972). It is important to realize that the open nonequilibrium nature of the biological system is only a necessary condition in order to defy locally the second law: Biosystems are not closed systems . . . which enables them to export entropy into their environment. But the fact that they are able to evade the degenerative (pessimistic) arrow of time does not explain how they comply with the progressive (optimistic) arrow. Freeing a system from the strictures of one law does not prove that it follows another. Many biologists make this mistake. They assume because they have discovered the above loophole in the second law, the progressive nature of biological evolution is explained. This is simply incorrect. (Davies, 1987)

Indeed, the overwhelming majority of open, nonequilibrium, chemical systems remain homogeneous. The new principle will constitute the sufficient condition for epigenesis. This new principle of nature can be taken to be Spencer’s principle of the instability of the homogeneous state.

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According to Spencer (1900), functional complexity, such as division of labor, whether in sociology or biology, would be based on the transition from an incoherent homogeneity to a coherent heterogeneity. Spencer’s principle can be taken as a useful forerunner to Turing’s idea of the instability of the homogeneous state in a system of reacting and diffusing chemicals. However, the failure to identify Turing’s morphogens despite an intensive search in past decades has led to a certain degree of disillusionment in the prospect of finding a biological morphogenetic Turing system. Typical of the remarks made with respect to the chance of ever finding such a Turing-type system, is: “I feel they [Turing systems] may not exist and certainly it is unlikely that we will ever find out what they are” (Bonner, 1989).Similarly, “Thus, at present the idea of a [Turing] morphogen directing pattern formation in embryos rests on about as solid a footing as the idea of a quark directing the formation of elementary particles in physics [because of a lack of evidence of actual material existence]” (Casti, 1991). We should indeed now focus our attention on the actual existence of a Turing biochemical mechanism which can result in the instability of the homogeneous state. We should not worry too much about the details of our (CAMP, ATP) Turing mechanism with respect to its effect on the geometry of Turing spatial patterns. Indeed, one major conclusion from my work in the early seventies on the bifurcation (nonlinear) theory of Turing reaction-diffusion systems in multiple-dimensional space [Schiffmann (1980, 1980) cited in Schiffmann (1991), and Schiffmann’s earlier work] was that the Turing spatial patterns are largely independent of the particular Turing chemical mechanism assumed, but are dependent on the geometry of the reaction-diffusion domain (e.g., the embryo) assumed. This is a fortunate state of affairs since although we know that the (CAMP, ATP) Turing mechanism involves very many chemicals and reaction steps, we can essentially remain assured that the geometry of the Turing spatial patterns will depend only on the geometry of the embryological reactiondiffusion systems and not on the details of these many chemicals and reaction steps. (Mathematically this situation arises from the following considerations: the Turing patterns depend on the solutions of the algebraic bifurcation equations, which in turn depend on the vanishing or nonvanishing of integrals over powers of the eigenfunctions of the Laplacian, which correspond to the geometry of the reaction-diffusion domain. This vanishing depends only on the geometry-dependent selection rules. Therefore the Turing patterns will largely depend on the geometry of the reaction-diffusion domain, for example, the geometry of the embryo, and not on the particular Turing kinetics assumed.) These earlier results of mine demonstrating the independence of the geometry of reaction-diffusion prepatterns with respect to the exact kinetic laws, can be considered as

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a realization of the goal of rational morphology, involving the search for universal laws of form (Casti, 1991). It is in particular interesting that the Turing chemicals turn out to be the most ubiquitous and universal molecules in biochemistry (Schiffmann, 1990, 1991), cAMP and ATP, and that the conditions for the instability of the homogeneous state turn out to be the conditions for rapid and economic energy management. Thus the instability (the spatial nonuniformity) is initiated by cAMP autocatalysis (f,> 0). However, because cAMP mobilizes the energy reserves to yield ATP (g, > 0), the same autocatalysis also contributes to rapid mobilization of the energy reserves upon extracellular instruction (increasing K i ) . Similarly, the economy in energy processing occurs not only because the increase in the concentration of ATP decreases the rate of ATP production ( g h < 0), but this economy is further enhanced because an increase in [ATP] also decreases the rate of production of CAMP (fh < 0)-the molecule that is responsible for the mobilization of the glycogen and lipid, thus further throttling unnecessary ATP production. We thus conclude that the evolution of the enzymes and their substrates as they currently are in the intermediary metabolism achieves simultaneously two major goals: The principle of the instability of the homogeneous state and effective energy management. We also recall here that the struggle against the degradation of energy has been considered by many scholars the mission of living organisms (Guye, 1942). The idea that differentiation and morphogenesis are dependent on the effectiveness of energy utilization also corresponds to the evolution of life and of metabolism as well as to development according to Child and to my theory. Indeed, Schidlowski (1976) points to the fact that organizational complexity, including multicellular life, had to wait for the advent of environmental oxygen pressure favorable for the more effective-compared with glycolysis-oxidative metabolism. He notes the coincidence during the Late Precambrian of the appearance of eukaryotic (notably multicellular) life with the incipient buildup of an environmental oxygen pressure (see also Tappan, 1974; Warburg, 1966; Broda, 1975; Raff and Kaufman, 1983). This coincidence in evolution corresponds to the spatially differential oxygen uptake observed by Child and others, which according to my theory is a manifestation of the universal Turing system responsible for all differentiation and morphogenesis. Thus Schidlowski is correct when he writes that biological differentiation and organic evolution, although they are highly improbable phenomena involving a decrease of entropy and an increase in order, nevertheless do occur due to oxidative metabolism, which he compares to the Maxwellian demon. This situation also corresponds to the dedifferentiation of cancer cells upon injury to respiration, as emphasized by Warburg (1956). In that

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sense the healing of cancer involves restoring the same effective energy metabolism, the appearance of which in evolution made development possible. I should emphasize here (Schidlowski, 1976) that the time when cyanobacteria (believed to be the first organisms to produce oxygen by photosynthesis) appeared is not the time when free molecular oxygen became available in the atmosphere. Indeed, as soon as photosynthetic oxygen was produced, it was instantaneously consumed by very effective reactions, thus becoming bound in sediments such as Fe,O, and SO:-. Only after the supply of ferrous iron in the ancient oceans was exhausted could free oxygen have accumulated in the sea and, consequently, in the atmosphere. This explains the very long delay between the appearance of cyanobacteria and the accumulation of free oxygen in the atmosphere. Why exactly is oxygen “the creator of differentiation” (Warburg, 1966)? Recall that glycolysis as well as other anaerobic biological oxidations (Wald, 1966) involve only dehydrogenations whereas respiration is unique in that the hydrogen atoms are separated into protons and electrons. Therefore, according to my theory, only respiration creates spatially differential electron transfer, which, I suggest, is the source of reduction fields that can affect transcription factors (TF) and other proteins in a spatially differential manner, as well as the source of electric fields, which are responsible for polarized transport of morphogenetic determinants, in particular in the egg; these effects explain why oxygen creates differentiation. While these ideas will be elaborated on later, I want at this stage to emphasize that Pasteur’s observation that a lack of oxygen results in the degeneration of structure in yeast, his hints that oxygen may be the source of differentiation and structure, and Warburg’s strong emphasis that oxygen creates differentiation (Warburg, 1966), can now for the first time be understood. Furthermore, the failure of oxygen-driven differentiation can come about in many ways. It can occur simply because of the absence of oxygen, as for example, in the early history of our planet, and is exemplified by the requirement of oxygen for the differentiation of slime moulds (Broda, 1975). In fact, oxidative phosphorylation seems to be a requirement for eukaryotic plants even though photophosphorylation is a plentiful source of ATP (Broda, 1975). A second failure of oxygendriven differentiation can occur when electron transfer is physiologically inhibited, as exemplified by the suppression of bristle differentiation in Drosophila upon the application of carbon monoxide (Wolsky, 1956). A third reason for the failure of oxygen-driven differentiation occurs when defective gene products interfere with normal respiration, which includes cancer-related dedifferentation (Warburg, 1956, 1966). It is the inability to generate spatially differential electron transfer that is common to these three different situations.

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The existence of aerobic electron transfer systems as well as glycolytic systems during all stages of development and not only in the adult organism (Wilde and Crawford, 1963) corresponds to these observations. Furthermore, the Turing instability conditions, the four inequalities in equation (2) in Schiffmann (1991) and the diffusion condition, hold for glycolysis alone as well as for the more complete respiratory metabolism. Therefore (CAMP,ATP) phosphorylation fields and nonphosphorylative ATP hydrolysis fields can be based on glycolysis alone. However, the conclusion in Wilde and Crawford (1963) that “the ability to undergo differentiation and morphogenesis appears to be dependent on aerobic metabolism” corresponds to the inability, according to my theory, to get spontaneous endogenous electrophoresis and reduction fields (see also later discussion) with glycolysis alone without the oxygen-based electron transfer. The very possibility for the instability of the homogeneous state and for a succession of metabolic patterns with an increasing number of nodes-which I suggested is the source of increasing spatial diversification (Schiffmann, 1991)-may have had to wait for the advent of atmospheric oxygen. Indeed, oxygen-based effective-energy metabolism may be responsible for the larger size of aerobic organisms (Fenchel and Finlay, 1994). However, one requirement for the instability of the homogeneous state is a minimal size of the organism, and it is also the case that as the size increases, metabolic patterns with an increasing number of nodes succeed each other (Schiffmann, 1991). 0,-sensitive enzymes and ion channels may be regulated by the reversible binding of 0, to coordination complexes formed by metal-protein sites, and indeed 0,-sensitive enzymes containing a heme prosthetic group are known (Lopez-Barneo, 1994). This direct regulation by 02,together with the possibility for spatially differential oxygen consumption predicted by the Turing instability and confirmed experimentally (Child, 1941), may contribute to a spatially differential activity of enzymes and channels. It should also be explicitly noted that the problem considered by Schidlowski (1976) has, until now, been the most intriguing problem in both paleontology and evolutionary biology (Mayr, 1982; Gould, 1991 ; Lewin, 1993), namely, the long delay in the evolution of metazoans (biological complexity) for almost 3 billions years after the first appearance of life, and the steep rise in the diversity of life at the beginning of the Cambrian Period, the so-called Cambrian explosion. This problem bothered Darwin himself (Mayr, 1982; Gould, 1991) since he believed in gradual evolution, and he tried to explain it away through the incompleteness of the fossil record. In fact, ever since Darwin, palaeontologists have strived to explain the Cambrian explosion, and it is not now attributed to an incomplete fossil record. Various hypotheses have been advanced (Lewin, 1993) to account for this Precambrian-Cambrian faunal discontinuity. Writers who do not declare a religious outlook and who come to the conclusion that

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Darwin was wrong and that there is a purposeful creator manifesting intelligent design (Cohen, 1985; Johnson, 1991) also enlist the Cambrian explosion against Darwinism; for example, “The single greatest problem which the fossil record poses for Darwinism is the ‘Cambrian explosion’ of around 600 million years ago. Nearly all the animal phyla appear in the rocks of this period, without a trace of the evolutionary ancestors that Darwinists require” (Johnson, 1991). It seems that if one takes into account the arguments presented earlier, that is, the coincidence of the buildup of biological complexity with the buildup of free molecular oxygen, the failure of the normally oxygen-driven differentiations, the increased efficiency of oxygen uptake by many evolutionary modifications (Tappan, 1974), and most important, understanding why oxygen is essential for differentiation, then the explanation for the Cambrian explosion can be seen to lie in the buildup of environmental oxygen pressure. In addition to the contributions to the autocatalytic cAMP production I suggested earlier (Schiffmann, 1991), we now have evidence that the phosphorylation by CAMP-dependent protein kinase of G-protein-inhibitor protein relieves the inhibition of the adenylyl cyclase system by the inhibitor protein, resulting in positive feedback in cAMP production. This may be a very general mechanism for autocatalytic cAMP production since this inhibitor protein is present in many tissue types (Bauer et al., 1992). The incubation of hydra in ATP solution results in the inhibition of regeneration and of induction in grafting experiments, and also in the abolition of morphology (Newman, 1973). These three phenomena support the identification of ATP as the inhibitor (Schiffmann, 1991). The symmetry breaking I predicted (Schiffmann, 1991) is confirmed by Bacskai er al. (1993), who show that the uniform application of the extracellular ligand serotonin to a single live Aplysia sensory neuron results in a steep intracellular spatial gradient of CAMP. It is usually assumed that developmental genes (which control determination, differentiation, and morphogenesis) are distinct from genes involved in housekeeping functions (Nijhout, 1990; Raff and Kaufman, 1983). However, the Turing mechanism of instability has to operate from the very beginning of development until its very end (Schiffmann, 1991). It therefore makes sense that this mechanism is based on ubiquitous household molecules such as those involved in universal metabolism and bioenergetics, and not on the rarer luxury molecules such as tissue-specific proteins. These observations also correspond to the observation that the hallmark of the phenomena of life is the transformation of high-grade energy into organization. The crucial question is how and by what mechanism this is done. Thus, for example, Ho (1989) observes that, the physical world is dominated by the second law of thermodynamics . . . order dissolves into disorder. . . . The biological world, by contrast, is capable of maintaining and reproducing organization on a macroscopic scale

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from a flow of energy and matter. The fundamental problem of life is that of how it can transform energy so efficiently, and, at the same time, organize matter so fruitfully. Significantly, Harold (1986) emphasizes that “living things convert energy into organization . . . , that life feeds on negative entropy. Make no mistake about it: [this is] the essence of life.” He hastens to add that “an experimental biologist will not be content with such abstractions . . . , one wants to know just how the trick is done.” Turing notes that The molecules which together make up the chemical waves are continually changing, though their concentrations in any particular cell are only undergoing small statistical fluctuations. Moreover, in order to maintain the wave pattern a continual supply of free energy is required. It is clear that this must be so since there is a continual degradation of energy through diffusion. This energy is supplied through the “fuel substances.” (Turing, 1952) Prigogine has emphasized the high dissipation of energy in early development and the flow of energy ( = dissipation of high-grade energy) through a system manifesting a dissipative structure (Nicolis and Prigogine, 1989; Prigogine, 1980, and earlier works). It is therefore interesting to note that the triggering of the universal positional information field, that is, the universal dissipative structure which is the source of all organization in living systems, involves precisely the triggering of the flow of energy through the system as anticipated (Maynard Smith, 1986). Indeed, the triggering of the instability of the homogeneous state involves increasing the bifurcation parameter KPC (Schiffmann, 1991). This removal of substrate inhibition of adenylate cyclase leads to an increase in the rate of production of CAMP [see equations ( 1 ) and (4) in Schiffmann (1991)l. This in turn leads to increased glycogen and lipid breakdown. Substrate oxidation, oxygen uptake, and C 0 2production will increase. The resulting increase in electron transfer will remove the substrate inhibition of the ATP synthase as well (Schiffmann, 1989),and ATP production will increase. We thus see that in the context of a reaction-diffusion system, triggering of catabolism and pattern formation merge. This triggering of catabolism by an increase in the bifurcation parameter KPC corresponds precisely to Turing’s intuition (Turing, 1952) that an increase in the “fuel supply” (corresponding to the increased glycogen and lipid breakdown noted earlier) will correspond to an increase in the bifurcation parameter that triggers the instability of the homogeneous state. Harold (1986) is being too pessimistic when he says that “there is certainly no unitary molecular mechanism of morphogenesis in the same sense that there is a single mechanism of oxidative phosphorylation”; in fact, there is a unitary molecular mechanism of morphogenesis, which furthermore is essentially oxidative phosphorylation! The emergence of

