Development Biology

Editors:

E. EDWARD BITTAR, Department of Physiology, University of Wisconsin, Madison NEVILLE BITTAR, Department of Medicine, University of Wisconsin, Madison

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Edited by E. EDWARD BITTAR Department of Physiology University of Wisconsin Madison, Wisconsin NEVILLE BITTAR

Department of Medicine University of Wisconsin Madison, Wisconsin

@ Greenwich, Connecticut

JAi PRESS INC.

Library of Congress Cataloging-in-Publication Data Developmental biology / edited by E. Edward Bittar, Neville Bittar. p. cm.-- (Principles of medical biology ; v. 11) Includes bibliographical references and index. ISBN 1-55938-816-1 1. Developmental biology. 2. Molecular biology. 3. Cells-Growth---Regulation. I. Bittar, E. Edwards. II. Bittar, Neville. III. Series. [DNLM: 1. Developmental Biology. 2. Molecular Biology. QH 491 D4868 1998] QH491.D4256 1998 571.8'1---dc21 DNLM/DLC for Library of Congress 98-18490 CIP

Copyright 9 1998 by JAI PRESS INC. 55 OM Post Road, No. 2 Greenwich, Conneticut 06836 JAI PRESS LTD. 38 Tavistock Street Covent Garden London WC2E 7PB England All rights reserved No part of this publication may be reproduced, stored on a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, filming, recording or otherwise withoutprior permission in writingfrom the publisher. 1SBN: 1-55938-816-1 Library of Congress Catalog No.: Manufactured in the United States of America

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The purpose of this module is to provide a survey of the rapidly expanding field of developmental biology and to introduce it to the student in a unifying way. In medical schools where courses in biochemistry, physiology, and pharmacology are already considerably intersecting, there is not surprisingly a rising demand in modern medical education for books emphasizing the interdisciplinary approach. In recent years, developmental biology has become a very vibrant and exciting field. The adoption of the interdisciplinary approach in this field has yielded enormous information about how DNA is able to produce a living organism from a fertilized egg. The discovery of 'master' genes in Drosophila that control spatial organization and share a segment of DNA, the so-called homeobox, and the discovery in C. elegans of genes controlling the timing of branching off of cell lineages are today recognized as milestones in molecular developmental biology. Because of space limitations and because of the information explosion, we have continued to pursue the policy of selecting broad topics but not in every case. This time, for example, though guided by the principle that a close connection exists between genes, adhesion, and morphogenesis, we opted to include certain topics such as cadherin--an adhesion molecule--rather than have the whole subject of adhesion dealt with in a single chapter. Substrate-adhering molecules (e.g., fibronectin) are touched upon in Chapter 5. In a similar manner, only one type of junction is discussed at length. Chapters 8, 9, and 10 border on the extraordinary, for they are together absorbingly interesting. The last chapter makes things more pragmatic.

The attention of the reader is drawn to the fact that several previous volumes of the compendium impinge on the present one. Chapters 25 and 26 in Volume 7B, in particular, have much to say on the subjects of extracellular matrix adhesion and intercellular communication. We would like to express our warmest thanks to the various contributors for their patience and scholarly work. We are also grateful to Mr. Fred Verhoeven and other staff members of JAI Press for their skill, courtesy, and sense of humor. E. EDWARD B ITTAR NEVILLE BITTAR

Introduction Methodology Cell Cycle Control Points: Genetics of the Cell Cycle Timer Controls Implications of Cell Cycle Research Future Research Notes

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The cell division cycle is the most fundamental of all developmental processes. Its outcome includes the proper replication and segregation of the genetic material during the formation of genetically identical daughter cells (Figure 1). The doubling in cell mass, and on average, of each type of cellular constituent, membrane component, and organelle, also relies on the efficient completion of this set of events (Nasmyth, 1996). The distinct stages of the overall process still bear the designations G 1, S, G 2, and M based on early cytological observations: S (for synthesis) is the phase of DNA replication; M is mitosis, in which segregation of cellular constituents precedes cell Principles of Medical Biology, Volume II Developmental Biology, pages 1-12. Copyright 9 1998 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-816-I

Figure 1. The eukaryotic cell division cycle. division G 1 and G 2 are the "gaps" between these two microscopically-observable stages. As well as identification of the biochemical steps of this complex dynamic process, both spatial and temporal controls need to be understood (Mackinnon and Gilbert, 1992; Lloyd and Gilbert, 1998). Only then will it become possible to fathom the sources and early consequences of derangement to normal cellular growth and division. We may ask why it is that in cultures of simple unicellular organisms cell division depends on the presence of every necessary nutrient; what of the more complex question of why, under similar nutrient status, stem cells (for epidermis or of bone marrow) continue to divide, whereas neurons do not. It has become clear that there are two major control sites that occur universally in the cell division cycles of many organisms from amoeba to humans. These regulate the progress of cells from stages G1 to S and from G2 to M. The time spent by any individual cell in each stage depends on many factors. As well as the necessity for all the nutrients normally required for growth (Lu and Means, 1993), the last two decades have seen the discovery of a host of growth factors specific for different cell types (e.g., epidermal growth factor, tumor growth promoting factor, etc., Wang, 1992). As is the case for a hormone, each of these has a specific cell surface receptor, and interaction with growth factor initiates a signal transduction pathway involving secondary messenger substances and eventually triggering gene transcription within the nucleus. As well as a requirement for extracellular information, progression through the cell cycle also needs positive inputs from an intracellular control network governed by the products of the cell division cycle genes. The importance of these enquiries can hardly be overstressed. Cancer cells frequently show genetic instabilities, and many of these lead to gene amplification and chromosomal rearrangements; tumorigenesis is a likely outcome (Hartwell, 1992).

Great strides have been made since 1980 in the demonstration of the fundamental uniformity that underlies the apparent diversity of dividing cell types. This comes about because of a conserved network of control processes that operates in all organisms from yeast to humans (Nurse, 1990), as well as in plants (Francis and Halford, 1995). This is so in spite of the fact that once embryonic mitoses are complete, the developmental functions of cell division are very different in animals and plants. Whereas continued mitotic activity is largely restricted in the latter to apical meristems, in adult vertebrates the maintenance and regeneration of tissues and circulating cells is reliant on mitoses in stem cells (Jacobs, 1992). The discovery that many, if not all, of the sites and agencies of cell cycle control are universal has enabled a highly successful search in "simpler" model systems such as yeast to be extended to the identification of homologous processes in higher organisms.

The organisms of growing cultures are extremely sensitive to small changes in external conditions of temperature, light, nutrient status, osmotic pressure, and other factors (Lloyd et al., 1982). Traditional methods for studying the cell division cycle relied on the induction by cyclic changes (usually of temperature or light) of synchronous division of each individual of the entire population. An alternative approach involves selection by filtration or centrifugation of a specific cell cycle stage cohort of organisms, usually on the basis of size (uniformly small and thus newlydivided cells). However, the rapid and marked responsiveness of cells to environmental alterations imposes severe limitations on permissible experimental procedures, and it has often proved difficult to distinguish between naturally-occurring cell cycle events and their responses to external perturbations. It is imperative that organisms do not lose sight of their nutrients or oxygen supply during the synchronizing procedure. Neither should they be exposed to extreme physical forces (e.g., during centrifugation). Several methods are available that may be regarded as minimally disturbing to normal cellular functions. For instance, the cell cycle of the budding yeast, Saccharomyces cerevisiae, is controlled in the G 1 phase by the event known as "Start". This step of the cell division cycle can be inhibited by the peptide mating pheromones, a-factor and ix-factor, the normal function of which is to synchronize opposite mating types prior to conjugation. Application of a synthetic pheromone thus provides a "natural" method for arrest of organisms in G 1. Another approach exploits the temperature sensitivity of many cdc (cell division cycle) mutants. The cell cycle of these cdc mutants is normal at the permissive temperature, whereas a shift to the nonpermissive (or restrictive, usually higher) temperature cause,:: arrest at that point in the cell cycle where the product of the mutated gene essential for cell cycle progress normally acts. Because large numbers of cell cycle genes have been identified, especially in the two yeast species, S. cerevisiae and Schizosaccharomyces pombe, enormous ad-

vances have been made in elucidation of the molecular basis of cell cycle control (Nurse, 1990). The yeasts provide the most convenient experimental systems for study; because central cell division control mechanisms are so highly conserved in evolution and thus so similar in all organisms from lower eukaryotes to humans, convenient experimental systems also provide excellent models for universal processes.

The bids to identify cell cycle genes by means of isolating cdc mutants were led by Hartwell working on budding yeast (S. cerevisiae) (Hartwell, 1978) and Nurse with fission yeast (S. pombe) (Lee and Nurse, 1988). In budding yeast the detection of cdc mutants relied on the fact that arrest at a specific stage of the cell cycle would result in cells with a uniform morphology. This uniform morphology was either one of the normal morphologies of the cell cycle (e.g., unbudded cells, cells with small buds, etc.) or an abnormal morphology resulting from a defect in an event of the normal cell cycle (e.g., multinucleate cells, multibudded cells, etc.). In fission yeast, cdc mutants were identified as producing abnormally elongated cells at the restrictive condition because although the cell cycle had stopped growth continued. Subsequent mutageneses showed a law of diminishing returns---only one or two new cell cycle genes were identified along with additional alleles of cdc mutations that had been isolated earlier. Nevertheless, steady progress continued and it became clear that the cell cycle is composed of a series of dependent pathways, i.e. that execution of a particular event required the prior completion of an earlier event(s). Cell cycle "maps" were produced, and newly isolated cdc mutations placed on the map by examining the order and dependence of the respective gene products with reference to other known cdc mutations and/or stage-specific chemical inhibitors. This sort of approach produced a view of the cell cycle as a cascade of events whose order could not be varied because late functions could not take place until the necessary earlier functions were completed. This application of classical mutational analyses to the understanding of the cell cycle led to the suggestion that most cell cycle genes would encode products that were "merely" (sic) required for cell cycle progression and not involved with regulation. It was argued that important regulatory genes would encode pacemaker functions and that, therefore, mutation of such genes would result in either acceleration or deceleration of the cell cycle (Nurse, 1981). A central control gene (cdc2 § in S. pombe was identified because it was essential for mitosis and because certain mutant alleles altered cell size at mitosis. The cdc2 § gene of S. pombe is functionally homologous to CDC28 in S. cerevisiae*, and homologs have been identified in all other eukaryotes examined. The protein encoded is a 34-kD serine/threonine protein kinase ("p34cdc2"). In S. pombe, binding between p34 cdc~and

cyclin B occurs as the former becomes dephosphorylated at tyrosine residue 15 and acquires protein kinase activity (Nurse, 1990) (Cyclins are a class of proteins originally observed in sea urchins, and now known to be ubiquitous and to appear and disappear at each mitosis). It is now clear that even in the simple yeasts the universal c d c 2 § ncoded kinase system is part of a n e t w o r k rather than being a single, isolated switch. In S. p o m b e , there are both positive and negative regulatory controls over the activity of the p34 Cdc2kinase. In this yeast, the "wee" phenotype is the result of premature mitosis, hence cell division occurs when the cells are smaller than in the wild type giving the unusually small ("wee") daughter cells. Overexpression of the activators c d c 2 5 § (protein phosphatase) and n i m l § (protein kinase), or deletion of the inhibitor w e e 1 § (protein kinase) advances cells into mitosis giving wee cells. Cells carrying dominant mutations in the c d c 2 § gene that result in a p34 cdc2with altered control features are also wee. The regulatory network controlling initiation of mitosis in S . p o m b e is outlined in Figure 2. There is more to the genetic control of the eukaryotic cell cycle than the control over entry in mitosis described thus far. It is now known that cells contain a multitude of cyclin-dependent kinases ("CDK's") and distinct classes of cyclins whose profiles of synthesis and destruction ensure that the appropriate CDK's are only activated at the correct occasions in the cell cycle (Nigg, 1995). Additionally, the CDKs and the CDK-cyclin complexes have matching inhibitors. Much interest is currently focussed on control of the proteolysis which ensures rapid destruction of the cyclins at the requisite times. In budding yeast, the G1 cyclins are also involved

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in the mating response. S. cerevisiae can exist as a haploid in either of two mating types called "a" and a. Each haploid type produces a distinct mating pheromone that has the effect of arresting the cells of opposite mating type in G1; (mating cannot take place in other phases of the cycle). The molecular details need not be recounted here, but the reader should note that species- or tissue-specific cyclins represent a general mechanism by which eukaryotic cells can switch out of the cell cycle into an alternative developmental state (see "Implications of the Cell Cycle" below). We must now address why in some cases late events depend on early events and in other cases they do not. Attempts to answer this question have invoked the concept of cell cycle "checkpoints" (Hartwell, 1991) which are envisaged as signal transduction pathways that generate an inhibitory signal when an upstream event is delayed so that the next downstream event can be delayed. (This is rather like a railway signalman telephoning to the next station to advise delaying the departure of a connecting train service because an earlier train is running late: the departure of the second train is not dependent upon the arrival of the first, but can be delayed until the first train arrives). Again, genetics can be applied because if a mutation can remove the dependence of certain cell cycle events, then a checkpoint exists. Checkpoints are used to ensure that DNA replication is completed, DNA repaired and chromosomes segregated.

A number of other controls are coupled to those already described; it seems that mechanisms for monitoring time as well as cell mass and growth rate must operate in order to ensure orderly progress of cellular growth and division (Murray and Kirschner, 1989). Various models have been proposedmsome consisting of fixed series of reactions that take a definite time for completion, and others involving a self-sustained oscillatory mechanism of the type provided by a limit-cycle (Figure 3). The state of a limit-cycle oscillator depends on two variables, and has four minimal requirements: (i) that A is converted to B; (ii) negative feedback (that B inactivates or destabilizes A); (iii) that B is itself unstable and thus has a characteristic half-life; and (iv) that the attainment of a critical threshold of A provides the system's signal output. There have been many candidates for the role of mitotic oscillator. The currently favored one involves cyclin-cdc2 kinase interaction (Hyuver and Le Guyader, 1990; Goldbeter, 1991; Norel and Agur, 1991; Novak and Tyson, 1993; Tyson et al., 1996). Although it is sometimes referred to as the cell cycle clock, the mitotic oscillator is not a clock in the strict sense. This is because a clock has to be temperaturecompensated whereas the cell division cycle is highly temperature-dependent (Lloyd, 1992; 1997). The best known cases of strict timekeeping of cell division are

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The cell division cycle as a limit-cycle oscillator. those of lower eukaryotes (e.g., planktonic algae) that divide each day at dawn. Cell division in these organisms is controlled (or "gated") by a circadian rhythm with a period of about 24 hours (Hastings et al., 1991). A similar subservience to circadian time is seen in the gated cell division of the ge.nerative tissues of higher plants and animals. A short period (ultradian) clock exerts dominant control in lower eukaryotic organisms that are growing and dividing more rapidly elsewhere than in the natural environment (e.g., in laboratory cultures close to optimum growth conditions of temperature and nutrient supply). Here cell division times are primarily determined by the mitotic oscillator, but interaction with regular signals from the ultradian clock at slightly suboptimum conditions give a series of permitted division times with fixed increments over and above the fundamental cell cycle duration (Lloyd and Kippert, 1994). These "quantal" increases in cell cycle time correspond to the period of the ultradian clock (Figure 4). Thus the ultradian clock has an output which can act to control cycle progression. Work with cell division cy-

cle mutants of Schizosaccharomyces pombe indicates that interaction with events of the mitotic cycle occurs at the key cdc2 § control network. Thus the wee1 § protein kinase is an essential component of the pathway coupling mitosis to the ultradian clock.

Much of the driving force to understand the eukaryotic cell cycle and cellular proliferation has been the desire to understand and cure cancer. Clearly, a cancer arises where cellular proliferation has become uncontrolled and the cancerous cells continue to proliferate when they should not. It is axiomatic that if we can understand all there is to know about control of the normal cell cycle, then we will have a complete understanding of cancer. Normal mammalian cells in culture have several features in common. Mostly they need a substratum for growth (so they will grow as a monolayer); they require certain complex growth factors (depending upon the origin of the cells); they have defined cell shape (characteristic of the particular cell type); have a defined lifetime (i.e., it is only possible to make a certain number of passages before the cells will no longer proliferate); and they show density-dependent inhibition of growth and/or contact inhibition. By contrast, cells which have undergone malignant transformation lose these properties and hence they: lose anchorage dependence and will grow as suspension cultures; become growth factor independent, and immortalized; lose inhibition phenomena (hence invasiveness of tumors in vivo);

have an altered karyotypemfrequently being aneuploid; lose their characteristic cell shape; and are no longer nutritionally fastidious. An exhaustive review of the origins of cancer is beyond the remit of this chapter, but the various agents capable of carcinogenesis (certain chemicals, ionizing radiations, oncogenic viruses) must all act via the same cellular machinery. Studies of oncogenic viruses have allowed the identification of both cellular and viral oncogenes. Cellular oncogenes encode normal cellular products, which only generate malignancy when their expression is hijacked by virus. Activated r a s genes are found in many human cancers (see Figure 5). Another example is where the oncogene encodes a growth factor, hence the transformed cell produces its own growth factor and keeps stimulating itself to proliferate. Development and differentiation involve a "choice" between another round of the cell cycle or switching to an alternative developmental pathway. Almost invariably, whether this involves differentiation in a metazoan or a developmental change in a unicellular eukaryote, the switch only occurs in G 1. Stationary phase

arrest also involves a switch out of G 1 into a noncycling state (often referred to as "Go", "senescence", or "quiescence"). Stationary phase is now recognized as a genetically distinct state because it is possible to isolate two different sorts of mut a n t s - t h o s e which are unable to arrest and those which are "stuck" in stationary phase and unable to restart proliferation (Filipak et al., 1992). Clearly, both events must be carefully controlled. In the adult mammal the majority of cells are not proliferating and are noncycling. Nevertheless certain classes of cells (e.g., lymphocytes) must be able to proliferate if stimulated to do so. Early cell cycle studies on mammalian cells in vitro frequently employed either lymphocytes, stimulated to proliferate by the addition of a mitogen, or quiescent fibroblasts, stimulated by the addition of fresh serum. Many now recognize that recovering cells from Go back into the cell cycle is not the same as when cycling cells pass through G 1 into S phase.

One aspect of current research emphasizes the need for cell cycle quality control. The highly complex and multistage processes of replication and segregation of DNA as well as that of all the cellular machinery and architecture are subjected to feedback controls. These can respond to inadequate completion and arrest the progress of the cell cycle at one of several "checkpoints" (Hartwell, 1992; Murray, 1992; Weinert and Lydall, 1993). For example the regulator ofG 1progression in fission yeast rum1 § determines the length of G1, the dependence of S phase upon the completion of mitosis, and the restraint of mitosis until G 1is finished (Moreno and Nurse, 1974). The process of mitosis is, as viewed by phase contrast microscopy, an extremely dynamic one that involves rapid reorganization of the cytoskeleton and the establishment of two opposing cellular poles. These then act as foci for orientation of replicated chromosomes. As yet, basic understanding of the mechanism and control of this massive spatial reorganization is inits infancy (Lew and Reed, 1993). Therefore current insights into the pathology of defective cell division are still severely limited at the level of cell ultrastructure. As noted earlier, cell cycle genes can encode either major regulators or components necessary for cell cycle progression. Identification of regulators has had a high profile in recent years with the resultant implication that work on "the mere mechanics" of cell cycle progression is less important, or that such research is not worthy of the attention of the best laboratories. It will be lamentable if, for reasons of intellectual elitism, the full capabilities of modem molecular biology and the necessary resources are not devoted to plugging these huge gaps in our knowledge. It is important to obtain a complete description of all of the molecular processes and interactions required for one turn of the cell cycle. After all, it should be remembered that genes encoding control functions represent a very small proportion of all of the cell cycle genes identified thus far.

Programmed cell death occupies a special place midway between the cell cycle, development, and aging (Bowen, 1993; Bowen et al., 1997). In mammalian cells, it appears that special genes are reserved to ensure termination of a specific cell at a pre-ordained time. There is a growing interest in identifying such genes, some of which have been identified in simpler eukaryotes such as baker's yeast, which also shows aging (Kennedy and Guarente, 1996). In a higher plant, xylem is composed of cells that are mostly dead, but nonetheless essential to the life of the plant. Xylem cells develop from procambial or cambial origin by a process in which programmed cell death is clearly essential. Some of the genes involved in xylem differentiation have now been identified (Fukada, 1996). The medical implications have considerable potential. For example, targeted controlled induction of apoptosis in the cells of a tumor would presumably be better than the chemotherapy or radiotherapy currently used. Clearly, there is much opportunity for further research here. To date, most approaches to understanding control of the cell cycle have started either with the assumption that the cell cycle is a set of dependent reactions or that there is an underlying timer. Although this dichotomy has been recognized (e.g., M u r r a y and Kirschner, 1989), only r e c e n t l y have a t t e m p t s b e e n m a d e to r e s o l v e it ( L l o y d and Gilbert, 1998). T h e r e is still great s c o p e here.

* The genetic conventions of the two yeasts are different. In S. cerevisiae, upper case italics (e.g., CDC28) denotes a gene and lower case italics (cdc28) a mutation in that gene. In S. pombe, wild-type (cdc2 § and mutant (cdc2) alleles are denoted as indicated, although occasionally other devices (e.g., cdc2 and cdc2 respectively) are used by some authors.

Bowen, I. D. (1993). Apoptosis or programmed cell death? Cell Biol. Intl. 17, 461-476. Bowen, I. D., Bowen, M. & Jones, A. (1997) Mitosis and Apoptosis. Chapman and Hall, London. Filipak, M., Drebot, M. A., Ireland, L. S., Singer, R. A. & Johnston, G. C. (1992). Mitochondrial DNA loss by yeast reentry-mutant cells conditionally unable to proliferate from stationary phase. Curr. Genet. 22, 471-477. Francis, D. & Halford, N.G. (1995). The plant cell cycle. Physiol. Plant 93, 365-374. Fukada, M. (1996) Xylogenesis: Initiation, progression, and cell death. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 299-325. Goldbeter, A. ( 1991). A minimal cascade model for the mitotic oscillator involving cyclin and cdc2 kinase. Proc. Natl. Acad. Sci. USA 88, 9107-9111. Hartwell, L. H. (1978). Cell division from a genetic perspective. J. Cell Biol. 77, 627-637. Hartwell, L. H. (1991). Twenty-five years of cell cycle genetics. Genetics 129, 975-980. Hartwell, L. H. (1992). Defects in cell cycle checkpoint may be responsible for the genomic instability of cancer cells. Cell 71,543-546. Hastings, J. W., Rusak, B. & Boulso, Z. (1991). Circadian rhythms: The physiology of biological timing. In: Neural Integrative Animal Physiology (Prosser, C. L., Ed.), p.439. Wiley-Liss, New York.

J. Hyuver, C. & Le Guyader, H. (1990). MPF and cyclin: Modelling of the cell cycle minimum oscillator. BioSystems 24, 85-90. Jacobs, T. (1992). Control of the cell cycle. Develop. Biol. 153, 1-15, Kennedy, B. & Guarente, L. (1996). Genetics analysis of ageing in Saccharomyces cerevisiae. Trends Genet. 12, 355-359. Lee, M. & Nurse, P. (1988). Cell cycle control genes in fission yeast and mammalian cells. Trends Genet. 4, 287-290. Lew, D. J. & Reed, S. I. (1993). Morphogenesis in the yeast cell cycle: Regulation by cdc28 and cyclins. J. Cell Biol. 120, 1305-1320. Lloyd, D. (1992). lntracellular timekeeping: Epigenetic oscillations reveal the functions of an ultradian clock. In: Ultradian Rhythms in Life Processes (Lloyd, D. & Rossi, E. R., Eds.) pp. 5-22. Springer, London. Lloyd, D. (1997). Circadian and ultradian clock-controlled rhythms in unicellular organisms. Adv. Microb. Physiol. (Poole, R.K., ed.) Vol. 39, Academic Press, London. Lloyd, D. & Kippert, F. (1994). lntracellular coordination by the ultradian clock. Cell Biol. Internat. 17, 1047-1052. Lloyd, D. & Gilbert, D.A. (1998). Temporal organisation of the cell division cycle in eukaryotic microbes. Symp. Soc. Gen. Microbiol. Microbial Responses to Light and Time (Hodgson, D.A., Eds.) Cambridge University Press. Lloyd, D., Poole, R. K. & Edwards, S. W. (1982). The Cell Division Cycle: Temporal Cellular Growth and Reproduction, pp. 523. Academic Press, London. Lu, K. P. & Means, A. R. (1993). Regulation of the cell cycle by calcium and calmodulin. Endocrine Rev. 14, 40-58. Mackinnon, H. & Gilbert, D. A. (1992). To divide or not to divide? That is the question. In: Fundamentals of Medical Cell Biology, Developmental Biology, Vol. 7, pp. 1-14. JAI Press, Greenwich. Moreno, S. & Nurse, P. (1994). Regulation of progression through the G~ phase of the cell cycle of the ruml" gene. Nature 367, 236-242. Murray, A. M. (1992). Creative blocks: Cell-cycle checkpoints and feedback controls. Nature 359, 599-604. Murray, A. M. & Kirschner, M. W. (1989). Dominos and clocks: The union of two views of the cell cycle. Science 246, 614-621. Nasmyth, K. (1996). Viewpoint: Putting the cell cycle in order. Science 274, 1643-1645. Nigg, E.A. (1995) Cyclin-dependent protein kinases: Key regulators of the eukaryotic cell cycle. BioEssays 17, 471-500. Norel, R. & Agur, Z. (1991). A model for the adjustment of the mitotic clock by cyclin and MPF levels. Science 251, 1076-1078. Novak, B. & Tyson, J. J. (1993). Quantitative analysis of a molecular model of mitotic control in fission yeast. J. Theor. Biol. 173, 283-305. Nurse, P. (1981). Genetic analysis of the cell cycle. In: Genetics as a Tool in Microbiology. SGM Symposium 31. (Glover, S. W. & Hapwood, D. A., Eds). Cambridge University Press, Cambridge. Nurse, P. (1990). Universal control mechanism regulating onset of M-phase. Nature 344, 503-508. Tyson, J.J., Novak, B., Odell, G.M., Chen, K. & Thron, C.D. (1996). Chemical kinetic theory: Understanding cell-cycle regulation. Trend. Biochem. Sci. 21, 89-96. Wang, J. Y. J. (1992). Oncoprotein phosphorylation and cell cycle control. Biochim. Biophys. Acta I 114, 179-192. Weinert, T. & Lydall, D. (1993). Cell cycle checkpoints, genetic instability, and cancer. Cancer Biol. 4, 129-140.

Introduction Invertebrate Cell Lineages

Caenorhabditis elegans Other Invertebrates Techniques Used to Study Cell Lineages Cell Lineage Studies in Vertebrate Embryos

Myoblast Cell Lineages Neural Crest Cell Lineages Summary

13 15 15 19 21 22 23 24 26

One of the central problems of developmental biology is how the single-celled fertilized egg is able to give rise to a multicellular embryo composed of many discreet populations of differentiated cells. In generating the embryo, cells which have undergone the process of cell division are faced with a choice of whether to continue as part of the same population of differentiated cells as the parent cell, or

Principles of Medical Biology, Volume 11 Developmental Biology, pages 13-28. Copyright 9 1998 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-816-1

13

whether to diverge and found a new population with different characteristics. To understand this program of cell differentiation in development, it is necessary to have a detailed knowledge of the family trees of as many cells as possible. Cell lineage analysis traces the ancestry of individual cells in developing embryos. This may be done in a number of ways, such as by injecting a tracer dye into an early cell and then looking for the tracer in the progeny of the cell; or by infecting a cell with a non-replicating retrovirus and then following the presence of virus-bearing cells over several generations. In translucent embryos much may even be learned by direct observation of the dividing cells. The results of these very exacting studies have indicated that although some populations of vertebrate embryonic cells differentiate according to a definite and identifiable lineage program, in many instances the lineage program has a degree of random variability so that no two individuals are precisely alike. This variability obviously makes detailed cell-by-cell analysis of lineage very difficult. However, in certain invertebrates the program is simple and largely invariant, and may apply to the entire organism and not just to specific populations. In these organisms, cell-cell interactions are highly reproducible between individuals because, unlike the situation in vertebrates, extensive cellular migration is much less common. Cells tend to be born in roughly their final location and are subject to predictable signals from neighbors and from their environment. Indeed, the presence of a relatively invariant lineage program, in the absence of extensive cell migrations, provides a simple means of achieving the spatial and temporal organization that is necessary in these early embryos. This being so, it is not difficult to see that cell lineage in these organisms is inextricably linked with cell fate. In other words, the developmental origin of a cell can define its fate, as, over the generations, its line becomes committed to founding descendants of only one differentiated type. On the other hand, it cannot be assumed from this that all differentiated cell types are founded from only one cell line or clone; most differentiated cell populations are in fact polyclonal (Stent, 1985). The invertebrate species whose cell lineages have been best studied is the nematode worm, Caenorhabditis elegans. Although far removed from vertebrates, this organism has taught us much about the biology and genetic control of cell lineages in development (Sternberg, 1991). The lineage analysis of C. elegans is complete for every cell in the entire adult organism. This thorough knowledge allows the next step in the analysis, which is to identify which genes are responsible for the control of body pattern in development. This is being done by examination of mutant organisms and organisms in which the genes have been manipulated or ablated. The principles of genetic control learned by study of this primitive animal are now being applied to the more complex differentiating populations of cells in vertebrates so that it may become possible to identify genes, the perturbation of which results in the appearance of anomalous body patterns.

15

In order to fully appreciate the potential and complexity of lineage studies in vertebrate development, it is necessary first to spend some time examining such studies carried out with invertebrate embryos.