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spontaneous self-organization via increased energy dissipation conforms to the vision of Child, Turing, and Prigogine, but others objected to a possible connection between metabolism and pattern formation. Thus, for example, Dworkin and Dworkin-Rastl (1991) feel that “it appears unlikely that differences in metabolism across the embryo play a determinative role in pattern formation.” Similarly it is stated, “Unfortunately . . . these findings were interpreted in terms of ‘physiological gradients of metabolic activity’ (Child, 1941) and may have contributed to the loss of interest in gradient fields by modern biologists” (De Robertis et af., 1991). For Crick (1970), the metabolic gradient seemed more likely to be the result of development rather than its cause. For Slack (1987), invoking the metabolic gradient to explain regeneration was an expression of the trend to invoke the respiratory metabolism in the interwar period. This trend is also criticized by J. Brachet (Horder et al., 1986). For Wolpert (1986, 19891, Child and his contemporaries were obsessed with energy metabolism, which Wolpert considers to be unrelated to pattern formation, whereas according to him, they should have been occupied with information. Ironically, this obsession of Child’s is blamed by Wolpert for delaying the emergence of new ideas, whereas in fact it is the nonappreciation of the value of Child’s work and ideas that delayed progress in the field. In fact, it is precisely because the “self-organizing capacity undoubtedly forms the most basic aspect of morphogenesis” (Nieuwkoop, 1992), and because the metabolic patterns of Child and others not only present such self-organization but also have been available since around the beginning of the century, that it is so striking that the proper interpretation and understanding, in biochemical and mathematical terms, of this already existing experimental work, were missing for such a long time. The often unjustified and unsubstantiated criticism of the work of Child and others on metabolic patterns, by many of the pillars of embryology such as H. Spemann, J. Needham, and J. Brachet, contributed significantly to the delay in the understanding of development. Meinhardt (1982, cited in Schiffmann, 1991) does not realize that his hypothetical activator-inhibitor system resides in Child’s metabolic gradients: “A biological fact is that most tissues have an intrinsic asymmetry, a polarity. Axial differentials in respiration, in oxidation-reduction reactions, in the permeability, or in electric potentials have been detected in protozoa, eggs, embryos, hydroids, and some algae (see Child, 1929, 1941).” Not realizing that all these polarities are just different manifestations of the same physiological gradient [this was already perceived long ago, e.g., “That the different gradients described by Child and others are only different manifestations of the same physiological gradient under different experimental procedures is highly probable” (Rulon, 1935, cited

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in Child, 1941)],that is, the same ( a = CAMP,h = ATP) reaction-diffusion system, he relegates these polarities to the status of a time-independent, shallow space-dependent parameter (asymmetry) in his dynamic equations. This parameter-“the graded source density” in his formulation-only orients the pattern which is itself generated by autocatalysis and lateral inhibition, by some (a, h) as yet unknown. From this reasoning he draws the conclusion that “in most biological cases, pattern formation does not involve symmetry breaking (although the proposed mechanism can perform this), since the tissue or its environment is asymmetric. The asymmetric organism forms an asymmetric egg and the orientation of the developing organism is therefore predictable.” However, 1 do not think that the role of the activator-inhibitor system is only to amplify preexisting polarities (which is indeed an important function in itself). In fact, in the overwhelming majority of cases, there is true symmetry breaking, true epigenesis, and an absence of systematic differentials in either tissue or environment-an opinion shared by P. Weiss, H. Driesch, and J. H. Woodger. Robust development must involve true symmetry breaking, again and again, in one ontogeny. It is useful to keep in mind the Boltzmann explanation that a uniform distribution is much more probable than a nonuniform one with a smaller number of microstates (Prigogine, 1980).It is therefore intuitively expected that the conditions to obtain the improbable nonuniform state, the conditions for the instability of the homogeneous state, will also be improbable. Indeed, if each f,,g , , f h , gh can be positive, negative, or zero, the odds against obtaining the set {f,> 0, g, > O , f h < 0, g h < 0}, [equation (2) in Schiffmann, 19911, are 34 = 81 to one. However, most two randomly chosen biochemicals do not influence each other’s production; for them f, = g, = f, = gh = 0. So the odds are infinitely greater than 81 to 1 . Furthermore, choosing, for example, the condition gl, < 0, it holds not only because the enzyme phosphofructokinase is inhibited by ATP, but also so is pyruvate dehydrogenase and many other enzymes that are arranged in series and in fact it was enough that only one of these enzymes would be inhibited by ATP. This leads to a robust and error-proof fulfillment of the condition g , < 0. This redundancy can also explain the stability of the prepattern with respect to many mutations in genetic mosaics experiments (Ursprung, 1963). Overall it is clear that the a pviovi chances of the random emergence of such a chemically improbable system are very small, and even in view of natural selection and accelerated autocatalytic polymerization (see later discussion), its emergence must have required a very long time indeed. The universality and the uniqueness of the prepattern which emerges from the genetic mosaics experiments (Ursprung, 1963) can be understood in the light of the chemically improbable nature of the Turing metabolic system.

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The realization of this improbable combination is important not only to demonstrate the uniqueness of the Turing couple, but also because many formulations in the literature may imply the inevitability of chemical instability occurring in general under far-from-equilibrium conditions, whereas most far-from-equilibrium chemical systems will not present the Turing instability, which requires a very unusual chemistry. It is the sufficient condition for the instability-not the necessary condition of being far from equilibrium-that manifests the “wisdom of the body,” at which we should marvel. Consider the other condition for the instability, that the diffusion coefficient for the inhibitor will be greater than that for the activator. The fulfillment of this condition was recognized as a difficulty with the Turing model (Hunding and Sorensen, 1988; Pearson and Bruno, 1992). Furthermore, the fulfillment of this condition through a difference in molecular weights will jeopardize the possibility of diffusion via the gap junctions. So nature’s resourcefulness solves this problem by choosing as Turing morphogens two molecules of similar molecular weight, but the diffusion of the inhibitor (ATP) will be greater than that of the activator (CAMP) because of the increase in negative charge on ATP in relation to CAMP and because (ATP) + (CAMP)(Bowen and Martin, 1964). The rare, improbable, and chemically nonrandom nature of the biochemical (CAMP, ATP) dissipative structure is further emphasized when it is contrasted with other types of dissipative structures (also called flow structures or process structures). For example, Benard cells (Nicolis and Prigogine, 1989) or the vortex created when water goes down a drain (Maynard Smith, 1986), will occur with other liquids and is independent of the shapes of the constituent molecules. This kind of universality is akin to the decrease of entropy obtained when ordered structures in equilibrium are created by lowering the temperature or increasing the pressure, which is also a universal phenomenon occurring independently of the nature of the constituent molecules. The biochemical dissipative structure does not, by contrast, occur independently of the constituent molecules and does not enjoy the universality of other types of dissipative structures and of structures that are created in (equilibrium) phase transitions. Thus when a case is made against the reduction of development to DNA by invoking the analogy that “knowing the structure of H,O gives you no clue as to why water goes down a plughole in a vortex” (B. Goodwin, in Lewin, 1993), it should then be remarked that the analogy is inappropriate since the vortex and the biochemical dissipative structure are different kinds of dissipative structures. For example, the binding of ATP to an allosteric site of phosphofructokinase, which thus contributes to g , < 0, depends very much on the structure of both ATP and phosphofructokinase, and the situation is as antici-

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pated by Monod (1972). Similar reservations apply to the statement “the shape of a rhino, unlike that of a ribosome, is independent of the shapes of its constituent molecules” (Casti, 1991). It is unfortunate to divide the mechanisms of morphogenesis and pattern formation in the context of development into generic physical mechanisms and genetic mechanisms, and to include the reaction-diffusion mechanism within the generic physical mechanisms, particularly when also stating that development is not inscribed in the genes (Newman and Comper, 1990), whereas I believe that the reaction-diffusion mechanism that governs development is written in the genes. If indeed, as suggested here, one Turing couple alone has to defy the tendency to disorder, one would expect evolutionary pressure to adapt processes downstream to this one Turing system so that they can benefit from the improbable decrease of entropy, the spatial nonuniformity , provided by the Turing couple. This in turn may lead to further rare occurrences. Consider the suggested spatial differentiation according to the overall time of exposure to the (CAMP,ATP) phosphorylation and to how recent the exposure has been (Schiffmann, 1991). This would require that CAMP-dependent protein kinase act on many substrate proteins and also that there.would be differing levels of substrate efficacy, resulting in a temporal order of protein phosphorylation in response to the CAMPsignal. These requirements indeed present rare occurrences, since the bulk of known enzymes (more than 90%) catalyze a single reaction with unique substrates (Walsh et al., 1992). Even the fewer enzymes with multiple substrates often present the same mode of interaction with their multiple substrates, thus not favoring temporal order (Walsh et al., 1992). The required rare properties do indeed occur for CAMP-dependent protein kinase. The variety of phosphorylation sequences and the many different modes of interaction with different substrates would result in a broad range of substrate efficacies that will regulate the order of substrate phosphorylation (Walsh et al., 1992). So we see that there is a good reason why many protein substrates of the CAMP-dependent protein kinase have not evolved to be as good a substrate as possible. The CAMP-dependent protein kinase is indeed rare among enzymes, as anticipated by the general considerations discussed here. For the biochemical processes to be organized in a spatially differential manner, molecules involved in these processes have to be affected by the molecules which are part of the Turing mechanism itself. The Turing mechanism can affect the proteins involved in these processes in three ways: by ATP hydrolysis without phosphorylation, by ATP hydrolysis with phosphorylation-dephosphorylation,and by reducing the proteins. All the experimental data point to the fact that these three effects occur in every biochemical process. Indeed, proteins involved in all biochemical processes are affected by at least one of these effects, thus changing their

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conformation, charge, etc. and altering their activities and so modulating the process of which they are part. Thus, the possibility for spontaneous emergence of spatial colocalized nonuniformity in CAMP and ATP is precisely what one would demand if one was not aware that (CAMP,ATP) is a Turing system anyhow. Furthermore, all three spatially differential actions of the Turing field-the nonphosphorylative ATP hydrolysis, the phosphorylative ATP hydrolysis, and the reduction effect-can act simultaneously on proteins involved in all biochemical processes, thus correlating them in space and in time. The problem of the coordination of biochemical processes in time and space has been considered the most formidable problem in various fields of biology. An example of particular interest (see later discussion) is the orchestration in time and space of the translation, stability, and localization of maternal RNAs (Macdonald, 1992, and the other essays in the same issue). The very requirement of such a correlation points to the existence of only one Turing system. If there were several Turing systems-as some authors, for example, Meinhardt and Newman, believe-there would be the additional burden of coordination among the various systems. Also, in order to correlate all biochemical processes, we need molecules that are involved in all biochemical processes. This in turn suggests that the universal molecule, ATP, and also CAMP, are the most likely candidates to be the Turing morphogens. The correlation provided by the (CAMP, ATP) Turing system can explain the coherence of the phenomena of life, a feature considered the hallmark of life (Monod, 1972; Spencer, 1900; Prigogine, 1980; Nicolis and Prigogine, 1989; Davies, 1987; Weiss, 1968, 1969). Many other essentially equivalent expressions have been used, such as long-range order, functional order, the counteracting of randomness, but behind all that there is a formidable compounding of improbabilities. Not only is the decrease of entropy provided by the Turing system in itself so improbable, but the coupling of all biochemical processes to this particular decrease of entropy increases enormously the improbability of the coherence phenomenon in the phenomena of life. An example of such a coupling (dealt with later) is the coupling of ATP hydrolysis and ion translocation. ABC transporters can couple to ATP hydrolysis a transmembrane-active transport of almost every conceivable class of substrate, including ions, peptides, sugars, polypeptides, and polysaccharides. However, while this pumping is substrate-specific, according to the three-dimensional structure of the substrate binding site, the ATP binding sites are, by contrast, highly conserved throughout the superfamily of transporters (Higgins, 1993, and other papers in this issue). We can interpret this as a reflection of nature’s enabling the coupling of the transport of a multitude of substrates to the particular entropy decrease provided by the one Turing couple. The odds against the occurrence of a chemical Turing system and all these couplings are

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such that the coherence of life would be very improbable if it occurred only as a result of random processes. It has been argued by some scientists that the probability of the random occurrence of millions of nucleotides in very specific sequences, such as those found in the DNA of cells, is so small that the planet Earth has not been in existence for long enough, and therefore the specifically ordered DNA sequences can only be explained by an outside predetermining intelligence (Cohen, 1985).However, other scientists have pointed out that one should not assume the de novo formation of polymers from their constituent subunits, and that autocatalytic polymerization (self-organization) together with natural selection, substantially reduce the time for the polymerization of specific sequences [see Peacocke (1983) for a discussion of these arguments and the presentation of Eigen’s autocatalytic polymerization schemes; also Kauffman, ( 1993)l. The opinion that natural selection is not of great significance in the problem of the genesis of form, for example, as expressed by B. Goodwin in Lewin (1993), follows naturally from the opinion that there is no genetic program for development. Indeed, the school of rational morphology (Lewin, 1993) never put the emphasis on heredity but rather on the continuous nature of living phenomena (Thom, 1975). For a vivid comparison of the school of rational morphology with that of molecular biology, see Casti, (1991). However, since I have reduced the problem of the genesis of form to molecular genetics, I do need to recognize the importance of Darwinian natural selection (together with self-organization in polymerization) in order to overcome the very high improbabilities involved in the generation of the genetic program of the Turing system and its couplings. I also note that there is one element of preformation in development, also recognized by Huxley and De Beer (1934), Child (1941), and Wilson and others in Horder et al. (1986), namely, the transmission of the hereditary material with its potential for development, a potential which we now understand includes the potential for spatial self-organization via the transmitted code for the Turing system and the couplings; it is this preformation that is the reason for the “practical impossibility [so far] to derive life but only from life itself” (Guye, 1942). In fact, natural selection is the additional element in biology which results in biology not being reducible to chemistry and physics (Mayr, 1982; Harold, 1986). If for each episode of development we had to wait until the DNA responsible for the Turing system and the couplings were to assemble de novo,then we would produce only one individual per eon. It is because development is reduced to DNA and genetics, and because it takes geological time for the required and improbable DNA for development to evolve, that fundamentally, Darwinian natural selection is centrally responsible for development in opposition to some statements in Lewin (1993). Nevertheless because we now understand better accelerated polymerization via autocatalysis, we

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can envisage for the first time the creation in the laboratory of complex multicellular organisms from inanimate matter and not, as up till now, only from life itself. Ours will be the outside intelligence (Cohen, 1985) that will shortcut Darwinian evolution.