Such free-living soil nematodes as C. elegans develop through a short embryonic period of 13-15 hours to produce a juvenile or larva, followed by a three-day, postembryonic period of development, which includes 4 larval molts, to generate an adult approximately lmm in length, (Sternberg, 1991). The adults exist in two forms" a self-fertilizing hermaphrodite consisting of 959 somatic cells and about 2500 germ cells, and rarely, a male consisting of 1031 somatic cells and about 1000 germ cells. The embryonic cell lineages of C. elegans are known for every cell in the organism from the single-celled zygote to the hatching of the larva with 558 cells (Sulston et al., 1983) on through the postembryonic period to the adult (Sulston and Horvitz, 1977; Kimble and Hirsh, 1979). These original works should be consulted in order to appreciate the astonishing detail in which the development of this organism is now known. The fertilized egg begins to cleave unequally, and by the 28-cell stage of development has produced 6 progenitor or founder cells, (Schierenberg, 1986) (Figure 1). Five of these are somatic founder cells, and the remaining one (the P4cell, a cell of the fourth generation from the fertilized egg) is the founder of the germ cell lineage, which has a greater degree of variability than the largely invariant somatic lineages (Strome et al., 1994). The five somatic founder cells (AB, MS, E, C, and D) generate the body tissues including intestines (from founder cell E only), muscle, neurons, and the outer layer of the body wall (the hypodermis), (Figure 2). Muscle is generated from founder cells AB, MS, C, and D; neurons from cells AB, MS, and C; and hypodermis from cells AB and C only. With the exception of the intestinal cells, therefore, somatic cells of the same differentiated type are not necessarily closely related in the lineage tree. On the other hand, different cell types, such as a muscle cell and a neuron, may be sisters. This implies that the genetic instructions for a particular differentiated fate are autonomous or programmed, and that the program can be activated regardless of position in the lineage tree and independently of cell-cell interaction. Such autonomous differentiation is termed "determinate" and is distinct from the situation in early mammalian embryos, for example where development is "indeterminate" and dependent on cellular interaction. In the latter case, cells do not make early, irrevocable, developmental choices, and manipulation of the embryo results in "regulative" adjustments and normal development. In other words, there is no early cell "commitment", and cells remain pluripotent over this period. In the highly determinate embryos of nematodes, by contrast, ablation

of a cell early in development generally results in the absence of the progeny of that cell with little or no compensation. This indicates that the surrounding cells have become committed to the generation of descendants of only one cell type and are incapable of altering their fate. Similarly, isolated early cells from C. elegans undergo cell divisions in a relatively normal temporal sequence, again suggesting the autonomous or intrinsic character of the lineage program. This is not to say that cellular interactions are not involved at all in early nematode development. Difficult experiments involving physical manipulation of the early embryo suggest that interactions among the founder cells and their immediate descendants may be required to "induce" their neighbors to pursue certain directions of differentiation (Schierenberg, 1986). Although differentiation in the embryonic period of C. elegans is essentially invariant and autonomous, a few cells in the postembryonic period appear to be nonautonomous. For instance, certain pairs of cells in the postembryonic male tail are normally positioned symmetrically to the left and fight of the mid-line, but which of the pair becomes disposed to which side depends not on ancestry, but on their relative antero-posterior positions. Such cells in which alternative fates are possible are termed an "equivalence group". Another example of an equivalence group occurs in the development of the genitalia of the hermaphrodite, in which each member of a cell pair will become, with equal likelihood, either a terminally differentiated "anchor cell" (AC) or a "ventral uterine precursor cell" (VU). The bi-

nary "AC or VU" decision that each of the cells has to make appears to be controlled genetically and also through cell-to-cell interaction, because in the absence of all other gonad cells either member of the pair will become AC only; the VU fate, how~ver, only comes about after interaction of the second cell with the AC cell (Greenwald, 1989). So, it is important to understand that the idea of"invariant" lineage and "autonomous" differentiation do not necessarily mean that these processes are independent of cellular interaction and unalterable. How are the lineage programs and cell fates controlled and invoked? The answer appears to lie in the activity of "developmental control genes". These genes have been identified through the study of mutants of C. elegans in which specific defects in cell lineage lead to temporo-spatial perturbations of cell fate and consequent abnormal morphology or behavior. Examination of the genetic mutation then gives insight into the normal activity of the gene in question and its role in lineage control in the wild type. A large number of such mutants and control genes are now known (see Sternberg, 1991, for detailed discussion). For example, the "AC or VU" decision described above seems to be under the control of the lin-12 locus (Greenwald and Seydoux, 1990). Mutations that raise lin-12 activity result in a VU decision in the AC/VU cells; mutations that lower lin-12 activity result in an AC decision. Because lin-12 seems to control structural patterning in development, it may be described as a "homeotic" gene. Significantly, the lin-12 product belongs to a family of transmembrane proteins believed to be involved in intercellular signalling in Drosophila and in vertebrates. These proteins include mammalian epidermal growth factor (EGF; Greenwald, 1985). This leads to the speculation that lin-12 may specify a cell surface component that acts as a receptor involved in mediating cell--cell interaction and transmembrane signal transduction. In this way, it would be possible for each of the cells in the AC/VU pair to "know" their positions relative to one another, and make their differentiative switch accordingly. Thus, the genetic control can be explained at the level of cellto-cell interaction. It is also important to note that, as in many other developmental situations, cell death is scheduled into the lineage program, so that here, as elsewhere, cell death becomes an active process in differentiation (Driscoll and Chalfie, 1992). In C. elegans, 131 cells undergo cell death at specific times and locations. As in other developmental systems, the neuronal cell lineages are particularly susceptible to programmed cell death, 105 nerve cells being fated to die. This death is under the control of ced-3 and ced-4 genes, without which the cells fated to die will survive and differentiate in a similar manner to cells closely related in the lineage. Death is followed by phagocytosis, which is also under the direction of a series of ced genes. The mode of action of the ced gene products is not at all clear, but in the case of ced-3 and ced-4 the cytotoxic gene products appear to act in the cells in which they are produced. Both ced-3 and ced-4 may be inhibited by the activity of another gene called ced-9, which prevents programmed cell death, ced-9 is similar to the human gene bcl-2, and in a very elegant demonstration, Vaux et al. (1992) have used trans-

19

genic C. elegans embryos containing the human bcl-2 gene to show that this human gene can reduce cell death in the nematode. It is easy to see how C. elegans can provide some important keys to our understanding of cell lineage and cell fate in higher organisms. The relative simplicity and the detailed knowledge of the cell lineages in this nematode worm make it a good subject for genetic manipulations designed to reveal some fundamental mechanisms of decision-making during differentiation.

C. elegans is not the only invertebrate species to be used in cell lineage analysis, although it is by far the best understood. Cross-species comparisons, including comparisons between invertebrates and vertebrates, reveal commonalities in the mechanisms regulating such early embryonic events as cleavage patterns, axis formation, and lineage determination. Many of the principles described for C. elegans are found to apply also to the sea urchin (Davidson, 1989) because these embryos also have invariant early cell lineages derived from founder cells ttiat give rise to populations expressing lineage-specific genes. As with the nematode, cell-to-cell interactions appear to be involved in fixing the fates of the founder cells. Several lineage-specific genes have been identified, and their patterns of expression relative to the various embryonic "territories" are established as are their times of activation (Cameron and Davidson, 1991). The mechanism of specific gene activation in cells in the different territories, both at the spatial and temporal levels, has been examined by incorporating copies of a gene, suitably constructed with a reporter, into unfertilized eggs. One such gene called CyllIa is a marker for the aboral ectoderm territory. This gene appears to be controlled by diffusible DNA-binding proteins present within each nucleus, and there may be many such factors interacting with the DNA at different times and in different places. The appearance of the appropriate DNA-binding factors in the founder cells is presumed to be induced by the cellular interactions occurring during cleavage, and this is responsible for the establishment of the embryonic territories. Interference with these factors may reduce gene activity or may cause expression of the gene in inappropriate cell lineages. The development of segmented invertebrates, from the annelid and arthropod phyla, provide an interesting problem in cell lineage because structures are reiterated down the body in an identical or modified pattern in each segment. However, superficial similarities between, for example, leeches and insects are not necessarily reflections of similar lineage mechanisms. In leech development, the founder cells all derive from one blastomere called D' into which a special region of yolkdepleted, RNA-rich cytoplasm or "teloplasm" is concentrated during the cleavage process (Nelson and Weisblat, 1992). The D cell line then gives rise, by a series of invariant cleavages, to a set of five bilateral pairs of founder "teloblasts". The teloblasts then divide many times to form longitudinally arrayed bands of segmentally arranged founder cells from which the segmental tissues arise (Figure 3). So each of

21

the original founder cells contributes descendants to the tissues in each segment. The role of the teloplasm in establishing cell lineages is not understood. The D' cell, which contains most of the teloplasm, divides to form the DM cell, normally fated to produce cells of mesodermal lineages, and the DNOPQ cell, normally fated to produce cells of the ectodermal lineages. Nelson and Weisblat (1992) have shown that while mesodermal determinants are present solely within the teloplasm of the DM cell, the DNOPQ cell also requires factors from the cell cortex in order to found ectodermal lineages. As with C. elegans, leech development also involves cell death and cell-to-cell interactions, particularly in the nervous system. Though the segmental neurons differentiate according to their ancestry from founder cells, there is the occurrence of certain asymmetries and segment-specific differentiation, which appear to come about by "competition" between pairs of apparently equivalent cells (Shankland et al., 1991; 1995). How do cell lineages relate to segmentation in these organisms? Progeny of the different founder teloblast cells in each longitudinal band contribute to each segment, but the progeny are not confined to one segment only. In other words, in the leech, each segment is not simply a set of clones derived from the teloblast cells; the descendants of the teloblast cells cross the segmental boundaries and are found in more than one segment (Weisblat et al., 1988). This contrasts with the situation in insects, for example, where the segments or "compartments" are "polyclones" (Crick and Lawrence, 1975) in which the cells, all descendants of a small group of founder cells, do not intermix across the compartment boundaries. A "compartment" in this sense is an underlying subdivision of the body axis, founded by clones of progenitor cells, which may, or may not, correspond with the observable segmentation. By definition, the clones do not cross the compartment boundaries, and there is evidence that the concept of serially arranged compartments applies to vertebrate as well as invertebrate embryos (Lawrence, 1990). In insects, it appears that cells become committed to a particular compartment during the cleavage process in the formation of the syncytial blastoderm without being constrained to a specific lineage; once allocated to a compartment, any cell can generate the entire compartment. In leeches, by contrast, where holoblastic cleavage divides the entire zygote, the teloblasts become committed to a lineage but not to a particular segment (Shankland et al., 1991). Thus, in two evolutionarily related phyla of segmented organisms, the role of cell lineages in the formation of the segmented pattern is quite different.

Before passing on to the discussion of lineage studies in vertebrates, it is worth considering briefly the methods used in these investigations. As mentioned earlier, the simplest approach, feasible only in transparent embryos such as C. elegans, is direct

observation by light microscopy of dividing cells. In most vertebrate embryos this is not possible, because the embryos are usually not transparent and because the progenitor cells, which may be embedded in tissues, are not individually identifiable. A number of methods are available for marking the putative progenitor cells and their descendants. One that has been particularly valuable involves the microinjection into cells of fluorescently labeled dextrans (Wetts and Fraser, 1991). Dextrans are useful because they are nontoxic and too large to pass through gap junctions, thus they are confined to the cytoplasm of the injected cell and its progeny. Further, after in vivo examination, the dextrans are fixable, allowing subsequent analysis of sectioned material or observation of whole-mount material with the confocal microscope. Dextrans labelled with fluorescein or rhodamine have been used, for example, in studies of cell lineage in the zebrafish embryo (Kimmel and Warga, 1986) and in the vertebrate embryonic nervous system (Wetts and Fraser, 1991). One way to permanently and stably identify individual cells in embryos is to use genetic markers. In some ways this approach has been used for a long time in the production of chimeras, for example, by grafting small groups of quail cells into chick embryos and taking advantage of the distinctive nucleoli of the quail cells to follow the movement and proliferation of the grafted cells (Le Douarin, 1986). However, it has more recently been possible to do this at the level of individual cells by infecting them with retroviruses which integrate into the host genome (Sanes, 1989). If the viruses are replication defective, as is often the case, they will not be able to spread within the cell population except by the normal DNA replication process of the host cell prior to mitosis. The viruses, usually constructed with a reporter gene that expresses 13-galactosidase, are introduced into the target cell and the progeny of that cell are then easily detected by histochemical or immunocytochemical methods which reveal the presence of 13-galactosidase. Viral markers have the advantage over fluorescent markers in that they are not diluted out by each cell division. On the other hand, care needs to be exercised in the interpretation of negative results because some cells may not be able to express the viral marker. Retroviral lineage markers have been used successfully in many studies, most notably in lineage analysis in the mammalian retina and cerebral cortex (Price et al., 1987), in the nervous system of the chick embryo (Frank and Sanes, 1991), and in lineage analysis of the preimplantation mouse embryo (Soriano and Jaenisch, 1986).

The fluorescent and viral marking techniques have now been applied to several different populations of differentiating cells in vertebrate development. Of necessity, the lineages are not as well defined as those in invertebrates; in particular the cellby-cell analysis that is possible in lower organisms is usually not possible in vertebrate embryos. Indeed, the word "lineage" is often not used in the strict sense of in-

23 dividually identified cells that it is, for example, in C. elegans. Further, the analyses are undertaken only during the relatively short phases of development that are appropriate to the differentiation of a particular cell population, and so one does not see complete analyses from the fertilized zygote onwards.

The early development of muscle is characterized by the sequential appearance of several different classes of myoblasts (Stockdale, 1992). These cell types, called embryonic, fetal and adult myoblasts, are perceived to differentiate from several distinct myogenic lineages, although the data on this is still incomplete and the lineal relationships between the different myoblast types are far from clear. While it is possible to identify progenitor cells in the early blastoderm prior to gastrulation, the myoblasts themselves are first identifiable in the mesodermal somites of the early embryo, and from there migrate to form the elements of the axial and appendicular musculature, (Figure 4). The myoblasts differentiate from fibroblasts to which they

\ t i

t

are morphologically identical, and this switch appears to be under the control of a series of"myogenic regulatory factors" (MRFs) of which the best known is MyoD (Olson, 1992). MyoD is a tissue-specific regulatory gene encoding DNA-binding proteins that are expressed early in muscle development. Transfection of the MyoD 1 gene into fibroblasts can elicit the stable differentiation of myoblasts, and there is evidence that MRFs, acting in combination, may indeed be lineagedetermining genes for myoblasts. In vitro, myoblasts from each of the three classes appear to give rise to muscle fiber colonies of different types characterized by the type of myosin heavy chain (MHC) they carry, i.e., fast, slow, or fast/slow fibers. For example, cloned avian embryonic myoblasts may give rise to primary muscle fibers showing any one of the three MHC types, but each colony expresses only one of the types, indicating that each myoblast is limited to only one developmental fate. So it has been proposed that in vitro, the phenotype of the muscle fibers is related to the lineage of the myoblasts from which they are derived. However, recent results from experiments in which myoblasts have been marked retrovirally suggest that the relationship in vivo is not that straightforward, and that extrinsic factors, such as MRFs, can prevail over any commitment imposed by lineage (Hughes and Blau, 1992). All this becomes clinically relevant in the light of the procedure called "myoblast transfer therapy" in which normal myoblasts are grafted into dystrophic muscle in an attempt to correct the abnormal expression of the gene product "dystrophin" (Partridge, 1991). This technique relies on the fact that muscle fibers develop and regenerate by myoblast fusion to form multinucleated fibers in which the products of each nucleus are incorporated into the cytoplasm. Fusion of donated normal myoblasts with the abnormal host cells, and the consequent introduction of normal nuclei, is therefore a potential method for rescuing dystrophic muscle. Clearly an understanding of the lineal relationships between the different myoblast types and between the myoblasts and the differentiation of the muscle fibers is important if this interesting procedure is to fulfill its promise.

Neural crest cells are a transient and pluripotent population of cells arising in the early vertebrate embryo from the lateral margins of the neural plate at the time of neural tube closure. The cells emerge from the neuroepithelium, transform into mesenchymal morphology, and migrate away from the neural tube to differentiate into a large array of different cell types (Le Douarin, 1992). These derivatives include: neurons and glia of the cranial ganglia, of the sensory and sympathetic ganglia, and of the enteric ganglia; facial cartilage and bone; melanocytes; chromaffin cells of the adrenal medulla; and elements of the developing heart. Naturally, with all these cell types deriving from an apparently homogeneous initial population of neural crest cells, questions arise regarding the role of lineage in the commitment of the cells to a particular differentiated state.

The answers to these questions are only now beginning to be addressed, and it is becoming apparent that there is a complex interaction between environmental regulation and an intrinsic or programmed regulation. The environment of the neural crest cells, as they migrate along well-defined routes, has been extensively studied for a number of years, and it has emerged that extracellular matrix molecules such as fibronectin and laminin, and soluble growth factors such as bFGF and TGFI3, encountered by the crest cells during migration and are able to influence their commitment. In vivo and in vitro perturbation of these signals, or of the cells' receptors for the molecules, can accordingly influence cellular potentialities. The questions being asked are: "To what extent are cell lineages determined before the cells leave the neural tube?" and "Are cells of a specific differentiated type clonally related to each other or can individual neural crest cells give rise to several types of descendant?" Three approaches have been taken to answer these questions. Firstly, early crest cells in vivo, both before and during migration, have been injected with lysinated rhodamine-dextran, and the progeny examined (BronnerFraser and Fraser, 1991). Secondly, an in vitro system has been used in which clonal cultures of isolated crest cells are subjected to various culture conditions (Baroffio et al., 1988; Sieber-Blum, 1989). Finally, the chick/quail chimera system has been used to graft identified fragments of neural primordia between species (Le Douarin, 1992). Each of these approaches seem to give a similar answer~although the potentiality of crest cells may be partially constrained by lineage, most cells are pluripotent at the time of migration. However, some cells may be committed early, such as those of the sympathoadrenal lineage (Anderson, 1993), yet some also appear to be totipotent and able to give rise to all derivative cell types. So, although neuronal, mesenchymal, and melanocytic lineages appear not to be segregated at the time of crest cell dissemination, at the same time, the migrating population of neural crest is not composed of a homogeneous population of equally pluripotent cells. This type of analysis will benefit greatly from the development of early markers of lineage that can identify progenitor cells of a particular line of differentiation as they leave the neural tube. Such work is proceeding with markers for certain neuronal precursor cells (Marusich and Weston, 1992). A model that has emerge is one in which cells gradually and sequentially become committed as they divide during the migration process, and one in which environmental influences are extremely important in determining outcomes by permitting the expression of certain potentialities but not others (Weston, 1991). For example, in gangliogenesis, putative "survival factors" emanating from the neural tube and somatic mesenchyme may be influential in selecting for the expression of potentialities---either neuronal or glial~probably from a common precursor cell. The actual commitment to cell fate therefore must occur during the last stages of migration, or upon arrival in the ganglion (Sieber-Blum, 1989). As with myoblasts lineages, a thorough knowledge of neural crest cell lineages may have clinical value (Anderson, 1993). Not only are cell replacement therapies

being contemplated for treatment of neurodegenerative diseases, but neuroblastomas and pheochromocytomas are tumors of the sympathoadrenal neural crest lineage. Cells in these tumors show varying phenotypes, which may correspond with a varying outcome of the disease. The different phenotypes may also be related to the state of differentiation in the neural crest lineage that the cells had reached at the time of transformation. Detailed understanding of the lineage relationships among crest cells, and the factors required for their normal differentiation, in particular nerve growth factor (NGF), could therefore be of value in diagnosis and treatment of these diseases.

The detailed understanding of the embryonic cell lineages of several invertebrate species, most notably C. elegans, has generated many new concepts and ideas as to the genetic control of differentiation and pattern formation. Although our knowledge of cell lineages in vertebrate embryos is rudimentary by comparison with those in invertebrates, the same concepts are beginning to be applied to well-defined populations of cells during relatively narrow windows of development in vertebrates. Knowledge of the genetic mechanisms of cell determination and c o m m i t m e n t may become important in new treatments as therapy turns more and more to cellular and molecular events for solutions to medical problems.

Anderson, D.J. (I 993). Molecularcontrol of cell fate in the neural crest: The sympathoadrenallineage. Ann. Rev. Neurosci. 16, 129-158. Baroffio, A., Dupin, E. & Le Douarin, N.M. (1988). Clone-formingability and differentiationpotential of migratory neural crest cells. Proc. Nat. Acad. Sci. USA 85, 5325-5329. Bronner-Fraser, M. & Fraser, S.E. (1991). Cell lineageanalysis of the avian neural crest. Development (Suppl.) 2, 17-22. Cameron, R.A. & Davidson, E.H. (1991). Cell type specification during sea urchin development. Trends Genet. 7, 212-218. Crick, F.H.C. & Lawrence, P.A. (1975). Compartmentsand polyclonesin insect development.Science 189, 340-347. Davidson, E.H. (1989). Lineage-specificgene expressionand the regulativecapacitiesof the sea urchin embryo: A proposed mechanism. Development 105, 421--445. Driscoll, M. & Chalfie, M. (1992). Developmental and abnormal cell death in C. elegans. Trends Neurosci. 15, 15-19. Frank, E. & Sanes,J.R. (1991). Lineageof neurons and gliain chickdorsalroot ganglia:Analysis in vivo with a recombinantretrovirus. Development 111,895-908. Greenwald, I. (1985). lin-12, a nematodehomeoticgene, is homologousto a set of mammalianproteins that includes epidermal growth factor. Cell 43,583-590. Greenwald, I. & Seydoux, G. (1990). Analysis of gain-of-function mutations of the lin-12 gene of Caenorhabditis elegans. Nature 346, 197-199.

27 Hughes, S.M. & Blau, H.M. (1992). Muscle fibre pattern is independent of cell lineage in postnatal rodent development. Cell 68, 659-671. Kimble, J. & Hirsh, D. (1979). Postembryonic cell lineages of the hermaphrodite and male gonads in Caenorhabditis elegans. Dev. Biol. 70, 396-417. Kimmel, C.B. & Warga, R.M. (1986). Tissue-specific cell lineages originate in the gastrula of the zebrafish. Science 231,365-368. Lawrence, P. (1990). Compartments in vertebrates? Nature 344, 382-383. Le Douarin, N.M. (1986). Cell line segregation during peripheral nervous system ontogeny. Science 231, 1515-1522. Le Douarin, N.M. (1992). New insights into the development of neural crest derivatives. Intl. Rev. Cytol. 138, 269-314. Marusich, M.F. & Weston, J.A. (1992). Identification of early neurogenic cells in the neural crest lineage. Dev. Biol. 149, 295-306. Nelson, B.H. & Weisblat, D.A. (1992). Cytoplasmic and cortical determinants interact to specify ectoderm and mesoderm in the leech embryo. Development 115, 103-115. Olson, E.N. (1992). Interplay between proliferation and differentiation within the myogenic lineage. Dev. Biol. 154, 261-272. Partridge, T.A. (1991). Myoblast transfer: A possible therapy for inherited myopathies? Muscle and Nerve 14, 197-212. Price, J., Turner, D., Cepko, C. (1987). Lineage analysis in the vertebrate nervous system by retrovirus-mediated gene transfer. Proc. Natl. Acad. Sci. USA 84, 156-160. Sanes, J.R. (1989). Analyzing cell lineage with a recombinant retrovirus. Trends Neurosci. 12, 21-28. Schierenberg, E. (1986). Developmental strategies during early embryogenesis of Caenorhabditis elegans. J. Embryol. Exp. Morph. 97 (Suppl.), 31-44. Shankland, M., Martindale, M.Q., Nardelli-Haefliger, D., Baxter, E. & Price, D.J. (1991). Origin of segmental identity in the development of the leech nervous system. Development Suppl. 2, 29-38. Shankland, M. (1995). Formation and specification of neurons during the development of the leech central nervous system. J. Neurobiol. 27, 294-309. Sieber-Blum, M. (1989). Commitment of neural crest cells to the sensory neuron lineage: Science 243, 1608-1611. Soriano, P. & Jaenisch, R. (1986). Retroviruses as probes for mammalian development: Allocation of cells to the somatic and germ cell lineages. Cell 46, 19-29. Stent, G.S. (1985). The role of cell lineage in development. Phil. Trans. Roy. Soc. B 312, 3-19. Sternberg, P.W. (1991). Control of cell lineage and cell fate during nematode development. Curr. Top. Dev. Biol. 25, 177-225. Stockdale, F.E. (1992). Myogenic cell lineages. Dev. Biol. 154, 284-298. Strome, S., Garvin, C., Paulsen, J., Capowski, E., Martin, P. & Beanan, M. (1994). Specification and development of the germline in Caenorhabditis elegans. Ciba Foundation Symposium 182, 31-45. Sulston, J. & Horvitz, H.R. (1977). Postembryonic cell lineages of the nematode Caenorhabditis elegans. Dev. Biol. 56, 110-156. Sulston, J.E. Schierenberg, E., White, J.G. & Thomson, J.N. (1983). The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev. Biol. 100, 64-119. Vaux, D.L., Weissman, I.L. & Kim, S.K. (1992). Prevention of programmed cell death in Caenorhabditis elegans by human bcl-2. Science 258, 1955-1957. Weston, J.A. (1991). Sequential segregation and fate of developmentally restricted intermediate cell populations in the neural crest lineage. Curr. Top, Dev. Biol. 25, 133-153. Wetts, R. & Fraser, S.E. (1991). Microinjection of fluorescent tracers to study neural crest cell lineages. Development Suppl. 2, 1-8.

Blau, H.M. & Hughes, S.M. (1990). Cell lineage in vertebrate development. Curr. Opin. Cell Biol. 2, 981-985. Davidson, E.H. (1990). How embryos work: A comparative view of diverse modes of cell fate specification. Development 108, 365-389. Greenwald, I. (1989). Cell-cell interactions that specify certain cell fates in C. elegans development. Trends Genet. 5, 237-241. Griggs, R.C., Karpati, G. (Eds.) (1990). Myoblast Transfer Therapy, Plenum Press, New York. Kenyon, C. (1985). Cell lineage and the control of Caenorhabditis elegans development. Phil. Trans. Roy. Soc. Lond. B 312, 21-38. Le Douarin, N.M. (1993). Cell lineage analysis in neural crest ontogeny. J. Neurobiol. 24, 146-161. Mikawa T. & Fischman, D.A. (1996). The polyclonal origin of myocyte lineages. Ann. Rev. Phys. 58, 509-521. Price, J. (1987). Retroviruses and the study of cell lineage. Development 101,409--419. Sassoon, D.A. (1993). Myogenic regulatory factors: Dissecting their role and regulation during vertebrate embryogenesis. Dev. Biol. 156, 11-23. Stemberg, P.W. & Felix, M.A. (1997). Evolution of cell lineage. Curr. Opin. Gen. Dev. 7(4), 543-550. Strome, S. (1989). Generation of cell diversity during early embryogenesis in the nematode Caenorhabditis elegans. Intl. Rev. Cytol. 114, 81-123. Weisblat, D.A., Price, D.J. & Wedeen, C.J. (1988). Segmentation in leech development. Development 104, Suppl., 161-168.

Introduction Experimental Identification of Cadherins The Search for Cell Adhesion Molecules Identification of Cadherins by Biochemical, Immunological, and Functional Assays Identification of Cadherins by Molecular Genetic Techniques Cadherin Diversity and Phylogeny Analysis of Cadherin Structure and Biosynthesis Basic Organization of Cadherin Proteins Cadherin Biosynthesis Calcium-Binding Sites Adhesive Recognition Sites Modulation of Cadherin Structure and Function by Posttranslational Modifications Glycosylation Sulfation Phosphorylation Proteolysis Interactions of Cadherins with the Cytoskeleton and Other Proteins Cytoskeletal Interactions and the Catenins

Principles of Medical Biology, Volume 11 Developmental Biology, pages 29-67. Copyright 9 1998 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-816-1 29

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Direct Cytoskeletal Interactions of Cadherins Are not Necessary for All Cadherin Functions Cadherin Interactions with Other Proteins

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Organization and Regulation of Cadherin Genes

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Genomic Organization Transcriptional Regulation of Cadherins Expression and Function of Cadherins during Development

Cadherins are Expressed in Complex Patterns during Development E-cadherin is Expressed Early in Development and Remains Expressed in Epithelia N-cadherin is Expressed in the Development of Numerous Tissues Cadherin Expression is Similar but not Identical across Species Perturbation Studies Shed Light on Additional Cadherin Functions Multiple Cadherins are Expressed during Development of the Nervous System Multiple Cadherins are Expressed during Muscle Development Cadherins are Involved in Functions beyond Adhesion such as Intracellular Signaling Alterations of Cadherin Expression and Function May Contribute to Disease Processes

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Cancer and Metastasis Other Pathological Conditions

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Conclusion and Future Directions

57

Fundamental to the evolution and development of metazoan organisms is the phenomenon of cell adhesion. Cell-cell adhesion is the mechanism by which cells physically interact and form associations with one another in the formation of solid tissues. Cell-matrix adhesion is the mechanism by which cells physically interact with noncellular elements of their environment such as extracellular matrix and basement membranes. Cell adhesive interactions may be transient, as when during embryonic development or tissue repair, changes in cell and tissue shape and position occur. Cell adhesive interactions may also be stable, as occurs during the formation and maintenance of mature tissues. Cell and molecular biological research has led to the identification of a large number of cell adhesion molecules whose ranks continue to grow. These molecules can be classified on the basis of their structural and functional relationships into a number of multigene families such as the cadherins, integrins, and immunoglobulin-related proteins. A broad general discussion of the discovery and analysis of these various classes of cell adhesion molecules and their importance in cell function, both during embryonic development and in disease, has previously appeared in a companion series to this compendium (Grunwald, 1992), and several chapters in this volume contain related material.

In order to provide the reader with an updated and more in-depth discussion of cell adhesion molecules and their importance in medical cell biology, this chapter will specifically focus on the cell and molecular biological analysis of cadherin cell adhesion molecule structure and function. The cadherins are cell surface integral membrane glycoproteins found in a wide variety of organisms throughout the animal kingdom, and that mediate calcium-dependent adhesive interactions critical for the stable associations of cells in solid tissues. The numerous members of the cadherin gene superfamily loom large at all stages of the life cycle of mulficellular organisms. From compaction of the morula, the first discernible morphogenetic event in mammalian embryogenesis, to the establishment of discrete germ layers, tissues, and organs during subsequent development, the cadherins play a prominent role in orchestrating the cellular rearrangements, which form the basis of morphogenesis. Cadherins also have important functions in the maintenance of mature tissues throughout the life of an individual, and their malfunction or misexpression may contribute to unfortunate pathogenetic changes that may herald disease and premature demise. The classical cadherins are those members of the cadherin gene superfamily, which include the first described and most structurally conserved of these proteins, and which to date have been found only in vertebrate animals. However, the classical cadherins are part of a growing cadherin gene superfamily comprising a phylogenetically ancient, large, and diverse group of molecules with a rich variety of structural motifs, spatiotemporal expression patterns, and functional roles in the development and mature function of vertebrate and invertebrate organisms alike. Additional perspectives on the cadherins are available from several recent reviews (Cunningham and Edelman, 1990; Ranscht, 1991; Geiger and Ayalon, 1992; Grunwald, 1992, 1993; Kemler, 1992; Takeichi, 1993.)

Cell adhesion research has a long and rich history, and the conceptual and experimental background to the century of vertebrate embryonic cell adhesion research that led to the identification of cadherin and other intercellular adhesion molecules has been reviewed (Grunwald, 1991). Holtfreter's classic experiments with recombined tissue fragments and subsequently with mixtures of dissociated cells, both derived from amphibian early embryonic tissues, clearly established the existence of cell-autonomous, surface-mediated affinities, which resulted in their assuming nonrandom associations and sorting out into appropriate tissue-like patterns (Holtfreter, 1939; Townes and Holtfreter, 1955). Later experiments using trypsin-dissociated tissues from more developmentally advanced avian and mammalian embryos demonstrated that cells from even well-differentiated tissues possessed similar selective adhesive properties (Moscona, 1962). Many early

experiments had suggested that calcium was essential for the maintenance of fight cell associations, and this was also observed to be the case in these cell aggregation studies. Additionally, it had become the practice to carry out trypsin dissociation of tissues in the absence of calcium because this resulted in a more efficient preparation of single cells. The inclusion of calcium during trypsinization resulted in the production of a population of cells that retained a greater degree of residual adhesiveness (Steinberg et al., 1973). The biochemical basis for this was first demonstrated by Takeichi (1977), who found that the presence of calcium during tryptic digestion of cells and tissues prevented the removal of specific cell surface proteins, apparently by affecting the protein conformation and subsequent tryptic sensitivity. Retention of these proteins on the cell surface correlated with the ability of the cells to adhere in a calcium-dependent manner. In the absence of calcium, even low levels of trypsin inactivated this adhesive system, while leaving intact a functionally distinct calcium-independent cell-cell adhesive system. The expression of such dual adhesive systems among vertebrate embryonic cells was described by several laboratories (Urishihara et al., 1979; Grunwald et al., 1980; Brackenbury et al., 1981; Magnani et al., 1981). These two adhesive systems, it was demonstrated that the calcium-dependent system seemed to play the dominant role in forming tighter adhesions that were essential for the formation of histotypic cell interactions, and that this adhesive system was subject to developmental regulation (Grunwald et al., 1981). It is now recognized that the calcium-dependent interactions are mediated by the cadherins, while the calcium-independent interactions are mediated in large part by adhesion proteins that are members of the immunoglobulin gene superfamily (Buck, 1992).

The first cadherins to be identified were found through a variety of approaches in a number of different experimental systems. The protection of cadherins by calcium against proteolytic digestion was used to advantage in the identification of the original cadherins. Cell surface proteins whose presence correlated with the expression of the calcium-dependent adhesive system were identified by direct biochemical comparisons of cell surface protein patterns on adhesive and non-adhesive cells that had been trypsinized in the presence and absence of calcium, respectively. In conjunction with many of these studies, the use of adhesion function-blocking antisera, and later addition of monoclonal antibodies, to these proteins further implicated them as the molecules responsible for mediating calcium-dependent cell adhesive interactions, and they were dubbed "cadherins" (Yoshido-Noro et al., 1984). Among the earliest cadherins identified by this combined functional, biochemical, and immunochemical approach was E-cadherin, so named for its prevalent expression in epithelial cells. It was identified both in teratocarcinoma cells as E-cadherin (Yoshida and Takeichi, 1982; Yoshida-Noro et al.,

Cadherin Adhesion Molecules

33

1984) and independently identified and named uvomorulin due to its demonstrated role in mammalian embryo compaction (Kemler et al., 1977; Hyafil et al., 1980; Peyrieras et al., 1983). Human E-cadherin was named cell-CAM 120/80 upon its identification in mammary carcinoma cells (Damsky et al., 1983) and as Arc-1 in MDCK cells (Behrens et al., 1985). The presumed avian form of E-cadherin, referred to as L-CAM, was named following its identification in chick embryo liver cells (Bertelloti et al., 1980; Gallin et al., 1983). N-cadherin was also identified by combined functional, biochemical, and immunological criteria, and was originally described in the embryonic chick neural retina and referred to as gp 130/4.8 (Grunwald et al., 1982; Cook and Lilien, 1982; Lagunowich et al., 1989) and later as NcalCAM (Crittenden et al., 1987). N-cadherin was so named upon its identification with a monoclonal antibody, and because of its prevalent expression in neural tissues (Hatta et al., 1985; Hatta and Takeichi, 1986). In the interim, it had also been identified as A-CAM, an adherens junction protein from chick heart muscle cell intercalated discs (Volk and Geiger, 1984, 1986). Monoclonal antibodies that blocked adhesion of extraembryonic cells led to the identification of P-cadherin, found in the placenta (Nose and Takeichi, 1986). Immunological and peptide mapping studies led to the identification of a close analogue of N-cadherin called CRM-L for cadherin-related molecule in liver (Crittenden et al., 1988).

Following the identification of cadherin proteins, partial amino acid sequence data and immunological screening of cDNA expression libraries led to the cloning of cDNAs for L-CAM (Gallin et al., 1987), E-cadherin (Schuh et al., 1986; Nagafuchi et al., 1987); P-cadherin (Nose et al., 1987); and N-cadherin (Hatta et al., 1988). This permitted the determination of basic cadherin protein structures and clearly indicated that these various proteins were closely related members of a multigene family. This high homology among cadherins also provided for the development of probes for cDNA library screening and the identification of both known cadherins in additional species and of novel cadherins. The chick N-cadherin cDNA was used as a probe to clone the murine N-cadherin cDNA (Miyatani et al., 1989) and the xenopus EP-cadherin (E- and P-cadherin-like) cDNA (Ginsberg et al., 1991). Similarly, a nucleotide probe corresponding to the cytoplasmic domain of mouse E-cadherin was used to screen chick embryo retina cDNA libraries and identified clones for R-cadherin (Inuzuka et al., 1991; Matsunami et al., 1993). A PCR-generated human N-cadherin probe was used to clone the full-length human N-cadherin cDNA (Reid and Hemperly, 1991). Library screening with antibodies prepared against a detergent-insoluble, concanavalin A-binding glycoprotein from chick brain led to the cloning of the unique truncated T-cadherin (Ranscht and Dours-Zimmerman, 1991; Vestal and Ranscht, 1992; Sacristan et al., 1993). An antiserum raised against the synthetic peptide corresponding to the cytoplasmic domain of L-CAM was used to screen an embryonic chick brain cDNA library and led

to the identification of B-cadherin (Napolitano et al., 1991). Naturally occurring antibodies from patients with pemphigus vulgaris, an autoimmune epidermal blistering disease, were used to screen human keratinocyte cDNA libraries and identify PVA, the pemphigus vulgaris antigen, which was identified as a cadherin (Amagai et al., 1991). As above for cDNA cloning, each advance in molecular genetics has been in turn applied to cadherins, the most recent case of which has been PCR analysis to search for previously unidentified cadherins. PCR analysis led to the identification of Mcadherin, expressed in muscle (Donalies et al., 1991), and of bovine N- and Pcadherin in endothelial cells (Liaw et al., 1990). The most striking application of this approach has been the identification of 11 putative cadherins in adult rat brain, three of which corresponded to the previously identified N-, E-, and P-cadherins, but eight of which appear to be novel (Suzuki et al., 1991; Tanihara et al., 1994). Northern blot analysis indicated that these putative cadherins were indeed expressed in the brain. PCR analysis also led to the identification of the Drosophila fat gene product as a cadherin-like protein (Mahoney et al., 1991). This large transmembrane protein, which represents the first identified invertebrate cadherin, contains 34 cadherin domains as well as other structural motifs such as EGF repeats. The previously known gene was named fat because mutations at this locus lead to overgrowth of embryonic tissues, suggesting the protein may have tumorsuppresser function. Structural analyses of independently identified proteins and cDNAs have also identified members of the cadherin family through sequence homologies, as in the case of the desmosomal components desmoglein and desmocollin (Buxton and Magee, 1992; Legan et al., 1992). These cadherin gene superfamily members, while about 30% homologous to N-cadherin at the amino acid level, differ significantly in structure from the classical cadherins, and they are discussed elsewhere (Buxton and Magee, 1992). Recently, scanning of cDNA sequence databanks indicated that the human protooncogene c-ret is structurally related to the cadherins (Schneider, 1992; Iwamoto et al., 1993). While it is not clear if c-ret functions as a cadherin, it is of interest because the cytoplasmic domain contains a tyrosine kinase domain, which is found in no other known cadherins.

As discussed above, the original family of the three classical E-, N-, and Pcadherins has now expanded to include several more classical cadherins and a number of interesting variants on the classical motif. In addition to these, enough distant cadherin relatives have been identified to now permit reference to the cadherin gene superfamily. Computer analysis of sequence homologies has led to the construction of a cadherin family tree (Pouliot, 1992). This analysis sorts the cadherins into three groups: (1) N- and R-cadherins; (2) P-, E-, EP,- and B-cadherins; and (3) the distinct M- and T-cadherins. Evolution of the cadherins seems to have proceeded through a series of gene duplications, perhaps preceded by tandem du-

!

f ;~nc~;;;;?:

protocadherin a !

I DrosophilaI

[T'cadherinl,/

plications of an ancestral single extracellular domain containing both a single calcium binding and adhesive recognition site. Such an ancestral protein, comparable to Thy- 1 of the immunoglobulin gene superfamily, has yet to be identified, if it still exists. A list of the presently known classical cadherins and their more distant relations is presented in Figure 1.

Based mainly upon comparisons of the structures of the original three N-, E-, and P-cadherins and their homologues as derived from cDNA-deduced amino acid sequences described in the above references, the classical cadherins are single polypeptide transmembrane glycoproteins with a narrow range of molecular weights averaging 130 kD and composed of 723-748 amino acids. Each cadherin consists of a large extracellular segment and smaller transmembrane and cytoplasmic segments (Figure 2). The extracellular segment is composed of five distinct repeateo domains of about 110 amino acids in a range called EC 1 to EC5 that starts with the N-terminus. The first four EC domains are highly homologous to one an-

~ Figure 2.

other, while the fifth is the least conserved. Among the extracellular domains, EC 1 is the most highly conserved between different cadherins and contains the critical functional site for adhesive recognition. This domain is also the target of most, but not all, adhesion function-blocking antibodies. However, the cytoplasmic domain was found to be the most highly conserved region of the cadherins overall, which immediately suggested an important role integrating extracelluar and intracellular cadherin functions. The functions of these various cadherin regions are discussed in detail below.

Proteolysis of cadherin precursors at the amino terminus prior to their mobilization to the cell surface appears to be a typical component of cadherin biosynthesis. As studied in detail for E-cadherin, this results in conversion of a 140-kDa precursor into the mature 120-kDa protein (Ozawa and Kemler, 1990). In this study, sitedirected mutagenesis indicated that failure of this cleavage to occur results in the production of inactive cadherins, which are mobilized to the cell surface but unable

to mediate adhesions. These cadherins could be activated by removal of the prepeptide region by exogenous proteases. As opposed to the mature protein, the precursor regions of the cadherins have little homology between cadherin subtypes. Biosynthesis and turnover has been studied for both E- and N-cadherin, which show similar profiles. Pulse--chase analysis of E-cadherin in MDCK cells indicated a peak accumulation time of one to two hours and a half-life of about five hours (Shore and Nelson, 1991). Similar studies of N-cadherin in the embryonic chick retina also indicated it takes about two hours for the peak accumulation of labeled N-cadherin, which then disappears with a half-life of about five hours (Paradies and Grunwald, 1993). As discussed below, N-cadherin turnover appears to be mediated by a cell surface metalloproteolytic activity that results in the release of a soluble 90-kDa N-terminal fragment called NCAD90, which retains biological activity (Roark et al., 1992; Paradies and Grunwald, 1993). It has been suggested from experiments on E-cadherin synthesis, transport, and turnover that differential turnover of E-cadherin at different regions of cells may contribute to the development of epithelial cell polarity because E-cadherin delivered to the apical membrane was rapidly removed, while E-cadherin delivered to the basolateral cell membrane had a longer half-life (Wollner et al., 1992).

As discussed above, the ability of cadherins to bind calcium in the extracellular domain, resulting in conformational changes that affect adhesive function and protease resistance, were fundamental in the initial identification of cadherins. Putative calcium-binding sites rich in asparagine residues were identified in the extracellular domain (Ringwald et al., 1987). The function of these sites has been explored through the use of synthetic peptides and site-directed mutagenesis studies (Ozawa et al., 1990). Synthetic peptides corresponding to a single calciumbinding domain were shown to bind calcium and undergo conformation changes upon binding. This binding was shown to be dependent on a critical aspartic acid residue. Furthermore, when this residue at site 134 in the EC2 domain of intact Ecadherin was changed to lysine, the expressed cadherin was found to be much more sensitive to proteolysis and inactive in formation of adhesive bonds. This calciumbinding site is highly conserved in all cadherins (Table 1).

During the several decades of cell adhesion research preceding the identification of discrete adhesion molecules, a major debate ensued over the relative importance of qualitative factors (Moscona, 1962) versus quantitative factors (Steinberg, 1963) in the determination of adhesive preferences among cells (see Grunwald, 1991). Analyses of cadherin function have shed light on this issue, and these studies indicate that both parameters may influence cell adhesive preferences.

Table 1. Amino Acid Homologies Among Cadherins Species Source RecognitionSite Calcium Site

N N N N

Chick Human Mouse Bovine Chick Mouse

RE GA RA RA RA RA YS

HAV DV HAV DI HAV DI HAV DI HAV DV HAV DM HAV SS

qMIAI TVTAI TVTAI TVTAI TVTAI TVTAN KVSAT

D D D D D D D

ADDP ADDP ADDP ADDP GDDP ADDS ADDD

N

Xenopus

R E L-CAM

Chick

LS

HAV

TVNAT

D

ADDA

SA

The differential expression of cadherin subtypes has clearly been demonstrated to affect such adhesive choices. The first direct evidence that cadherins function as adhesion molecules was demonstrated using cadherin cDNA-transfected fibroblastic cells, which, in the absence of exogenously expressed cadherins, are not mutually adhesive in a calcium-dependent fashion. Cells transfected to express cadherins became mutually adhesive and were calcium-dependent, and this could be inhibited with the corresponding cadherin type-specific antibody (Nagafuchi et al., 1987; Edelman et al., 1987; Mege et al., 1988; Miyatani et al., 1989). Such cells transfected to express either murine N-, E-, or P-cadherin showed preferential homophilic adhesion, while cells expressing murine N-cadherin or chicken N-cadherin formed mixed aggregates (Miyatani et al., 1989), further indicating the specific homophilic nature of cadherin interaction. However, some potential for heterophilic interaction has been demonstrated between N-cadherin and L-CAM (Volk et al., 1987). In these experiments, in which lens cells expressing N-cadherin and liver cells expressing L-CAM were mixed, the composite aggregates that formed showed a preferential segregation into the expected homophilic contacts. However, antibody staining demonstrated the presence of some heterophilic contacts containing both cadherin types. More recently, it has been demonstrated that significant heterophilic adhesive affinities exist between N- and R-cadherin, although among these two cadherins homophilic interactions are still predominant (Inuzuka et al., 1991a). As N- and R-cadherin are sig-

nificantly more homologous than N-cadherin and L-CAM, this indicates that heterophilic cadherin affinities may vary over a continuum that depends in part upon the sequence similarities in the adhesive binding domain and other regions. Most antibodies that inhibit cadherin function bind near the N-terminus. The specific regions of cadherins that mediate adhesive recognition, and their amino acid sequences, have been identified by molecular genetic modifications that either delete specific cadherin domains or result in the exchange of domains between different cadherins. Chimeric cadherins representing various combinations of E- and P-cadherin domains, and expressed in transfected cells, demonstrated that the cadherin-type selectivity was largely determined by sequences within the 113-amino-acid, N-terminal domain (Nose et al., 1990). Swapping of domains for E- and P-cadherin resulted in the generation of chimeric molecules for which adhesive selectivity was dependent on the extracellular EC1 domain. Point mutations ftulher located the key regions for adhesive binding specificity between amino acid residues 78-83, and the targets of adhesion-inhibiting antibodies at amino acids 16 or 31, depending on the antibody and cadherin subtype' The amino acid sequence histidine-alanine-valine (HAV) was found to be conserved in the classical cadherins within the binding specificity region, and site-directed mutagenesis, which altered the immediately flanking amino acid residues, resulted in alterations in cadherin subclass recognition (Table 1). The HAV-containing regions of cadherins have homologies to members of the fibroblast growth factor receptor family and influenza hemagglutinins, suggesting that these motifs may be involved in protein-protein interactions among a wide variety of molecules (Blaschuk et al., 1990; Byers et al., 1992). Further supporting the importance of these sites, peptides containing the HAV domain have been shown to inhibit cadherin-mediated processes such as mouse embryo compaction as well as neurite growth on glial substrates (Blaschuk et al., 1990). While these distal cadherin regions are clearly involved in adhesive recognition and binding, aUosteric effects also appear to be involved. Structural alterations in other regions of the molecules, such as the extracellular domain just outside the membrane (Ozawa et al., 1990b) or the cytoplasmic domain (see below), also affect cadherin binding activity. The above studies clearly indicate that qualitative differences in adhesion molecule expression can mediate selective adhesion and cell sorting. However, it has also been demonstrated that quantitative differences in cadherin expression may also contribute to variations in adhesive interactions and cell sorting. This was demonstrated by cDNA transfection of fibroblastic cells, and the resulting cell lines with a range of cadherin expression were found to sort out within mixed aggregates (Friedlander et al., 1989).

Cadherins have been known to be glycoproteins since their initial discovery (Cook and Lilien, 1982; Damsky et al., 1983, Vestweber and Kemler, 1984) but the

functional significance of these carbohydrate moieties remains unknown. While sequence analysis of cadherins has indicated the general presence of multiple potential glycosylation sites, structural details are available for only a few cadherins. For example, both E- and N-cadherin have been shown to possess at least four Nasparagine linked oligosaccharides in the extracellular domain with differing endoglycosidase sensitivities (Cunningham et al., 1984; Crittenden et al., 1987). However, pharmacological studies suggest that inhibition of glycosylation has no effect on cadherin function or stability (Vestweber and Kemler, 1984; Shirayoshi et al., 1986).

Metabolic labeling studies have shown that E-cadherin (Vestweber and Kemler, 1984) and N-cadherin (Lagunowich and Grunwald, 1991) are sulfated, although the functional significance of cadherin sulfation is not known.

Cadherins have been known for some time to be phosphoproteins. E-cadherin isolated from human mammary carcinoma cells is phosphorylated on serine (Wheelock et al., 1987), while L-CAM from chick liver is phosphorylated on both serine and threonine (Cunningham et al., 1984). More recently, phosphorylation has been implicated in functional regulation of cadherins. As mentioned earlier, Ecadherin had been implicated in the process of mouse embryo compaction because anti-E-cadherin antibodies were found to inhibit this process. However, E-cadherin was known to be expressed prior to compaction, which indicated that perhaps posttranslational modifications could be responsible. Activation of protein kinase C was shown to induce premature compaction and cause a redistribution of Ecadherin to cell--cell junctions, while inhibition of protein kinase C prevented normal compaction (Winkel et al., 1990). The premature compaction was still inhibited by anti-E-cadherin antibodies. This work did not explore whether E-cadherin was a direct target of PKC-induced changes. A subsequent study explored the phosphorylation state of E-cadherin in early mouse embryos and found that, while Ecadherin is only weakly phosphorylated up to the four-cell stage, it becomes phosphorylated at the eight-cell stage just prior to compaction (Sefton et al., 1992). Again, the total level of E-cadherin changed little over this period. These results, which link changes in cell adhesiveness, E-cadherin distribution, and phosphorylation state, suggest that posttranslational control of E-cadherin may be critical to aspects of its function. A number of studies utilizing Rous sarcoma virus (RSV) transformed cells, which express the potent tyrosine kinase v-src, have explored the relationship between phosphorylation state of proteins and cell adhesive interactions. Lens cells express high levels of N-cadherin, which is localized to adherens junctions. When

Cadherin Adhesion Molecules

41

chick embryo lens cells were transformed with temperature sensitive RSV and shifted to the permissive temperature, these cells that are normally epithelial, assumed a fibroblastic appearance and lost adherens junctions, and N-cadherin was redistributed to a diffuse cell-surface organization (Volberg et al., 1991). These studies indicated that protein components of adherens junctions were major locations of phosphotyrosine-containing proteins, although N-cadherin was concluded not to be one of them because it was not bound to anti-phosphotyrosine immunoaffinity supports. The effect of v-src and v-fos on cadherin-mediated adhesion was also explored among rat fibroblasts, which express endogenous P-cadherin, as well as on their transfected counterparts, which also expressed E-cadherin (Matsuyoshi et al., 1992). Again, transformation was found to have no effect on overall levels of expression of the cadherins, and no effect was observed on the rate of cell--cell adhesion as measured in suspension assays. However, the morphology of cell aggregates was altered resulting in looser connections between cells. These effects were attributed to changes in tyrosine kinase activity because it was found that among the transformed cells inhibitors of these enzymes induced tighter cell associations, while tyrosine phosphatase inhibitors reduced cadherin-mediated adhesion. In this study, cadherin and catenin phosphorylation were examined by immunoblotting with anti-phosphotyrosine antibodies, and it was found that, whereas control cells exhibited no tyrosine phosphate on cadherins and little on catenins, transformed cells were found to have highly tyrosine-phosphorylated 13-catenin and weakly phosphorylated E-cadherin. A subsequent study of chick embryo fibroblasts, which express N-cadherin, demonstrated that RSV-transformation of these cells did not alter expression of Ncadherin but did suppress N-cadherin function (Hamaguchi et al., 1993). Transformation also resulted in enhanced tyrosine phosphorylation of both N-cadherin and catenins, but these proteins were still found to exist in a complex, suggesting that the inhibitory effects of phosphorylation on adhesive interactions occur via modulation of other aspects of cadherin-catenin function. Similar results have been reported for v-src-transformed MDCK cells (Behrens et al., 1993). A direct demonstration of cadherin adhesive function by phosphorylation changes will require specific identification of the modification sites and their alteration through site-directed mutagenesis. However, the above results would predict that kinase inhibitors might serve to stabilize cell adhesions. Indeed, it has been demonstrated in MDCK cells that pharmacologic inhibition of kinase activity prevents the disruption of cell-cell contacts and the cadherin redistribution normally induced by calcium depletion from the medium (Citi, 1992). These studies strongly indicate that tyrosine phosphorylation of one or more members of cadherin-catenin complexes may be an important control point for the association and function of these molecules. Studies currently underway in this author's laboratory are directed at addressing these issues in primary embryonic cells as opposed to cell lines that have been virally transformed and express high levels of exogenous tyrosine kinases. N-cadherin was shown to be phosphorylated in retina, brain, heart, and lens,

which express high levels of this protein (Lagunowich and Grunwald, 1991). In contrast to the above studies on src-transformed cells, this study suggested that a positive correlation existed between the extent of phosphorylation and the insolubility of N-cadherin in non-ionic detergents and junctional association. More recent studies have directly analyzed the phosphorylation of N-cadherin in these tissues, and indicate that in primary embryonic cells, N-cadherin is constitutively phosphorylated on serine, with peptide digests indicating at least two independent sites (Lee and Grunwald, unpublished). Treatments with tyrosine phosphatase inhibitors indicate that N-cadherin is also phosphorylated on tyrosine on a third independent peptide site, which correlates with enhanced turnover of N-cadherin to produce NCAD90. This indicates that N-cadherin is a substrate for multiple endogenous kinases, including both serine/threonine and tyrosine kinases, and that the stability and functional state of cadherins may depend on the balance between a number of potential phosphorylation sites.

Several types of proteolytic processing have been described for the cadherins, and they appear to be targets of proteolysis both intracellularly and extracellularly and by exogenous as well as endogenous proteases. Intracellular proteolysis of cadherin precursors to yield the mature protein has already been described above. With respect to the extracellular domain, as mentioned previously, removal of calcium renders cadherins sensitive to digestion by exogenously added proteases. The above cited studies had shown that in the presence of calcium, cadherins may be completely resistant to proteolysis or may release an approximately 80-90-kDa soluble N-terminal fragment, depending on the cadherin and whether intact tissues or isolated cells are treated. Antibodies to various cadherins obtained from tissue homogenates often detect a l l0-kDa membrane-associated fragment on immunoblots. Cadherins yield such a fragment upon cleavage at intracellular sites by calcium-activated proteases in tissue homogenates (Covault et al., 1991), although it is not clear if this type of processing occurs in intact cells or tissues. Cleavage of mature N-cadherin by endogenous proteases has been suggested to be a mechanism for regulation of its expression and function. Cultured lens cells produce a 78-kDa membrane-bound fragment when maintained in low-calcium medium, while a 97kDa fragment is released into the culture medium even in the presence of calcium (Volk et al., 1990). This study further correlated proteolytic turnover of N-cadherin with the dispersion of epithelial somites in developing chick embryos. Intact chick embryo retinas were known to release a 90-kDa fragment of N-cadherin into the medium in culture (Grunwald et al., 1982). The precursor-product relationship of this fragment and 130-kDa N-cadherin was further indicated by turnover studies of iodinated retinal cell surface proteins (Cook et al., 1984). The down-regulation of N-cadherin, which occurs during retinal development, was shown to be proteinsynthesis dependent, and inhibitor studies indicated that proteolytic activity was re-

Cadherin Adhesion Molecules

43

sponsible for N-cadherin turnover yielding a soluble 90-kDa N-terminal fragment (Roark et al., 1992). More recent pulse-chase metabolic studies have demonstrated that there is a direct precursor-product relationship between N-cadherin and this NCAD90 protein, that NCAD90 occurs in vivo, and that this turnover is mediated by a cell surface-associated metalloprotease (Paradies and Grunwald, 1993; Ferreira and Grunwald, unpublished). As mentioned above, these studies showed that purified NCAD90 retains biological activity and can mediate cell adhesion and neurite growth when attached to a solid substrate (Paradies and Grunwald, 1993). The exact nature of the endogenous proteases responsible for cleavage of cadherins are not known. Recently, however, urokinase plasminogen activator has been shown to be co-distributed with E-cadherin in keratinocytes where it is in a position to influence epithelial cell interactions (Jensen and Wheelock, 1992).

Many of the above-mentioned studies indicated that among the functions of cadherins were associations with cell junctions and the cytoskeleton because adherens junctions and calcium-dependent adhesiveness were both resistant to proteolysis in the presence of calcium, (Grunwald et al., 1981) and because cadherins were found to co-localize with adherens junctions and actin bundles (Boiler et al., 1985; Volk and Geiger, 1984; Volk and Geiger, 1986; Hirano et al., 1987). The highly conserved nature of the cytoplasmic domain amino acid sequences of cadherins, revealed by the above mentioned cloning studies, further implicated this region in subserving an important function. Such a linkage was strongly indicated by studies utilizing expression of E-cadherin constructs, which were truncated to various degrees in the cytoplasmic domain (Nagafuchi ad Takeichi, 1988). These studies indicated that truncated E-cadherin was expressed at the cell surface, but that it could no longer mediate cell-cell binding and that it could now be more efficiently extracted with non-ionic detergents. However, while proteins co-immunoprecipitating with cadherins had been reported previously, their identification and the specific mechanism by which cadherins could directly link to the cytoskeleton remained unclear. Subsequent studies identified three novel proteins of molecular weights 102, 88-92, and 80 kDa, termed c~, 13,and ~,catenins, which were found to be conserved in a wide variety of species and whose interaction with E-cadherin was dependent on an intact cytoplasmic domain (Ozawa et al., 1989; Nagafuchi and Takeichi, 1989). c~-catenin has been cloned and shown to be related to vinculin and to associate with several different cadherins (Nagafuchi et al., 1991; Herrenknecht et al., 1991). ~-catenin has also been cloned and shown to be related to both the drosophila protein armadillo and the mammalian junctional protein plakoglobin, which is

identical to y-catenin (McCrea et al., 1991; Kemler, 1993). Armadillo is a segment polarity gene, suggesting that catenins may be involved in developmental pattern formation. However, these three proteins may not be true homologues but rather may be members of a multigene family (Peifer et al., 1992). The ~ catenin gene has not yet been cloned, but immunological and biochemical studies suggest that it may be more closely related to plakoglobin than to 13-catenin (Knudsen and Wheelock, 1992). The above studies indicate that catenin--cadherin association appears to be universal, having been demonstrated with numerous cadherins and in different cell types and species including differentiated tissues such as muscle (Wheelock and Knudsen, 1991). Biochemical studies have indicated that the stoichiometry of cadherin-catenin association may be one molecule of E-cadherin, one or two molecules of 13-catenin, and one molecule of a-catenin (Ozawa and Kemler, 1992). These studies furthermore indicated that ~-catenin may be the more direct link with cadherins, and that it becomes associated with E-cadherin even while the latter is still not fully processed, ot-Catenin becomes associated later, and ~-catenin is the most weakly associated. Some experiments indicate that little if any catenin-free cadherins exist within cells (Ozawa and Kemler, 1992). Multiple cadherins can be simultaneously expressed by cells, and it has been shown that in cultured cells these can be differentially expressed with diffuse pericellular expression as well as in discrete junctional complexes (Salomon et al., 1992). Such different distributions of cadherins may be regulated by catenins, as changes in expression of the latter occur during changes in individual cell polarity, as well as during embryonic development (Wheelock, 1990; DeMarais and Moon, 1992). Transfection of cells with cadherin cDNAs has been shown to up-regulate expression of ct-catenin protein while not affecting mRNA levels, thus indicating a possible translational regulatory mechanism for maintaining a balanced expression of cadherins and catenins (Nagafuchi et al., 1991). The catenin story is likely to become more complex, as recently a novel predominantly neural subtype of ot-catenin has been described (Hirano et al., 1992). In this study a line of lung carcinoma cells, which expresses E-cadherin and fl-catenin, but no ot-catenin, and normally grows as single cells, was induced to form aggregates when transfected to express neural a-catenin. Thus the catenins, like the cadherins with which they interact, may also be members of multigene families, raising the potential for complex regulatory cross-interactions among these two protein families. The importance of cytoplasmic interactions for cadherin function was made clear in two series of experiments where mutant N-cadherin molecules, which lack extracellular domains, were generated and expressed in Xenopus embryos (Kintner, 1992) and in a keratinocyte cell line (Fujimori and Takeichi, 1993). In both instances, cell interactions were disrupted, leading to abnormal embryonic development of epithelial tissues in vivo and decreased cell interactions in vitro, respectively. However, these authors differ in their interpretation of the results, one set of experiments suggested inhibition mediated by competition for catenins and

inhibition of their interaction with endogenous cadherins (Kintner, 1992). The other set suggested no inhibition of catenin interaction but rather a displacement of endogenous cadherins from their normal junctional sites, at which the mutant cadherin was found, without competition for catenins (Fujimori and Takeichi, 1993). While these different outcomes may simply reflect the use of different experimental systems, these data could also be reconciled if further studies indicate that the resuits lie along a continuum of possible outcomes that depend on the relative amounts and subtypes of cadherins and catenins competing within a cell. Another recent study utilized fibroblastic cells transfected with chimeric cDNAs to express a chimeric receptor containing the cytoplasmic and transmembrane domains of 131-integrin and the extracellular domain of N-cadherin (Geiger et al., 1992). The expressed protein was found to preferentially localize to extracellular matrix adhesion sites, indicating that the cytoplasmic integrin domain was guiding cytoskeletal associations and subcellular distribution. However, transfectants expressing a high level of the chimeric protein assumed a more epithelial morphology and the protein was also found in cell--cell junctions. Interestingly, talin, a cytoskeletal component normally found in cell-matrix junctions, was recruited into these latter junctions. Thus the localization of adhesion molecules into different types of specialized junctions, and the attendant association with distinct cytoskeletal elements, appears to depend on a combination of factors mediated by interactions with both extracellular and intracellular ligands for such adhesion receptors.