IV. Dorsoventral and Terminal Systems in Drosophila

The establishment of the dorsoventral polarity and terminal development in Drosophila embryos both involve a uniformly distributed transmembrane receptor (Toll and torso respectively; see also Fig. 2). It is also believed that the source of localization in both maternal systems involves a prior differentiation within the somatic follicular epithelium, so that the ligand for torso is localized in the terminals and the ligand for Toll is localized ventrally (Lipshitz, 1991). The source of these localizations, if they exist, is not clear. Consider, for example, ventral localization. It is suggested that although the torpedo receptor is uniformly distributed in all follicle cells, its ligand is localized because it becomes more concentrated at the dorsal side of the oocyte. So it is suggested that this latter localization in the oocyte is ultimately responsible for the later ventral localization of Toll ligand (Pawson and Bernstein, 1990; Govind and Steward, 1991; Shilo, 1992). However, the origin of the dorsal localization of the torpedo ligand is not explained. There is an a priori reason against a mechanism based on ligand localization. Even if extracellular ligand localization occurs-and this is far from proven-it is too fragile to support the localization involved in differentiation and morphogenesis (Schiffmann, 1991).In this context it is interesting to note that even relatively large molecules such as bovine serum albumin (BSA) can diffuse rapidly within the perivitelline space (Stein et al., 1991; Warn and Magrath, 1982). Also, perivitelline fluid taken from the dorsal side of Toll- embryos contains the same amount of polarizing activity as fluid taken from the ventral side (Stein et al., 1991). This paradox (St Johnston and Nusslein-Volhard, 1992)-that on the one hand the Toll receptor is uniformly distributed and on the other that the ligand seems to diffuse freely and is not inclined to preserve its hypothesized localization-is solved by assuming a limited amount of ligand and an excess of Toll so that it can sequester the ligand before it becomes delocalized (Stein et al., 1991;St Johnson and Nusslein-Volhard, 1992).A similar explanation is advanced for the terminal system (Sprenger and Nusslein-Volhard, 1992). Thus, quite apart from the original dubious localization of the ligand, the preservation of such a localization is a fragile business. A further addition to the fragility arises from the large temporal delay between the

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production of the inducing signal within the follicle cells and the fertilized egg response at a time when the follicle cells have long disappeared. The mature eggs can be held for up to 15 days before being fertilized, so the localization and activity of the follicle cell signals must remain stable for long periods of time between their synthesis during oogenesis and their activation after the egg has been laid (St Johnston and Nusslein-Volhard, 1992). In view of not only the time delay but also the replacement of follicle cells by egg coverings, the chorion, and the vitelline membrane, the preservation of localization is unlikely. The fundamental change of structure and the randomizing effects, such as thermal agitation and mechanical movement, will have destroyed the earlier localization, if there ever was one. These arguments apply to both the terminal and dorsoventral systems, and I suggest that not only the receptors but also the ligands for both systems are homogeneously distributed in the perivitelline compartment (see Fig. 2). The increase in the concentration of the ligand constitutes the bifurcation parameter, the trigger, the cause, the inducing signal that will result in the loss of stability in the homogeneous distribution of CAMP and ATP, according to the principles I stated in an earlier work (Schiffman 1991). The resulting nonuniform distribution of CAMP and ATP in the periphery of the syncytial blastoderm will be the source of localization (=nonrandomness, low entropy, order, etc.) down the line. Thus there will be no prior localization. The localization, the spatial asymmetry, will be generated spontaneously as a result of the internal instability mechanism (Schiffmann, 1991) in response to a change in a spatially homogeneous parameter. The fact that in some of the experiments in Stein et al. (1991) and Stein and Nusslein-Volhard (1992), one dorsoventral polarity is obtained and in some of the experiments the reversed-polarity pattern is obtained is not very significant from my point of view. Indeed, if the syncytial blastoderm was a perfect prolate spheroid and all the other conditions of the experiments (or real life) were perfectly symmetrical, we would expect that in 50% of the cases, one dorsoventral polarity will appear, and in 50% of the cases its reversed-polarity pattern will appear. Even a slight asymmetry in the nonperfectly symmetric syncytium or in the other conditions will favor one dorsoventral polarity or its reversed-polarity counterpart. This is not fundamental from the point of view here, which is to emphasize that the cause can be essentially symmetric and homogeneous and the effect can be of lesser symmetry and nonhomogeneous: we can have a situation that violates Curie’s principle and common intuition, that is, symmetry breaking does occur, and is the best explanation of the experimental results. Both the cause, the perivitelline space, and the effect, the responding tissue, are originally homogeneous, but the syncy-

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tium becomes nonhomogeneous spontaneously, with no prior localization in the cause, which remains homogeneous. The spontaneous emergence in many lower animals prior to gastrulation of higher metabolic activity in the ventral than in the dorsal side found by Child and others where metabolism was measured by many methods that agreed on the location of the metabolic activity, is a direct verification of my theory (Schiffmann, 1991). See, for example, Child (1941) with respect to sea urchins. Note in particular that the pattern of metabolic activity develops tangentially in the two-dimensional surface of the blastula (see the figures in Child, 1941). Another example is that of the annelids. Child and Rulon (1936; cited and discussed in Child, 1941) find for many species of annelids a very distinct ventrodorsal reduction gradient. Reduction of dyes occurs first in the midventral region with the rate of reduction decreasing from the midventral region laterally and dorsally. The reduction is particularly strong in the region of the ventral nerve cord. It is interesting that early chordate development, by contrast, manifests higher metabolism in the dorsal side (Child, 1941), which may correspond to the late arrival of the chordates and t o their having a notochord or backbone. According to Needham (19421, axiation begins on that surface of the embryo that will form the central nervous system. According to Child’s (194 1) general conclusion, all localized major development is preceded by localized higher metabolic activity. The generalization that Hox cluster and other genes are first expressed in the head region, then in the opposite pole, and later still in the intermediate region (Slack et al., 1993) follows precisely the time course of Child’s (1941) metabolic patterns as well as the predictions of the bifurcation theory of reaction-diffusion systems (Schiffmann, 1991). The observation that as the size of the induced tissue increases, more cell types and more structures become selforganized, and that below a certain minimum size only an amorphous mass of cells emerges (Nieuwkoop, 1992; Nieuwkoop, 1973, cited therein) corresponds precisely to Child’s findings and my theory (Schiffman, 1991), and to the fact that regional differences in oxygen consumption are abolished when an embryo is cut into pieces (Raddatz and Kucera, 1983). The common ancestry and homology (Slack et al., 1993) is dictated by the common metabolic Turing-Child system of positional information. Also, major differences among phyla, in particular the expression of Hox and other genes in the dorsal or ventral side (Slack et al., 1993), correspond precisely to whether the higher metabolism is on the dorsal or ventral side respectively. Returning to insects in general and Drosophila in particular, it is interesting to note that for the beetle, from egg-laying to 6 1/2 hours later, the region of highest metabolic activity is on the ventral surface, and the area

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of highest metabolism gradually becomes localized in the median ventral, prothoracic-maxillary region. The locus of highest metabolic activity in the ventral prothoracic region is the position of the differentiation center of Seidel (Child, 1941). Also, “this spread of visible differentiation from the presumptive prothorax is a very general phenomenon in insects . . . so the differentiation centre is probably very general also” (Needham, 1942). Of particular interest are the corresponding results for Drosophila hydei, in which the full-grown egg cell manifests a ventrodorsal metabolic gradient, again with the high point on the ventral side (Child, 1941). I expect that areas of high metabolic activity in the sense of Child-and it appears that in general the organizers and the differentiation centers of the classical literature are such areas-will also be the areas of high activity of nonphosphorylative ATP hydrolysis, (CAMP,ATP)-dependent phosphorylation and reduction, affecting simultaneously a variety of endogenous proteins, which will correlate and colocalize many effects. Thus, it is mainly there that transcription factors and other proteins will be phosphorylated, but it is also the place where mechanical deformation, such a invagination, will occur through the localized action of ATP or (CAMP,ATP)-dependent phosphorylation, on the apical belt of actin and myosin, or other localized effects on the cytoskeleton (see later discussion). For the sea urchin, see Fig. 49 in Child (1941), the chemical metabolic prepattern precedes and dictates the localization of the mechanical deformation, and the high points of metabolic activity correspond to the points of invagination. Exactly the same will apply universally. Thus, for Drosophila, in complete analogy to the sea urchin example, the higher metabolic activity in the median ventral region, as indicated from Child’s experiments, will result in the onset of ventral furrow formation (see Fig. l in Parks and Wieschaus, 1991), and in addition to this action on the cytoskeleton, the same field will act on other proteins, including in particular the phosphorylation (and reduction, see later discussion) of the dorsal protein, resulting in the localized formation of mesoderm. The ventrodorsal metabolic gradient will initiate simultaneously differentiation along the ventrodorsal direction (starting from the ventral side, mesoderm, neurogenic ectoderm, dorsal ectoderm, amniosersa), via graded nuclear uptake of the dorsal protein, and the invagination involved in gastrulation. The ventral midline cells will be the high-activity region for nuclear localization of the dorsal protein and invagination, and for the same reason. It is chemistry (the second messenger field) that dictates simultaneously the spatially differentia1mechanical deformation, the spatially differential gene expression, and the other spatially differential effects. A Toll receptor is structurally similar to the interleukin 1 receptor which operates via CAMP, and so it is reasonable to expect that Toll will also

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operate via CAMP. Similarly, IL-1 causes the transcription factor NF-KB to be translocated from the cytoplasm into the nucleus, where it is active. Dorsal not only manifests similar translocation, but it also manifests sequence similarity to NF-KB. Furthermore, IL-1 enhances not only the cAMP level (Shirakawa et a / . , 1989), but also protein kinase activity (Zhang et a / . , 1988). There is now evidence that the signaling pathway from Toll to dorsal involves the activation of PKA, which phosphorylates the dorsal protein and thus disrupts the dorsal-cactus complex and sets dorsal free to move into the nucleus (Norris and Manley, 1992). The involvement of a Ga-like protein in order to obtain the apical constriction necessary for the formation of the ventral furrow in gastrulation is also in accord with CAMP-dependent gastrulation (Parks and Wieschaus, 19911, and is analogous to the fact that the effect of IL-1 on cAMP accumulation presumably occurs via receptor-G-protein interaction (Zhang et al., 1988). These observations taken together indicate strongly that the signal transduction pathway that emerges for the dorsal-ventral system is precisely the one I postulated as required for the Turing instability mechanism (Schiffmann, 1991). It is also interesting to note that a similar signal transduction pathway (the interleukin system) is used in a reaction-only system to obtain activation and memory, and in a reaction-diffusion system, to obtain instability of the homogeneous state (Schiffman, 1991, Appendix). These recent molecular and genetic results, together with the observations in the classical literature for insects on the spread of visible differentiation and on a metabolic gradient from the presumptive prothorax in the ventral side, and the fragility of possible localization in the perivitelline space, indicate very strongly that dorsal-ventral polarity is established by the Turing instability mechanism I suggested (Schiffmann, 1991). One could further confirm my theory by collapsing the metabolic pattern with various agents such as cyanide (see the section on symmetry breaking and its failure in Schiffmann, 1991) and observing the collapse in the localization of the morphogenetic determinants such as the dorsal. The importance of the CAMP-system in Drosophila development is also supported by morphological defects in the development of Drosophila with a mutated cAMP system (Whitehouse-Hills el ul., 1992). Localization in the somatic follicular epithelium needs to be explained irrespective of whether it is the source of localization in the early embryo. We suggest that the Turing-Child metabolic localization is the cause of the restriction of gene expression to a subpopulation of the follicle cells. It would thus be interesting to verify that the torso-like bipolar pattern in the follicular epithelium (Martin et a / . , 1994) is caused by a bipolar metabolic pattern, which we predict will exist not only in the wild type but also in the loss-of-function mutant; such an experiment will eliminate the possibility that the bipolar torso-like pattern is the cause of the bipolar metabolic pattern.

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V. Spontaneous Endogenous Electrophoresis Polarized transport of charged protein and RNA may be of central importance in the formation of the anterior-posterior axis, in insects, for example, and my theory predicts the spontaneous formation of an endogenous electric potential gradient. Indeed, the instability of the homogeneous state results in a spatially nonuniform distribution of cAMP and ATP, which in turn implies a spatially nonuniform distribution of the rate of cAMP and ATP production [as can be seen from the substitution of the spatially nonuniform mathematical solutions u and u in equation ( 1 ) in Schiffmann (1991)l. However, from the modern field of bioenergetics (for a good review of the field, see Harold, 1986) in homogeneous systems we know that ATP synthesis is coupled to the catabolism that makes it possible. The essence of this coupling is that ATP synthesis does not occur without the catabolism, but also the catabolism does not occur without ATP synthesis. Therefore the predicted spatially nonuniform [ATP] predicts spatially nonuniform catabolism. Spatially nonuniform catabolism includes spatially nonuniform activity of the electron-transport chain, which in turn implies spatially nonuniform electric potential. This reasoning predicts that if we uncouple electron transfer from ATP synthesis, for example with dinitrophenol, the electric potential gradient will decrease. This is indeed the case (Telfer, 1975). I thus have proved that the instability of the homogeneous state in the (CAMP, ATP) reaction-diffusion system implies an electric potential gradient. It is worth noting that the intensity of electron transfer is measured in modern bioenergetics by the reduction of various electron acceptors, for example, by the reduction of Fe3+ to Fe”. It is the same spatially nonuniform electron pressute, that is, spatially nonuniform electron transfer activity, that will be responsible for both the spatially nonuniform rate of reduction of an electron acceptor and the spatially nonuniform electric potential. I also expect that regions of the embryo characterized by higher metabolic activity will be more electronegative. My conclusion corresponds to the opinions and experimental results of “Child’s school” (Child, 1941; Hyman and Bellamy, 1922) that the electric potential gradient is one expression of, and correlated with, the various manifestations of the gradient in metabolic activity. Thus, for example, the decrease of electric potential difference and of oxygen consumption caused by cyanide (Child, 1941) and, similarly, the decrease of potential difference with azide (Woodruff, 1989) results from the inhibition of the cytochrome c oxidase by these molecules, with the ensuing inhibition of electron transfer, oxygen consumption and ATP synthesis; that is, the instability of the homogeneous condition g , > 0 (Schiffmann, 1991) fails.

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The patterns of electric potential parallel the metabolic patterns and manifest similar polarity, bipolarity, and inversion, and the regions of high metabolic activity are electronegative with respect to other regions (Hyman and Bellamy, 1922; Child, 1941), as my theory predicts. For example, the oscular region in sponges is not only characterized by a higher rate of reduction of permanganate, higher oxygen uptake and CO, production, but also by higher electric negativity (Child, 1941; Hyman and Bellamy, 1922). Similarly, for the polar gradient in hydrozoa it is the apical end that manifests high susceptibility, oxygen uptake, CO, production, and reduction of electron acceptors such as permanganate, methylene blue, and indophenol. However, this higher rate of metabolic activity in the apical region also corresponds to higher electronegativity in this region (Hyman and Bellamy, 1922; Child, 1941). For annelids, a bipolar metabolic pattern follows the polar pattern, so when the two ends manifest higher metabolic activity, as measured by differential susceptibility, oxygen uptake, CO, production, and dye reduction, the two ends are also negative with respect to an intermediate region (Child, 1941; Hyman and Bellamy, 1922). An association of high metabolic activity with high electronegativity that is of particular interest to us is the determination for Drosophila that nurse cell cytoplasm is electrically more negative than the oocyte cytoplasm (Woodruff et al., 1988; Woodruff, 1989). On the other hand, the rate of reduction of methylene blue and Janus green is higher in the nurse cells than in the oocyte (Child, 1941). This experimental correlation predicted by my theory may represent a general situation. For Cecropia, again the nurse cell is negative relative to the oocyte (Woodruff and Telfer, 1980), and similarly for telotrophic ovaries, as well as for polychaetes (Nuccitelli, 1988). I predict that Child’s results (1941) for the Drosophila egg chamber-that there is a basipetal gradient of decreasing rate of reduction, where the oocyte is at the low end of this gradient-will be found in all the cases where a gradient in the electric potential has been found. I suggest that in general the battery that drives the gradient in the electric potential (Telfer et al., 1981) is the metabolic phenomenon of the Turing instability in the homogeneous distribution of the Turing morphogens CAMP and ATP. This gradient in the electric potential may provide a general mechanism which may be responsible for polarized transport of charged endogenous proteins and RNAs, in particular from the nurse cells to the oocyte. The observations that positive probe molecules move only from the oocyte to the nurse cells whereas negative molecules move in the opposite direction (Woodruff et al., 1988; Woodruff, 1989; Woodruff and Telfer, 1980; Nuccitelli, 1988; Telfer et al., 1981) conform to my theory and Child’s results. That the emergence of a pronounced