The above cited experiments in which small peptides containing the HAV domain were found to inhibit cadherin binding indicate that the isolated binding region may retain biological activity. This is also supported by the observation that the soluble extracellular 80-kDa domain of E-cadherin also competitively inhibits cadherin function (Wheelock et al., 1987). These experiments do not address the question of whether positive adhesive interactions can be mediated in the absence of a cytoplasmic domain. Nature has addressed this point in providing an interesting variation on the classical cadherin structural theme in the form of T-cadherin, a truncated cadherin, which contains the typical extracellular cadherin domain organization but lacks both the HAV sequence and the cytoplasmic domain (Ranscht and Dours-Zimmerman, 1991). T-cadherin is linked to the cell surface by a phosphatidylinositol glycan linkage. T-cadherin can mediate homophilic calciumdependent adhesions among cells in suspension that have been transfected to express it, although it does not accumulate at intercellular boundaries among these cells when they are grown as a monolayer (Vestal and Ranscht, 1992). This study also indicated that T-cadherin appears to be relatively resistant to proteolysis even in the absence of calcium, and is not affected by drugs that perturb the cytoskeleton, which is not surprising because it lacks a cytoplasmic domain.

Additional evidence for cadherin interactions in the absence of a cytoplasmic domain was obtained in experiments where the 90-kDa extracellular domain of Ncadherin, which occurs in vh, o as a naturally occurring soluble proteolytic turnover product of N-cadherin (Roark et al., 1992), was purified and bound to a solid substrate in adhesion assays (Paradies and Grunwald, 1993). This study demonstrated that the isolated extracellular domain, referred to as NCAD90, could promote both adhesion and neurite growth among retinal neurons. More recently, direct homophilic NCAD90 binding has been demonstrated using covalently modified latex microbeads (Chagin and Grunwald, unpublished). Interestingly, as opposed to intercellular cadherinmediated adhesions, this NCAD90-mediated bead adhesion was found to be calciumindependent, although NCAD90 binds and is protected by calcium against trypsin. This raises the possibility that the calcium dependence of cadherin-mediated cell adhesion may be due in part to intracellular signaling or other functions than that mediated by the extracellular calcium-binding domains, as discussed below.

N-cadherin has been demonstrated to be a substrate for a cell surface Nacetylgalactosaminylphosphotrasferase (GalNAcPTase) of which the spatiotemporal expression, like that of N-cadherin, changes during development (Balsamo et al., 1986; Balsamo and Lilien, 1990). This enzyme appears to be structurally coupled to cadherins as the two proteins co-immunoprecipitate. Furthermore, a functional interaction between the GalNAcPTase and cadherins has been demonstrated in several systems. Antibodies to the enzyme uncouple cadherins from the cytoskeleton and inhibit N-cadherin-mediated adhesion among retinal cells (Balsamo et al., 1991). Furthermore, antibodies to the enzyme inhibit E-cadherin-mediated adhesion among pancreatic islet cells (Bauer et al., 1992). While the interaction with N-cadherin appears to be direct as part of a macromolecular complex, antibody perturbation of the enzyme affects not only N-cadherin-mediated neurite growth but that promoted by integrin and immunoglobulin family adhesion molecules as well (Gaya-Gonzalez et al., 1991). While no direct interactions between cadherins and other cell adhesion molecules have been demonstrated, indirect influences have been suggested by a number of experiments. Conflicting data exist on N-CAM-N-cadherin interaction, since cadherin function could be modulated by N-CAM-mediated interactions among membrane vesicles (Rutishauser et al., 1988), although such interaction was not observed among intact neurons (Doherty et al., 1991 b). While the classical cadherins are a major component of adherens junctions, and the related desmogleins and desmocollins are major components of desmosomes, they may act at least indirectly to regulate the formation of other types of intercellular junctions such as gap junctions and fight junctions (Mege et al., 1988; Gumbiner et al., 1988). The connexins, which form gap junctions, are not structurally related to cadherins. However, among cells that express both connexins and N-cadherin and possess both

adherens and gap junctions, antibodies to either protein alone were found to inhibit formation ofboth types ofjunctions (Meyer et al., 1992). In addition to the catenins, cadherin-containing protein complexes have been shown to contain ankyrin and fodrin (Nelson et al., 1990). Expression of exogenous E-cadherin in fibroblasts that, as previously mentioned, results in their assuming a more epithelial appearance, also results in the redistribution of Na§247 from a diffuse to a polarized membrane distribution, which parallels the normal expression in mesenchymal and epithelial cells, respectively (McNeill et al., 1990). Such a redistribution was not observed in cells transfected to express truncated E-cadherin lacking the cytoskeletal domain. Thus the cadherins have the potential to affect a wide variety of cellular molecules and overall morphology via their coordination of extracellular and intracellular events.

Among those cadherins whose gene organization has been explored, results suggest that each cadherin type is encoded by distinct single-copy genes. A possible exception to this appears to be Xenopus N-cadherin, which possesses two pseudoalleles that are both expressed, although apparently in similar patterns (Simonneau et al., 1992). Detailed information is available on the genomic organization of N-, E-, and P-cadherin, the original three classic cadherins. The structures of the chicken L-CAM (Sorkin et al., 1988) and mouse E-cadherin (Ringwald et al., 1991) genes are highly homologous in terms of exon-intron organization, although the E-cadherin gene is much larger. The E-cadherin gene is located on mouse chromosome 8, and is encoded by 16 exons encompassing over 40 kb ofDNA (Ringwald et al., 1991). These studies suggested that the exon structure does not correspond in any obvious way to functional domains of the protein. E-cadherin promoter analysis indicated that tissue-specific epithelial expression is controlled in part within the 5' upstream region, which contains motifs for several known transcription factors. Control of epithelial expression has been more closely localized to two regions, one GC-rich basic promoter and a neighboring palindromic enhancer which is structur' ally and functionally homologous to keratin gene promoter elements (Behrens et al., 1991). More recent analysis of the chick L-CAM gene revealed another cadherin gene upstream named K-CAM, which by sequence analysis is highly homologous to chicken B-cadherin (Sorkin et al., 1991). The mouse P-cadherin gene is encoded by 15 exons that extend over 45 kb of DNA, which includes a very large first intron of 23 kb (Hatta et al., 1991). Strong homologies exist between the Pcadherin gene and that of E-cadherin and L-CAM, and like E-cadherin, P-cadherin is also located on mouse chromosome 8. The M-cadherin gene has also been localized to mouse chromosome 8, which links it to murine E- and P-cadherin, and to hu-

man chromosome 16, which also links it to E-cadherin (Kaupmann et al., 1992). The structure of the mouse N-cadherin gene has also been analyzed and, unlike Eand P-cadherin, is located on mouse chromosome 18 (Miyatani et al., 1992), while human N-cadherin has been placed on human chromosome 18 (Walsh et al., 1990). Again, highly homologous exon-intron organization was found when compared to those of other cadherins. However, the murine N-cadherin gene is huge, encompassing over 200 kb of DNA which include 16 exons. The large size of the gene is due in part to a large first intron, as is the case with P-cadherin, but in addition there is a uniquely large second intron of over 100 kb in size. There is also a highly conserved extra 16th exon that appears to have arisen by a recent duplication, but is of unknown function.

No evidence exists for differential splicing among the classical cadherins, although it has been described for their desmosomal relatives, the desmocollins (Parker et al., 1991). In general, single major mRNA species have been detected on northern blots, which correspond to the single major protein species detected on western blots, and there is no evidence to suggest that the minor additional mRNA species detected are transcribed into identifiable gene products. Nevertheless, the appearance of multiple mRNA bands on Northern blots has been a common observation. For example, the murine N-cadherin mRNA is 4.3 kb in fetal mouse brain and heart, with minor bands at 5.2 and 3.5 kb (Walsh et al., 1990). This study also found N-cadherin mRNAs at 5.2, 4.3, and 4.0 kb in C6 glioma cells; at 4.3 and 4.0 kb in human fetal muscle; and at 4.3 kb in human fetal brain. Expression of cadherin mRNA's have been examined in the development of a variety of rat tissues, including retina (Roark et al., 1992), testis (Cyr et al., 1992), hippocampus (Wagner et al., 1992), and whole rat embryos (Chen et al., 1991). These studies indicate both quantitative and qualitative developmental changes in cadherin mRNA expression. Although several minor species of messages were identified in many of these studies, it is not yet clear if this variation is due to species, tissue, or age differences, or due to cross-reactivity with other cadherins, or structural variations such as differential polyadenylation.

The availability of immunological and molecular genetic probes has permitted extensive analyses of cadherin expression both during development and in mature tissues. These studies have indicated that cadherins are expressed in spatially and

temporally dynamic patterns, which suggest roles in the histogenesis of a wide variety of embryonic structures, with major changes occurring during active periods of rearrangement of cells resulting in new assemblies and segregations. The largest body of information exists for the three originally identified cadherins, E-, N- and P-cadherin. Such descriptive studies, of which selected examples follow, have demonstrated that most cadherin names are misnomers because they are based upon the first tissue in which the respective cadherins were identified. Most cadherins have a complex pattern of expression and can be found in multiple tissues. Also, many tissues express multiple cadherins. While cadherins are undoubtedly involved in cell signaling events, there is no data to suggest that they play a direct role in early embryonic inductive interactions. Rather, their distinct patterns of expression appear to be a result of such interactions. For example, studies in Xenopus show that N-cadherin expression results from the induction of ectoderm prior to neurulation, and no N-cadherin mRNA is detected in early embryos before induction or in isolated ectoderm, which does not undergo induction (Detrick et al., 1990). However, in these experiments, Ncadherin could be induced in isolated ectoderm using a heterologous inducer such as Hensen's node from the chick embryo. Thus N-cadherin expression appears to follow embryonic induction and to precede the morphogenetic changes associated with neural tube formation.

E-cadherin and its homologues are the first cadherins expressed in avian and mammalian embryonic tissues during development and can be detected in single cell mouse embryos (Vestweber et al., 1987; Sefton et al., 1992). As determined in both mammalian and avian embryos, it is initially expressed in all three germ layers, but is generally lost from regions of ectoderm giving rise to neural derivatives and from mesoderm as it assumes a mesenchymal behavior (Thiery et al., 1984; Damjanov et al., 1986). It remains prominently expressed in ectodermal derivatives such as skin and in endodermal epithelia. Antibodies to E-cadherin have been shown to disrupt normal epidermal morphogenesis through blocking feather development (Gallin et al., 1986) and keratinocyte stratification (Wheelock and Jensen, 1992). Langerhans cells, the leukocytes of epidermis, have been suggested to localize and interact with epidermal cells through the expression of E-cadherin because they express E-cadherin and anti-E-cadherin antibodies inhibit their interaction with keratinocytes (Tang et al., 1993). This is the first description of cadherin expression among leukocytes, and it indicates that cadherins may play important roles in the biology of a wider variety of cell types than previously demonstrated. While most studies of cadherin expression during early development have focused on the embryo proper, the cadherins play a role in the development of extraembryonic structures as well. E-cadherin is expressed by human trophoblast

cells during fusion and is down-regulated following their formation of syncitial trophoblast (Coutifaris et al., 1991). Syncitium formation was inhibited by anti-Ecadherin antibodies, suggesting a role for this protein in aggregation and fusion of trophoblast cells. P-cadherin is detected in extraembryonic tissues of implantation-stage mouse embryos and subsequently in maternal uterine tissue, suggesting it may play a role in implantation (Nose and Takeichi, 1986).

As discussed below, N-cadherin plays a dominant role in the development of the nervous system. However, N-cadherin has been found to be widely expressed during early stages of avian embryogenesis, in derivatives of all three germ layers (Duband et al., 1987, 1988; Hatta and Takeichi, 1986; Hatta et al., 1987). Expression was first detected on mesodermal cells which had migrated through the primitive streak, and many mesodermal derivatives including notochord, somites, nephrotome and lateral plate all expressed N-cadherin, with polarized expression during development of epithelial organization, and diffuse distribution on mesenchymal cells. Changes in the expression of N-cadherin were found during the subsequent development of these structures, with the most striking changes occurring in concert with changes in cell segregation patterns, such as tubulation to form the Mullerian ducts, or epithelialization of somites. Among ectodermal derivatives the most prominent site of N-cadherin expression was the developing nervous system, with additional expression found in the developing otic, olfactory, and lens placodes. The switch from E-cadherin to N-cadherin expression in these ectodermal derivatives correlates with the segregation and involution of the neural plate and lens and otic placodes. Among endodermal derivatives, the visceral pouches expressed high levels of N-cadherin, although most other endodermal derivatives expressed weakly if at all.

Comparison of studies of cadherin expression indicates that the observed patterns are often conserved but not identical across species. For example, in Xenopus development, E-cadherin expression is first detected in non-neural ectoderm of late gastrula stage embryos concomitantly with expression of N-cadherin in the neural plate (Levi et al., 1991 a). At earlier stages, another cadherin, EP-cadherin, is found in all cells from oocytes to the late blastula stage (Levi et al., 1991b). However, EPcadherin is lost from most of the cells contributing to the nervous system, while it continues to be expressed in non-neural ectoderm and endoderm where E-cadherin is found as well. It is also expressed in the somites and later in muscle. P-cadherin, which is expressed at high levels in the mouse placenta, is not detected in the bovine placenta (Liaw et al., 1990). Immunoblotting studies with an antiserumrecognizing N-cadherin in several species indicated that, in general expression, pat-

terns are conserved between avian and murine tissues during development (Lagunowich et al., 1990). The exceptions to this cited here suggest that in some cases relative differences in cadherin expression are perhaps more important than the absolute expression of a particular cadherin for the segregation of tissues, and that some cadherins may substitute for one another to accomplish similar morphogenetic ends.

The earliest expression of N-cadherin in several embryonic systems occurs following neural induction when it is found expressed in the neural plate (Hatta and Takeichi, 1986). The role of N-cadherin in early development has been explored via both negative and positive changes in expression. By injection of N-cadherin mRNA into Xenopus embryos, premature ectopic expression was obtained. This result led to the development of abnormal cell boundaries because of altered cell mixing and severe morphological defects, including abnormal development of the ectoderm and neural tube (Detrick et al., 1990; Fujimori et al., 1990). Negative perturbation of N-cadherin in early development has been carried out by the expression, in early Xenopus embryos, in vivo of a truncated form of N-cadherin lacking the external domains (Kintner, 1992). The construct was expressed by injection into blastomeres of mRNA encoding the transmembrane and cytoplasmic domains. The expressed protein was found to inhibit cell adhesion and to inhibit catenin binding to E-cadherin. In injected embryos, the integrity of the ectoderm was disturbed, and abnormal gastrulation tookplace. Interestingly, a series of deletion constructs demonstrated that at least two distinct sites on the truncated protein were able to interfere with cadherin function. One of these corresponds to the site involved in catenin binding, while the other is closer to the membrane and may be involved in cadherin clustering.

Given the complexity of the nervous system, it is perhaps not surprising that it is the site of multiple cadherin expression. N-cadherin is prominently expressed in the neural tube from its formation until late stages in neural histogenesis, when its expression becomes more restricted. A study of N-cadherin expression during chick spinal cord development indicated that, as in other CNS regions, N-cadherin is down-regulated but remains expressed at high levels in the floor plate in association with commissural segments of axons (Shiga and Oppenheim, 1991). At later stages of chick retinal development, N-cadherin expression is down-regulated and remains expressed at a high level only at the retinal outer limiting membrane, which is composed of adherens junctions (Matsunaga et al., 1988; Lagunowich and Grunwald, 1989). While initially widely expressed in the chick embryo brain during later

phases of histogenesis, expression becomes restricted to cells lining the ventricles and also in choroid plexus, most likely ependymal cells (Lagunowich et al., 1992). This study also demonstrated that N-cadherin expression remained high in the brainstem and spinal cord floorplate, a region which may serve as a guidepost for axonal migration during development. These protein studies have been complemented by studies ofN-cadherin mRNA expression by in situ hybridization (Redies and Takeichi, 1993). The role of N-cadherin in chick retinal histogenesis was explored by incubation of retinal explants in vitro with antibodies directed against N-cadherin (Matsunaga et al., 1988). While control explants developed the characteristic multilaminar appearance of retinal tissues, the antibody-treated tissues became disordered with abnormal laminations. This abnormal development was especially pronounced in the region of photoreceptors, perhaps reflecting the normally high expression of Ncadherin at the outer limiting membrane. However, there did not appear to be any effect on differentiation per se as determined by expression of photoreceptor marker antigens. While N-cadherin is the predominant cadherin expressed in developing neural tissues, other cadherins have been described within the nervous system as well. Even E-cadherin, which had long been considered to not be expressed there, has been shown to be expressed transiently in restricted regions of the metencephalon, mesencephalon, diencephalon, and cerebellum, persisting longest in the roof plate (Shimamura and Takeichi, 1992). Explants of E-cadherin-positive brain regions, when treated with anti-E-cadherin antibodies, underwent alterations in morphology. E-cadherin is also expressed in sensory neurons of mouse dorsal root ganglia, satellite, and Schwann cells during development and into adulthood (Shimamura et al., 1992). Two interesting observations made as part of this study were that only a subset within each class of cell expressed E-cadherin, and that the target region of the spinal cord to which the E-cadherin-positive DRG neurons projected was restricted to a region of lamina II of the dorsal horn, which extended all the way rostrally to include part of the trigeminal nucleus. This may indicate a specific role for this cadherin in establishment of sensory pathways in the developing CNS. This interpretation is also supported by comparative studies of R- and Ncadherin expression. R-cadherin has been identified in chick embryos (Inuzuka et al., 1991a,b). While retina and brain were identified as expressing the highest levels, it was found to be expressed, as is N-cadherin, in all neural tissues. Interestingly, the temporal pattern of expression of R- and N-cadherin are reciprocal, in N-cadherin is expressed very early and is then down-regulated, while R-cadherin expression increases during development. In addition, expression of N- and Rcadherin even within tissues in which they are expressed at the same time was not identical. As with N-cadherin, R-cadherin is also not restricted to neural tissues, as it was also found to be expressed in notochord, myotome, and early skeletal muscle. In a later study, the detailed expression of N- and R-cadherin were compared during the development of sensory and motor axon systems of the embryonic chick (Re-

dies et al., 1992; 1993). It was observed that these molecules are often expressed in a reciprocal fashion, as in the hindbrain where N-cadherin is preferentially expressed on sensory axons while R-cadherin is found on motor axons. N- and R-cadherin may differ in other functional ways as well. The developmental loss of the in vitro histogenetic potential of dissociated retinal cells parallels the loss of N-cadherin expression (Grunwald et al., 1981; Matsunaga et al., 1988; Lagunowich and Grunwald, 1989), even while R-cadherin expression increases. This suggests that N- and R-cadherin play very different roles in retinal histogenesis, or that other factors than cadherin expression become limiting as the retina differentiates. N-cadherin expression is dynamically regulated during neural crest development, as revealed by the earlier cited antibody localization studies. More recently, the role of N-cadherin in neural crest development has been examined in the embryonic chick (Akitaya and Bronner-Fraser, 1992; Bronner-Fraser et al., 1992). In agreement with earlier studies, N-cadherin was detected on the neural folds prior to tube closure and on the closed neural tube. However, no immunoreactivity was detected on migrating neural crest cells that had left the neural tube, although it was expressed on neural crest cells during condensation into ganglia. The functional role of N-cadherin in this process was explored by microinjection of anti-Ncadherin antibodies into the cranial cavity. This led to the development of abnormal and misshapen or open neural tubes and the formation of ectopic aggregates of neural crest cells. The expression of T-cadherin suggests it may play a role in determining neural crest migration patterns (Ranscht and Bronner-Fraser, 1991). Finally, in the nervous system, cadherins are expressed not only in nervous tissue proper, but also in associated structures. E-cadherin is found in arachnoid villi (Yamashima et al., 1992) and N- and B-cadherin are expressed in choroid plexus (Napolitano et al., 1991; Lagunowich et al., 1992).

As mentioned above, immunohistochemical studies of embryonic development indicated that N-cadherin is prominently expressed in the mesoderm of embryos and early in muscle development, but is down-regulated during later stages of myogenesis (Hatta et al., 1987). N-cadherin is expressed on the surface of avian skeletal myoblasts (Soler and Knudsen, 1991), and antibodies against N-cadherin inhibit myoblast fusion (Knudsen et al., 1990; Pouliot et al., 1990). Myoblast fusion is inhibited by synthetic peptides containing the cadherin HAV recognition sequence and with antibodies directed against N-cadherin (Mege et al, 1992). The more recently identified M-cadherin, also expressed in muscle, is found at low levels in myoblasts but is up-regulated during myotube formation (Donalies et al., 1991). Thus it is likely that at least two cadherins contribute to the process of myogenesis, although two more, B- and T-cadherin, have been identified in muscle as well (Ranscht and Dours-Zimmerman, 1991; Napolitano et al., 1991). Interestingly, Ncadherin mRNA and protein expression in skeletal muscle appears to be under neu-

ral regulation, with down-regulation occurring upon innervation and reexpression occurring following denervation (Hahn and Covault, 1992). This study further indicated that the level of N-cadherin expressed in mature muscle fibers may depend in part on the muscle fiber type and on the level of motor activity. In addition to its already discussed role in adherens junctions of heart cells, Ncadherin may play important roles in other aspects of cardiac development. Recent studies of N-cadherin expression indicate it may play a role in formation of the pericardial coelom, and in later stages is down-regulated in the endocardium but continues to be expressed in the formation of the myocardium (Linask, 1992). A confocal scanning laser study of heart development indicated that N-cadherin may serve to align myofibrils between adjacent myocytes and play a role in the important morphogenetic event of cardiac looping (Shiraishi et al., 1993).

The prominent expression of N-cadherin during nervous system development has led to many studies of its expression and function during neurogenesis, with particular attention paid to its involvement in neurite growth. Many adhesion molecules are expressed on neurites, and the experimental abolition of neurite growth over complex substrates such as other neurons or glial cells has been shown to require simultaneous inhibition of several different classes of adhesion molecules. For example, antibody inhibition studies of ciliary ganglion cell neurite growth on astrocytes indicated that both cadherins and integrins were involved in process outgrowth (Tomaselli et al., 1988). Similar studies of growth of motor neuron axons over Schwann cells suggested that integrins, immunoglobulin gene superfamily members, and cadherins all functioned together in an additive fashion to promote neurite outgrowth (Bixby et al, 1988). Developmental changes in the relative roles of different adhesive receptors were also found in these studies, indicating that the promotion of neurite outgrowth is a highly context-dependent process exhibiting both cell-type and age-dependent specificity. Studies using transfected fibroblastic cell lines showed that when such cells expressed N-cadherin, they could serve as a substrate for neurite growth (Matsunaga et al., 1988). Such transfected cells have been used to analyze aspects of the mechanism of cadherin-mediated neurite growth. Using a variety of neurons including retinal and cerebellar cells, the response to N-cadherin expressed by transfected 3T3 fibroblasts appeared to increase linearly with increased expression of Ncadherin. In contrast to this, the response to N-CAM was highly cooperative, and the two adhesion molecules were found to potentiate each other, although the developmental profiles of response to these adhesion molecules differed (Doherty et al., 199 l b). N-cadherin-expressing, transfected fibroblasts were found to induce the differentiation and neurite extension of PC12 cells (Doherty et al., 1991 a). This re-

sponse was inhibited by pertussis toxin and calcium channel blockers, could be modulated by drugs that affect protein kinases, and occurred in a transcriptionindependent fashion. This appeared to be mediated by a pathway independent of that induced by nerve growth factor. These observations clearly indicate that transmembrane signaling events triggered by cell adhesion molecules can regulate aspects of neuronal differentiation. However, the extent to which this occurred independently of adhesive function per se remained in question. This was further examined using a similar experimental paradigm in which it was found that PC12 cells growing on a 3T3 monolayer could be induced to differentiate in a manner similar to that induced by N-cadherin by potassium-induced depolarization or agonist-induced activation of calcium channels (Saffel et al., 1992). Like the Ncadherin stimulated growth, the potassium-effect was blocked by calcium channel antagonists and kinase inhibitors; however, pertussis toxin had no effect. These resuits support the idea that N-cadherin may affect PC12 cells by activating secondmessenger systems rather than acting as an adhesion protein per se, and that the chain of events proceeds from adhesion receptor activation, to activation of calcium channels via a G-protein-mediated process, and through a kinase-dependent step to affect neurite growth. N-cadherin promotion of neurite growth may be modulated by a number of factors such as incorporation of exogenous ganglioside GM 1 into PC 12 cells (Doherty et al., 1992a). Fibroblasts expressing N-cadherin and N-CAM can both serve as a substrate for neurite growth among hippocampal neurons, although over the course of development from late embryonic to early postnatal days, responsiveness to NCAM decreases while that for N-cadherin increases (Doherty et al., 1992b). Neurite outgrowth is also inhibited by antibodies against the Nacetylgalactosaminylphosphotransferase which, as described above, is functionally and structurally closely associated with N-cadherin (Gaya-Gonzalez et al., 1991). While many of the above studies examine the function of N-cadherin in complex settings such as when expressed by transfected cells, other studies have evaluated the biological activity of isolated N-cadherin. Purified N-cadherin was found to be a potent substrate for neurite growth when directly coated onto a nitrocellulose support and used as a substrate for ciliary ganglion neurons (Bixby and Zhang, 1990). This approach has permitted the further analysis of N-cadherin function where it was found that, as opposed to integrin-mediated neurite growth, which is inhibited by protein kinase inhibitors, N-cadherin-mediated outgrowth was stimulated by these drugs (Bixby and Jhabvala, 1990). However, this effect was transient as the potentiation seemed to affect initiation of neurite growth rather than its maintenance, and the protein kinase C agonist TPA was found to stimulate N.cadherinmediated neurite growth. These results suggest that the involvement of intracellular signaling systems may depend on the adhesion molecule, as well as the stage in the process. Purified NCAD90, the naturally occurring soluble extracellular fragment of N-cadherin discussed above, also functions in a similar manner when immobi-

lized on nitrocellulose as a promoter of neuronal adhesion and neurite outgrowth (Paradies and Grunwald, 1993). Studies utilizing purified adhesion molecules including N-cadherin as adhesive substrates have examined growth cone behavior, and have led to the conclusion that different adhesion molecules have different effects on growth cone morphology (Payne et al., 1992), and that relative adhesiveness does not appear to be the major determinant with respect to axonal pathway choice, growth rate, or extent of fasciculation (Lemmon et al, 1992). In addition, recent studies have provided a direct demonstration of elevation of intracellular calcium levels in neuronal growth cones through N-cadherin interactions (Bixby et al., 1994).

Most studies of cadherins have been done in the context of embryonic development. However, cadherin expression persists in adult organisms and presumably plays important roles in the function and stability of mature tissues. It is therefore likely that misexpression of cadherins may be involved in a variety of pathologies. This has been examined most extensively in cancer cells, since the role of cadherins as adhesion molecules prompted examination of their possible role in metastasis. This work has focused on E-cadherin and carcinomas. Several studies have indicated that an inverse correlation exists between expression of E-cadherin and cell invasiveness and metastasis. Among a large panel of human squamous cell skin carcinomas, the highly differentiated tumors expressed as much E-cadherin as normal epithelia, while E-cadherin levels were much reduced in the less welldifferentiated tumors (Schipper et al., 1991). Among tumor cells which had infiltrated lymph nodes, low E-cadherin expression was found. Similarly, decreased expression of E-cadherin has been associated with progression of rat prostatic cancer (Bussemakers et al., 1992), mouse skin cancer (Ruggeri et al., 1992), and human meningiomas (Tohma et al., 1992). Genetic elements which regulate Ecadherin expression are less active in poorly differentiated tumor cells than in well-differentiated cells (Behrens et al., 1991). While most attention has been focused on E-cadherin and cancer, one study of N-cadherin expression in tumor cells has been done where expression of this neural cadherin was examined in a series of human retinoblastoma cell lines (Schiffman and Grunwald, 1992). This study indicated that N-cadherin is expressed by retinoblastoma cells, although both qualitative and quantitative variations in expression were observed between the various cell lines. Interestingly, while not absolute, a correlation existed between the level of expression and the extent of calcium-dependent adhesiveness and morphology of adhesions between the cells. However, it is important to note

that the expression level of cadherins is not likely to be the only factor determining changes in the behavior of these cells. For example, the fibroblast growth factor-induced dispersion of rat bladder carcinoma cells that results in their transition from an epithelial to mesenchymal behavior, results in a redistribution of cell surface E-cadherin without a reduction in its total expression or the calciumdependent adhesiveness of the cells (Boyer et al., 1992). In addition, a line of human lung cancer cells with defective cell-cell adhesion was shown to express Ecadherin, but to be defective in expression of a-catenin (Shimoyama et al., 1992). Finally, transformation of cells may result not only in the loss of expression of some cadherins, but the expression of inappropriate cadherins of a less mature phenotype, as in pancreatic tumors (Moiler et al., 1992).

Cadherin misexpression has been suggested to play a role in other pathogenetic processes. A possible role for cadherins has been suggested as the basis for epithelial abnormalities in hereditary hair loss in children (Baden et al., 1992). Cadherin misexpression has also been associated with Darier's and Hailey-Hailey diseases, which result in abnormal keratinocyte adhesion (Burge and Schomberg, 1992). A desmosomal cadherin called PVA has been identified as the target antigen in the autoimmune skin blistering disease pemphigus vulgaris (Amagai et al., 1991). Passive transfer of anti-PVA antibodies caused pemphigus-like disturbances in the skin of experimental mice (Amagai et al., 1992). As calcium-binding proteins, the cadherins are potential targets for calcium antagonists. Indeed it has been demonstrated that cadmium alters the distribution of E-cadherin and formation of cell junctions among renal epithelial cells (Prozialek and Niewenhuis, 1991).

The cadherins have been identified as key molecules in the regulation of cell interactions during development and are now implicated in a number of pathological processes. While much has been learned in the last decade about cadherin structure and function, the growing diversity of the cadherin gene superfamily and the emerging complexity of regulatory mechanisms governing cadherin expression and function demonstrate that much remains to be discovered regarding the cadherins. How many cadherins are there? How is cadherin expression regulated genetically and epigenetically? Why do tissues express multiple cadherins and how do their functions differ? What types of information are communicated between cadherins and the cytoskeleton and via cadherin-mediated second messenger pathways? Can the analysis of cadherins be of diagnostic and prognostic use in clinical situations? These and other questions are and will continue to be addressed as more is learned about the biology of these fascinating molecules.

The author' s work is supported by grants EY06658 from the National Institutes of Health and BNS9021703 from the National Science Foundation.

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Yamada, K.M. & Gumbiner, B.M. (1993). Cell-to-cell contact and extracellular matrix. Curr. Opinion in Cell Biol. 5, 769-939. (An entire volume of this journal devoted to reviews of the current status of cell adhesion research.) Garrod, D.R. (1996). Epithelial development and differentiation: The role of desmosomes. J. Roy. Cell. Physicians. 30(4) 366-373. Gilbert, S.F. (1994). Developmental Biology, 4th edn. Sinauer, Sunderland, MA. (An excellent comprehensive text dealing with aspects of both descriptive and experimental embryology, which provides additional background about developmental events many of which are regulated in part by cadherin function.)

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Introduction Sorting of Membrane Proteins in the Secretory Pathway of Epithelial Cells Membrane-Cytoskeletal Proteins Interact with Specialized Epithelial Cell Junctions The Brush Border The Tight Junction Intercellular Adhesion Roles for the Fodrin-Based Membrane-Cytoskeletal Complex and Cadherin-Mediated Cell-Cell Contact in Generating Polarized Distributions of Membrane Proteins Concluding Remarks

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Epithelial cells arise by a series of developmental events in which "nonpolarized" precursor cells (often mesenchyme) differentiate and form a layer of cells that perPrinciples of Medical Biology, Volume 11 Developmental Biology, pages 69-87. Copyright 9 1998 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-816-1 69

Figure 1. Morphogenetic processes involved in generating epithelial structures during histogenesis and organogenesis. Mesenchymal (undifferentiated) cells often give rise to epithelial tissues. For example, during kidney development, mesenchymal cells are induced by the growing ureter bud, and this mesenchyme condenses (Condensation), resulting in epithelial cell differentiation. Epithelial cells develop a polarized phenotype (see text), and fluid transport may result in lumen formation (Lumen Formation). Continued differentiation and histogenesis produces functional kidney tubule segments that perform specific functions. In other cases, epithelial tissues are produced by rearrangement and differentiation of existing epithelial tissues. By gross morphogenetic changes, an epithelial structure may invaginate and pinch off a cyst or tube structure (Cell Shape Change). Examples of this type of process are neurulation and lens formation. form specialized functions in the animal. Simple epithelial cells (for example, kidney or intestinal epithelia) separate two biological compartments, and maintain the ionic composition of the fluids in these biological compartments by vectorial transport of ions, solutes, and water. To perform these functions, epithelial cells must establish and maintain distinct protein compositions at two plasma membrane domains, termed apical and basal-lateral, that face the different biological compartments. The apical domain is almost always a free cell surface (not in contact with any other cell layer) that faces a lumen. The basal-lateral plasma membrane domain forms contacts with other cells in the monolayer (lateral plasma membrane) and with the basement membrane (basal plasma membrane), and usually faces the serosa. The apical and basal-lateral membrane domains are separated by the fight junction. This junctional complex regulates the passive diffusion of ions, solutes, and fluid between cells (paracellular transport). Also, the tight junction acts as "fence", preventing lateral diffusion of proteins and some lipids between the api-

71 cal and basal-lateral plasma membrane domains. Through a variety of mechanisms that will be discussed in this chapter, epithelial cells establish and maintain differences in the composition of ion transporters, ion channels, and enzymes between the apical and basal-lateral membrane domains, which determine the vectorial transport of ions and solutes across the epithelium. Mesenchymal cells must undergo a fundamental change in cell form and cell identity to organize the plasma membrane into domains that perform cell-type specific functions (Figure 1), and this phenotype must be maintained over the lifetime of the cell. Numerous studies have revealed mechanisms that work in concert to develop a polarized cell phenotype (Mostov et al., 1992; Nelson, 1992; RodriguezBoulan and Powell, 1992). Significantly, some of these mechanisms are altered as a consequence of certain pathological conditions (Avner et al., 1992; Fish and Molitoris, 1994). Three major mechanisms are used to establish and maintain polarized distributions for membrane proteins (ion transporters, ion channels, and enzymes) and lipids: (i) restricting lateral diffusion between membrane domains by assembly of the tight junction; (ii) polarized delivery from the trans-Golgi network to the plasma membrane, and (iii) retention of proteins at the proper plasma membrane domain. In this chapter, we will summarize the important role that sorting of membrane proteins plays in generating cell polarity that has been characterized in recent years, and we will outline the current understanding of how structural elements (cytoskeleton and junctional complexes) in epithelial cells function and give rise to the polarized cell phenotype.

There are at least three intracellular sites for membrane protein sorting in polarized epithelial cells, which appear to be located in the Golgi complex, endosomes, and at the plasma membrane. In Madin-Darby canine kidney (MDCK) cells, a cell line derived from polarized renal epithelia, proteins are generally sorted into different vesicle populations in the trans-Golgi network and delivered directly to either the apical or basal-lateral membrane (Figure 2). In hepatocytes, and to a lesser extent in the intestinal cell line, Caco-2 proteins are first delivered to the basal-lateral membrane; basal-lateral proteins are selectively retained, while apical proteins are sorted and selectively targeted to the apical plasma membrane (Simons and Wandinger-Ness, 1990; Rodriguez-Boulan and Powell, 1992). A major line of investigation in cell polarity research has been to identify signals within proteins that are used in sorting of membrane proteins to their destination at the apical or basal-lateral membrane domains. These sorting signals are generally recognized in the trans-Golgi network, but also at the cell surface or in endosomes.

In MDCK cells, sorting of one class of apical proteins in the trans-Golgi network may be mediated by a glycophosphatidylinositol (GPI) anchor. Because glycosphingolipids are also sorted in the trans-Golgi network to the apical membrane, it has been proposed that GPI-anchored proteins are sorted in the trans-Golgi network by incorporation into glycosphingolipid rafts (Simons and Wandinger-Ness, 1990). This hypothesis has been tested by blocking glycosphingolipid synthesis us-

ing the drug fumonisin B 1. In these drug-treated cells, normally apically-targeted, GPI-anchored membrane proteins were delivered randomly to both the apical and basal-lateral plasma membrane domains (Mays et al., 1995), supporting the proposed role of glycosphingolipid clustering in apical sorting. For non-GPI anchored apical proteins, sorting information in the ectodomain could be recognized by an apical sorting receptor that interacts with glycosphingolipids (Simons and Wandinger-Ness, 1990). Glycosylation moieties on the ectodomains of membrane proteins have been shown to act as apical sorting information (Scheiffele et al., 1995). The glycosylation modification may be recognized by lectin-like proteins in the trans-Golgi network (Fiedler et al., 1994). Sorting signals for basal-lateral type I membrane proteins are characterized by a short cytoplasmic amino acid sequence located close to the transmembrane domain (Rodriguez-Boulan and Powell, 1992). Nearly all the basal-lateral sorting signals examined contain critical tyrosine residues in the cytoplasmic domain and may include a cluster of three negatively charged amino acids, C-terminal to the critical tyrosines. Removal of the basal-lateral sorting signal results in the protein being transported directly from the trans-Golgi network to the apical membrane. These results suggest that there is a hierarchy of sorting determinants within a single protein. The critical tyrosine in the basal-lateral sorting signal is reminiscent of the endocytosis signal, xNPxYx, which forms a tight 13-turnrequired for coated pit localization (Eberle et al., 1991). Although the basal-lateral sorting signal of the poly IgA receptor does not contain a "critical" tyrosine residue, the three dimensional structure of the basal-lateral signal also forms a tight 13-turn (Aroeti et al., 1993). Based upon these results, it has been suggested that the sorting machinery for basal-lateral proteins recognize a common structural motif in proteins, and that endosomes may also be sorting sites for polarized proteins (Figure 2). In vitro reconstitution of protein sorting and formation of transport vesicles in the trans-Golgi network is providing insight into the cellular machinery involved (Pimplikar et al., 1994; Lafont et al., 1994; Ikonen et al., 1995). However, there is a significant gap in our understanding of protein sorting because the mechanism involved in trafficking and docking of transport vesicles to specific domains of the plasma membrane is largely unknown (for example, see Mays et al., 1995; Ikonen et al., 1995). Apical and basal-lateral sorting signals are not restricted to proteins in polarized cells. Similar sorting pathways may exist in "nonpolarized" cells; however, because the membrane is "undifferentiated" in these cells, proteins may be delivered randomly to the cell surface. An important distinction between many polarized and nonpolarized cells is the formation of specialized cell-cell adhesion junctional complexes. The recruitment of membrane proteins and membranecytoskeletal proteins to sites of cell-cell adhesion results in formation of specialized membrane domains in many cell types. Examples include epithelia (see below), the synapse, the neuro-muscularjunction, and activated cells of the immune system.

Early ultrastructural observations gave clues about specializations of the plasma membrane that distinguish polarized from nonpolarized epithelial cells. These include brush border, tight junction, adherens junction, desmosome and hemidesmosome (Figure 3). These plasma membrane specializations are intimately associated with specific cytoskeletal structures (microfilaments or intermediate filaments), suggesting that they are organized by the cytoskeleton, and provide stable membrane attachment sites for the cytoskeleton. Furthermore, epithelial isoforms of

membrane-cytoskeletal proteins originally identified in the erythrocyte (ankyrin and spectrin) were shown to play important roles for establishment and maintenance of plasma membrane domains.

The brush border is a specialized and highly organized assemblage of microvilli that is characteristic of the apical plasma membrane in some epithelial cells (Mooseker, 1985). Early ultrastructural studies showed that the major structural component of the microvillus is the actin microfilament. Actin binding proteins (villin, fimbrin, myosin I; see below) organize these actin microfilaments into bundles that insert at the tip of each microvillus and extend downward into the cytoplasm, where the filaments end at the terminal web. The terminal web is a meshwork of actin and intermediate filaments at the level of the adherens junction that attaches to and stabilizes, actin filaments of the microvilli. The molecular components of the microvillus and terminal web have been partially described (Mooseker, 1985; Bretscher, 1991). Myosin I, villin, fimbrin, and ezrin are the best characterized components of the microvillus. These components bind actin filaments directly (Bretscher, 1991; Algrain et al., 1993; Turunen et al., 1994). Villin and fimbrin crosslink actin filaments to produce the actin filament bundle. Ezrin interacts with membrane proteins and actin (Tsukita et al., 1994). Myosin I can bind both actin and lipids, which may give rise to the tight association of membrane around the actin bundle and the microvillus membrane projection. Myosin I may also serve to deliver Golgi-derived vesicles to the apical surface/brush border (Fath and Burgess, 1993). Indeed, there have been numerous myosin types discovered recently, and some of these myosins will probably be shown to be important for epithelial structure and function (Mooseker and Cheney, 1995). The terminal web meshwork of actin filaments are crosslinked by accessory proteins that include a terminal web isoform of fodrin (Bretscher, 1991). Isoforms of the sarcomeric proteins titin and nebulin have also been described in the terminal web of the brush border (Eilertsen and Keller, 1992), and numerous other proteins will no doubt be shown to play roles in maintaining the structural and functional properties of the brush border.

The principal function of epithelial cells is the formation of fluid-tight barriers between different biological compartments, thus allowing efficient transport of ions and solutes across that barrier. The barrier function is performed by the tight junction, or zonula occludens (Gumbiner, 1993). The tight junction may have selective permeability to certain ions ("gate" function) indicating that tightjunction permeability is regulated (Madara, 1990). Tight junction permeability varies significantly over the length of the kidney nephron (weak tight junction

function in the proximal tubule to very tight in the distal tubule), suggesting that tight junction function is regulated in vivo. The tight junction restricts the mixing of proteins and lipids between the membrane domains ("fence" function) (Mandel et al., 1993). A number of cytoplasmic proteins (ZO-1, ZO-2, cingulin, 7H6) have been identified as tight junction-associated proteins; they have provided molecular markers for the tight junction and their characterization is leading to a greater understanding of how the tight junction is regulated (Gumbiner, 1993). Recent cloning of the ZO-1 and ZO-2 proteins has revealed significant amino acid sequence identity with the Drosophila protein, discs large giant (dlg), which functions as a tumor suppresser gene for imaginal disc epithelial cells (Willott et al., 1993; Woods and Bryant, 1993; Jesaitis and Goodenough, 1994). In fact, ZO-1, ZO-2 and dlg gene product are members of a large family of proteins that share a common domain structure, the membrane-associated guanylate kinase (MAGuK) family; many MAGuK family members are localized to intracellular junctional complexes (e.g., tight junction) and are involved in developmental events (Kim, 1995). This suggests that there may be intracellular signaling properties associated with the tight junction complex. Such signaling events may also regulate paracellular transport. An integral membrane protein component of the tight junction (occludin) has been cloned and characterized (Furuse et al., 1993). The occludin cytoplasmic domain binds ZO- 1 directly (Furuse et al., 1994), thus raising many new experimental avenues for investigating the mechanisms involved in regulating paracellular ion and fluid transport. Evidence strongly indicates that integral membrane proteins have important roles in the "fence and gate" functions of the tight junction (Mandel et al., 1993), but the mechanism involved is unknown.

Epithelial cells form close cell-cell contacts in the monolayer that are largely mediated by Ca2+-dependent cell adhesion molecules of the cadherin superfamily. These proteins include a subset of cadherins, which are components of the adherens junction (classical cadherins), and others that are components of morphologically distinct structures termed desmosomes (desmosomal cadherins) (Kemler, 1992). Adherens junction cadherins typically bind to the same cadherin family member on the opposing cell, a process termed homophilic adhesion. Cadherin-mediated cell-cell adhesion is essential; disruption of the E-cadherin gene showed that this protein is necessary for early mouse development (Larue et al., 1994). Embryos lacking zygotic E-cadherin fail to hatch from the zona pellucida and die. Cadherins perform many roles in epithelial cells beyond simple adhesive function. Cadherins generate intracellular signals and organize membrane domains through linkage to the membrane cytoskeleton and other integral membrane proteins (Doherty et al., 1991; Nelson, 1992; Drubin and Nelson, 1996).

77 Adherens junctional and desmosomal cadherins bind different cytoplasmic, membrane-cytoskeletal proteins, and the different plaque structures of the two junctional complexes provide attachment sites for different cytoskeletal filament systems, actin (adherens junction), or intermediate filaments (desmosome) (Buxton and Magee, 1992; Nathke et al., 1993). Classical cadherins have three tightly associated membrane-cytoskeletal proteins termed the catenins (tx-catenin, [3-catenin and T-catenin). The hierarchy of binding catenins to cadherins has been established (Aberle et al. 1994; Jou et al., 1995). Cadherin cytoplasmic domains bind directly to either I]-catenin or T-catenin, but not both-the binding is mutually exclusive (Hinck et al., 1994). In turn, [3-catenin and T-catenin bind directly to tx-catenin, which in turn binds actin directly (Rimm et al., 1995). Therefore, the binding of catenins to classical cadherins is required for association with the actin cytoskeleton (Nagafuchi et al., 1994; Rimm et al., 1995). Classical cadherins also associate with the fodrin/ankyrin membrane-cytoskeleton network (nonerythroid isoforms of the spectrin/ankyrin red cell membrane-cytoskeleton; see below; Nelson, 1992). Numerous protein-protein associations are probably involved in adherens junction plaque assembly, and some of the proteins described may play structural (e.g., tx-actinin, vinculin, and radixin) or signaling (e.g., protein tyrosine kinases) roles (Tsukita et al., 1992). The catenins (a-catenin, ~-catenin and T-catenin) have been molecularly characterized (reviewed in Kemler, 1993). Alpha-catenin is related to vinculin and acts to link cadherins to the actin cytoskeleton (Rimm et al., 1995). Alpha-catenin was also shown to be required for morphoregulatory functions of cadherins, that is, an tx-catenin mutant cell line was unable to form three-dimensional tissue-like structures until these cells were rescued by tx-catenin cDNA transfection (Hirano et al., 1992; Watabe et al., 1994). Gamma-catenin was shown to be identical to the previously characterized protein plakoglobin. Beta-catenin and T-catenin share amino acid sequence homology with one another and with the Drosophila segment polarity gene armadillo. In Drosophila, armadillo is downstream in the segment polarity signaling pathway from the wingless gene; vertebrate homologs of wingless are termed "wnts". The sequence similarity between armadillo, 13-,and T-catenin suggested that all these proteins function in intracellular signaling and developmental pathways. Experiments involving tissue culture cells have shown that the wingless homolog, the wnt- 1 proto-oncogene, regulates protein levels for 13-catenin and controis the avidity of cell-cell adhesion (Bradley et al., 1993, Hinck et al., 1994). Xenopus dorsal-ventral polarity during early embryogenesis is controlled by the wnt signaling pathway, and 13-and T-catenin were shown to be involved (Gumbiner, 1995). Recent gene disruption experiments show that 13-catenin is required for early mouse development; embryos lacking 13-catenin fail to gastrulate (Haegel et al., 1995). Catenins have been shown to associate with proteins other than cadherins within epithelial cells. Most notably, [3-catenin associates directly with the product of the APC gene (adenomatous polyposis coli gene, a tumor suppresser gene

associated with colorectal cancer susceptibility; Rubinfeld et al., 1993; Su et al., 1993). It is not known whether APC regulates 13-catenin function during tumorogenesis in colon epithelial cells. APC also interacts with tubulin (Munemitsu et al., 1994; Smith et al., 1994; Nathke et al., 1996), and the human homolog of the discs large tumor suppresser protein (dlg; Matsumine et al., 1996). These data suggest that APC regulates junctional complex assembly, cell migration, and cell growth. Desmosomal cadherins provide Ca2§ cell-cell adhesion and nucleate desmosomal plaque assembly providing membrane attachment sites for intermediate filaments (Buxton and Magee, 1992). Desmosomal cadherins are less well characterized than classical cadherins; for instance, it is unknown whether these cadherins form homophilic or heterophilic complexes with other desmosomal cadherins. There are two classes of desmosomal cadherins: desmocollins and desmogleins (Buxton and Magee, 1992). Desmosomal cadherins interact with plakoglobin (y-catenin; Mathur et al., 1994; Troyanovsky et al., 1994). However, other protein components of the desmosomal plaque are very different from those of the adherens junction (Jones and Green, 1991). Indeed, it was shown that plakoglobin bound to desmosomal cadherin can not bind a-catenin (Wahl et al., 1996; Witcher et al., 1996), which is consistent with the desmosome providing membrane linkage for the intermediate filament cytoskeleton rather than microfilaments. A peripheral desmosome component, desmoplakin, binds directly to intermediate filament proteins (Kouklis eta!., 1994). Hemidesmosomes are cell-substratum contacts that form plaque structures that resemble desmosomes morphologically and associate with intermediate filaments, but are composed of a different set of proteins (Jones and Green, 1991). One membrane component of hemidesmosomes is the 0[,6~ 4 integrin, which attaches the cell to the extracellular matrix (Kurpakus et al., 1991). The presence of an integrin in the hemidesmosome is in contrast to most integrin attachments (such as focal contacts), which provide membrane attachments for actin filaments (Pavalko and Otey, 1994). An intermediate filament binding protein has also been shown to associate with both desmosome and hemidesmosome, providing a possible molecular linkage for the filament attachment (Skalli et al., 1994). In studies of desmosomes and hemidesmosomes, autoimmune sera from patients with a variety of skin-blistering diseases have been exploited (Jones and Green, 1991). One of these diseases, bullous pemphigoid (BP), affects hemidesmosomes. BP patients produce autoimmune sera that recognize a set of antigens. One major antigen is bullous pemphigoid antigen 1 (BPAG1), which shows sequence and structural similarity with desmoplakins (an intermediate filament binding protein in the desmosome; Kowalczyk et al., 1994). Genetic knockout experiments have shown that mice lacking BPAG 1 lose attachment to intermediate filaments and have blistering disease (Guo et al., 1995), suggesting that BPAG1 binds intermediate filaments and that this is required for normal epidermal tissue integrity.

Mechanisms for generating polarized distributions of membrane proteins in epithelial cells are being actively investigated. As noted above, proteins can be directly targeted from the Golgi complex to either the apical or basal-lateral membrane domains (Rodriguez-Boulan and Powell, 1992). Upon arrival in the proper location in the cell, tight junctions act like a fence to keep membrane proteins from mixing between the apical and basal-lateral membrane domains (Mandel et al., 1993). At the plasma membrane, membrane proteins are continually subject to internalization by endocytosis, and endocytosis could serve to retrieve a protein from one membrane domain and, through transcytosis, to be sent to the other membrane domain (Mostov and Simister, 1985). Alternatively, proteins internalized may be sent to the lysosome and degraded. It has been suggested that association with the fodrin-based membrane-cytoskeleton immobilizes membrane proteins and sequesters them from endocytosis and the potential consequences of this process, thereby acting as a retention system for membrane proteins (Nelson, 1992). This hypothesis is supported by early studies in red cells showing that loss of interactions between membrane proteins and the spectrin-based membrane-cytoskeleton results in increased mobility of proteins in the plane of the lipid bilayer (Sheetz et al., 1980). Membrane-cytoskeleton components related to those found in the erythrocyte have been identified in most cell types including simple epithelial cells (Bennett, 1990). This fodrin (spectrin)-based membrane-cytoskeleton forms a cytoskeletal meshwork attached by another cytoskeletal protein, termed ankyrin, to integral membrane proteins. Several integral plasma membrane proteins have been identified that are linked to the membrane-cytoskeleton, including the anion exchanger (AE1 or band 3), Na+-K+-ATPase, the voltage-sensitive sodium channel, neurofascilin, and CD45 (Bennett, 1990). Na+-K+-ATPase is located in the basal-lateral plasma membrane in most epithelial cells (Figure 4), for example, the renal epithelium, where it functions in sodium and water reabsorption. Ankyrin binds directly to Na+-K+-ATPase with high affinity (Koob et al., 1987, Morrow et al., 1989, Nelson and Veshnock, 1987; Davis and Bennett, 1990,). Assembly of Na+-K+-ATPase with the membrane-cytoskeleton appears to increase Na+-K+-ATPase metabolic stability, suggesting that it is protected from endocytosis and degradation (Nelson, 1992). This is supported by the observation that Na+-K+-ATPase delivered to the basal-lateral plasma membrane in Madin-Darby canine kidney (MDCK) cells has a much longer residence time (i. e., is more metabolically stable) than that which arrives at the apical plasma membrane (Hammerton et al., 1991).

Figure 4. Na+-K+-ATPase and membrane-cytoskeleton distribution in different epithelial cell types. In the kidney (and most epithelia), Na+-K+-ATPase is localized to the basal-lateral plasma membrane domain. In these epithelia, membrane-cytoskeletal proteins (ankyrin and fodrin) co-localize with Na+-K+-ATPase at the basal-lateral plasma membrane. In contrast, Na+-K+-ATPase is localized to the apical plasma membrane domain in the choroid plexus epithelium, but membrane--cytoskeletal protein distribution does not completely coincide with that of Na+-K+-ATPase. Ankyrin and fodrin are localized to the apical plasma membrane (and form a multiprotein complex with Na+-K+-ATPase), but additionally they are localized to the lateral plasma membrane (probably associating with the cell adhesion molecule B-cadherin. Cadherin-mediated, cell-cell adhesion may play critical roles in the induction of epithelial cell polarity. E-cadherin binds the catenins (see above), but in addition, E-cadherin also interacts with the fodrin-based membrane-cytoskeleton (Nelson et al., 1990) although the protein interactions involved are not well understood. Coassembly of E-cadherin with Na§ into the membrane--cytoskeleton resuits in co-localization of these two proteins in the basal-lateral plasma membrane of epithelial cells (McNeill et al., 1990; Nelson et al., 1990). This was demonstrated by the expression of full-length E-cadherin in "non-polarized" fibroblasts. Expression of E-cadherin resulted in a reorganization of Na+-K§ and fodrin from a diffuse distribution to regions of E-cadherin-mediated, cell-cell contact. This distribution was dependent on extracellular calcium and was specific for Na+-K+-ATPase because bulk membrane glycoproteins (visualized using a fluorescent lectin,

wheat germ agglutinin) were diffusely distributed with, or without E-cadherin expression. Deletion/truncation of the cytoplasmic domain of E-cadherin inhibited membrane--cytoskeletal associations resulting in loss of induction of Na§ TPase reorganization. This result specifically implicates the cytoplasmic domain and cytoskeletal association of E-cadherin in the determination of Na§247 polarity in epithelial cells. It was concluded from these studies that Na§247 becomes restricted at sites of cell--cell contact by co-assembly with E-cadherin in the membrane-cytoskeleton (McNeill et al., 1990). Numerous experiments have strongly supported this conclusion. For example, dominant, negative spectrin constructs were expressed in epithelial cells, and these mutant spectrins cause the mislocalization of Na§247 (Hu et al., 1995). In contrast to the basal-lateral polarity of Na§247 in most epithelial cells, Na§247 is localized to the apical plasma membrane of the choroid plexus and retinal pigmented epithelial cells (Figure 4). Fodrin and ankyrin are also localized to the apical plasma membrane of the choroid plexus and retinal pigmented epithelial cells (Gundersen et al., 1991; Marrs et al., 1993); Na§247 is associated with the membrane-cytoskeleton (Gundersen et al., 1991, Marrs et al., 1993) in a complex similar to that found in renal epithelial cells. Mechanisms involved in localizing Na§247 to the apical membrane of these cells are not well understood, but cytoskeletal involvement appears to be critical. Na+-K§ may also be directly delivered to the apical plasma membrane in these epithelia, in contrast to that seen in other epithelia. However, it is noteworthy that polarity of choroid plexus and retinal pigmented epithelia are not completely reversed. In retinal pigment epithelial cells, viral glycoproteins (VSV G-protein and influenza HA protein) are sorted to the same membrane domains as those in other epithelia (such as MDCK cells) that have a basal-lateral Na§247 (Gundersen et al., 1991). These and other studies have led to the proposal that there is a hierarchy of mechanisms that result in the polarized distribution of membrane proteins, including Na§247 (Mays et al., 1995). Apical Na§247 polarity seemed to be at odds with the cadherin-induced basal-lateral polarity of the Na§247 of most epithelia, but a different cadherin is expressed in the choroid plexus and retinal pigment epithelium (Bcadherin) than that expressed in other epithelia (E-cadherin). E-cadherin was neither detected in thechoroid plexus nor in the retinal pigmented epithelium (Gundersen et al., 1991; Marrs et al., 1993). Instead, both of these epithelia express B-cadherin, another member of the cadherin superfamily of proteins (Napolitano et al., 1991; Murphy-Erdosh et al., 1994). B-cadherin is closely related to E-cadherin, especially in the cytoplasmic domain in which these two proteins share 88% amino acid identity (Napolitano et al., 1991). The ability of B-cadherin to induce a polarized distribution of Na§247 in transfected fibroblasts was tested. Unlike Ecadherin transfected fibroblasts, B-cadherin transfected fibroblasts displayed an unpolarized distribution of Na§247 (and fodrin), similar to untransfected

fibroblasts (Marrs et al., 1993). These data demonstrate that E- and B-cadherin differ in their ability to induce sodium pump polarity. At present, it is not known how E- and B-cadherin have different effects on the membrane-cytoskeleton and Na+-K+-ATPase distribution. That B-cadherin does not induce a polarized distribution of Na+-K+-ATPase is consistent with the apical distribution of Na+-K+-ATPase in these epithelia. Recent studies on the role of different cadherins in the induction of the polarized epithelial cell phenotype in retinal pigment epithelial cells have greatly extended our initial observations. In situ, retinal pigment epithelial cells have an apical distribution of Na+-K§ (Gundersen et al., 1991), but in primary culture and in a cultured cell line (RPE-J), Na+-K+-ATPase has nonpolarized distribution (Gundersen et al., 1991, Nabi et al., 1993). Upon transfection of E-cadherin into RPE-J cells (RPE-J+EC cells), Na+-K+-ATPase becomes polarized to the basal-lateral membrane (Marrs et al., 1995). Significantly, newly synthesized Na§ was delivered from the Golgi complex of RPE-J+EC cells to both membrane domains. As in MDCK cells, Na+-K+-ATPase that arrived at the basal-lateral plasma membrane was stabilized by selective retention, whereas Na§247 delivered to the apical membrane is degraded rapidly. Additionally, the induction of basal-lateral polarity of Na+-K§ by Ecadherin was found to be accompanied by the formation of desmosomes and an induction of the accumulation of a new ankyrin isoform in the basal-lateral domain of the RPE-J+EC cells (Marrs et al., 1995). The correlation of selective retention of Na§ at the basal-lateral plasma membrane domain, and induction of a new ankyrin isoform in the same domain, indicates that Na+-K+-ATPase is being stabilized in the basal-lateral membrane domain by interaction with the membrane-cytoskeleton. These data also suggest that cadherins act as morphoregulatory molecules in epithelial cells (and probably other cell types), controlling differentiation.

The generation and maintenance of polarized distributions of ion transporting proteins is required for the many specialized cellular functions of epithelial cells. Differentiation of epithelial cells, and thus the generation of polarized distribution of membrane proteins, is accompanied by the reorganization and remodeling of the cytoskeleton. Evidence suggests that cadherin-mediated cell--cell adhesion and the membrane-cytoskeleton serve to generate and maintain polarity of Na§247 in epithelial cells. Oppositely polarized distributions of Na§247 in different epithelia demonstrate plasticity in the mechanisms used to generate cell polarity. A general mechanism for establishing the polarity of other membrane proteins may involve similar roles for the membrane-cytoskeleton in the remodeling of epithelial cells during cellular differentiation.