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tropharium-oocyte potential difference is fundamentally a truly epigenetic symmetry-breaking phenomenon is further supported by the requirement of homogeneous juvenile hormone (Telfer er al., 1981). Just as a basic (positive) protein will move only to the negative nurse cell and an acidic (negative) protein will move only to the positive oocyte, I would expect that basic proteins will move to the animal (apical) pole of an egg, since this pole is usually the more metabolically active (Child, 1941), and therefore more negative, whereas the acidic proteins will move to the vegetal (basal) pole. This expectation is fulfilled. Thus in the egg of the polychaete Nereis, the early stage manifests a polar metabolic gradient with a high point in the apical pole (Child, 1941) while a migration to opposite poles occurs in such a way that the basic substances accumulate in the apical region and the acid substances accumulate in the basal region, as expected; the phenomenon is attributed to the electric field based on physiological differential (Child, 1941). In molluscs as well, the apical region of the egg becomes more alkaline than the basal and metabolic polarity is also observed (Child, 1941). Similarly, for fish at the time of maturation, basic colloids accumulate apically, in the animal pole blastodisc, which becomes distinctly marked off from the acid remainder (Child, 1941). The migration of basic and acidic substances to opposite polar regions has been observed for other eggs (Child, 1941; Wall, 1990). The localization of maternal An2 mRNA to the (negative) animal pole of Xenopus eggs may correspond to the abundance of positively charged amino acids and the absence of negatively charged amino acids in the corresponding protein (Weeks and Melton, 1987). This positive charge will contribute to the localization of the mRNA-protein complex. It is significant that numb, whose asymmetric localization within the mother cell is responsible for the different fates of the daughter cells, is a highly basic protein (Posakony, 1994). It can thus be affected by the electric field, and we predict, that it is localized at the high point of an intracellular metabolic gradient. For many developing biological systems, Child’s school finds a metabolic gradient with a high activity at one pole in an early stage, and in a later stage a second gradient, opposite in direction to the original gradient, with a region of high metabolic activity at the opposite pole. Often also the bipolar metabolic pattern is such that this second gradient becomes more distinct than the first. This is, for example, the case for hydrozoans (Child, 1925, 19411, annelids, Fucus, and echinoderms (Child, 1941). A similar unequal bipolar metabolic pattern is observed in the early Drosophila embryo (Akiyama and Okada, 1992). Vital staining with rhodamine 123, as a measure of electron transport, points to the strongest respiratory activity in the posterior pole region and the weakest in the middle region. This spatially differential metabolic activity is not due to nonuniform distribution of mitochondria (Akiyama and Okada, 1992). The occurrence

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of spatially nonuniform metabolic activity, even though mitochondria1 distribution remains uniform [a similar situation is observed for the frog (Weeks and Melton, 1987) and the sea urchin (Wall, 1990)l is in accord with my theory that it is the (CAMP,ATP) Turing nonuniformity that is responsible for the spatially nonuniform metabolic activity. Consider now the spatial localization of the staufen protein. The protein is fairly basic (St Johnston et al., 1991), so we expect it to localize strongly in the posterior pole, to a lesser extent in the anterior pole, and even less in the middle. This expectation is fulfilled for the same early stage of the embryo (St Johnston et al., 1991).

VI. Localized Activity instead of Localized Distribution of Pumps and Channels

The bioelectric phenomena under discussion and in particular the origin of the electric potential gradient in eggs and embryos are currently explained by assuming the segregation of ion pumps from ion leaks in the plasma membrane and thus the generation of a transcellular ion current. It is the ion current thus generated that is considered responsible for the electric potential gradient in the egg, syncytium, or embryo, which may be used for polarized transport (for reviews of the field, see Nuccitelli, 1984, 1988; Harold, 1986). However, no experimental evidence for such a segregation of plasma membrane proteins is cited. In fact, there is no reason to assume that such a segregation actually occurs since there is no reason to assume that these plasma membrane proteins are part of a Turing system. Nongenetic, nonchemical models for the creation of nonuniformity of membrane proteins based on electrical autocatalysis are considered in Nuccitelli (1984) and Larter and Ortoleva (1982). By contrast, the explanation advanced earlier that the instability of the homogeneous state of the (CAMP,ATP) system leads to metabolic gradients that in turn lead to electric potential gradients, is experimentally supported, as we saw (in particular recall the experimental correlation between the metabolic spatial nonuniformity and the electric potential spatial nonuniformity). My theory can also explain naturally the localization of influx sites and of the efflux sites involved in the transcellular ionic current. This follows both from the geometric distribution of the metabolic electric field and the isomorphous fields of membrane permeability or activated pumps. Indeed, I noted (Schiffmann, 1991)that localized (CAMP,ATP) phosphorylation of channel proteins allows localized permeability. There is also the possibility that localized metabolic energy in the form of ATP will locally energize the pump, the ion-translocating ATPase. In fact, localized

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phosphorylation will regulate the pumps (Harold, 1986). It is important to recall that pump ATPases are unlike the F,F,ATPase, which is not phosphorylated (Harold, 1986), and is a part of the metabolic Turing system. The pump ATPases are acted upon in a spatially differential manner by the Turing system. The ion-motive ATPase, the pump, couples the hydrolysis of ATP and the active translocation of one or more ions across the membrane; this coupling is such that neither process can proceed without the other (Harold, 1986). A region with a localized high rate of ATP production, as predicted by my theory and verified by Child-type metabolic experiments, will be the region where the metabolic energy is transduced to a gradient of electrochemical potential, the region where the ion will cross the membrane against its gradient, and such a region will determine the spatial pattern of ion currents measured around eggs and embryos. It is the coupling between ATP hydrolysis and ion translocation which allows the Turing-Child metabolic pattern to impose an isomorphous spatial pattern of ion translocation across (plasma) membranes. This kind of coupling is responsible for the property of spatial and temporal coherence (nonrandomness, order, organization, low entropy) which is the hallmark of the phenomena of life. The “improbable” evolution of a Turing system would not have been so productive on its own in spatially and temporally correlating life processes if in addition such molecular coupling had not evolved as well. The coupling between ATP hydrolysis by myosin ATPase and the binding of actin, and similarly for other molecular motors (Hackney, 1992), can also occur locally because of the Turing instability. Thus, instead of a segregated ion pump, we get a locally activated ion pump. Furthermore, the localized phosphorylation and ATP may locally affect the cytoskeleton. Thus, for the prototype example of the Fucus (Harold, 1986; Nuccitelli, 1984), the site of the rhizoid outgrowth involves a localized assembly of the cytoskeleton (Harold, 1986) and is also the site of high metabolic activity (Child, 194 1); localized cytoskeleton is discussed later. Just as the positive current enters the site of the prospective rhizoid outgrowth (Harold, 1986; Nuccitelli, 1984), which is also the site of high metabolic activity and high negativity in Fucus, and a similar positive current enters the animal pole of the Xenopus oocyte (Browder, 1983, which is also a site of high metabolic activity (Child, 1941) and hence high negativity, so it enters the ventral side of the cockroach oocyte, the site where the future embryo is going to start its development (Kunkel, 1986). This corresponds to the results for the beetle cited earlier and to Child’s suggestive results for Drosophila, which we expect to be true in general for insects (unlike chordates, in which the location of the differentiation center appears to be universally on the dorsal side), that is, that the differentiation center on the ventral side is also the site of high metabolic activity. My theory explains in general why in many cases (Nuccitelli,

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1984) the entry region of the positive current predicts the site of outgrowth since in general this site is also the site of high metabolic activity. It is interesting to note that for the oocyte-nurse cell complex of the Cecropia moth, a positive current first enters the anterior pole but later it enters both the anterior and posterior poles (De Loof, 1983). This can be explained by my theory and the experimental results of Child’s school, both of which show that a bipolar metabolic (electronegative)pattern will follow a polar pattern. It is sometimes thought that an external localization, such as unidirectional light in the prototypical case of algal zygote, is responsible for the emergence of polarity (Nuccitelli, 1984; Harold, 1986). In fact, such a polarity can emerge in complete darkness without any evident relation to external factors (Child, 1941). Thus, the prototypical Fucus egg, for example, presents a striking case of symmetry breaking of a homogeneous spherical system in a homogeneous environment. This occurs only because of the presence of the Turing system in the Fucus, as is clear from Child’s (1941) experimental results. cAMP has been reported in algae (Danchin, 1993). When unidirectional light is involved in the emergence of polarity, its role is only to favor a particular spatial fluctuation around the homogeneous state, so that the rhizoid will develop on the side away from the light. However, a Turing system is still essential to amplify even this particular favored fluctuation. So in general a Turing system is an absolute requirement to amplify a random or any favored fluctuation around the homogeneous state. The attempt to extend the main concept in the current theory of electrobiology, namely, the segregation of ion leaks and ion pumps in the plasma membrane in a single cell, to multicellular embryos (Nuccitelli, 19841, is difficult and far from straightforward. Yet it is observed that “both single cells and epithelia appear to display their most active growth and pattern formation in regions of ion leaks . . . . In general, these embryonic ion currents are associated with the overall polarity of the embryo” (Nuccitelli, 1984). My theory, by contrast, is largely indifferent to whether the system is a single cell or multicellular, since, with respect to cAMP and ATP, the system is a syncytium, that is, cytoplasmically continuous due to gap junctions. The above-noted association between the location of active growth, pattern formation, and polarity of the embryo with the geometry of the ion currents simply follows from the fact that for both a single-cell and a multicellular system, it is the metabolic pattern that determines the spatial pattern of growth, differentiation, and morphogenesis, and the geometry of the ion currents. For example, strong electrical currents leave the primitive streak of chick embryos, and similar phenomena and geometry are observed for mouse embryos (Winkel and Nuccitelli, 1989); this corresponds to the geometry of the metabolic patterns in chick embryos, for example, the reduction of dyes and oxygen consumption

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are also localized at the primitive streak of chick embryos (Needham, 1931; Child, 1941; Raddatz and Kucera, 1983). Furthermore, corresponding to my theory, Zivkovic et al. (1990, 1991) find in eggs and embryos of Moflusca, localized activity [instead of the nonhomogeneous distribution of ion channels and pumps in the plasma membrane of egg cells considered in their earlier work (Zivkovic and Dohmen, 1989)] of Ca2+/MgATPase and Ca2+-stimulated ATPase. The origin of the colocalized ion fluxes and enzymatic activity lies precisely (I suggest) in a spatially nonuniform Turing-Child (CAMP,ATP) phosphorylation field that can indeed phosphorylate some Ca2+channels and pumps and alter their activity in a localized manner. It is this one Turing-Child underlying field that is responsible for the various temporal correlations noted in Zivkovic et al. (1991), for the polarity in enzyme activity and in the currents found by Zivkovic et al. (1990, 1991)along the animal-vegetal axis, and for the reversal of polarity and bipolarity also observed; all these are predicted by Turing-Child fields. Indeed, metabolic gradients oriented along the animal-vegetal axis of eggs and early embryos in molluscs have been observed (Child, 1941), and it would be interesting to repeat these experiments and to correlate these gradients in time and space with the events reported by Zivkovic et a f .

VII. Sufficiency of Child’s Results The theory of the instability of the homogeneous state of the (CAMP, ATP) system implies not only the nonuniform spatial codistribution of the rate of synthesis of CAMP and ATP, as we saw earlier, but also-because modern bioenergetics tells us that higher ATP synthesis necessarily entails higher catabolism and electron transfer in particular-an isomorphous spatial nonuniformity of catabolic activity (electron transfer in particular) and an isomorphous electronegative field (as we saw earlier). Nonuniform catabolic and electric fields are indeed found experimentally by Child’s school, as the theory predicts. Consider now the other direction of the coupling between ATP synthesis and electron transfer, namely, that electron transfer necessarily implies ATP synthesis. Many spatially nonuniform electron-transfer activities are observed by Child’s school. Therefore it is necessarily true that spatially isomorphous nonuniform ATP synthesis also occurs. This spatially nonuniform ATP synthesis is a direct prediction of the Turing instability (Schiffman, 1991). The results of Child’s school thus already provide strong experimental support for the spatially nonuniform ATP synthesis. I expect that in general a region of the embryo characterized by higher metabolism as found by Child’s various methods will also present higher ATP synthesis.

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Also, since we now know that electron transfer is often a reflection of the presence of CAMP, which is responsible for glycogen or lipid breakdown, in particular in the absence of amino acids as energy sources, Child’s type of result about spatial nonuniformity in electron transfer intensity is a very good indication for spatial nonuniformity in the rate of cAMP synthesis, which is a prediction of the Turing instability (Schiffmann, 1991). Indeed some of Child’s contemporaries reported an isomorphous (i.e., isomorphous to electron transfer intensity pattern), spatially nonuniform, glycogen-depletion pattern, which modern biochemistry would explain as a reflection of an isomorphous, spatially nonuniform pattern in the rate of cAMP production. It is important to keep in mind modern knowledge about the universal coupling between electron transfer and ATP synthesis (that one does not occur without the other), and also the universal function of cAMP in mobilizing the fuel macromolecules, in order to appreciate that the enormous body of experimental results on metabolic patterns from Child’s school and others provides strong support for my theory about the Turing instability of the (CAMP, ATP) homogeneous state (Schiffmann, 1991).

VIII. Reduction Fields

We know that regions higher in free energy production (higher rate of ATP synthesis) will also be higher in (CAMP,ATP)-dependent phosphorylation potential, in electron negativity, and in reduction potential. Just as we have argued (Schiffmann, 1991)that a spatially differential phosphorylation field can differentially affect endogenous proteins such as enzymes, channels, pumps, and transcription factors, one wonders if the spatially differential reduction potential, predicted by my theory and indeed discovered by Child’s school through the spatially differential reduction of probes such as permanganate and various dyes, can also affect endogenous proteins in a spatially differential manner, and thus constitute an additional spatially isomorphous source of positional information in addition to the phosphorylation and ATP fields (Schiffmann, 1991). This is the case and indeed not only can enzymes switch their activity according to their redox state (Li et af., 1991), but also so can TFs. Thus the AP-1 DNA binding activity of Fos and Jun is enhanced by the redox factor protein mediating the reduction of Fos and Jun (see Xanthoudakis and Curran, 1992, who also review the emerging field of redox regulation of TFs). It is interesting to note that reduction is also needed to convert the inactive cytosolic NF-KB to an active DNA binding form (Toledano and Leonard, 1991). Indeed, the dorsal protein is similar to NK-KB, and as we saw, there are indications for the beetle, cockroach, and Drosoph-

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ila-which may well be true for all insects-that the high point of reduction and phosphorylation potential occurs precisely on the ventral side, so the activation of the dorsal and analogous proteins for other insects may well involve localized phosphorylation and reduction in the ventral side. I also expect that typically the answer to the question for C . elegans of what might cause lin-11 to be active in only one of the two sister cells (Horvitz and Herskowitz, 1992) lies in a spatially differential reduction field (to be checked experimentally) acting post-translationally on a uniform distribution of the TF lin-1 1 , which indeed can be affected by such a field since it is a redox-sensitive TF (Li et al., 1991). All these conclusions are exciting not only because they further support the (CAMP, ATP) Turing instability theory (Schiffmann, 1991), which explains why and how spatially differential metabolism can arise spontaneously, but also because they correspond exactly to Child’s vision that a spatially differential quantitative and not a specific difference in metabolism will lead to a qualitative difference in a cell’s genetic fate. That quantitative difference (difference in the rate of the same metabolism) is probably the most important factor in early development was emphasized in every work of his (e.g., Child, 1925, 1941), and in all the results of his co-workers (e.g., Hyman and Bellamy, 1922). It is perhaps this point that was the least understood by Child’s critics (e.g., Spemann, 1938). Phosphorylation is central to translation (Traugh, 1989, cited in Schiffmann, 1991), and reduction may also be important in translation (Wall, 1973, cited in Wall, 1990). Thus the possibility for the spontaneous emergence of gradients in phosphorylation, reduction, and ATP may be precisely what is required for localized translation, even when the (masked) RNA is homogeneously distributed. It should be kept in mind that localized electron transfer is a direct prediction of the Turing instability and is not a result of localized translation or transcription. Consider the following spatial and temporal correlations in the sea urchin: the early blastula manifests higher rates of reduction, RNA synthesis, and protein synthesis in the animal pole; the late blastula manifests a bipolar pattern in the rates of reduction, RNA synthesis, and protein synthesis, with a dominant vegetal pole. Amphibians exhibit similar parallelisms. First there is an animal-vegetal gradient in the rates of metabolism and the synthesis of RNA and of protein. Later, a perpendicular dorsoventral gradient in the rates of metabolism and the synthesis of RNA and of protein superimposes itself on the initial anterior-posterior gradient. In general, gradients in oxygen consupmtion and glycogenolysis, as well as the sequence in time of the localization of morphogenetic movements (for example, in the amphibian, invagination begins earlier in the dorsal than in the ventral lip), also parallel the reduction gradients. For these and other such correlations, see Wall (1990), Wall (1973, cited in