Dr. Marrs acknowledges support by the NIH and the American Cancer Society. Dr. Nelson also acknowledges support by the NIH and the American Cancer Society. We thank Megan Troxell for her critical reading of the manuscript.

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Introduction The Molecules of the Extracellular Matrix Proteoglycans Collagens Fibronectin Laminin Tenascin/Cytotactin Integrins Cellular Interactions with the Extracellular Matrix in Development Cell Attachment and Cell Movement Cell Differentiation Summary

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The extracellular environment of cells in developing embryos is not merely a passive supporting scaffolding or "connective tissue". Instead, the matrix in which the

Principles of Medical Biology, Volume 11 Developmental Biology, pages 89-101. Copyright 9 1998 by JAI Press Inc.

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cells are embedded exerts profound influences on both cell movement and cell differentiation in embryos. During development, cells migrate or rearrange their positions relative to one another, and these movements, of which there are many examples, are initiated at specific times in development, are often highly directed, and cease at precise times and locations. The precision of these movements is thought to be generated by programmed changes in the activity of the cytoskeleton in combination with changes in the ability of cells to interact with elements of their extracellular environment (Sanders, 1989; Damsky et al., 1993). This extracellular environment, consisting of a matrix of primarily proteins and glycoproteins, is itself constantly changing, providing developmentally regulated cues for movement to which the matrix receptors present on the cell surface can respond. The interaction between the extracellular matrix and the receptors is then thought to activate the cytoskeleton and thereby initiate or inhibit cell movement. The extracellular matrix may also regulate cell differentiation independently of cell migration or cell shape changes. Contact of cells with specific molecules of the matrix during narrow windows of sensitivity may elicit tissue-specific gene expression in the responding cells. In this way, the matrix is able to positively or negatively influence differentiation during development (Adams and Watt, 1993; Lin and Bissell, 1993) either directly or via the effects of soluble growth factors that are bound by the matrix. In all types of matrix-mediated influence, the external signal must be transmitted to the cytoplasm. This is thought to occur through the activity of transmembrane proteins, called integrins, which bear matrix receptors on their extracellular domains, and intracellular domains that interact with the cytoskeleton or with cytoplasmic signal transduction pathways. The integrins have been classed as "cell adhesion molecules", since they are present at the cell surface and are clearly involved in cell-to-matrix adhesive processes. As such, they are part of a greater group of cell adhesion molecules (CAMs), many of which are implicated in cellto-cell adhesion. Although of fundamental importance to embryonic morphogenesis (Edelman and Crossin, 1991), the CAMs as a group will not be considered here. This chapter will survey some of the molecules present in the extracellular matrix, the receptor-bearing integrins that cells use to recognize them, and some well established examples of matrix-mediated cell migration and differentiation.

Proteoglycans are complex macromolecules that contain a core protein with one or many covalently bound glycosaminoglycan (GAG) chains (Kjellen and Lindahl, 1991; Wight et al., 1991). GAGs are unbranched saccharide polymers with varying degrees of sulfation that consist of repeating dimers in which an amino sugar (e.g.,

N-acetylglucosamine) typically alternates with uronic acid residues (e.g., glucuronic acid). In proteoglycans, GAGs such as chondroitin sulfate, dermatan sulfate, heparan sulfate, or keratan sulfate are linked to serine residues of the core protein. Cartilage matrix, for example, is very rich in chondroitin sulfate and keratan sulfate proteoglycans. Hyaluronan (hyaluronic acid), a very important matrix constituent, is an unusual GAG because it is unsulfated and exists unbound to a protein core. It's large hydrated volume enables hyaluronan to occupy and open up tissue spaces and maintain cell separation (see Figure 1). Because there are cell surface binding proteins for hyaluronan, and because hyaluronan is able to influence cell adhesion and locomotion, it is thought to contribute significantly to embryonic cell migration and differentiation (Toole, 1991; Turley, 1992). The proteoglycans represent a highly heterogeneous class of extracellular macromolecules, able to interact in the matrix

with the GAGs and with glycoproteins in ways that are still incompletely understood. Proteoglycans have been difficult to study, and this is reflected in the uncertainties regarding their functions. Although they are clearly involved in many aspects of cell adhesion, movement, and differentiation, precise roles have often been difficult to assign. The heparan sulfate proteoglycans (HSPGs) are a large class of molecules that are associated integrally and peripherally with the cell membrane as well as the extracellular matrix and basement membranes. Among these, the syndecans, which are cell surface HSPGs, have been especially implicated in developmental processes (Bernfield et al., 1993). These molecules are developmentally regulated, appearing temporally and spatially at certain key phases of development, and are able to bind other matrix molecules and also growth factors. As with the other proteoglycans, however, the precise function of the syndecans has yet to be determined.

The collagens comprise a large family of molecules all of which possess a characteristic triple helical structure (van der Rest and Garrone, 1991). They are classified according to the nature of the polypeptide chains that make up the homo- or heterotrimer, but are grouped according to the macromolecular form of the protein. Several groups are now recognized (Mayne and Brewton, 1993), but the principal ones are as follows: those that form fibrils (collagen types I, II, III, V, and XI); those that are associated with fibrils (types IX, XII, and XIV); those that anchor basement membranes into the underlying stroma (type VII); and those that form networks in basement membranes (type IV). Many other distinct types of collagen have now been identified. Progress has been made in understanding the roles of some of these groups in differentiation, in particular those from the first and last groups. Type I collagen is the most common interstitial collagen, found in skin, bone, and tendon, while type II is the collagen of hyaline cartilage, and both of these have an influence on the differentiation of the tissues with which they interact (Hay, 1991). For example, the sclerotome is a mesenchymal tissue derived from the somites that is the progenitor of chondrogenic tissue that will form the axial cartilage. The induction of chondrogenesis is dependent on the presence of neighboring collagen-producing tissues (notochord or neural tube) or on the presence of types I or II collagen itself. Another developmental phenomenon that appears to be collagen-dependent is epithelial-mesenchymal transformationmthe production of a mesenchymal tissue from an epithelial one. In vitro and in vivo evidence suggests that the presence of collagen surrounding cells can promote transformation between these two states (Hay, 1991). Type IV collagen is, together with laminin (see below), a characteristic component of basement membranes. In this location it not only provides a network structure upon which the basement membrane is constructed, but it also seems to have instructive or permissive roles (reviewed by Sanders, 1989). The collagen compo-

nent of basement membranes appears to stabilize the integrity and apico-basal polarity of the overlying epithelium, and its presence or absence determines sites of epithelial folds during glandular morphogenesis. In addition, it can be demonstrated that changes in the constituents of the basement membrane are correlated with several inductive interactions that occur between the mesenchyme on one side and the epithelium on the other. For example, during tooth development the oral epithelium gives rise to ameloblasts that will secrete enamel, while on the other side of the basement membrane the mesenchyme gives rise to odontoblasts that will secrete dentin. These tissues undergo multiple, sequential, and reciprocal interactions during their differentiation that are mirrored by changes in the composition of the intervening basement membrane. This suggests that the basement membrane is more than just a passive barrier, and that it can assume an active and instructive role in development.

Of all the extracellular glycoprotein, fibronectin is the most widespread and the best understood in both biochemical and physiological terms. This molecule forms fibrillar arrays on the surfaces of cells (Figure 1D) as well as being present in the matrix, where it serves an adhesive function and is implicated in cell spreading, migration, and interaction. The molecule consists of two similar polypeptide chains, each approximately 220-kDa, containing domains with binding sites both for other matrix molecules, such as collagen and heparan sulfate proteoglycan, and for cell surfaces (Yamada et al., 1992; Potts and Campbell, 1994). This means that the molecule functions importantly both to integrate the matrix and as a substratum for cell adhesion, and is but one component of an array of such molecules. Fibronectin has several cell-binding sites, but the best studied is the one comprising the peptide sequence Arg-Gly-Asp-Ser (RGDS), which appears in many matrix molecules and is a motif of major importance in cell-matrix interaction. RGDS is recognized by the cell surface fibronectin receptor (or integrin, see below), which is a transmembrane protein linking to the cytoskeleton via a series of intermediate proteins. In this way, fibronectin-mediated cell attachment can initiate the cell-shape changes associated with cell movement. The importance of the RGDS sequence has been demonstrated by providing cells with a fibronectin substratum and incubating them, in vitro or in vivo, with synthetic peptides with this sequence and showing the consequent cell detachment and loss of motility (Yamada et al., 1992). Its widespread occurrence probably means that fibronectin has more of a permissive rather than instructive role in developmental processes (Sanders, 1989; Thiery et al., 1989). In other words, it provides a hospitable substratum to which cells can attach, and its fibrils may impart some directionality to cell movement, but fibronectin is probably not a major determinant of cell migration routes. Some specific examples of the role of fibronectin and its receptor in migratory processes will be considered later.

An important recent development is the beginning of our understanding of the ways in which the functions of extracellular matrix molecules are integrated with those of soluble growth factors. Growth factors are now appreciated as a major influence on cell proliferation and differentiation during development (Hamilton and Millis, 1990), and one of their roles appears to be to regulate the expression of extracellular matrix proteins. For example, transforming growth factor-13 (TGFI3) is able to stimulate cells to synthesize fibronectin and collagen, which are then incorporated into the matrix. Even the expression of the cell surface integrins for these molecules may be increased. The mechanism of this effect is unclear, but the implications are very important. Developmentally regulated expression of TGFI3 could modulate the cells' environment, thereby influencing the cell adhesion and movement of subsequent populations of cells occupying that region. It is suspected that the integration of growth factor and matrix activity is a major, and as yet poorly understood, determinant of cellular interaction in development (Adams and Watt, 1993).

Laminin is the major noncollagenous glycoprotein in basement membranes. It is a multidomain molecule of about 800-kDa, consisting of three polypeptide chains---one large A chain and two smaller B chains--organized in the form of an asymmetric cross (Paulsson, 1992; Tryggvason, 1993). A number of isoforms are now recognized, but all have a number of functional domains in a similar way to fibronectin. Accordingly, there are cell-binding domains that are especially recognized by epithelial cells, matrix-binding domains, and a neurite-outgrowthpromoting domain. The cell attachment domains include an RGDS sequence, but also a laminin-specific pentapeptide, YIGSR, located close to the intersection of the three chains. Laminin is assembled into basement membranes by its interaction with type IV collagen, heparan sulfate proteoglycan, and another basement membrane-specific glycoprotein called entactin/nidogen. The self-assembly of these molecules onto the type IV collagen lattice produces the basement membrane mesh (Yurchenco et al., 1990). As mentioned above, basement membranes are synthesized largely by epithelia and serve as a supporting structure for epithelial sheets and, as such, the overlying epithelial cells have receptors for laminin, polarized to their basal surface. These receptors, some of which are integrins, recognize RGDS, YIGSR, or other laminin domains (Mecham, 1991). In addition to contributing to the general functions of basement membranes, laminin has some special functional properties. One of the most interesting of these is its ability to promote the attachment and outgrowth of neurites from nerve cells (Sanes, 1989). Neuronal outgrowth during development is characterized by the directed extension and migration of neurites followed by synaptogenesis. The extracellular matrix is perceived to provide cues that guide the growth cones in their migrations to their target tissues, and laminin appears to be the most important ma-

trix constituent in this process. In vitro, laminin promotes the extension of neurites better than other matrix molecules, and in vivo laminin is present along migration pathways. The mechanism of the guidance is unclear, but neurons have several receptors that can interact with laminin and these presumably mediate a preferential adhesiveness of the filopodia to laminin in the pathways of migration. Also after injury, the laminin-rich basement membranes of the damaged Schwann cells remain and appear to serve as pathways for regenerating nerves. Contact with the regrowing nerves then provides a stimulus for the Schwann cells to proliferate and regenerate new basement membranes around the regenerated nerves.

Tenascin (also called cytotactin), is a large hexameric extracellular glycoprotein that is present in distinctive temporo-spatial distributions during development (Chiquet-Ehrismann, 1995). It occurs coincidentally with several important morphogenetic tissue interactions and, because its distribution is more restricted than fibronectin and laminin tenascin, is likely to be an important local influence on cell-to-matrix adhesion. Tenascin interacts with a number of other matrix molecules; most particularly it appears to interfere with the ability of cells to adhere to fibronectin, and thus it becomes what has been called an "antiadhesive" molecule (Chiquet-Ehrismann, 1995). Tenascin itself is also not a good adhesive substratum for cells in vitro, and so its presence in the embryo may indicate a region that migrating cells avoid by virtue of nonadherence. Also, tenascin is present at the time of certain epithelial-mesenchymal interactions and may be involved in signaling across the basement membrane at these times, though the mechanism of such interactions is unclear because little is known of cellular receptors for tenascin.

The integrins are a large class of transmembrane glycoproteins that cells use as receptors to attach to molecules of the extracellular matrix (Hynes, 1992). As such they form a transmembrane link between the extracellular environment and the cytoplasm of the cells, where they are associated with elements of the cytoskeleton (Figure 2). This arrangement allows the passage of signals not only from the extracellular matrix to the cytoskeleton, thus influencing cell movement and proliferation, but also from the cytoplasm to the cell surface, thus influencing the expression, activation, and modulation of the integrins themselves (Damsky and Werb, 1992; Juliano and Haskill, 1993). Each integrin type is a heterodimer composed of an ct and a 13subunit in which the extracellular domains of each subunit associate with one another. At the time of writing, eight 13 subunits and 16 a subunits had been identified (Hynes, 1992; Stewart et al., 1995). These various subunits preferentially associate, giving over

Schematic model of the otsl31fibronectin receptor. The cz5subunit is at the left and the ~1 subunit at the right. Each subunit has a transmembrane segment and a short cytoplasmic tail. The external portion of this receptor is 20-22 nm long, with a head that is 12-15 nm in diameter. Reprinted from Yamada et al., (1992) with permission from the Cold Spring Harbor Laboratory Press. twenty different integrin dimers, each of which is assigned an extracellular matrix ligand. There is redundancy in the system, however, because several ligands bind to more than one integrin, although not necessarily via the same cell-binding site in the ligand. Thus, fibronectin is able to bind to ot3ot~, ot413~, and ot513~integrins, but not all via the RGD site, while laminin and the collagens also each bind to several different integrins. Indeed, at the last count, both fibronectin and laminin were each recognized by six different integrin heterodimers (Hynes, 1992). The ligandbinding site in the integrin dimer is thought to be positioned at the extracellular tip, at or near the point of contact between the two subunits. The situation is made more complicated by observations that indicate that although a given heterodimer is capable of binding a particular ligand in one cell type, the same integrin may not be so capable on a different cell (Hynes, 1992). Furthermore, the activity of a particular integrin may not always be the same within a given cell. This has given rise to the idea that integrins can be activated by means of a conformational change, and that the activation, or de-activation, is controlled in some way by cytoplasmic or nuclear events. This modulation of receptor activity, by

inside-out signaling, is supplemented by another level of control brought about by receptor co-operativity at the cell surface (Damsky and Werb, 1992). This cooperativity can involve integrins together with growth factors and their receptors, or with nonintegrin matrix receptors such as HSPG at the cell surface. Cell-to-cell variations in these co-operative effects could explain the different affinities of the same integrin dimer in different cell types. The association of integrins with elements of the cytoskeleton provides one means by which signals may be transmitted "outside-in", to affect cell shape, cell movement, and proliferation (Miyamoto et al., 1995). At the so-called "focal contacts" in fibroblasts, for example, there is evidence for the interaction of a5131 integrin, most probably by the 13 subunit, with microfilaments via linking proteins such as talin, ot-actinin, and vinculin. How, or if, extracellular signals are transmitted to the nucleus via the cytoskeleton is unclear, although it is known that intermediate filaments are associated with the nuclear membrane. What is clear, however, is that the extracellular matrix does have an influence on the nucleus, because changes in the matrix surrounding cells can radically affect the expression of various proteins, and thus differentiation itself (Adams and Watt, 1993). The.signal transduction pathways involved in this transmission are beginning to be worked out and appear to involve not only cytoskeletal re-organization, but also changes in tyrosine phosphorylation events involving cytoplasmic protein kinases (Juliano and Haskill, 1993; Schaller and Parsons, 1994). Indeed, there appear to be several kinases present in the focal contacts that cells make with matrix proteins. The result of these transmembrane interactions is that ligand binding to integrins can apparently influence gene expression and cell differentiation by means of second messenger pathways, thus receptor-ligand interactions are brought into line with the more conventional ones known in the fields of cellular physiology and pharmacology.

There are several examples in early embryogenesis where cell attachment and movement have been indirectly correlated with changes in the spatio-temporal distribution of specific extracellular matrix molecules. In some cases this circumstantial relationship has been supported by in vivo and in vitro experimental evidence that the matrix molecule, most often fibronectin, is directly responsible for promoting some aspect of cell migration (Sanders, 1989; Thiery et al., 1989). One of the earliest morphogenetic processes is gastrulation, in which the two-layered embryo is converted to a three-layered structure by the invagination or ingression of cells from the surface of the embryo to the interior to form the mesodermal cell layer. The mesodermal cells are fibroblast-like and move away from their point of formation

as individuals or loosely associated as a sheet (see Figure 1). In amphibian and chicken embryos, where this phenomenon has been most heavily studied, the mesoderm cells use the matrix as a substratum for their movement (Figure 1C). This early matrix consists primarily of a network of fibronectin filaments often layered on a basement membrane. The fibronectin network is not of uniform texture and topography throughout the region of mesoderm cell migration, giving rise to the idea that patterns in the network can influence patterns of cell movement. Direct evidence that cells rely on the fibronectin, at least for attachment and movement, has been obtained by injection of antifibronectin antibodies and soluble peptide fragments containing the RGD cell-binding site of fibronectin (Brown and Sanders, 1991). These agents cause cell detachment and consequent arrest of movement. A similar approach has been taken with cells of the neural crest, which undergo very extensive and elaborate migrations in the early embryo and, upon arrival at their destination, differentiate into a wide array of different cell types (BronnerFraser, 1993). A number of different extracellular matrix molecules have been described in the migration pathways of the crest cells, including fibronectin, laminin, and tenascin. As with mesoderm cell movement, these molecules are thought to provide a permissive substratum, and possibly to influence migration, either initiation, directionality, or cessation, although direct evidence has been difficult to obtain. The effects of fibronectin in these circumstances may be one of "contact guidance" in which oriented fibrils of matrix impart orientation and perhaps directionality to the cell movement, or "haptotaxis" in which an adhesive gradient of matrix influences directionality. In some cases it is also necessary to explain not only why the neural crest cells preferentially choose a particular migration route, but also why they are excluded from other potential routes. For example, in the trunk, the crest cells are excluded from the caudal, but not the rostral, portion of each somite; they also avoid the region in the immediate vicinity ofthe notochord. So, work in this field concentrates on matrix molecules that both promote and discourage cell attachment. Microinjection of antimatrix antibodies and cell-binding fragments from matrix molecules disrupt the cranial neural crest cell migration routes, as do antibodies against integrin receptors (Bronner-Fraser, 1993), providing more evidence that, at least in some regions of the embryo, receptor-matrix interactions provide directional cues for cell movement. The most remarkable long-range migrations during early development are those undertaken by the primordial germ cells (PGCs), which arise in locations remote from their final destination in the genital ridges. Although difficult to study, these migrations are perceived also to rely to some extent on the fibronectin-rich substratum on which these cells move (Sanders, 1989). In vitro studies have shown that PGCs are strongly oriented by fibrils of fibronectin, suggesting that contact guidance may be important. Other evidence suggests that the PGCs vary in their response to fibronectin before, during, and after the migratory phase so that the cells' migratory response to this molecule can change, despite relatively constant levels of fibronectin in the matrix (ffrench-Constant et al., 1991).

Matrix in

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In addition to roles in cell movement, the extracellular matrix can exert profound effects on cell differentiation and tissue-specific gene expression. An example of how differentiation can be manipulated by changes in the extracellular environment is provided by experiments on mammary epithelial cells (Lin and B issell, 1993). Like all epithelial cells, these are exposed, in situ, to matrix in a polarized fashion, with differing components Confronting the apical and basal surfaces. When these cells are cultured in vitro, they are dependent on the type of substratum and its topography if they are to secrete milk proteins such as 13-casein; the cells fail to differentiate if they are merely cultured on plastic or thin layers of matrix. Expression of this protein is, however, elicited on a substratum composed of a thick gel of type I collagen, but only if the substratum is floating in culture medium thus allowing access of the medium to both sides of the epithelial sheet. Furthermore, culturing the cells on a laminin-rich matrix derived from basement membranes allows the formation of hollow spherical structures similar to those seen in situ, and the expression of other tissue-specific proteins. It appears that the interaction between laminin and its integrin receptor is essential for the expression of ~-casein genes. Another example of matrix modulation of cell differentiation is seen in the in vitro differentiation of pluripotent cells from the tail of the early chick embryo (Griffith and Sanders, 1991). These cells, from the tail bud of the two-day embryo, look like a homogeneous population of fibroblast-like cells, but when they are cultured they have the ability to differentiate into a large number of different cell types including myoblasts, neurons, melanocytes, and chondrocytes, depending on the substratum. Several substrata favor the appearance of certain cells; for example, fibronectin and type I collagen favor the appearance of myoblasts, while laminin promotes neuron and melanocyte differentiation.

The extracellular matrix exerts profound effects on both cell movement and cell differentiation during development. The influences of the matrix are integrated with those of the differentiative lineage program of the cell, and with the cell-to-cell interactions that occur during morphogenesis. The matrix itself is composed of an array of multidomain glycoproteins, including fibronectin and various collagens, which form an integrated extracellular environment that has both permissive and informational roles in cell movement and differentiation. Basement membranes are specialized regions of the matrix that serve a number of roles, including the maintenance of epithelial polarity, acting as a substratum for cell movement, and in the mediation of epithelial-mesenchymal inductive interactions. The extracellular matrix is subject to developmental regulation in that its composition can be shown to vary during different phases of development. Cells are able to respond to changes in the

extracellular matrix by means of cell surface receptors, including the t r a n s m e m brane integrins and p l a s m a m e m b r a n e - a s s o c i a t e d p r o t e o g l y c a n s such as heparan sulfate proteoglycan. Signals from the matrix are transduced into the cell via transm e m b r a n e c o m p l e x e s that are linked at the cytoplasmic face to e l e m e n t s of the cytoskeleton. E v i d e n c e is also accumulating for the participation o f second m e s s e n g e r p a t h w a y s in the signal transduction pathways, although the details and m e c h a n i s m s of these p a t h w a y s are poorly u n d e r s t o o d at present.

Adams, J.C. & Watt, F.M. (1993). Regulation of development and differentiation by the extracellular matrix. Development 117, 1183-1198. Bernfield, M., Hinkes, M.T. & Gallo, R.L. (1993). Developmental expression of the syndecans: Possible function and regulation. Development (Suppl.) 205-212. Bronner-Fraser, M. (1993). Environmental influences on neural crest cell migration. J. Neurobiol. 24, 233-247. Brown, A.J. & Sanders, E.J. (1991). Interactions between mesoderm cells and the extracellular matrix following gastrulation in the chick embryo. J.Cell Sci. 99, 431--441. Chiquet-Ehrismann, R. (1995). Tenascins, a growing family of extracellular matrix proteins. Experientia 51,853-862. Damsky, C.H. & Werb, Z. (1992). Signal transduction by integrin receptors for extracellular matrix: Cooperative processing of extracellular information. Curt. Opin. Cell Biol. 4, 772-781. Damsky, C., Sutherland, A. & Fisher, S. (1993). Extracellular matrix 5: Adhesive interactions in early mammalian embryogenesis, implantation, and placentation. FASEB J. 7, 1320-1329. Edelman, G.M. & Crossin, K.L. (1991). Cell adhesion molecules: implications for a molecular histology. Ann. Rev. Biochem. 60, 155-190. ffrench-Constant, C., Hollingsworth, A., Heasman, J. & Wylie, C.C. (1991 ). Response to fibronectin of mouse primordial germ cells before, during, and after migration. Development 113,1365-1373. Griffith, C.M. & Sanders, E.J. (1991). Effects of extracellular matrix components on the differentiation of chick embryo tail bud mesenchyme in culture. Differentiation 47, 61-68. Hamilton, R.T. & Millis, A.J.T. (1990). Developmental roles for growth factor-related secreted proteins. Curt. Top. Dev. Biol. 24, 193-218. Hay, E.D. (1991). Collagen and other matrix glycoproteins in embryogenesis. In: Cell Biology of Extracellular Matrix, 2rid edn. (Hay, E.D., Ed.), pp. 419-462, Plenum Press, New York. Hynes, R.O. (1992). Integrins: Versatility, modulation, and signalling in cell adhesion. Cell 69,11-25. Juliano, R.L. & Haskill, S. (1993). Signal transduction from the extracellular matrix. J. Cell Biol. 120, 577-585. Kjellen, L. & Lindahl, U. (1991). Proteoglycans: Structures and interactions. Ann. Rev. Biochem. 60, 443-475. Lin, C.Q. & BisseU, M.J. (1993). Multi-faceted regulation of cell differentiation by extracellular matrix. FASEB J. 7, 737-743. Mayne, R. & Brewton, R.G. (1993). New members of the collagen superfamily. Cuff. Opin. Cell Biol. 5, 883-890. Mecham, R.P. (1991). Laminin receptors. Ann. Rev. Cell Biol. 7, 71-91. Miyamoto, S., Teramoto, H., Coso, O.A., Gutkind, J.S., Burbelo, P.D., Akiyama, S.K. & Yamada, K.M. (1995). J. Cell Biol. 131,791-805. Paulsson, M. (1992). Basement membrane proteins: Structure, assembly, and cellular interactions. Crit. Revs. Biochem. Mol. Biol. 27, 93-127.

Potts, J.R. & Campbell, I.D. (1994). Fibronectin structure and assembly. Curr. Opin. Cell Biol. 6, 648-655. Sanders, E.J. (1989). The Cell Surface in Embryogenesis and Carcinogenesis. The Telford Press, Caldwell, New Jersey. Sanes, J.R. (1989). Extracellular matrix molecules that influence neural development. Ann. Rev. Neurosci. 12, 491-516. Schaller, M.D. & Parsons, J.T. (1994). Focal adhesion kinase and associated proteins. Curr. Opin. Cell Biol. 6, 705-710. Stewart, M., Thiel, M. & Hogg, N. (1995). Leukocyte integrins. Curr. Opin. Cell Biol. 7, 690-696. Thiery, J-P., Duband, J-L., Dufour, S., Savagner, P. & Imhof, B.A. (1989). Roles of fibronectin in embryogenesis. In: Fibronectin, (Mosher, D.F., Ed.), pp. 181-212, Academic Press, San Diego. Toole, B.P. (1991). Proteoglycans and hyaluronan in morphogenesis and differentiation. In: Cell Biology of Extracellular Matrix, 2nd edn. (Hay, E.D., Ed.), pp. 305-341, Plenum Press, New York. Tryggvason, K. (1993). The laminin family. Curr. Opin. Cell Biol. 5, 877-882. Tudey, E.A. (1992). Hyaluronan and cell locomotion. Canc. Metast. Revs. 11, 21-30. van der Rest, M. & Garrone, R. (1991). Collagen family of proteins. FASEB J. 5, 2814-2823. Wight, T.N., Heinegard, D.K. & Hascall, V.C. (1991). Proteoglycans: structure and function. In: Cell Biology of Extracellular Matrix, 2nd edn. (Hay, E.D., Ed.), pp. 45-78, Plenum Press, New York. Yamada, K.M., Aota, S., Akiyama, S.K. & LaFlamme, S.E. (1992). Mechanisms of fibronectin and integrin function during cell adhesion and migration. Cold Spring Harbor Symposia on Quantitative Biology 57, 203-212. Yurchenco, P.D. & Schittny, J.C. (1990). Molecular achitecture of basement membranes. FASEB J. 4, 1577-1590.

DeSimone, D.W. (1994). Adhesion and matrix in vertebrate development. Curr. Opin. Cell Biol. 6, 747-751. Gullberg, D. & Ecklom, P. (1995). Extracellular matrix and its receptors during development. Intl. J. Dev. Biol. 39(5), 845-854. Hardingham, T.E. & Fosang, A.J. (1992). Proteoglycans: Many forms and many functions. FASEB J. 6, 861-870. Heidemann, S.R. (1993). A new twist on integrins and the cytoskeleton. Science 260, 1080-1081. Lin, C.Q. & Bissell, M.J. (1993). Multifaceted regulation of cell differentiation by extracellular matrix. FASEB J. 7(9), 735-736. Wight, T.N., Kinsella, M.G. & Qwarnstrom, E.E. (1992). The role of proteoglycans in cell adhesion, migration, and proliferation. Curr. Opin. Cell Biol. 4, 793-801. Yamada, K.M. (1991). Fibronectin and other cell interactive glycoproteins. In: Cell Biology of Extracellular Matrix, 2nd edn. (Hay, E.D., Ed.), pp. 111-146, Plenum Press, New York. Yamada, Y. & Kleinman, H.K. (1992). Functional domains of cell adhesion molecules. Curr. Opin. Cell Biol. 4, 819-823.

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Gap Junctions

Introduction

The Gap Junction Regulation of Gap Junctional Communication Development and Patterning

Gap Junctions during Early Development The Frog Embryo Patterning in Hydra The Chick Limb The Mouse Embryo The Mammalian Heart Human Gap Junction Gene Defects Summary and Conclusions

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A characteristic of multicellular organisms is the need to coordinate the behavior of cells and tissues during development and patterning. Direct intercellular communication is one mechanism that allows this. Gap junctions are plasma membrane specializations providing the pathway for this direct cell-to-cell communication,

Principles of Medical Biology, Volume II Developmental Biology, pages I03-121. Copyright 9 1998 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-816-1

permitting the transfer of ions and small molecules from the cytoplasm of one cell to that of its neighbor without recourse to the extracellular space. The purpose of this chapter is to demonstrate the importance of gap junctions in patterning and development using various animal model systems and the mammalian heart as exampies. It will then cover some specific developmental defects in the human that appear to result from gap junction gene perturbations.