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Wall, 1990), Brachet and Alexandre (1986), and Child (1941). On the basis of my work, one can now say that the autonomous metabolic field-one important manifestation of which is the reduction field-not only parallels the gradients in the synthesis of RNA and protein and the localization of morphogenetic movements, but also is their cause and driving force. The results for the sea urchin showing that the micromeres are the most active in transcription, but that all the cell types present similar rates when the cells are separated (Wall, 1990), correlate with the disappearance of the metabolic gradients in small fragments, and are explained by the dependence of the metabolic patterns on the size of the reaction-diffusion system (Schiffmann, 1991). The spatially periodic reduction fields discovered by Child and his coworkers provide strong experimental support for my theory, and I suggest that they underlie metamerism and segmentation in general. Consider Fig. 1. The peaks represent locations of high cAMP and ATP concentration and high rates of cAMP and ATP production. These peaks are also the locations of high electron-transfer activity in the mitochondria1 inner membrane and high reduction rates for endogenous proteins such as T F and indicators such as oxidized dyes. There is increased electron-transfer activity (which is responsible for the increased reduction) in these peaks because the higher rate of ATP synthesis in the peaks implies higher electron-transfer activity owing to the coupling between electron transfer and ATP synthesis, and because the higher rate of cAMP production in the peaks implies a higher glycogen and lipid breakdown, therefore a higher supply of oxidizable substrate. The troughs in Fig. 1 represent locations of low cAMP and ATP concentration, low rate of production, low degree and rate of reduction, and thus low reduction potential. Thus the peaks represent regularly spaced centers of both higher reduction and higher phosphorylation potential which locally affect, for example, reducible or phosphorylable TFs. The twelve-somite stage chick embryo provides such a periodic reduction pattern (see Fig. 3A adapted from Fig. 8 in Rulon, 1935 and cited and discussed in Child, 1941). Spatially periodic reduction of other agents such as tetrazole during somitogenesis in chicks is also confirmed in the works of N. T. Spratt (cited in Raddatz and Kucera, 1983). The figures in Hyman (1927), cited and discussed in Child (1941), also show various stages in chick somitogenesis; the periodic susceptibility patterns observed are isomorphic to the reduction patterns. The emergence of tentacles in a Hydra provides another example of a periodic reduction pattern around a ring. Figure 3C is an adaptation of Fig. 30C from Child (1941) and is the historic example cited by Turing (1952). Note that the patches of reduced dye arise at the points where the tentacles are subsequently to appear; they precede any visible morphological change. They arise at

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FIG. 3 Metabolic patterns and metabolic periodicity. (A and B) Differential reduction of Janus green by the chick embryo. When reduced. the blue-green oxidized form becomes red, which is indicated in the figure by stippling and darker regions. (A) The twelve-somite stage. Note the spatial periodicity of the reduction centers at the region of the somites. and that the lateral regions in the anterior part, which are to form the optic vesicles, are characterized by deep staining and by rapid reduction. (B) The primitive-streak stage. Note the perpendicular cephalocaudal and mediolateral gradients. (C and D) Symmetry breaking of circular symmetry into polygonal symmetry. (C) The reduction pattern that arises for the hydroid Corymorpha palma when permanganate, methylene blue, or other agents are used. Note the circular periodicity. (D) Similar circularly periodic pattern for Tubularia, adapted from Child (1941). (E and F) Periodic reduction patterns in annelids. (E) The development of a cosinusoidal wave reduction pattern in a two-zooid chain annelid; the horizontal baseline indicates no visible reduction; the height of the curve indicates the degree of reduction; anterior end at the left; F indicates fission zone. (F) A stage in the segmentation of an annelid. The periodicity of the segments is preceded by a periodic susceptibility pattern. Courtesy of University of Chicago Press and John Wiley & Sons, Inc.

the widened head end, where indeed instability is predicted to occur by the reaction-diffusion theory. Similar periodicity around a ring is seen in Fig. 3D for the hydroid Tubulariu, and was also observed in the medusa buds of Pennaria. The differential reduction of the vital dyes, Janus green and methylene blue, by annelids (Child and Rulon, 1936, cited and discussed in Child, 1941) allows one to visualize experimentally instability in action as in Fig. 1. For example, in the very early stages of zooid development, reduction

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(staining) is uniform, but as development progresses the cosinusoidal shape of the reduction field emerges (Fig. 3E). Annelid development also demonstrates metabolic spatial periodicity on a finer scale, that of segments (see, for example, Fig. 3F, adapted from Hyman, 1916 and cited and discussed in Child, 1941). This periodic pattern is observed with susceptibility methods, which we know in general from all of Child’s work produce patterns isomorphic to the reduction patterns. In all the experimental cases considered here, the number of peaks of reduction increases with the length of the linear system or with the circumference of the ring, as predicted by reaction-diffusion theory. It is reasonable to assume that the segmentation in the anterior-posterior axis of the Drosophila is also based on periodic metabolic patterns. Such patterns can result in periodic cytoskeleton patterns (see Section IX), which are indeed observed (Callaini, 1989). The (CAMP, ATP) reactiondiffusion system is operative before and after cellularization (Schiffmann, 1991). What is needed now is to continue Child’s (1941) experiments on Drosophila. Using reduction of methylene blue and Janus green, he discovered the polar reduction gradient in oogenesis, which I suggest is the basis of localizations, such as bicoid. I predict that the periodicity of the pair-rule and the segment-polarity genes is also based on the (CAMP, ATP) system. Periodicity in reduction or in CAMP (e.g., by the method in Bacskai et al., 1993) and ATP synthesis should be experimentally confirmed. The earlier metabolic patterns can account for the nonequivalence of the segments. The potential of reaction-diffusion systems in general to distinguish among similar segments is discussed in Nagorcka (1989; cited in Schiffmann, 1991). Spatial periodicity, such as in segmentation, zooid formation, somitogenesis, rhombomeres, and tentacles on a ring, as considered earlier, is but one reflection of the reduction field as the primary cause of differentiation and morphogenesis. The works on the chick mentioned (in particular those of L. H . Hyman and 0. Rulon, cited and discussed in Child, 1941) and of Raddatz and Kucera (1983)and the works cited therein, in particular those of N. T. Spratt, illustrate that the reduction fields initiate every developmental activity in the chick embryo. Thus, for example, in the primitive-streak stage, the rate of reduction decreases posteriorly from the region of the node and also laterally from the median region (see Fig. 3B, adapted from Child, 1941). The same geometry of anterior-posterior, mediolateral gradient is observed with many susceptibility methods (Child, 1941) in the pattern of oxygen consumption (Raddatz and Kucera, 1983) and the expression of a gene (Hume and Dodd, 1993), as we would expect. This situation is general and applies to the whole embryo and to organs; it also applies to other vertebrates such as the frog and the fish (Child, 1941).

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Continuing with the example of the chick, the works mentioned here show that a high reduction (and susceptibility) precedes and locally and temporarily characterizes the appearance of the optic vesicles (Fig. 3A), the otic primordia, gill invaginations, feather germs, tail bud, the heart, limb buds, and so on. When organs such as the heart or limb buds grow, they present their own typical gradients, and here too the gradient occurs before visible differentiation of the organs. Regions of greater curvature, invagination, bending, folding, torsion, and flexion are invariably preceded and characterized by higher reduction. For example, before torsion occurs in the chick embryo, the location at which it will occur is temporarily a region of high reduction (and susceptibility). In light of the present-day discussion as to whether the primary driving force is chemistry or mechanics, it is interesting to note that as early as Hyman’s (1927, cited in Child, 1941) work on metabolic gradients in vertebrate embryos, it is stated that “such developmental processes as the closure of the neural folds . . . are the result primarily of cell activity and not a consequence of mechanical conditions as supposed by early embryologists.” Spratt (1958, cited in Raddatz and Kucera, 1983) showed that the reduction patterns represent quantitative differences in intracellular enzyme activity of individual cells of the different regions and are not the result of differences in the density of cells or the thickness of the blastoderm in different regions, nor are they the result of differential permeabilities of the cells to substrates or the vital dyes. Rulon (1935, cited in Child, 1941) has already emphasized the importance of the reduction patterns observed in living embryos. Corresponding with the prediction of my theory that the regions with a high amount of reduction will coincide with those of a high reduction rate, is the observation (e.g., by Rulon, 1935, using Janus green) that these active regions show deeper red (reduced) color, and that the red color first appears in these regions and spreads most rapidly from them. An advantage of a symmetric embryonic system is that the metabolic pattern is also endowed with some of this symmetry, since it is essentially equal to an eigenfunction of the Laplacian (equivalently, a normal mode of vibration, a Chladni figure familiar from the theory of vibration of plates and membranes), or to a well-defined superposition of such normal modes. [The mathematical existence, construction, and stability of two-dimensional reaction-diffusion patterns is given in detail in Schiffmann (1975) and summarized in Schiffmann (1978)l. The pattern of gene expression will be either isomorphic to a normal mode, or to some superposition of the normal modes of the particular geometry of the embryonic system in question. Recall (Schiffmann, 1991) that earlier metabolic patterns left their trace in the patterns of the TFs that they “excited” (via a patterned post-translational modification of preexisting TFs and a pat-

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terned gene expression of new TFs) and that these patterns also mirror the normal modes of the geometry in question. A normal mode of an ellipsoid is represented in Fig. 4,which is indeed isomorphic to the Drosophila Kruppel gene expression on the Drosophila blastoderm (Schiffmann, 1991). I predict that there is an underlying metabolic pattern of the same shape present in both the wild type and in the mutant, to be checked experimentally. Similarly, some normal modes of the circular membrane are represented in Fig. 4. The existence of metabolic patterns corresponding to these normal modes can be experimentally checked in the leg imaginal disc of Drosophila. The black areas in the patterns correspond to areas of high CAMP and ATP concentration and rate of production, and a high degree and rate of reduction; that is, these areas are simultaneously areas of high phosphorylation, reduction, and nonphosphorylative ATP hydrolysis potential. Thus, for example, with Janus green, the red color will appear first in these areas, which will also be the areas with the most intense coloration. Similarly, since these areas

Metabolic normal modes

6B 0

Gene expression patterns

FIG. 4 Normal modes (eigenfunctions) of the circular membrane and patterns of gene expression in the leg disc of Drosophila. The black and gray areas correspond to expression of different genes.

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are characterized by high CAMP concentration and rate of production, they can be discerned by the method of Bacskai ef al. (1993). My theory predicts that the patterns of gene expression will either be isomorphic to the metabolic patterns, such as shown in Fig. 4, or to a superposition of them, which means gene expression in the shape of single rings, concentric rings, radial sectors, and arcs. This is indeed the experimental case (Bryant, 1993). Some of the gene expression patterns adapted from Bryant (1993) are shown in Fig. 4, which indicates that the gene expression is isomorphic to a normal mode or a combination of such modes. This discussion poses the fundamental question of whether the oftenobserved spatial-temporal correlation between differential gene expression and visible differentiation and morphogenesis on the one hand, and metabolic differential activity on the other, means that the differential metabolic pattern is the cause of differential gene expression, differentiation, and morphogenesis, or whether it is only their by-product. This question parallels old discussions, for example, Centers of greater developmental activity are said to be characterized by higher rate of metabolic activity, and when the former subsides, the latter declines, too. Increased developmental activity, therefore, seems to imply increased metabolic rate. But again we must ask: Is it admissible to reverse this statement and claim, as has been done on occasions, that gradients of metabolic activity generate developmental activity? (Weiss, 1939, cited in Schiffmann, 1991) .

Indeed, throughout the century, criticism of Child ranged from the denial of the very existence of genuine metabolic gradients or of the correlation between the metabolic gradient and development, to claiming that the metabolic gradient is not a primary factor generating developmental activity, but merely the result of development. Child and his coworkers-for example in the series of papers by Hyman on metabolic gradients of vertebrates (cited in Child, 1941)-defended in many places their thesis that the metabolic gradients are the cause and the instruments of development. They pointed out that localized metabolic activity preceded localized (visible) differentiation and morphogenetic activity, such as an outgrowth of an organ. They also pointed out that an alteration of the metabolic gradient results in a corresponding alteration in the pattern of development, for example, a loss of symmetry or polarity by the metabolic pattern will result in a corresponding loss of the same symmetry or polarity in the resulting organism. Not only can one obliterate the pattern of development by obliterating the metabolic pattern, but an induction of a new metabolic pattern will yield a corresponding new pattern of development. Many aspects of my theory (Schiffmann, 1991) further substantiate the thesis that the metabolic pattern is the cause of development. My understanding that the source of the metabolic gradient is the insta-

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bility of the homogeneous state further confirms that differential metabolism is the cause of differential gene activity and differentiation and morphogenesis in general, is not a by-product of development, and that differential gene expression and morphogenesis are not the cause of differential metabolism. Indeed, the Turing instability leading to the metabolic nonuniform pattern resides in the organization of the bioenergetic system: for example, glycolysis, the Krebs cycle, the respiratory chain, and of the signal transduction pathways. As I argued before, the metabolic system is a very improbable one and therefore unique and universal, and it simultaneously takes care of the instability of the homogeneous state and of energy homeostasis. I note here that it is essentially an autonomous system that affects the other systems via phosphorylation, reduction, and nonphosphorylative ATP hydrolysis, but that it is not specifically affected by them. For example, it affects the cytoskeleton (hence chemistry is the primary factor in morphogenesis, being the cause of the differential mechanical-elastic effects, because these effects are only derived from chemistry), but the cytoskeleton does not affect the bioenergetic and signal transduction system. The same applies to the system of TFs associated with the luxury proteins. Here too there is a one-sided relationship; TF activity can be modulated in a spatially differential fashion by the metabolic system, and there is no reason to believe that spatially differential gene expression-ostensibly caused in some as-yet-unknown way-is the cause of the spatially differential metabolic activity. My understanding of how metabolic nonuniformity arises via Turing instability further confirms that we do not need differential gene activity to obtain metabolic spatial nonuniformity. One can further verify this theory by experimentally observing that the same metabolic pattern will occur in the wild type and in various mutants associated with luxury proteins; this will also tie in with the autonomy and stability of Stern’s prepattern in genetic mosaics experiments (Ursprung, 1963). On the other hand, I predict that, analogously to what Child did, metabolic patterns can be altered or obliterated and this will result in a corresponding alteration or obliteration of differential gene expression as determined by modern methods. These suggested experiments can be visualized in Fig. 4. I predict that exactly the same metabolic patterns depicted there will be obtained for the wild type and mutants in the leg disc. The morphogenetic gene PS-1 is expressed in a restricted portion of the dorsal region of the Drosophilu wing disc, whereas the apterus ( a p ) gene is expressed in all the cells of the dorsal region of the disc. Preliminary results indicate that up is involved in the regulation of PS-f (Williams and Carroll, 1993), and this can be explained by the fact that u p encodes a T F which contains both a homeodomain and an LIM (/in-fl,Zsl-1, and mec-3)domain. The successive reduction patterns progrekivelysubdivide

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the disc (which should be checked experimentally), resulting in a differential reduction within the dorsal region, in a manner analogous to the differential phosphorylation described in Schiffmann (1991), and this will affect up in a spatially differential manner, due to its redox-sensitive LIM domain. This would be another example of differential metabolism dictating morphogenetic movement. An increasing number of TFs with the LIM domain are being discovered and are involved in differentiation (Barnes et al., 1994). It is reasonable to assume that in all these cases the LIM metallodomain functions in a redox-sensitive regulation of transcription (Li et al., 1991).It is satisfying to conclude in general that the reduction gradient, which was only one manifestation of Child’s physiological gradient, is directly instrumental in obtaining differential gene expression.

IX. The Metabolic Field and Cytoskeleton Localization

The generation of asymmetry in a syncytium or a cell like an oocyte or an egg, through the localization of cytoplasmic determinants such as mRNA, can lead, by subsequent cleavage, to blastomeres with different fates. There has been great interest in the involvement of the cytoskeleton in this localization (Jeffery, 1989; Gottlieb, 1990; Singer, 1992; Bearer, 1992). Three obvious models (which are not mutually exclusive since different types of cytoskeleton may be involved simultaneously) can be considered (Strome, 1986a): (1) The determinants move along oriented bundles, which raises the question of where the direction of the polymerization of the cytoskeleton monomers originates. (2) The determinants, although moving randomly, adhere to localized cytoskeleton, for example via a 3‘ untranslated portion of mRNA capable of forming extensive secondary structure (Macdonald and Struhl, 1988). (3) The determinants are repelled from the localized cytoskeleton. Models (2) and (3), corresponding to the pull-and-push models of Strome (1986a), raise the question of the origin of the spatially differential cytoskeleton. Why should the cytoskeleton assemble in one particular region of the egg and not in another? While all three events in ( I ) , (2), and (3) seem to occur, the general conclusion is that (2) is the most prevalent. Thus Jeffery (1989) concludes that mRNA tends to be localized in regions rich in cytoskeletal architecture and that regions of mRNA localization appear to coincide with egg cytoskeletal domains. The prototypical case of Drosophila bcd RNA presents such features: “the sites of accumulation of bcd message . . . are the sites of highest microtubule density” (Pokrywka and Stephenson, 1991). “The gradient in microtubule density during stage 8 through 10 closely mirrors the distribution of bicoid mRNA in the oocyte . . . thus, microtubule

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binding alone could be the basis for bicoid localization” (Theurkauf er a / . , 1992). It is thus most important to find the source of the spatially differential localization (assembly) of the cytoskeleton. I suggest that the Turing (CAMP, ATP) field is precisely of the kind that can be and is indeed responsible for such a localization. Indeed, it is emphasized (Korn et a / . , 1987; Carlier, 1992; Mitchison, 1992) that actin filaments and microtubules are not equilibrium structures. Whereas expenditure of metabolic energy by the hydrolysis of nucleoside triphosphates is not usual in noncovalent assembly processes in biology, such as in the polymerization of hemoglobin or the assembly of virus coats, for example, the assembly and maintenance of microtubules and actin filaments require continuous energy input, which ultimately derives from ATP hydrolysis. The need for continued hydrolysis of ATP results from the requirement of ATPfor the polymerization and the fact that ATP is not resynthesized when the filament depolymerizes. Thus, unlike many ATPases that are also ATP synthase, the cytoskeletal polymers are only ATPase. With this in mind, recall that one manifestation of the Turing-Child metabolic pattern is a spatially differential rate of ATP synthesis. Thus we expect that in any region where Child finds high metabolic activity, by any of his methods, invariably that will also be a region with a high rate of ATP synthesis, which is also a region favorable to the assembly of microtubules and actin filaments. This localized assembly will be a steady-state, nonequilibrium dissipative structure, surviving on the localized higher rate of ATP synthesis and persisting only as long as this localized higher metabolic activity persists. The spatially differential metabolic pattern changes with time (Schiffmann, 1991) and this will dictate the spatial and temporal course of the cytoskeletal assemblies. The statement “In contrast to most other biological polymers, the major functions of both microfilaments and microtubules require spatially and temporally regulated depolymerization as well as polymerization” (Korn et al., 1987) compliments the fact that depolymerization is essential for the rapid following of the cytoskeleton pattern of the underlying changing metabolic pattern. This dynamic nature of the cytoskeleton spatial pattern, namely, that cytoskeletal structure appears here and disappears there, has been emphasized by many authors, for example, Harold (1986), and is of great practical importance, since the localized cytoskeleton has t o disappear once it has done its directive task. This also follows from the fact that I believe that the same Turing-Child fields are responsible for the orientation and localization of the cytoskeleton required in mitosis and cytokinesis in germ and somatic cells. Thus, for example, it is obvious that the mitotic spindle and the contractile ring should promptly disappear once they have done theirjob. The polymeriza-

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tion of actin filaments requires ATP, whereas the polymerization of microtubules requires GTP, but the Turing-Child field involves localized ATP synthesis. However, this does not detract from my theory because a localized high rate of ATP synthesis will imply a localized spatially isomorphous high rate of GTP synthesis, since there will be a higher rate of localized transfer of the phosphate group from ATP to GDP. So far I have considered the effect of the ATP field, that is, a spatially nonuniform rate of ATP hydrolysis, without phosphorylation. However, the other manifestation of the same Turing-Child field is the (CAMP,ATPIdependent phosphorylation field. This field is also precisely of a kind to affect cytoskeletal organization in a differential manner. Indeed, (CAMP, ATP) phosphorylation operates on microtubule-associated proteins and thus may result in enhanced microtubule nucleation, stabilization, and elongation and may affect the interaction of microtubules with other types of cytoskeleton and other cell ,components (Dustin, 1984; Olmsted, 1986). Of particular interest is the growing recognition that intermediate filaments are also dynamic structures that also involve continuous incorporation of subunits and that (CAMP, ATP) phosphorylation is of central importance in the assembly of IFs and their interaction with other proteins, organelles, and other cytoskeletal elements. Evidently, the reversibility of the phosphorylation contributes to the dynamic nature of the IF network. The IFs undergo dramatic structural modifications during different phases of the cell cycle or during differentiation, and such changes in IF organization coincide temporally with changes in the extent of IF phosphorylation. One such change is the organization of the IF network during mitosis into a cage-like structure that surrounds the spindle. In general, phosphorylation of IF may be involved in the compartmentalization by IF of regulatory molecules (Skalli et al., 1992; Eriksson et al., 1992; Schliwa, 1986). These conclusions also correspond to Jeffery’s (1989) general conclusion about morphogenetic mRNA determinants: “Presently, IF are the most reasonable candidates for a localization receptor.” The possibility of spatially differential (CAMP, ATP) phosphorylation potential (Schiffmann, 1991) accounts precisely for these general conclusions with respect to the role of IF in the localization of cytoplasmic components. It has been suggested (Gordon and Brodland, 1987) that the initial event in differentiation is mechanical followed by the synthesis of specific proteins. Similarly, it has been suggested (Goodwin and Trainor, 1985) that it is the mechanochemical instability that is responsible for the spatially differential metabolism. By contrast, I suggested (Schiffmann, 1991) that it is the chemical (metabolic) spatial nonuniformity-as expressed by the possibility of instability of the homogeneous state in the (CAMP, ATP) system-that primarily dictates the mechanical spatial nonuniformity, and

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not the other way round. One example of this general thesis is the possibility of localized contraction of an apical belt of actin and myosin (Schiffmann, 1991). A further example is my postulate that it is the Turing-Child metabolic field that determines the spatial patterns of expression of adhesion molecules, which may in turn determine, at least in part, the pattern of morphogenetic movements and association among cells in the early embryo. IFs provide another example (Skalli et al., 1992; Eriksson rt al., 1992). IFs such as vimentin, whose assembly is precisely regulated by (CAMP,ATP) phosphorylation, present unique viscoelastic properties, and therefore spatially differential extents of phosphorylation would result in spatially differential stiffness, that is, the chemical spatial pattern determines the mechanical spatial pattern (morphogenesis). My theory in which the electrical (as we saw earlier) and mechanical fields are primarily dictated by the chemical field is in the spirit of Turing’s work and is the opposite of “Morphogenesis without Morphogens” (Goodwin, 19851, where the primary initiation of the pattern, the instability, derives from the mechanics and not from the chemistry. These OsterMurray-Goodwin continuum mechanical theories of morphogenesis sprang at least partially from the inability to identify the Turing morphogens: “It is possible that primary morphogenesis [dynamic instabilities] occurs solely as a result of the field properties of the cytoskeleton and associated modulators of its state (e.g., calcium), and that there are no morphogens of Turing type, thus explaining why they have been so elusive” (Goodwin, 1985). Similarly: “A serious drawback of the reactiondiffusion approach has been the elusive nature of the morphogens involved. . . . With the drawbacks of the reaction-diffusion approach. . . . Oster and Murray et al. proposed a mechanical approach to biological pattern formation” (Bentil and Murray, 1993). If indeed it is the case that the instability is derived from the mechanics rather than from the chemistry, then my claim that there is a genetic program for development and that development is reduced to molecular genetics would be more difficult to sustain. It is said that explaining morphogenesis by invoking a mechanochemical instability has the advantage that it “eliminates the hypothetical ‘reading’ or ‘interpretation’ of prepatterns which is poorly defined in current models” (Goodwin and Trainor, 1985). Even if this were the explanation of morphogenesis, it is hard to envisage how this mechanochemical instability can explain differentiation; in fact, the explanation of differentiation does require the interpretation of prepatterns. I suggest that chemical instability is the primary cause of both morphogenesis and differentiation and is responsible for the correlation between them in space and time. To what extent does the spatial localization of the Turing-Child meta-

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bolic pattern correlate with the spatial localization of the cytoskeleton as predicted by my theory? The prototypical case of an alga such as Fucus can be invoked again to provide an example of such a correlation. Compare the metabolic patterns for the Fucus in Child (1941) with the localized patterns of F-actin in Brawley and Robinson (1985). There is complete correspondence. Not only do the polar patterns with localization in the presumptive rhizoid pole correspond, but so also do bipolar patterns with localization in both the presumptive rhizoid pole and the presumptive thallus pole. In fact, sometimes an even greater amount of F-actin was found in the thallus pole, which corresponds to the observation that often the second metabolic pole becomes even stronger than the first. Keeping in mind that the dotted regions in Fig. 26 of Child (1941) are also regions of enhanced rate of ATP synthesis (because of the coupling between electron transfer and ATP synthesis), which is in turn responsible for locally enhanced microfilament polymerization, this explanation replaces the one advanced in Brawley and Robinson (1985) and Harold (1986). The often-observed polarity of the metabolic, gradient along the animalvegetal axis (Child, 1941) would, according to my theory, dictate that the polarization of cytoskeletal organization would also be along this same axis. This prediction is indeed observed; see the animal-vegetal gradient in cytokeratin organization for the frog (Fig. 15, Jeffery, 1989) and this gradient in IF organization is indeed, at least in part, responsible for the localization of VgZ RNA (Pondel and King, 1988). Also for Xenopus oocytes a strong correlation between the size of an oocyte and the degree of localization of V g l mRNA is observed (Yisraeli et al., 1990, and earlier works). Below an oocyte diameter of 0.55 mm, V g l RNA is uniform and above this threshold it begins to localize. This situation corresponds to my theory (Schiffmann, 1991), where we saw that in order to get an instability of the homogeneous state, according to the bifurcation theory of reaction-diffusion systems, the system needs to be of a minimum critical size. We also saw in Schiffmann (1991) that Child found that to get a polar metabolic gradient he too needed to have a system of a minimum critical size. In connection with the statement that “it is interesting that oocytes cultured in medium alone without any serum are unable to localize VgZ mRNA. How serum stimulates localization of the message remains a mystery” (Yisraeli et al., 1990), I note that one function of the serum might be to provide an external factor such as growth factor (GF) which is the homogeneous bifurcation parameter, that is, the cause, for the localization and symmetry breaking. The experiments in vitro and in viuo in Yisraeli et al. (1990),in which VgZ mRNA (as well as other components) is homogeneously distributed in the early oocyte, and the ambient solution

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is also homogeneous and yet localization of V g l mRNA occurs, manifest the occurrence of the “unthinkable” that drove Driesch to vitalism, but I can now explain them (Schiffmann, 1991). Among the ways to further verify my theory is to collapse the Turing-Child metabolic gradient (for example, with N3-, CN- or CO; see the section, “Symmetry breaking and its failure” in Schiffmann, 1991), and to observe that V g l RNA localization as well as the gradients in cytoskeletal organization will also collapse. Similar experiments can be carried out for other systems such as Drosophila. I expect that the collapse of the metabolic patterns will also result in the collapse of the localization of all other cytoplasmic determinants such as bicoid mRNA as well as the collapse of cytoskeletal localizations, such as the gradient in microtubule density referred to earlier. So far I have dealt with models (2) and (3) mentioned in the beginning of this section. However, my theory can also explain model ( l ) , namely, the origin of the orientation of the bundles. Indeed, if the continued polymerization requires ATP (or GTP, which is ultimately a requirement for ATP as we saw earlier) or (CAMP, ATP) phosphorylation, then the polymerization will preferentially occur in the spatial direction of maximum increase in the rate of ATP synthesis or in the rate of (CAMP, ATP) phosphorylation. Corresponding to this explanation are the animal-vegetal orientation and the anterior-posterior orientation of the metabolic gradients for the frog and Drosophila respectively (Child, 1941), which also correlate with the localization of different determinants in the animal and vegetal poles (Weeks and Melton, 1987), and similarly in the anterior and posterior poles (St Johnston and Nusselin-Volhard, 1992). It seems that indeed in general the orientation of the filaments is in the direction of the steepest gradient of the metabolic field (Madden et al., 1992; Harold, 1986). The polarity of the filament will also be a dynamic and changeable property following the metabolic patterns. Here too we can check experimentally whether collapsing the metabolic gradients with CN- will abolish the polarity and orientation of the filament.

X. Metabolism of Proliferation versus Metabolism of Differentiation and Morphogenesis In differentiation, the DNA is used for differential transcription whereas at the cleavage stage it is used for rapid synchronous cycles of DNA replication, and it makes sense to assume that it is difficult for both to occur simultaneously (Lamb et al., 1991). Dworkin and Dworkin-Rastl (1991) find for the frog that during the cleavage stage, amino acids are the

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main energy and carbon source, and glycogen catabolism begins near the onset of gastrulation. Their concluding paragraph states: Glycolysis is activated in embryos at approximately the onset of gastrulation and before morphological differentiation, and we speculate that these may not be independent events. In particular, some aspect of the metabolism of the cleaving embryo may not be permissive for differentiation, while a glycolytic metabolism is permissive. Thus, the significance of pyruvate formation from mitochondria1 amino acids in cleaving embryos may be the ability of this metabolism to support high rates ofcell division while simultaneously repressing differentiation.