Gap junctions consist of an aggregate of channels directly linking the cytoplasm of one cell with it's neighbor. In thin section electron microscopy, the membranes of the adjacent cells run closely together with a 2-nm gap spanned by the junctional channels (Figure la). In freeze-fracture electron microscopy the junctions appear as a plaque or array of particles on one membrane face (pits on the other), each representing a single channel (Figure lb). Junction size varies enormously as do junction numbers in different tissues. The channels have a 1-2-nm diameter central pore

(Figure 1c) allowing molecules up to about 1000 MW to pass through as evidenced by dye transfer experiments. Larger polypeptides and nucleic acids are restricted and each cell thus retains its own identity. Each channel is made up of two hemichannels termed connexons, one contributed by each of the adjoining cells. Each connexon is made up of six protein subunits, the connexins, which in most cases are identical to each other. A gap junction channel will therefore consist of 12 protein subunits (Figure 2). Several connexin genes have been characterized (Table 1), but there is still a high level of conservation. The proteins encoded in each case have a similar topology; they all have four a-helical transmembrane spans, have both the amino- and carboxyl-termini on the cytoplasmic side, and have two extracellular loops (Zimmer et al., 1987; Milks et al., 1988). The transmembrane regions are highly conserved, with the third transmembrane span having several hydrophilic residues and therefore being thought to line the hydrophilic channels linking the adjacent cells (Milks et al., 1988). The greatest variability between connexins is in the cytoplasmic loop and carboxyl terminal tail, with sequenced connexins having molecular weights between 26 kDa and 50 kDa (Table 1). Connexins do vary between species, and particularly between phylogenetic classes (for further details, see Bennett et al., 1991; Kumar and Gilula, 1992; Paul, 1995). One reason for multiple connexin expression may be to facilitate the formation of communication compartments, considered vital for developmental processes.

Cx37

Connexin 31, for example, seems able to form functional channels only with itself while connexin 40 is unable to form channels with connexin 43 (Elfgang et al., 1995). In the heart, this may be important in separating the fast conduction pathways (Bundle of His, Purkinje fibers) expressing connexin 40 from the working myocardium expressing connexin 43 (Gourdie et al., 1993). Another benefit of connexin variability may be channel specificity. Both electrophysiological studies (Bennett et al., 1991) and dye transfer studies (Elfgang et al., 1995) show clear channel permeability differences (size, charge) between connexin types. During development, the passage of messenger molecules can therefore be regulated spacially by differential connexin expression, but it could also be regulated in the direction of travel (Robinson et al., 1993). This may be facilitated by asymmetrical

junctions formed with connexons made up of different connexin types (heterotypic junctions; Bukauskas et al., 1995), or even multiple connexin types within a connexon (heteromeric junctions; Stauffer, 1995).

In addition to the physical presence of a gap junction, the level of coupling through the channels can be regulated. The channels themselves may be opened or closed and the presence of gap junctions observed ultrastructurally or by immunohistochemical techniques, or the amount of protein demonstrated by immunoblotting, may not accurately reflect the level of cell-cell communication. Furthermore, the number of gap junction channels in a membrane is not stable and may be affected by the balance between transcriptional and/or translational levels and protein degradation during junction turnover, which can be very rapid. In cultured hepatocytes, the half-life of the protein may be as short as one and a half hours (Traub et al., 1989) and quantitative changes in gap junction expression appear to play an important role during cell proliferation (Dermietzel et al., 1987). Gap junctions can also form very rapidly when cultured cells are brought into contact indicating that junctional protein is already in or near the membrane. This means that in some cases at least, the formation and assembly of gap junctions may be independent of transcriptional and translational events (Kojima et al., 1994). Several factors have been implicated in junctional regulation and these are summarized in Table 2. Some of them appear to act directly on the junctional protein, for example hydrogen ions, which can have a rapid though reversible effect (Turin and Warner, 1977). However, the effect of the range of factors varies from tissue to tissue; in liver and heart a lowering of intracellular pH to 6.5 causes an uncoupling of cells, but in Xenopus blastomeres a small drop from pH 7.6 to only pH 7 will have the same effect. Similarly, increases in calcium ion concentration lowers junctional permeability in some species, but embryonic tissues may be less sensitive (Spray et al., 1982). One major importance of calcium sensitivity may be the uncoupling of cells that would occur following cell injury, restricting the damage effect (Asada and Bennett, 1971). Gap junction voltage sensitivity also varies with cell or tissue type and may be asymmetrical as in the crayfish rectifying synapse (Furshpan and Potter, 1959), or symmetrical as in amphibian embryos (Spray et al., 1981). Indirectly acting regulatory factors are even more difficult to analyze. Cyclic AMP, acting as a second messenger, is known to readily pass through gap junctions but can itself influence junctional communication. Cell communication studies of the insect epidermis have also shown a hormonal influence, but in this case there is no evidence that the effect is cyclic AMP mediated (Caveney, 1978). The evaluation of indirect regulatory factors is therefore complex, and it is necessary to realize that in developing organisms especially, several factors may be acting together or sequentially to influence junctional communication. This would allow specificity of

cell-cell interactions not just by the use of selected molecules, but by controlling the time and rates at which they are able to be transferred.

There is a need to regulate developmental and patterning processes, and considerable evidence suggests that gap junctions are playing a major role in this. Even the oocyte is in gap junctional contact with the granulosa cells of the cumulus oophorus, but coupling decreases and is lost at the time of ovulation indicating that communication regulates the maturation of the oocyte prior to ovulation (Gilula et al., 1978). Upon disruption of the coupling, the oocyte is released from meiotic arrest (Dekel et al., 1981). In the mouse embryo, intercellular communication coincides with blastomere compaction (Lee et al., 1987) and in Drosophila, embryos gap

GapJunctions junctions are formed and prepositioned even before the completion of cellularization of the blastoderm (Tepass and Hartenstein, 1994). As development proceeds, gap junctions appear to play a role in differentiation and patterning, as indicated by their presence between embryonic muscle cells prior to their fusion, in the embryonic nervous system (for review, see Caveney, 1985), and during initial "tasting" contacts between nerves and muscles. Cell-cell communication is not the same between all cells of an embryo though, with marked patterns of junctional communication being observed as early as the 32-cell stage in Xenopusembryos (see Warner et al., 1984). The most substantial evidence for the role of gap junctions in development in fact comes from the large number of studies now showing the presence of developmental communication compartments that function semiautonomously and give rise to different structures in the adult organism. Following compaction in the mouse embryo, at least two compartments can be detected on the basis of dye transfer experiments. These are the inner cell mass that develops into the embryo proper, and the trophectoderm, which gives rise to part of the placenta (Lo and Gilula, 1979). Similar compartments, which may be temporal as well as spacial, have been observed in molluscs where they correlate with determination of the mesentoblast and the establishment of dorso-ventral polarity (de Laat et al., 1980). In the imaginal wing discs of Drosophila, indications are that boundaries of reduced-gap junctional communication coincide with known lineage compartment borders (Weir and Lo, 1982). Fraser and Bryant (1985) have suggested that the compartmentalization may not be as clear cut as this, but none the less report that signaling molecules may be transferred through gap junctions of the wing disc over different ranges depending upon selective properties of the junctions and upon properties of the signaling molecules themselves (i.e., receptor affinity, synthesis, and degradation rates). Furthermore, gap junction distribution in wing discs in mutant strains does appear to differ from that observed in the wild type (Ryerse and Nagel, 1984). These studies indicate that alterations in the number or distribution of gap junctions may be very disruptive to normal growth and development. Studies of dye transfer properties at the segmental border in insect epidermis are even more revealing. Warner and Lawrence (1982) showed that cell-cell communication in the epidermis of milkweed bug and blowfly larvae was greater within a segment than across segmental borders. In both species, lucifer yellow (MW 450) moved from cell-to-cell within a segment, but only the smaller, more compact anion tracer lead-EDTA (MW 374) passed freely at the segmental border. Similar results were reported by Blennerhassatt and Caveney (1984), who found a specific cell type with reduced junctional permeability to be located at the segmental border. Both groups of workers proposed a modulation in junctional permeability was occurring, rather than a reduction in actual channel numbers. This could be an important factor during early development when very rapid changes in embryonic cell communication patterns are occurring. From these few selected examples, it is clear that gap junctional communication often correlates with a need for cells to be aware of their position and ultimate role

in a developing organism or during patterning. The major problem with studies of this type is the need to extrapolate from structural observations to functional roles, or from apparent embryonic communication patterns to adult structures. It is obvious that many cells may be coupled to different degrees, electrical coupling often persisting when dye coupling is not apparent, and as seen above, even the extent of dye coupling varies within different regions of an embryo. The development of antibody probes that specifically block junctional communication, and more recently, molecular biological developments, have therefore been of importance in allowing a more direct approach to studying the role of gap junctions.

The development of gap junction-specific antibodies that were able to perturb gap junctions specifically enabled a new approach to studying the role of gap junctions during development. The antibodies bound to epitopes on the cytoplasmic side of the junctions and, when loaded into cells, blocked communication, as revealed by both dye transfer block and electrophysiological studies. During early development, the Xenopus embryo does not have uniform dye transfer but has maximal transfer near the dorsal side. Warner and colleagues (1964) injected gap junction-specific antibodies into one of these cells on the dorsal side of the eightcell embryo, which in controls, would show widespread dye transfer to its neighbors. The progeny of this cell would normally give rise to both ectodermal and mesodermal derivative tissues on the right-hand side of the body. At the 32-cell stage, the embryos were tested for gap junctional communication, and cells derived from that injected were found to be noncommunicating. Other cells of the embryo were unaffected and could communicate with cells other than those containing antibodies. The embryos were then allowed to develop and a high proportion of those loaded with antibodies (some 63%) showed right/left asymmetry. In many cases, the right eye failed to develop and there was under-development of the brain on that side. Injection of preimmune antibodies, or antibodies to an extracellular protein, had no effect on development. Hence, blocking intercellular communication for a certain window of development led to a population of cells that did not receive developmental signals and failed to differentiate. More recently, a dominant-negative connexin mutant has been used to inhibit coupling between the developing Xenopus cells (Paul et al., 1995). In this case, the injection of inhibitor mRNA into the eightcell embryo led to ejection or absorption of the noncommunicating cells during gas~ trulation. This extrusion did not occur in the antibody block experiments, although the tadpole-stage embryos in the two experiments looked identical. In these experiments, the time period of block cannot be precisely determined and the developmental messages or mechanisms involved have not been determined. For this reason, a set of experiments on the fresh water coelenterate Hydrahave been useful in throwing light on developmental processes involving gap junctional communication.

In the fresh-water coelenterate Hydra, well developed models for patterning of this relatively simple, two-layered organism have been developed, largely on the basis of grafting and tissue regeneration experiments (for example, see MacWilliams, 1983a,b). Of interest here are a putative pair of gradients thought to control head formation. One, the head activator gradient, is quite stable and is thought to stimulate head generation. The second, the head inhibitor gradient, is thought to be formed by a labile molecule produced by existing head tissue and diffusing down the body column. If the existing head is removed, the inhibitor level rapidly falls below that of the activator and a new head then regenerates in the upper region of the animal where the activator levels are highest. A peptide with an activity consistent with that of the putative head activator has been isolated from Hydra(Schaller and Bodenmuller, 1981), and the candidate for the head inhibitor is a small, low molecular weight (less than 500 Da), hydrophilic molecule (Berking, 1979). The rate of diffusion of the inhibitor molecule in Hydrahas been calculated at 2 x 10.6 cm 2 (Macwilliams, 1983a), about 10 times slower than would be expected if it was diffusing through the aqueous medium. Its passage down the body column is therefore most likely to be from cell to cell via gap junctions, while that of the activator is more likely to be passed extracellularly or from membrane to membrane. Perturbation of junctional communication does, in fact, result in patterning defects consistent with an alteration to the inhibitor gradient (Bode et al., 1987; Fraser et al., 1987). In these experiments, pieces of tissue from near the head of a donor animal, and therefore containing high activator levels, were grafted just below the head region of a host animal that was either intact or decapitated, therefore having either normal or reduced inhibitor levels owing to the removal of the inhibitor source. Grafts to normal animals were almost always inhibited and the extra tissue was absorbed into the body column of the host, while those to decapitated animals resulted mainly in two-headed animals. This is because both the upper region of the decapitated host and the grafted tissue piece have high activator levels and two heads form simultaneously. After bulk loading gap junction protein antibody into host animals that blocked junctional communication for 12-14 hours, and then carrying out the grafting experiments, a significant number of secondary axes developed. Even though the host animal was intact, the result mimicked that obtained with a decapitated host, only in this case the source of the inhibitor had not been removed but its means of diffusion to the graft site restricted (Figure 3). There was no increase in secondary axis formation if preimmune antibodies were loaded as a control. These results thus provide good evidence that a putative patterning morphogen, the Hydrahead inhibitor, does move from cell-to-cell via gap junctions, and that the junctions are therefore playing a vital role during patterning. This concept can be further examined in a vertebrate system such as the developing chick limb bud, where gap junction expression can be correlated with interactions between groups of cells.

Figure 3. The result of patterning experiments in Hydra into which nonimmune or functional connexin specific antibodies have been loaded. The animal on the left was loaded with non-immune sera prior to tissue grafting experiments with high head activator level tissue from a donor animal (refer to text). The grafted tissue has moved down the body column and is being absorbed into the host animal (arrow). The animal on the right was loaded with connexin-specific antibodies blocking cell-to-cell communication. This prevented the movement of head inhibitor from the existing head to the grafted tissue and a secondary axis has developed.

As noted earlier, the formation of gap junctions between groups of cells can be controlled, providing a mechanism for defining domains of interacting cells. The early chick limb bud has a core of mesenchymal cells encased in an ectoderm with a thickened ridge at the tip, the apical ectodermal ridge. Gap junctions are expressed within the ridge and between cells of the mesenchyme (see Green et al., 1984). It has been shown that interaction between the apical ectodermal ridge and underlying mesenchyme is necessary for bud outgrowth with signals from the ridge maintaining the progress zone. This zone of undifferentiated mesenchymal cells at the tip of the growing bud generates the sequence of structures along the proximo-distal axis of the limb (Summerbell et al., 1973). In turn, signals from the mesenchyme appear to maintain the apical ridge (Tickle, 1991). Gap junctional communication appears vital for these interactions to occur. When the limb bud is manipulated in order to flatten the ridge, the gap junctions between the cells disappear and truncated limbs develop (Tickle et al., 1989); antibody block experiments suggest an involvement in a mesenchymal signaling system (Allen et al., 1990). In another set of experiments, Green et al. (1994) have demonstrated that the expression ofconnexin 43 in tissue atthe tip of the limb bud is

related to epithelial-mesenchymal interactions that mediate morphogenetic processes. In the chick limb, high levels of connexin 43 are expressed in the apical ridge and underlying mesenchymal cells during specific stages of development. In a first set of experiments the apical ectodermal ridge was removed, a procedure that inhibits outgrowth and results in truncated limbs. Gap junction expression in the underlying mesenchyme was seen to be reduced. The same effect on gap junction expression was observed when retinoic acid-soaked beads were implanted into the limb producing ridge flattening and truncated limbs, while in a reverse experiment the apical ridge from a donor embryo was grafted to a host limb bud resulting in double limb outgrowth within 40 hours. In this case, gap junction expression was induced in the mesenchyme underlying the grafted ridge where there would not normally be connexin 43 expression. The results indicate that at a time when the apical ridge is expressing high levels of connexin 43, it induces connexin 43 expression in the immediately neighboring mesenchymal tissue. In the mouse limb apical ridge, connexin 43 is also expressed, but in this species it is not connexin 43 that is induced in the underlying mesenchyme but presumably a different member of the connexin family. Green et al. (1994) demonstrated this by immunolabeling mouse limbs, and by transplanting mouse mesenchymal cells into the chick limb below the apical ridge. No labeling of junctions was observed in the transplanted mouse mesenchyme using the connexin 43 probes, although chick positive control cells continued to show connexin 43 expression. Hence, gap junctional communication is vital in outgrowth and patterning of the avian limb. In the mammals, gap junctional communication between cells of the developing embryo is just as vital, as shown by studies on the mouse.

Cells in the compacted eight-cell mouse zygote are coupled by gap junctions, as can be shown by dye transfer and electrical coupling experiments (Lee et al., 1987). If, however, the gap junction channels in these cells are blocked by the injection of gap junction protein antibody, they are subsequently extruded from the zygote. When antibody is injected earlier into one of the cells at the two-cell stage, the majority of the zygotes continue to divide normally until the eight-cell stage, but the cells containing the antibody (i.e., the progeny of that injected) do not compact. These experiments indicate that gap junction communication may be important for the maintenance of compaction in the developing mammalian embryo. Interestingly, reduced cell-cell communication has also been observed in zygotes derived by the fertilization of eggs from an inbred DDK strain with foreign sperm (Buehr et al., 1987). In these zygotes, dye transfer is significantly slower than it is in DDK/DDK controls or controls from other strains, and 95% ofthem die by the blastocyst stage. However, by treating the zygotes with the weak base methylamine, effectively raising intracellular pH, dye transfer became as fast as in controls. This is not inconsistent with nuclear transplantation experiments, which have indicated

that the incompatibility between strains involves the egg cytoplasm rather than the maternal pronucleus (Mann, 1986). More importantly though, zygotes cultured in methylamine from the four- to eight-cell stage through to the 16-cell stage, or from early to late 16-cell stage, were significantly rescued, surviving at least until the blastocyst stage. These defective embryos therefore show the same defects as normal embryos that have been injected with antibodies to specifically block cell-cell communication, and their rescue can be brought about by increasing cell-cell communication to normal levels. Hence, a specific mammalian developmental defect can, in this instance, be correlated directly with poor gap junctional communication. It is now known that the gap junction protein expressed in the early mouse embryo is connexin 43 (Figure 4) with precise temporal control of expression observed (De Sousa et al., 1993; Valdimarsson and Kidder, 1995). DNA replication at the two-cell stage precedes the first junctional plaques at the eight-cell stage and the onset of intercellular communication with compaction. In a separate set of experiments, Becker et al. (1992) followed junction expression in normal embryos and the DDK/C3H cross using immunohistochemical techniques. At the same time, they mimicked the DDK defect by lowering the cytoplasmic pH (using butyrate) in normal embryos and rescued the DDK embryos by raising the cytoplasmic pH (using methylamine or cyclic AMP) while following junction expression. Their experiments again demonstrate the vital role for gap junctions in maintaining compaction.

It is noteworthy, however, that a high level of compensation may exist in developing organisms. Targeted mutagenesis of connexin 43 in mice has shown that its absence still enables development to term (Reaume et al., 1995). Instead, these mice die at birth as a result of swelling and blockage of the right ventricular outflow tract, an area that undergoes extensive remodeling during development. It is possible that altered electrical conduction between cells places abnormal mechanical loads on this area, leading to the morphogenetic defects observed. Nonetheless, while the early embryo appears able to compensate during earlier development, by upregulating other connexin types or relying upon a level of redundancy, the proper development of the cardiovascular system is less flexible. This system is highly dependent upon the expression of connexin 43 during development and for subsequent function.

In the heart, gap junctions form the low resistance pathways for rapid impulse conduction and propagation, coordinating myocyte contraction. Connexin 43 junctions coordinate contraction of cardiac myocytes in the working myocardium, while connexin 40 provides electrical coupling between cells of the fast conduction pathways (i.e., Bundle of His, bundle branches, and Purkinje fiber system; Gourdie et al., 1993). Gap junctions again play a role during development and patterning, but are now required to maintain their electromechanical function at the same time. Even at birth and subsequently, major changes in heart development continue (e.g., closure of the foramen ovale; systemic pressure increase and left ventricle wall thickening). Gap junction expression has been mapped during development of the heart (Gourdie et al., 1992). In these studies, connexin 43 distribution was linked particularly with the development and establishment of contractile behavior, but the role of the junctions in patterning was also recognized. Gourdie et al. (1992) noted that where there were low levels of connexin 43 expression, it might be acting to restrict the movement of intercellular communication molecules as well as the passage of electrical current. The role of other connexin types also needs to be considered. An interesting aspect of cardiac development is the extent to which postnatal changes in gap junction distribution occur; up till about Day 21 in the rat, but up until the fifth or sixth year in the human (Figure 5; Peters et al., 1994). As noted earlier, it was at birth and in the heart that the major effects of a connexin 43 knockout in mice was observed (Reaume et al., 1995). Changes in gap junction distribution also occur as a result of cardiac disease with a reduced connexin 43 content in both hypertrophied and ischemic human heart tissue (Peters et al., 1993), and with a major rearrangement of junctions around ischemic infarct border zones (Smith et al., 1991). The age-related changes may diminish the ability of the heart to remodel in response to pathological or hemodynamic perturbation, and may have implications in the timing of surgery in children (Peters et al., 1994).

coordination of myocyte contraction is maintained. (Scale bar = 601am).

With the importance of gap junctional communication between cells during development and patterning, it is not surprising that gap junction gene defects might lead to developmental disorders. Two examples have now been clearly mapped: mutations in the connexin 32 gene in X-linked dominant Charcot-Marie-Tooth disease and mutations in the connexin 43 gap junction gene in patients with heart malformations and laterality. Charcot-Marie-Tooth disease (CMTX 1) is a peripheral neuropathy-type disease with disorders characterized by impaired motor and sensory functions followed by atrophy of the associated muscle groups. It has been mapped to the X chromosome and to aregion encoding the connexin 32 gene (Fairweather et al., 1994). Point mutations of the gene have then been shown to segregate with the disease. Two of the

mutations detected are predicted to produce truncated proteins owing to a premature stop codon. In one of these, the stop codon produced replaces one of the protein's extracellular loop cysteines that is normally highly conserved in the gap junction connexin family. Not only is a truncated protein produced, but connexon-connexon stability is likely to be affected. Another of the mutations occurs in the third transmembrane span and is likely to interfere with the channel wall lining. In all, eight out of nine families investigated showed mutations in the connexin 32 coding region. In the case of connexin 43 gap junction gene defects, children with syndromes that included complex heart malformations had residue substitutions (BritzCunningham et al., 1995). In some cases, two independent mutations were detected suggesting an autosomal, recessive disorder. In one other case, a completely different heart condition correlated with another point mutation in the connexin 43 gene. In this study, the researchers cloned the samples after sequencing, and in parallel with control connexin 43 sequences, transfected a cell line that normally showed low endogenous gap junctional communication. They then carried out microinjection studies proving that the defects noted in the human gene sequences do in fact result in reduced cell-to-cell communication compared to controls. None of the defects noted in this case would appear to effect channel formation but rather regulation of channel function. The resulting disturbances may be more insidious than those produced by complete inactivation of the gene (Reaume et al., 1995), but they may offer important clues to the timing and location of critical events in the early stages of human heart development (Britz-Cunningham et al., 1995).

Gap junctions play a major role during development and patterning, and in controlling cell proliferation and growth, providing the pathway and regulating the direct cell-to-cell exchange of signals. Their importance starts with the oocyte, but their influence persists through all stages of development as they are responsible for maintaining homeostasis in the adult. Antibody probes used in immunohistochemical studies have proven valuable in studying gap junction distribution in developing organisms; the greater overview provided has given many advantages over the ultrastructural techniques employed previously. In combination with dye transfer and electrophysiological experiments, the evidence for gap junctional roles has mounted. While much of the evidence for this is correlative, the development of functional antibody probes that specifically block cell-cell communication has enabled several experimental systems to be probed directly. More recently, molecular techniques (e.g., transfection, mutagenesis experiments, transgenic approaches, in situ hybridization) have made it possible to tie down the role of gap junctions during development and patterning with even greater precision. The links between connexin mutations and human pathologies have provided an exciting new avenue of

e x p l o r a t i o n and d e m o n s t r a t e d crucial roles for gap junctions. In virtually all systems, however, the regulatory signals involved have yet to be identified and the precise role for m u l t i p l e c o n n e x i n types and h o w they are regulated is not u n d e r s t o o d .

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Stauffer, K.A. (1995). The gap junction proteins ~,-connexin (connexin-32) and 132-connexin (connexin-26) can form heteromeric hemichannels. J. Biol. Chem. 270, 6768--6772. Sugiura, H., Toyama, J., Tsuboi, N., Kamiya, K. & Kodama, I. (1990). ATP directly affects junctional conductance between paired ventricular myocytes isolated from guinea pig heart. Circ. Res. 66, 1093-1102. Summerbell, D., Lewis, J. & Wolpert, L. (1973). Positional information in chick limb morphogenesis. Nature 224, 492-496. Tepass, U. & Hartenstein, V. (1994). The development of cellular junctions in the Drosophilaembryo. Devel. Biol. 161,563-596. Tickle, C., Crawley, A. & Farrar, J. (1989). Retinoic acid application to chick wing buds leads to a dose dependent reorganization of the apical ectodermal ridge that is mediated by the mesenchyme. Development 106, 691-705. Tickle, C. ( 1991). Retinoic caid and chick limb development. Development (suppl.) 113-121. Traub, O., Look, J., Dermietzel, R., Brummer, F., Hulser, D. & Willecke, K. (1989). Comparative characterization of the 21-kDa and 26-kDa gap junction proteins in murine liver and cultured hepatocytes. J. Cell Biol. 108, 1039-1051. Trosko, J.E., Jone, C., Aylsworth, C. & Tsushimoto, G. (1982). Elimination on metabolic cooperation is associated with the tumor promotors, oleic acid, and anthralin. Carcinogenesis 3, 1101-1103. Turin, L. & Warner, A.E. (1977). Carbon dioxide reversibly abolishes ionic communication between cells of early amphibian embryos. Nature 270, 56-57. Valdimarsson, G. & Kidder, G.M. (1995). Temporal control of gap junction assembly in preimplantation mouse embryos. J. Cell Sci. 108, 1715-1722. Warner, A.E., Guthrie, S.E. & Gilula, N.B. (1984). Antibodies to gap junctional protein selectively disrupt junctional communication in the early amphibian embryo. Nature 311,127-131. Warner, A.E. & Lawrence, P.A. (1982). Permeability of gap junctions at the segmental border in insect epidermis. Cell 28, 243-252. Watts, S.W., Tsai, M.-L., Loch-Caruso, R. & Webb, R.C. (1994). Gap junctional communication and vascular smooth muscle reactivity: Use of tetraethylammonium chloride. J. Vasc. Res. 31, 307-313. Weir, M.P. & Lo, C.W. (1982). Gap junctional communication compartments in the Drosophila wing disk. Proc. Natl. Acad. Sci. USA 79, 3232-3235. Yamasaki, H., Enomoto, T., Martel, N., Shiba, Y. & Kanno, Y. (1983). Tumor promoter-mediated reversible inhibition of cell-cell communication (electrical coupling). Exp. Cell Res. 146, 297-308. Zimmer, D.B., Green, C.R., Evans, W.H. & Gilula, N.B. (1987). Topological analysis of the major protein in isolated intact rat liver gap junctions and gap junction-derived single membrane structures. J. Biol. Chem. 262, 7751-7763.

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Introduction Receptors Vertebrate Development Mesoderm Induction Mesoderm-Inducing Factors Activin Vgl Nodal Bone Morphogenetic Protein (BMP) Fibroblast Growth Factor (FGF) Wnt Mesodermal Patterning Neural Induction and Patterning Vertebrate Limb Development Bone Morphogenesis Conclusions

Principles of Medical Biology, Volume 11

Developmental Biology, pages 123-151. Copyright 9 1998 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-816-1

124 125 130 132 134 135 136 136 137 137 139 140 143 145 148 149

Polypeptide growth factors (PGF) are small secreted proteins that control the viability, differentiation and proliferation of cells. They typically have a short or in-

termediate range of action and bind to specific high affinity receptors on the surface of responding cells. Although similar to peptide hormones in many respects, PGFs tend to act locally on adjacent cells (paracrine action) or on the secretory cell itself (autocrine action). In several cases this is a consequence of the PGF remaining attached to the surface of the cell that produced it, either bound to components of the extracellular matrix (ECM) or as an integral membrane protein. Apart from restricting diffusion, interactions between secreted PGFs and the ECM may also regulate their activity. This appears to be the case for FGF and TGFI3; access of both molecules to their high affinity receptors is enhanced by their prior association with membrane-bound proteoglycans. On the other hand, soluble forms of these proteoglycans may restrict access to the receptors. PGFs can be grouped into a small number of families that may contain as many as 30 different, but structurally related, molecules (Table 1). Perhaps the best known are the epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor-beta (TGFI3), insulin-like growth factor (IGF) and wingless/int 1 (Wnt) families. The names can be misleading, being based on the biological activity or assay system used for their original isolation, and it is now apparent that most are multifunctional molecules with a range of biological effects. Many of these molecules were not initially identified because of their growth-promoting activities; rather, they were identified through their ability to regulate other processes or through their sequence homology to previously identified PGFs. Some of these have provided definitive evidence that PGFs play important roles in embryonic development, influencing not only growth but also differentiation and morphogenesis. It is these latter aspects of PGF activity that we will concentrate on in this review.