This conclusion fits with my theory, since the triggering of the glycogen catabolism means the triggering of the (CAMP,ATP) Turing system used in the ensuing differentiation and morphogenesis. It is this major transition in metabolism that makes spatial nonuniformity in the same metabolism possible. The “organizer,” for example, the dorsal lip of the blastopore of the frog or Hensen’s node of the chick, is characterized by the highest rate of glycogen catabolism (Child, 1941; Needham, 1931; Schiffmann, 1991). The organizer of the frog, the chick, and the mouse is also the region of the maximum expression for the homeobox gene goosecoid (Izpisda-Belmonte et al., 1993). Thus the change in metabolism just prior to gastrulation may be universally responsible for the homologous morphogenetic movement and the homologous localized homeobox gene expression involved in the onset of gastrulation. A similar transition from amino acid metabolism to glycolytic metabolism is observed for other animals (Leese et af., 1993). Cancerous cells, like cleaving embryos, also manifest amino acid metabolism (Dworkin and Dworkin-Rastl, 1991). This corresponds to the many examples (Weiss and Strada, 1973; Friedman, 1976; Cho-Chung, 1992) in which cancer is characterized by a low level of cAMP and a defective response mechanism to CAMP, whereas differentiation agents involve a rise in cAMP and cessation of growth. Cancer can be considered as a regression to the cleavage stage of the embryo. In a very real sense it represents the failure of the biological defiance of the second law embodied in the lowering of entropy due to cell differentiation. “Differentiation therapy” (Cho-Chung, 1992) represents an attempt to reverse this failure precisely by restoring the Turing morphogenetic system. The view of cancer as a regression into a metabolism that is no longer able to provide for division of labor and specialization conforms to the view of life according to the philosophies of Spencer and Bergson invoked earlier. The inhibitory effect of (CAMP, ATP) on meiotic cell division, that is, on oocyte maturation, is analogous to the inhibitory effect of (CAMP, ATP) on normal cell division, that is, on somatic cell mitotic entry (Lamb et af., 1991). According to my theory, the initiation of oocyte maturation

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by progesterone, which involves the inactivation of the adenylate cyclase (Browder, 1985),also involves the deactivation of the (CAMP,ATP)Turing system. M y theory then predicts a decrease in the physiological gradients, and the differential properties, which as we saw earlier, depend on these gradients. Indeed, it is observed for the Xenopus oocyte that the current that enters the oocyte in the animal pole and exits from the vegetal pole decreases dramatically within minutes of treatment with progesterone (Browder, 1985). The possibility that in insects ecdysone may be the physiological equivalent of progesterone in amphibians has been suggested (De Loof. 1983). It thus seems that for Xenopus-and perhaps more generally-glycogen breakdown, and the accompanying activation of the (CAMP,ATP) Turing system, occur not only after the midblastula transition when the rapid cell division rate slows down, but also before the prophase of the first meiotic division. In between, glycogen is synthesized. This is also supported by the finding that very little UDP-glucose is formed in stage I1 Xenopirs oocytes, but that in stage VI oocytes, UDP-glucose does form (Ref. 1 1 in Dworkin and Dworkin-Rastl, 1991).

XI. Concluding Remarks: The Reducibility of Development t o Molecular Genetics

Returning to my assertion that development is reducible to molecular genetics, equivalently that there is a genetic program for development, I adopt P. Weiss’s definition for such reductionism, namely that “the true test of a consistent theory of reductionism is whether or not an ordered unitary system (a cell being such a system) can, after decomposition into a disordered pile of constituent parts, resurrect itself from the shambles by virtue solely of the properties inherent in the isolated pieces” (Weiss, 1968). I can indeed say that an equilibrium structure such as the ribosome is reducible to the DNA base sequence, since the latter is clearly responsible for the process of self-assembly (Maynard Smith, 1986; Casti, 1991; Lewin, 1984). However, self-assembly, Weiss emphasizes, deals with a static structural character

. . . neglecting the inseparable complementarity between structure and process in the living system, in which processed structure is but an outcome of structured processes. The fact that diverse activities of a definite pattern can coexist and go on concurrently in the space continuum of the cell even in the absence of tight compartmentalization, reveals that although only afraction of the cellular estate is strictly structured in a mechanical sense, there still is

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coordination among the diverse biochemical processes, which evidently must remain relatively segregated and localized. So, here we are back again at the question asked before: Coordination, how and by what? (Weiss, 1968) The spatially differential coherent and coordinated chemical processes cannot, according to Weiss, be explained by the molecular biology sequence DNA + RNA + protein which ‘‘still would leave us only with a random bag of molecular units milling around in thermal agitation” (Weiss, 1968, 1969). This corresponds, for example, to Nijhout’s statement that “genes are passive sources of materials” (Nijhout, 1990). Therefore according to Weiss and others, development and biological coherence are not reducible to molecular genetics. It is instructive to explicitly note that in fact development is reduced to DNA precisely according to Weiss’s own test cited earlier. Consider, for example, the most intriguing stage, the very beginning of development, and attempt to “answer the familiar question of how the first critical differentials may have come about when the egg was still a single cell, uniformly exposed, as it is in many cases, to an environment devoid of the kind of systematic differentials to which one could ascribe a differentiating effect” (Weiss, 1968). This initial heterogeneity of the egg is crucial not only for Weiss’s concept of development, but also even more so for recent authors, who attempt to explain epigenesis on the basis of inductive interactions. Even they still have to concede that “ ‘epigenesis’ implies an increase in complexity of the developing egg and embryo through interaction with its environment and among its constituent parts. However, ‘inductive interaction’ in addition presupposes a minimal heterogeneity in the egg in order that interaction may occur between its different moieties. This initial heterogeneity represents the indispensable ‘preformistic element’ in development” (Nieuwkoop et al., 1985), but we can dispense with this “preformistic” element within “molecular biology.” Weiss’s disordered pile of isolated pieces occur in fact in the beginning of every ontogeny if we consider the egg early enough, as Driesch has already observed. This homogeneity and randomness of the early egg is confirmed in all the cases in which it was checked at the molecular level, for example, by Melton and co-workers for Xenopus (Yisraeli et al., 1990, and earlier works), or for C. elegans by Strome (1986b). Also, the concept of a cortical map (Weiss, 1973; Wall, 1990) still presents great difficulties (Wall, 1990). This includes the fact that normal development is obtained after centrifugation that dislodges the cortical components, and the observation that not only are cytoplasmic components distributed randomly and homogeneously, but also that the oocyte surface components are uniformly distributed (Strome, 1986b). The more recent concept of localized signal transduction, for example, via a localized ligand, was dispensed with earlier. Therefore, true symmetry breaking in the egg must occur if

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Weiss’s test for reducibility is to be upheld. Furthermore, the “resurrection” in Weiss’s test is also upheld when we recall that often after centrifugation the normal egg structure is reestablished and that normal development is not disrupted (Morgan, 1927; Jeffery, 1989; Wall, 1990). The required symmetry breaking of an originally homogeneous or homogenized egg, or the reestablishment of centrifuged egg, will be effected by virtue of the principle of the instability of the homogeneous state. According to my theory, two conditions are required to activate this instability. The first is the activation of the respiratory metabolism, which can indeed occur at various stages of egg development such as maturation, ovulation, oviposition, or at fertilization (Wall, 1990). The second condition is that the system (the egg) must be above a minimal size; this condition is also experimentally verified, as we saw earlier, in particular in the case of the frog oocyte (Yisraeli et al., 1990, and earlier works). Mitochondria in eggs and early embryos are of maternal origin and their early distribution is uniform even though a spatially differential respiratory activity can be present (Weeks and Melton, 1987; Strome, 1986b; Akiyama and Okada, 1992; Wall, 1990). Thus the complete Turing system is already present in the egg even before fertilization and can be activated before fertilization in some cases. This can also account for “differentiation”, such as considerable animal-vegetal organization in unfertilized or even enucleated eggs (Wall, 1990). Oxygen is required for such “differentiation”; for example, pseudogastrulation does not occur without O2 (Smith and Ecker, 1970, cited in Wall, 1990). Significantly, it is only the fragment of the ascidian egg with the oxygen-consuming mitochondria that develops (Wall, 1990); mere production of ATP by glycolysis is not enough. Recall the discussion of the Warburg and Schidlowski thesis on “oxygen, the creator of differentiation.” I have thus shown that the principle of the instability of the homogeneous state embodied in the oxygen-consuming mitochondria can overcome the random thermal agitation which was the essence of Weiss’s objection to the reduction of development to the DNA. Weiss’s objection to reducibility includes the need to resurrect in a coordinated manner and simultaneously the localized structure and the localized process. Indeed, the instability of the homogeneous state results in a dissipative structure (Prigogine, 1980; Nicolis and Prigogine, 1989), significantly also called a process structure (Jantsch, 1980) or flow structure (Katchalsky, 1976), which by its very nature ties together the process and the structure as required by Weiss’s test. Also corresponding to this is Child’s emphasis (Child, 1941; see also Child, 1929, cited in Schiffmann, 1991) that his metabolic patterns are unlike a crystalline structure, and the pattern is dynamic and depends on continuing-chemical reactions, an aspect also emphasized by Turing, as we saw earlier. Furthermore, Weiss’s requirement that all biochemical processes be spatially correlated

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is fulfilled by virtue of the fact that the fundamental (CAMP,ATP) dissipative structure carries with itself reduction, phosphorylation, and free energy fields that act simultaneously on the proteins involved in all biochemical processes: this is the source of the spatial coordination that Weiss was looking for. Thus the Turing field embodied in spatially differential respiration is the sole source of nonrandomness and coherence from the very beginning of development. The beginning of development in an egg with mosaic properties presents a major spatial correlation problem (Wessells, 1982), namely, how the cytoplasmic localization and the cleavage pattern are so precisely correlated, which is of course essential for the differential distribution of the determinants to the blastomeres. Components of the cytoskeleton are manufactured during oogenesis; for instance, tubulin (the subunit of microtubules) is a major soluble protein of mature Drusuphilu eggs (Wessells, 1982). Similarly, the C . eleguns oocyte provides the components of microtubules and microfilaments (Strome, 1986b). Since I have argued that the Turing field will dictate the spatial pattern of cytoskeleton assembly, I can now say that the maternal components of cytoskeleton manufactured during oogenesis will be assembled in a spatial pattern such that it will simultaneously be responsible both for the localization of the cytoplasmic morphogenetic determinants in the egg (via their association with the egg cytoskeleton) and for the pattern of cleavage. Furthermore, it is the same Turing field, which also manifests spatially differential electric potential, that will also be responsible for the possibility of polarized electrophoretic transport of morphogenetic determinants within the egg. Thus it is the same Turing field that is responsible for all aspects of the spatial correlation in question. It is important to note that the self-organization of the underlying (CAMP, ATP) dissipative structure, and the localizations and processes it supports (the couplings discussed earlier), is akin to the self-assembly of equilibrium structures referred to earlier, in the sense that both depend only on the three-dimensional structure of proteins, that is, on the DNA base sequence. We have here exactly the fulfillment of Monod’s (1972) anticipation cited earlier. Thus, for example, one contribution to the condition of the Turing instability of the homogeneous state, gh < 0, depends on the inhibitory allosteric site of the phosphofructokinase to which ATP binds. However, this structure is dictated by the DNA sequence. My reduction of development to a genetic program corresponds also to the definition for such a reduction by Stent (in Lewin, 1981) and Stent (1982), namely, that there should be a one-to-one correspondence between the DNA base sequence and the phenomenon (development). It seems that Stent was led astray in concluding that development is not reducible to a genetic program by an overfascination with semantic information theory

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which attempts, in the context of linguistics, to complement information theory with meaning derived from the context-dependence. He analogously claims that only protein primary structure is programmatic since it is isomorphic to the DNA base sequence. However, the higher levels of protein structure as well as development, contends Stent, are not programmatic since there is no isomorphism with the DNA sequence because of the context (environment)-dependence. Stent’s arguments are adopted and cited by other workers who also object to a genetic program for development (Sheldrake, 1989; Goodwin, 1989; Newman and Leonard, 1983; Newman, 1988) and it is important to realize the irrelevance of these arguments. Indeed, all levels of protein structure are entirely determined by the DNA base sequence in normal physiological conditions, and it is irrelevant for the problem of reduction of development to a genetic program to consider extreme environmental conditions. Beyond protein structure, we have to invoke the principle of the instability of the homogeneous state (and the fact that the Turing field acts on the genes) in a manner analogous to invoking the principles of self-assembly in the case of equilibrium structures. Thus both equilibrium structures, such as a ribosome, and development are in one-to-one correspondence with the DNA, and in both situations there is a reduction to a genetic program according to Stent’s own test. I also note that the DNA dictates the equilibrium structure (e.g., phosphofructokinase as mentioned earlier and the rest of the molecular machinery that effects the instability of the homogeneous state) which in turn dictates a nonequilibrium dissipative structure, which in turn dictates development. One can refute all arguments raised in the literature against the reduction of development to a genetic program. For example: “It [the notion of a ‘genetic program’ for development] confuses the valid concept that hereditary phenotypic dgferences between organisms are correlated with particular genetic differences, with the erroneous deduction that development itself can be explained by the action of genes” (Goodwin, 1989). This argument is often raised to oppose the reduction of morphogenesis to genes and also appears in current genetics and in evolutionary biology as well as development (Nijhout, 1990, and references cited therein). The same argument had already been advanced and discussed in detail, for example in Weiss (1969), where it is argued that because it is true that the differences between a blue eye and a brown eye (for example) correspond to differences in the genetic material, it is erroneous to deduce that the “formative dynamics” that result in such an ordered structure as the eye itself, is also inscribed in the genes. In fact this argument of Weiss, Goodwin, and Nijhout against the reduction of development to a genetic program is itself invalid and erroneous

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Index

A ADP-ribosylation, neurofilament protein modulated by, 43 Amphibians, differentiation systems, 61-62 ATP, and instability of homogeneous state Child’s results, 346-34 colocalization role, 313 concentration, 31 1-312 Drosophila, 338 localized activity of pumps, 343- 345 metabolic field, 356-360 metabolism of proliferation, 361-362, 364-365 reduction fields, 347-348, 350, 352, 354 spontaneous endogenous electrophoresis, 340-342 Turing biochemical mechanism, 323-324, 326-327, 330-333 Turing-Child metabolic system, 310

C Calcium phosphorylation-altered activity, 345-346 post-translational modification, intermediate filament protein activation by, 46-47 Cecropia, instability of homogeneous state, localized activity, 344 Chromosomes DNA replication, eukaryotic, see DNA replication, in mammals sex, see Sex chromosomes, mammalian Cloning, of testis-determining factor SRY gene, 233-237

Cyclic AMP and instability of homogeneous state Child’s results, 346-347 colocalization role, 313 concentration, 31 1-312 Drosophila, 338-339 localized activity of channels, 343, 345 metabolic field, 356-360 metabolism of proliferation, 361-362, 364-365 reduction fields, 347-348, 350, 352-353 spontaneous endogenous electrophoresis, 340-342 Turing biochemical mechanism, 323-324, 326-328, 331-333 reverse transformation of malignant cells by, 44 Cytokeratin, filament role in differentiation systems, 54-57 Cytoplasmic determinants, localization, and metabolic field, 355-360 Cytoskeletal elements, gene-regulatory function, see Intermediate filament proteins, gene-regulatory function Cytoskeleton, localization of cytoplasmic determinants, instability of homogeneous state and, 355-360

D Desmin, muscle differentiation by, 49-52 Development and differentiation systems, 60-62 postnatal epithelial clear cells, 173-175 principal cells, 169-170

377

378 reducibility to molecular genetics, 362-367 DFR, see DNA fiber radioautography Diacyl glycerol, vimentin binding stimulated by, 45 Differentiation amphibian, 61-62 characterization of systems, 48-49, 62-64 cultured cells, 58-59 cytokerdtin filament role. 54-57 extension of cellular processes, 53-54 gonadal. see Gonadal differentiation leukemia cell, by vimentin, 52 mammalian, 60-61 metabolism of, versus metabolism of proliferation, 360-362 muscle, by desmin. 49-52 redundancy of intermediate filament protein function, 64-66 transgenic animals, 59 DNA and instability of homogeneous state metabolism of proliferation, 360 reduction fields, 347 Turing biochemical mechanism, 324 intermediate filament proteins and cytokeratin filaments, 54-57 cytoskeleton role in reverse transformation, 40 double nuclear membrane barrier, 27-28 evolutionary aspects, 69-70 loop formation, 23-24 as matrix elements in gene expression, 25-26 muscle differentiation, 51 nuclear functions, 5-7 nuclear organization, 7-12 post-translational modification, 48 redundancy of function, 65-66 repetitive DNA sequence interactions, 32-37 transcription factors and similarities, 13-19 methylation, gene inactivation by, 209, 212 and testis-determining factor genes, 230-23 I Z-DNA formation, 37

INDEX

DNA replication. in mammals chromosomal organization, 263-265 DNA fiber radioautography analysis basic principles. 265-269 critical data. 269-274 double-pulse labeling prior to, 274-280 method, 263 DNA organization, 301 molecular mechanisms, 26 1-252 process, 261 replication fork rate, 286-292 replication unit size, 277, 281-286 replicon model, 294-301 replicon termination, 292-294 DNA fiber radioautography analysis basic principles, 265-269 critical data, 269-274 double-pulse labeling prior to. 274-280 method, 263 replicon model, 296 Drosopliila instability of homogeneous state dorsoventral system, 335-339 localization, 322, 344 metabolic field. 310, 355, 360 oxygen-driven differentiation balance, 326 reduction fields, 347-348, 352, 354 terminal system, 335-339 testis-determining factor SRY generelated sex determination, 242 zeste gene product function, 65 matrix element, 24-26

E Efferent ducts testis, structure and function, 131, 137- I46 epithelial nonciliated cells, 137-146 Electrophoresis, spontaneous endogenous, analysis of instability of homogeneous state, 339-342 Endo B, expression by antisense RNA, 64 Endocytosis, sperm maturation role adsorptive endocytosis epithelial cells of efferent ducts, 137- 141

INDEX

rete epithelial cells. 131-134 Sertoli cells. 117-1 18 epididymal clear cells, 170-177 epithelial cells of efferent ducts quantitative studies, 145-146 sulfated glycoprotein-I, 139, 143- I45 sulfated glycoprotein-2. 139, 142 fluid-phase endocytosis epithelial cells of efferent ducts. 137-141 rete epithelial cells. 131-134 Sertoli cells, 116-1 18 germ cells, 128- I30 immobilin. 171, 175-176 process. 105 in Sertoli cells receptor-mediated at base. 120-122 secondary lysosomes. 118-120 sulfated glycoprotein-I. 134-135 sulfated glycoprotein-2. 134-135 transferrin. 135-137 vas deferens, 177- I78 Endoplasmic reticulum, epididymis caput epithelium, 159, 161-163 initial segment, 154 Epidermis bullosa simplex, intermediate filament protein disturbances characterized by, 54-55 Epididymis, sperm maturation, 147-156 cell types and functions. 146-177 endocytosis, epithelial clear cell role basal cells, 177 immobilin, 171. 175-176 narrow cells, 176-177 postnatal development, 173- 175 secretion by principal cell 147-156 caput epididymis, 159-169 cauda epididymis, 159-169 corpus epididymis. 159-169 immobilin. 157-158 intermediate zone, 157, 159 postnatal development, 169-170 SPG-2. 155, 157. 164, 166 sperm membrane in, modification during migration, 178-184 Epigenesis instability of homogeneous state as source, 309 and preformation, antithesis between, 3 13-322

379 Epithelial cells, rete testis, structure and function, I3 1-137 Epithelium, and sperm maturation epididymal clear cells, 170-177 vas deferens, 177-178 Epodermolytic hyperkeratosis. intermediate filament protein disturbances characterized by, 55 Eukaryotes. DNA replication of chromosomes, see DNA, replication in mammals E i t p l o ~ e seiirysfoinos, nuclear organization. 1 I Evolution gene-regulatory function of intermediate filament proteins. 66-70 sex chromosomes, mammalian, 202-206 testis-determining factor SRY gene organization. 24 1-244 X chromosome inactivation, 21 1-214

F Fricu.s, instability of homogeneous state localized activity, 344-345 metabolic patterns, 359

G GAGA factor, chromatin modifications by, 36 Genes regulatory function of intermediate filament proteins, see Intermediate filament proteins, gene-regulatory function sex-determining, see Sex-determining genes SR Y characterization, 233-237. 247 cloning. 233-237 demonstration of sex determination, 236, 238 evolution of function, 238-241 evolution of organization, 241-245 identification, 246 mechanism, 238-241 testis-determining factor, 229-233

380

INDEX

Genetics, instability of homogeneous state, 362-367 Germ cells, male endocytosis. 128- 130 secretory functions, 126, 128 GI ycoproteins sperm maturation role, in epididymal transit, 179-180 sulfated glycoprotein-l , see Sulfated glycoprotein-1 sulfated glycoprotein-2, see Sulfated glycoprotein-2 GI ycos ylation in epididymal transit, 180-184 intermediate filament protein function modulated by, 42-43 Glycosyl transferase, in epididymal transit, 180-181, 183 Golgi apparatus, of epididymis caput epithelium, 159-161, 163 initial segment, 154 Gonadal differentiation developmental steps, 247 higher vertebrates, 220-221 hormone function, 216-218 marsupials, 219-220 testis determination, mammalian, 22 1-222

H Histones evolutionary aspects of DNA interactions with, 70 intermediate filament protein interactions with, 7 Homogeneous state, instability channels, localized activity, 343-346 Child’s results, 346-347 colocalization, 313 cytoskeleton localization, 355-360 differentiation metabolism, 360-362 Turing biochemical mechanism, 324-327 Drosophila dorsoventral system, 335-339 terminal system, 335-339 electrophoresis, spontaneous endogenous, 339-342

epigenesis versus preformation, 3 13-322 lateral inhibition, 3 12 metabolic field, 355-360 morphogenesis metabolism, 360-362 Turing biochemical mechanism, 324-325, 328-329, 334 preformation versus epigenesis, 3 13-322 principle, 309-3 I 1 proliferation metabolism, 360-362 pumps, localized activity, 343-346 reducibility of development to molecular genetics, 362-367 reduction fields, 347-355 Turing biochemical mechanism, 322-335 Turing-Child metabolic system, 3 10 Hormones, and gonadal differentiation, 216-2 I8 Hydra, instability of homogeneous state, reduction fields, 348

I Immobilin, sperm maturation role in epidid ymis endocytosis, 171, 175-176 secretion, 157-158, 164, 166-168 Intermediate filament proteins, generegulatory function characteristics, 1-2, 70-71 cytoskeletal functions, 2-4 differentiation systems amphibian early development, 61-62 characterization, 48-49, 62-64 cultured cells, 58-59 cytokeratin filament role, 54-57 extension of cellular processes, 53-54 leukemia cell differentiation by vimentin, 52 mammalian early development, 60-61 muscle differentiation by desmin, 49-52 redundancy of function, 64-66 transgenic animals, 59 evolutionary aspects, 66-70 functions, 71-75 nuclear functions analysis, 4 cytoskeleton role, 38-41 DNA loop formation, 23-24

INDEX

381

double nuclear membrane barrier, 27-3 1 histones. interactions with, 5-7 matrix element. gene expression role. 24-26 nucleic acids, interactions with. 5-7 organization of nucleus, 7-13 physical associations, 26-37 post-translation modification, 41-48 repetitive DNA sequence interactions, 32-37 reverse transformation model, 38-48 transcription elongation. 20-23 and transcription factors, relationship, 13-18 transcription initiation, 18-20 nucleic acids, interactions with, 5-7 Intermediate filaments, phosphorylation. 357 Ion channels. localized activity versus localized distribution, 343-346 Ion pumps. localized activity versus localized distribution, 343-346

K Keratinocytes, mechanical integrity, cytokeratin filament role, 54-57

sex chromosomes, see Sex chromosomes, mammalian Marsupials gonadal differentiation, 219-220 sexual dimorphisms, 219-220 Matrix-associated regions binding proteins, 12 gene expression effects, 8 transcription elongation role, 21 Membranes, sperm, modification during epididymal transit. 178-184 Metabolic field. and cytoskeletal localization, 355-360 Metabolic system, Turing-Child, see Turing-Child metabolic system Mollitscu, instability of homogeneous state. localized activity, 345 Morphogenesis instability of homogeneous state as source, 309 metabolism of, versus metabolism of proliferation, 360-362 Mullerian inhibiting substance, gonadal dfferentiation role,2 18 Multivesicular bodies, sperm maturation role endocytic apparatus, 145 structural properties, 109 Muscles. differentiation, desmin role, 49-52

L Leukemia cells, differentiation by vimentin, 52 Lysosomes, secondary, of Sertoli cells, sulfated glycoprotein-1 synthesis, 118-120

M Male reproductive tract, and sperm maturation, see Sperm, maturation Malignant cells, reverse transformation, cytoskeleton role, 44 Mammals DNA replication. see DNA replication. in mammals early development, differentiation systems, 60-61

N Neurofilament protein ADP-ribosylation, 43 expression, subject to feedback control mechanism, 63 Nucleic acids, intermediate filament protein interactions with, 5-7

P Pennariu, instability of homogeneous

state, reduction fields, 349 Phagocytosis, Sertoli cell mechanism, 109, 116 processing of residual bodies, 1 1 1, 114, 116

382

INDEX

Phosphofructokinase. Turing biochemical mechanism affected by. 365 Preformation, and epigenesis, antithesis between, 313-322 Proliferation, metabolism of, versus metabolism of differentiation and morphogenesis. 360-362 Prosaposin. see Sulfated glycoprotein- 1 Protein kinase C, post-transitional modification, intermediate filament protein role. 44-46 Proteins, see also Glycoproteins acidic, instability of homogeneous state. electrophoretic analysis. 341 basic. instability of homogeneous state, electrophoretic analysis. 341-342 GAL4, transcription initiation role, 19 intermediate filament, see Intermediate filament proteins, gene-regulatory function of microtubule-associated, evolutionary conserved interaction with repetitive DNA sequences. 40 mouse epididymal characterization. 148 immunolocalization, 148 neurofilament ADP-ribosylation, 43 expression subject to feedback control mechanism, 63 glycosylation, 42 nuclear matrix protein 125, as carrier of vimentin molecules, 28, 31 RAPl, physical association with nucleus, 33 Rb. nuclear function. 12, 14 secretory, in sperm maturation, 105- 106, 126- 128

sperm membrane. in epididymal transit, 179

Replication, DNA, in mammals, see DNA, replication in mammals Replication fork barrier, 263 replicon termination, 292-293 Replication fork rate concept. 263 replicon model, 295. 299 replicon termination, 293 Reproductive tract, male, and sperm maturation, see Sperm, maturation Rete testis structure and function, 131-137 rete epithelial cells, 131-137 RNA and instability of homogeneous state metabolic field, 359-360 intermediate filament proteins. gene regulatory function. 5, 69 messenger and instability of homogeneous state cytoskeletal localization, 355 metabolic field, 359-360 SPG-I encoded by, 167

S Secretion, sperm maturation role, 105 germ cells. 126, 128 principal cells of epididymis, 149-170 immobilin, 157- 158, 164- I68 SPG-2, 155, 157, 164, 166 Sertoli cells proteins. 122- I23 sulfated glycoprotein-I, 123-124 sulfated glycoprotein-2, 124-127 vas deferens, 177-178 Seminiferous tubules, intermediate region, 130-131

telomere-binding, physical association with nucleus. 34

R

Sertoli cells, sperm maturation role endocytosis adsorptive, 117-1 18 fluid-phase, 116-1 18 receptor-mediated at base of cells, 120- 122

Radioautography, DNA fiber, see DNA fiber radioautography Reduction fields, and instability of homogeneous state, 347-355

in secondary lysosomes, 118-120 function, 106 phagocytosis of residual bodies, 109, I l l , 114, 116

'

INDEX

secretory functions in proteins. 122-123 sulfated glycoprotein-I. 123-124 sulfated glycoprotein-2. 124-127 ultrastructure. 107-1 15 Sex chromosomes. mammalian comparative studies. 246. 248 dosage compensation. 206. 214-216 evolution. 202-206 function. 191 gene content. 196-198 gonadal differentiation role higher vertebrates. 220-221 hormone function. 2 16-218 marsupials. 219-220 regional localization. 226-229 testis determination. 22 1-222 organization. 191- 195 testis-determining factor function, 222 regional localization. 226-229 Y chromosome as determinant, 223-226 testis-determining factor S R Y gene cloning. 233-237 demonstration of process, 236, 238 evolution of organization, 241-244 variations of. 195-202 X chromosome inactivation comparative studies, 21 1-212 cytological changes. 209-210 dosage compensation role, 214-216 evolutionary aspects. 211-212 gene expression, 207-209 mechanism, 206-207. 209-210 sex chromosome evolution, 212-214 Y chromosome as sex determinants, 223-226 Sex-determining genes characteristics. 191- 195 comparison, 244. 248 dosage compensation, 206-209, 214-2 I6 function, 246 gene dosage effects. 214 gonadal differentiation developmental steps. 247 higher vertebrates, 220-221 hormone function, 216-218 inactivation. 209-213

383 mapping studies, 196-202. 204. 206, 246 marsupials, 219-220 testis determination. mammalian. 221-222 sexual dimorphisms examples, 216 in marsupials. 219-220 and testis-determining factor function, 222 genetic research, 229-233 regional localization. 226-229 SR Y gene. 233-245 Y chromosome role, 223-226 Sexual dimorphisms examples, 216 in marsupials. 219-220 Sister chromatid exchange, formation. replication model, 293 Sperm. maturation. 105-106 conditions required, 184 efferent ducts, epithelial nonciliated cells of, 131. 137-146 endocytosis in. see Endocytosis epididymis, see Epididymis germ cells, 126. 128-130 membrane. modification during epididymal transit, 178-184 rete testis epithelial cells, 131-137 secretion role, see Secretion seminiferous tubule intermediate region. 130-13 1 Sertoli cells, see Sertoli cells vas deferens, 177-178 Sulfated glycoprotein-l , sperm maturation role endocytosis in nonciliated epithelial cells of efferent ducts, 139, 143-145 by rete epithelial cells, 134-135 epididymis, synthesis by, 167-169 secretion by Sertoli cells, 123-124 synthesis in secondary lysosomes of Sertoli cells, 118-120 Sulfated glycoprotein-2, in sperm maturation endocytosis in nonciliated epithelial cells of efferent ducts, 139, 142 by rete epithelial cells, 134-135 epididymis, 155. 157, 164. 166 secretion by Sertoli cells, 124-127

384

INDEX

T Telomeres, intermediate filament protein interactions with, 33 Testis, determination, mammalian, 221-222 Testis-determining factor function, 222 regional localization, 226-229 S R Y gene characterization, 233-237, 247 cloning, 233-237 demonstration of sex determination, 236. 238 evolution of function, 241-245 evolution of organization, 241-245 function mechanism, 238-241 genetic research, 229-233 identification, 246 Y chromosome role, 223-226 Testosterone, gonadal differentiation role. 218 Terrahyrnena, transcription factors related to. 17 Transcription factors instability of homogeneous state post-translational modification. 351 recox regulation, 347-348 spatially differentiated modulation of activity, 354 intermediate filament proteins related to, 13-18 Transferrin, sperm maturation role endoc ytosis, 135- 137 receptor-mediated endocytosis at base of Sertoli cells. 120, 122 Triturus udgaris, DNA fiber radioautography analysis, 272-273 Tubularia, instability of homogeneous state, reduction fields, 349 Turing biochemical mechanism characteristics, 322-335 phosphofructokinase effects, 365

Turing-Child metabolic system metabolic field, 356-359 metabolic pattern, 344 phosphorylation field, 345-346 principle, 310

W Vertebrates, gonadal differentiation, 220-22 I Vimentin double nuclear membrane barrier. 28-3 I leukemia cell differentiation by, 52 nucleic-acid binding potential, 5 nucleic acid interactions, 6 in reverse transformation model of gene regulation, 39, 41. 44-45

X X chromosomes, mammalian, see Sex chromosomes, mammalian Xenoprrs instability of homogeneous state localized activity, 344 metabolic field, 359 proliferation, metabolism, 362-363 keratin mRNA depletion in oocytes, 61

Y Y chromosomes, mammalian, see Sex chromosomes, mammalian

z zeste, gene product of Drosophila function, 65 matrix element, 24-26