PGFs are extracellular proteins that trigger many biochemical responses inside the cell. To achieve this, they interact with the extracellular domain of specific cell surface receptors that traverse the plasma membrane and transmit the signal to the cytoplasm (reviewed by Alberts et al., 1994). Members of a given PGF family are structurally quite similar, and in many cases they may interact with similar, if not identical receptors. For example, both EGF and the closely related TGFa bind to the same receptor with a similar efficiency; consequently they elicit a similar response from exposed cells. On the other hand, members of the TGFI3 family can initiate quite different responses, and this may be achieved through the differential affinities of a number of receptor subtypes. The response elicited by a given PGF is also determined by the cell type that is exposed to it; in the case of TGFI31, this can result in either the promotion or inhibition of cell proliferation. While the response is undoubtedly influenced by the presence or absence of other PGFs, it is also determined by the origin of the cell in question. This must reflect their different develop-

mental histories and the range of molecules, some of which are PGFs, to which they have previously been exposed. There is now a great deal of information concerning the response of different cell types to PGFs in vitro; however, it is not always clear what relevance this has to their roles in vivo. A large number of receptors have now been identified and, like the PGFs themselves, they can be placed into a small number of structurally related families. Broadly speaking two types of receptors have been identified: those that possess an intrinsic cytoplasmic protein kinase activity, an enzyme that catalyzes the transfer of the T-phosphate of ATP to specific residues on substrate proteins, and those that activate separate protein kinases (reviewed by Alberts et al., 1994). Whereas members of the FGF, EGF, PGF, and NGF families have receptors that possess a tyrosine-specific kinase, members of the TGFI3 family have receptors that possess a serine/threonine-specific kinase. In contrast, members of the Wnt family have receptors that lack an intrinsic kinase activity. Receptors for PGFs related to NGF, EGF, PDGF, and FGF, as well as IGF1 and hepatocyte growth factor (HGF), possess kinase domains that specifically phosphorylate tyrosine residues (reviewed by Alberts et al., 1994). Following PGFbinding, the receptors form homo-dimers and activate the cytoplasmic kinase domain, which subsequently phosphorylates specific substrate proteins (Figure 1). These include the receptors themselves as well as enzymes such as phospholipase cT, phosphotidylinositol 3'-kinase, the Src family of tyrosine kinases, the serine/threonine-specific Raf kinase, the tyrosine-specific Syp phosphatase (which removes phosphate from tyrosine residues) and GTPase-activating protein. These subsequently activate a number of other enzymes, many of them kinases, that are components of a number of different signal transduction pathways responsible for activating the various cellular responses. In terms of embryonic development, the best characterized pathway leads to the activation of the so-called mitogenactivated protein kinases (MAPK). Activated MAPKs can enter the nucleus and phosphorylate a number of transcription factors; they may therefore provide the link between receptor-mediated events and changes in gene expression in the nucleus (reviewed by Marshall, 1995). MAPKs are activated by a cascade of enzymatic reactions: as a result of receptor activation, the small GTPase Ras interacts with Raf kinase, which subsequently phosphorylates and activates MAPK kinases (Figure 1). These latter enzymes are responsible for activating MAPKs. This pathway is used by many different cell types to initiate many different responses including determination of cell fates, proliferation, and differentiation. Two structurally related receptor subtypes have been identified for the TGFI3 family, known as type I and type II (reviewed by Ten Dijke et al., 1996). TGFI31 and activin bind to their respective type II receptors first, which then form a hetero-oligomeric complex with the type I receptor. Within this complex the type II receptor phosphorylates and activates the type I receptor, which propagates the signal to downstream components (Figure 2). In contrast, BMP2 and BMP4 have equal affinity for both the type I and type II receptors, although they share the same require-

ment for hetero-oligomeric complex formation as TGF[31 and activin. All of these receptors possess a cytoplasmic kinase domain, which phosphorylates serine and/or threonine residues in substrate proteins. By comparison with other PGFs, very little is currently known zbout the cytoplasmic elements involved in the response to the TGFI3 family, largely because the receptors for this class of PGF have only recently been identified. One gene involved in transducing the signal for the TGF~ family member decapentaplegic, was identified by mutational analysis in the fruit fly Drosophila melanogaster. This gene was called mothers against decapentaplegic (MAD), and a number of structurally related proteins (known as SMADs) were subsequently isolated from vertebrates and the nematode worm Caenorhab' ditis elegans (reviewed by Wrana & Attisano, 1996). Receptor activation results in

these SMAD proteins being phosphorylated and translocated from the cytoplasm to the nucleus, in turn leading to changes in gene expression. Interestingly, different SMAD proteins appear to be activated by different members of the TGFI3 family; for example, whereas SMAD1 appears to transduce the signal for BMP2 and BMP4, SMAD2 appears to transduce the signal for activin (Figure 2). Receptors for the Wnt family of PGFs have recently been described in flies, nematodes, sea urchins, frogs, and mammals (reviewed by Orsulic and Peifer, 1996). These receptors are related to a Drosophila gene called frizzled 2, which is the receptor for the Drosophila Wnt family member wingless, and at least eight frizzled homologues have now been identified in mammals. In contrast to the receptors described above, frizzled belongs to the serpentine class of receptors that pass through the plasma membrane seven times and do not possess intrinsic kinase activity. Serpentine receptors are typically coupled to the cytoplasmic G-proteins, which

indirectly activate cytoplasmic kinases, but no frizzled receptor has yet been shown to couple to G-proteins. Analysis of Drosophila mutations with similar phenotypes to wingless has identified a number of genes encoding cytoplasmic components of the wingless signal transduction pathway including dishevelled, zeste-white 3 (zw3) and armadillo. Vertebrate homologues of all three genes have been identified--d/shevelled, glycogen synthase kinase 3 beta (gsk3~), and ~3-catenin respectively-and have been shown to act in Wnt signaling pathways (Figure 3). Activation of the Wnt signaling pathway leads to the stabilization of armadillo in

1 1

1 1 t

Drosophila and 13-catenin in vertebrates. Once stabilized these proteins enter the nucleus, perhaps as a complex with transcription factors, where they control the transcription of target genes. The molecular mechanisms whereby this is achieved are still unknown, but inactivation of ZW3/GSK313 by dishevelled is an essential component, ZW3/GSK313 being primarily responsible for targeting armadillo/13-catenin for breakdown.

There is now a considerable amount of evidence that PGFs play important roles during development, controlling not only localized rates of cellular proliferation but also differentiation and morphogenesis. A general theme is that PGFs act as signaling molecules, synthesized and secreted by one cell type to control the development of adjacent cells that express their receptors. In recent years, it has been shown that some PGFs are the inducing factors that embryologists had been trying to identify for nearly 70 years. Induction is probably the single most important mechanism involved in the generation of pattern within vertebrate embryos, being responsible for the formation of all the major tissue types and organs (reviewed by Slack, 1991). This has been apparent since 1924, when Hans Spemann and Hilde Mangold reported the result of transplanting the dorsal blastopore lip of an amphibian gastrula into the ventral region of a host embryo. Quite remarkably, this resulted in the formation of a second highly organized body axis on the ventral side. Within this second axis, host cells that would normally have given rise to epidermis or blood now formed neural tissue and muscle respectively. The capabilities of the dorsal blastopore lip so impressed Spemann that he called it the "organizer". Subsequent work concentrated on the ability of the organizer to induce neural differentiation in competent ectoderm, and a great deal of effort was put into identifying neural-inducing factors. However, it was soon discovered that many nonspecific agents could mimic the organizers' neural-inducing activity, suggesting that ectoderm was delicately balanced between epidermal and neural fates and that this balance could be easily tipped in the direction of neural tissue. In the last few years, amphibian embryologists have been much more successful in identifying inducing factors, especially those capable of directing ectoderm down a mesodermal pathway. Mesoderm inducing factors, as described below, are PGFs belonging to the TGFI3 and FGF families. Confirmation that PGFs have important functions during embryonic development has been provided by genetic studies on the fruit fly Drosophila melanogaster, the nematode worm Caenorhabditis elegans, and the laboratory mouse Mus musculus. While the studies on Drosophila and Caenorhabditis are particularly elegant, it is our intention to concentrate on vertebrate development and, therefore, we will only discuss genetic analyses performed in the mouse. A number of naturally occurring mouse mutations have been identified that both disrupt the activities of

B. Targeted/Insertional Mutations

Gene Products

Phenotype

PGFs and exhibit abnormal embryonic phenotypes; (listed in Table 2A). Additionally, a particularly powerful technique that can be applied to the mouse is targeted disruption of known genes, a method that produces loss-of-function mutations by inserting a piece of foreign DNA into the coding region. Many genes encoding PGFs or their receptors have now been mutated using this technique, and many have abnormal embryonic phenotypes, some of which are listed in Table 2B. In the following discussion we will concentrate on a small number of well studied examples that illuminate the functions of PGFs during embryonic development in vertebrates, including mesoderm formation in amphibians, neuralization in both amphibians and chicks, and limb formation in chicks. Where relevant, we will also discuss mouse mutations that demonstrate a role for PGFs in the process under discussion.

In amphibians, the fertilized egg is initially divided into two zones, a darkly pigmented animal hemisphere that is specified as ectoderm and a lightly pigmented vegetal hemisphere that is specified as endoderm. The third germ layer, mesoderm, is formed in the equatorial marginal zone of the blastula (32-5000 cells) as a result of inductive signals released by the vegetal hemisphere. This was first shown by Peter Nieuwkoop (reviewed by Slack, 1991), who juxtaposed animal and vegetal poles isolated from mid-blastulae; in isolation the two poles differentiated as epidermis and endoderm respectively, but when combined, the animal pole also differentiated into mesoderm (Figure 4). Using this type of experiment, two inducing regions have been identified in the vegetal hemisphere, a small dorsal region (usually called the Nieuwkoop center to honor Peter Nieuwkoop's discovery of mesoderm induction) that induces dorsal mesoderm (notochord and muscle) and a much

larger lateral and ventral region that induces ventral mesoderm (blood and mesothelium; see Figure 5). As a result of the activity of these two vegetal regions, only two types of mesoderm are initially specified in the marginal zone of the blastula. A small region of 60-90 ~ of circumference lying just above the Nieuwkoop center and equivalent to the organizer identified by Spemann &Mangold is specified as dorsal mesoderm, while all remaining regions (270-300 ~ of circumference) are specified as ventral mesoderm (Figure 5). To a certain extent these two mesodermal types can be seen in the distribution of mesoderm-specific transcripts in late blastulae. At this stage, we can see the initial expression patterns of genes that are directly activated by mesoderm induction, and these patterns should therefore reflect the spatial distribution of the signals that in-

Figure 5. Inductive interactions in mesoderm formation. Signals released by the vegetal pole during blastula stages induce the formation of mesoderm in the overlying marginal zone: while the signal(s)localized to the dorsal quadrant (DV) induce(s) the dorsal organizer (O), the signal(s) released by lateral and ventral quadrants (W) induce(s) ventral mesoderm (M4). Signals released by the organizer dorsalize the adjacent mesoderm, generating the full mesodermal complement (M1 to M4). Organizer signals also act on the overlying ectoderm inducing the formation of neural tissue (NT). Current evidence suggests that both dorsalizing and neuralizifig signals are mediated by the secreted proteins noggin, chordin and follistatin, which prevent BMP4 from activating its receptor. BMP4 is a potent inhibitor of both dorsal mesoderm and neural tissue. Dorsalization and neuralization occur during gastrula stages when the embryo is undergoing extensive cell and tissue rearrangements; these alter the relative positions of the organizer and both the mesoderm and ectoderm. The diagram is therefore a simplification of the overall process. duced them. Whereas some genes (e.g., brachyury) are expressed throughout the mesoderm, others (e.g., goosecoid) are only expressed in the organizer. This might suggest that a general mesoderm-inducing factor is released by all vegetal cells, inducing the expression of brachyury throughout the marginal zone, and that a dorsal signal released by the Nieuwkoop center is superimposed upon this general signal to induce the expression of goosecoid in the organizer. We will now discuss the PGFs thought to be involved in mesoderm induction in amphibians (Moon et al., 1993; Slack, 1994; Kessler and Melton, 1994; Hogan, 1996).

The best characterized mesoderm inducing factors are activin, a member of the TGFI3 family, and FGF2 (also called basic FGF). Addition of either protein to isolated animal pole explants induces the differentiation of mesodermal tissues such as notochord, muscle, and blood. Whereas activin tends to induce the differentiation

of dorsal mesoderm including notochord, FGF2 will only induce the differentiation of ventral mesoderm. This led to the suggestion that an FGF was the general mesoderm-inducing factor secreted by all vegetal cells and that activin was the dorsal signal secreted by the Nieuwkoop center. However, subsequent work has raised doubts as to whether either of these molecules is a natural mesoderm-inducing factor in amphibian embryos. Before any candidate inducing factor can be considered the natural signal, it must meet at least three criteria: (1) the factor must have the correct biological activity in an appropriate test; (2) it must be expressed in the right place and at the fight time; and (3) specific inhibition of its action must prevent the inductive process from occurring in vivo. In practice it has proven very difficult to provide conclusive evidence for any putative mesoderm-inducing factor with respect to these criteria.

Activin is a very potent mesoderm-inducing factor when added to animal caps, active down to concentrations of as little as 10 pM. At these low concentrations, activin induces ventral mesoderm, but with increasing concentrations posterolateral mesoderm, muscle, notochord, and finally organizer tissue are induced. That activin can act as a gradient to initiate transcription of mesoderm-specific genes has been demonstrated by recombining animal caps with a localized source of activin. When the source releases high concentrations of activin the cells closest to the source express the organizer-specific goosecoid gene, while cells further away express brachyury. If we assume that activin diffuses away from the localized source to set up an activin gradient, a reasonable but as yet unproven assumption, then brachyury would be activated by low concentrations of activin and goosecoid by high concentrations. Consistent with this is the fact that if the source only releases low concentrations of activin, then brachyury is now expressed by those cells closest to the source, and no cells express goosecoid. This is good evidence that a concentration gradient of a single PGF could account for the initial expression pattern of mesoderm-specific genes, high concentrations inducing organizer-specific genes, and low concentrations inducing ventral-specific genes. Evidence that activin may be the natural mesoderm-inducing factor was provided by Ali Hemmati-Brivanlou and Doug Melton who constructed a truncated activin receptor in which the cytoplasmic kinase domain had been deleted. When overexpressed in Xenopus animal caps, this truncated receptor blocked mesoderm formation in response to activin, but not FGF. Presumably this mutant receptor formed inactive complexes with the endogenous receptors, thereby inhibiting the activin-induced signal transduction pathway. In this respect, the truncated receptor is acting as a dominant-negative or antimorphic mutation. Of greater interest was their observation that this mutant receptor inhibited the formation of mesoderm in the marginal zone of the embryo, suggesting that it had inhibited the endogenous signal. However, it is now clear that this truncated receptor can also inhibit the sig-

naling pathway of other members of TGFI3 family including Vgl and BMP4. Furthermore, while the activin binding protein follistatin can block the mesoderm-inducing activity of activin in Xenopus animal caps, it has no effect on the endogenous signal. That activin may not be the endogenous mesoderminducing factor has also been suggested by experiments in the mouse in which the activin genes (activin fSA and activin 13B), or an activin receptor gene (actRII), have been mutated. Although all of these mutations give abnormal embryonic phenotypes, mesoderm formation is normal. Together, these results suggest that mesoderm formation in vertebrates does not require activin.

Vgl Like activin, Vg 1 is a member of the TGF~ family of PGFs. This molecule gets its name because it is localized to the vegetal hemisphere of Xenopus oocytes and early embryos, meeting the expression criteria for a natural mesoderm-inducing factor. Yet no biologically active protein has been detected in the embryo, and injection ofVgl mRNA into animal caps does not induce mesoderm. This would seem to make Vgl an unlikely candidate for the natural mesoderm-inducing factor, but recent work suggests otherwise. Biologically active Vg 1 has been produced from chimeric proteins between V gl and either activin or B MP, and is a potent mesoderm-inducing factor when applied to animal caps. Like activin, it can induce all forms of mesoderm in a concentration dependent manner, demonstrating that Vgl does have the correct biological activity. The problem we are left with is in trying to demonstrate that the Xenopus embryo actually produces biologically active Vgl. Interestingly, the zebrafish homologue of Vgl is processed in Xenopus embryos and does induce mesoderm in animal caps. Consistent with arole for Vgl in mesoderm induction is the observation that the dominant negative activin receptor inhibits the mesoderm-inducing activity of Vg 1. As described above this mutant receptor inhibits the natural signal released by the vegetal pole, although it also inhibits many other members of the TGFI3 family. In contrast, the activin-binding protein, follistatin, neither inhibits the natural signal nor Vgl, whereas it does inhibit activin. While this does not conclusively prove that Vgl is the natural mesoderm-inducing factor, it does make it a more attractive candidate than activin.

The nodal gene was originally identified as a mutation that severely disrupted mesoderm formation in early mouse embryos and was subsequently shown to encode a PGF with considerable homology to members of the TGFI3 family. Four nodal-related genes have also been isolated from the frog embryo, and two of these, XNr 1 and XNr2, induce dorsal mesoderm in animal caps. However, while they may meet the activity criteria, expression of both genes is activated by mesoderminducing factors with the highest levels of expression on the dorsal side of the gas-

trula, the organizer. In the mouse embryo, nodal transcripts are first detected throughout the embryonic ectoderm, but following the initiation of gastrulation, they become restricted to proximal ectoderm. Cells from this region will normally populate the posterior and extraembryonic mesoderm, the tissues most dramatically affected in nodal mutant embryos. Later in development, nodal expression is confined to the periphery of the mature node, the murine equivalent of the frog organizer. While these results demonstrate that nodal plays an important role in mesoderm formation in early vertebrate embryos, it is unlikely to be involved in the intial induction of mesoderm.

BMP2, BMP4, and BMP7 are PGFs with considerable homology to the TGFI3 family that also induce mesoderm in Xenopus animal caps following injection of their RNAs. In contrast to activin, Vg 1, XNrl, and XNr2, these proteins only induce ventral mesoderm and suppress the differentiation of dorsal mesoderm when overexpressed in embryos or in animal caps treated with activin. This might suggest a role for BMPs in the induction of ventral mesoderm, and transcripts for bmp2 and bmp7 are present at the time of mesoderm induction although they show no localization to the vegetal hemisphere, bmp4 is only expressed from late blastula stages, suggesting a role later in development (see below). Consistent with a later role, a truncated receptor for BMP2 and BMP4 that lacks the cytoplasmic kinase domain does not inhibit mesoderm induction but converts ventral mesoderm into dorsal mesoderm (reviewed by Harland, 1994). Mouse mutations in either the bmp4 gene orthe gene encoding a receptor for both BMP2 and BMP4 (bmprl) result in the failure of mesodermal development and the absence of brachyury expression. While this is consistent with a role for BMP4 in mesoderm induction in the mouse embryo, this phenotype may be a secondary consequence of a reduced rate of cell proliferation in these mutant embryos.

The first PGF to be properly identified as a mesoderm-inducing factor was FGF2, which is one of ten structurally related proteins that constitute the FGF family. Most of these appear to have mesoderm-inducing activity. Like activin, the effect of FGF2 is concentration dependent, as little as 50 pM being required to divert animal caps from an epidermal fate towards that of ventral mesoderm. With increasing concentration more dorsal tissues, such as muscle, are differentiated, but, unlike activin, FGFs rarely induce notochord. FGF2 has been shown to interact with activin in a rather interesting way--a low, but constant concentration of FGF2 increasing the response of animal caps to activin. As a consequence, muscle and notochord are induced at lower concentrations of activin in the presence of FGF2 than in its absence. This could suggest that one role for FGF is to control the

response of the animal hemisphere to mesoderm-inducing factors released by the vegetal hemisphere. Consistent with such a role, maternalfg~ is localized to the responding animal hemisphere, and expression in the vegetal hemisphere following injection of f g ~ RNA induces ectopic expression of mesodermal marker genes. Evidence that FGFs play a role in the formation of mesoderm has been provided by a truncated receptor that lacks the cytoplasmic kinase domain. This receptor inhibits the response of animal caps to FGFs and disrupts mesoderm formation when injected into embryos, although the phenotype is not as severe as that obtained with the mutant activin receptor described above. Typically these embryos have normal heads but severely deformed trunks and no tail, defects reflected in the loss of notochord and muscle. Identical results have been obtained following the injection of RNAs encoding dominant negative forms of either Rafl kinase or Syp-phosphatase, or of normal MAP kinase phosphatase. All three molecules act to block the cytoplasmic signal transduction pathway for FGF that leads to the activation of MAP kinase, but they have no effect on the signaling pathway for activin. At the molecular level, the dominant negative FGF receptor inhibits expression of the brachyury gene but has little effect on the expression of other mesoderm-specific genes such as goosecoid. This is consistent with the activity of FGF2, which induces the expression of brachyury but not goosecoid in animal caps. The inhibitory effects of this dominant-negative receptor are not restricted to FGF; it also inhibits the ability of activin to induce the expression of brachyury but not goosecoid. This might suggest that FGFs act downstream of the endogenous mesoderm-inducing factor, and recent evidence suggests that they are required to maintain the expression of brachyury. When Xenopus mesoderm is disaggregated at the beginning of gastrulation, then expression of brachyury is lost but then restored by adding FGF to the incubation medium. Because brachyury activates the expression of Xenopus efgf, which in turn maintains the expression of brachyury, an autocatalytic loop has been suggested. Consistent with this efgfis the expression in the circumblastoporal mesoderm and posterior notochord during gastrulation of an expression pattern that substantially overlaps with

brachyury. That FGFs might might not be involved in the initial induction of mesoderm is also suggested by the embryonic phenotype of mice carrying mutations in an FGF receptor (fgfrl) gene (Deng et al., 1994; Yamaguchi et al., 1994). Although these embryos displayed early growth defects, they were still capable of forming mesoderm and brachyury was expressed. However, mesodermal patterning was aberrant and embryos frequently lacked paraxial mesoderm (somites) and in some cases exhibited truncations of the abdomen and tail. Superficially these embryos resemble those obtained in the frog by expressing the dominant inhibitory mutation of this receptor. The results confirm that FGFs do play a role in mesoderm formation in vertebrate embryos, especially in posterior regions.

Members of the wnt gene family have very little mesoderm-inducing activity when expressed in Xenopus animal caps, suggesting that they are not mesoderm inducing factors, but some are capable of altering the response of animal caps to mesoderm-inducing factors such as FGF2 and activin (reviewed by Moon et al., 1993). For example, whereas FGF2 alone will induce ventral mesoderm in animal caps, FGF2 plus XWnt8 induces dorsal mesoderm including a notochord. This suggests that Wnts might normally act to modify the response of animal cap cells to vegetal inducers, rather than induce mesoderm themselves. One consequence of this modifying activity is that localized expression of XWnt8 on the ventral side of the Xenopus blastula results in the formation of a second dorsal axis including notochord and head structures. Because injected cells need not contribute to the secondary axis, it is clear that XWnt8 can mimic the Nieuwkoop center, which induces the Spemann organizer. In Xenopus, xwnt5a, xwnt8b, and xwnt11, are all maternally expressed, which is consistent with a role in mesoderm induction, but only xwnt11 is localized to the vegetal hemisphere, xwnt11 is later expressed in Spemann's organizer, an expression pattern that is consistent with a role in dorsal mesoderm formation. However, ventral expression of xwnt11 does not induce a complete secondary axis, which typically lacks a notochord and head structures, casting doubt on any involvement of this PGF in mesoderm-induction. In contrast, xwnt8b is localized to the animal hemisphere of Xenopus blastulae and may be involved in modulating the response to vegetal signals (Cui et al., 1995). Recently, Randy Moon and his colleagues have generated a dominantnegative form of xwnt8a, which, when coexpressed with wild-type xwnt8 or wntl, inhibits their ability to induce a secondary axis inXenopus embryos (Hoppier et al., 1996). However, dominant-negative xwnt8 has no effect on the expression of Spemann organizer genes such as goosecoid or the formation of the notochord, although it does suppress the development of axial muscle, when injected alone. Similarly, a dominant-negative form of Xenopus dishevelled, an intracellular component of the Wnt signaling pathway (Figure 3), inhibits the ability of coinjected Wnts to induce a secondary axis but has no effect on the development of dorsal mesoderm when injected alone (Sokol, 1996). Together these results suggest that the Wnt family may not be involved in the induction of Spemann's organizer during normal development. One explanation for these resuits is that components of the Wnt signaling pathway are activated during dorsal mesoderm formation in a Wnt-independent manner. For example, GSK313 (see Figure 3) is inactivated by several extracellular signaling molecules apart from Wnts, including EGF and insulin. In this model, expression of Wnts in ventral blastomeres would activate part of a signaling pathway normally used in dorsal blastomeres by a different signaling molecule, the endogenous dorsal mesoderm-inducing factor.

Embryological experiments in the frog have shown that patterning within the mesoderm is the result of inductive signal(s) emanating from the dorsal organizer (Figure 5), a process called dorsalization (reviewed by Slack, 1991). For example, in fate maps made at the 32-cell stage, about 60% of all muscle cells are derived from the ventral half of the embryo; yet if isolated during blastula stages, these ventral cells will differentiate into blood rather than muscle. Somehow they must be instructed to adopt a more dorsal fate. This can be achieved experimentally by recombining ventral fragments with a dorsal organizer fragment at gastrula stages; in these circumstances, the ventral mesoderm cells will differentiate as muscle. The most dramatic demonstration of dorsalization is the organizer transplantation experiment of Spemann and Mangold, as described above, where the resulting embryo often forms a complete secondary axis in which ventral mesoderm has been re-specified as muscle. Recent evidence suggests that the dorsalizing signal may act by inhibiting the activity of BMP4, a PGF related to the TGFI3 superfamily that is expressed in lateral and ventral sectors of the mesoderm and is absent from the organizer during gastrulation. When bmp4 is expressed in the organizer, it completely blocks dorsal development and promotes the development of ventral mesoderm (reviewed by Hogan, 1966). Despite the eventual absence of dorsal mesoderm in these embryos, they appear to develop normally until the early gastrula stage, forming a dorsal blastopore lip and expressing organizer-specific genes such as goosecoid. However, later gastrulation movements are severely disrupted and goosecoid expression is rapidly down-regulated. This suggests that BMP4 may be acting during the early phases of gastrulation to specify ventral mesoderm. Consistent with this is the observation that BMP4 will induce ventral mesoderm in isolated animal caps, and a truncated BMP4 receptor lacking the cytoplasmic kinase domain will promote the development of dorsal mesoderm when expressed in ventral blastomeres. One explanation for these observations is that BMP4 acts as an inhibitor of dorsal development and that the function of the dorsalizing signal is to overcome this inhibition. Recently, two secreted proteins called noggin and chordin have been identified as binding directly to BMP4, thereby inhibiting its activity (Piccolo et al., 1996; Zimmerman et al., 1996). Both proteins are synthesised in the dorsal organizer, will dorsalize ventral mesoderm in vitro, and will induce a secondary axis when expressed in ventral blastomeres. Xwnt8a may also be involved in regulating the activity of dorsalizing signals; this gene is expressed in ventral and lateral mesoderm of Xenopus gastrulae but is absent from the organizer. Ectopic expression of this gene in the organizer suppresses goosecoid expression and promotes the development of ventral and lateral mesoderm at the expense of dorsal mesoderm (reviewed by Moon et al., 1993). The resulting embryos typically lack head structures and fail to differentiate the notochord, a phenotype resembling that observed following expression of bmp4 in the

organizer. In contrast, expression of a dominant-negative XWnt8a protein in lateral sectors of the gastrula mesoderm, where xwnt8a is normally expressed, results in the loss of dorsal muscle (Hoppler et al., 1996). These results suggest a model in which the function of endogenous XWnt8a is to induce expression of genes involved in the specification of ventral and lateral mesoderm. In the mouse, Wnt3a may also be involved in mesodermal patterning; embryos homozygous for a mutation in this gene lack posterior somites, have a disrupted notochord, and fail to form a tailbud (Takada et al., 1994). PGFs also appear to be involved in patterning the somites following the completion of gastrulation. Somites are blocks of mesoderm that bud off from the paraxial mesoderm in a head to tail sequence, and in human embryos, 40 to 42 somites are formed on each side of the notochord. While the ventral portion of each somite subsequently forms the sclerotome, which ultimately gives rise to the axial skeleton, the dorsal portion becomes the dermomyotome, the precursor of striated muscles and dermis (Figure 6A). Each somite can also be subdivided into a medial compartment that gives rise to the axial muscles and a lateral compartment that gives rise to the muscles of the limbs and body wall. Specification of the different somite territories is controlled by signals released by adjacent tissues such as the notochord, neural tube, and lateral plate mesoderm (Figure 6B). Signals released by the notochord and floor plate of the neural tube induce cells of the ventral somite to become the sclerotome, and in the absence of these tissues ventral somite cells adopt a more dorsal fate. Furthermore, if an additional notochord is grafted adjacent to the dorsal region of a chick somite, then these dorsal cells adopt a more ventral fate. The signal released by the notochord and floor plate is probably sonic hedgehog (Shh), a vertebrate PGF highly homologous to the Drosophila protein hedgehog; shh is expressed in the notochord and floor plate at the relevant time of development, and in chick embryos, ectopic expression induces dorsal somitic cells to adopt a sclerotomal fate. Consistent with this proposed role for Shh, is the observation that mice lacking the shh gene exhibit severe defects in the paraxial mesoderm and subsequently have skeletal abnormalities (Chiang et al., 1996). In contrast, signals released by the lateral plate mesoderm induce cells of the lateral somitic compartment to express the single-minded gene; in the absence of these signals single-minded is not expressed in the somite (reviewed by Hogan, 1996). In the chick embryo, the lateral plate signal appears to be BMP4 because this gene is expressed in the lateral plate mesoderm at the relevant time of development, and ectopic expression of BMP4 induces ectopic expression of single-minded and inhibits expression of myoD, a gene expressed in the medial myotome. The results suggest that BMP4 induces somite cells to adopt a lateral fate as apposed to a medial fate. Another growth factor that is involved in mesodermal patterning is platelet derived growth factor A (PDGFA): this gene is expressed in the ectoderm of frog embryos and is inactivated by mesoderm-inducing signals. In contrast, the tx receptor for PDGF is expressed in the mesoderm and is one of the first genes to be activated

A)

Neural Tube

t Myotome IEpaxial) Dermatome

(Hypaxial) (Limb) Notochord

B)

BMP4r

6. Somite and neural patterning. (A) Diagram through the dorsal axis of a vertebrate embryo illustrating the relative positions of the notochord, neural tube, and somites. Division of the somites into sclerotome, dermatome, and both medial and lateral myotome are indicated. (B) Patterning of the somite and neural tube are mediated by signals released from adjacent tissues, especially sonic hedgehog (Shh) from the notochord and floor plate, dorsalin 1 (Dsll) from the dorsal neural tube, bone morphogenetic proteins 4 and 7 (BMP4 and BMPT) from the dorsal epidermis and lateral plate mesoderm, and platelet-derived-growth-factor A (PDGFA) from the dorsal epidermis.

by mesoderm-inducing signals. Transcriptional control of these genes appears to be designed to ensure their reciprocal expression patterns and suggests the mesoderm may require signals from the adjacent ectoderm. That PDGF is required for mesoderm formation has been demonstrated by the murine Patch mutation, which is a naturally occurring deletion of the PDGF ot receptor (reviewed by Betsholtz, 1995).

As in the frog, the murine ct receptor is expressed in mesodermal tissues whereas PDGF A is expressed in the overlying ectoderm, and this mutation disrupts the development of most mesodermal tissues. For example, mutant embryos have small irregularly shaped somites that fail to differentiate into dermamyotome or sclerotome demonstrating a requirement for signaling between the epidermis and somites.

In amphibians, grafting the dorsal organizer into the ventral side of a gastrula can result in the formation of a complete secondary axis that includes neural tube. This was first demonstrated by Spemann and Mangold who also showed that the secondary neural tube was derived from the host embryo. The secondary neural tube was induced in the host ectoderm by signals released from the grafted organizer, and many unsuccessful attempts were made to identify the molecules responsible for these signals (reviewed by Kelly & Melton, 1995). However, recent studies suggest that neuralizing signals may be antagonists of BMP4, which appears to promote epidermal differentiation at the expense of neural differentiation. When ectoderm isolated from Xenopus gastrulae is dissociated, then all the cells adopt a neural fate, but addition of BMP4 to the medium directs these cells to form epidermis. During gastrulation, transcripts for bmp4 are lost from presumptive neural tissue but are retained in presumptive epidermis, probably as a consequence of yet unknown neuralizing signals released by the organizer. Inactivation of BMP4 appears to be sufficient for neural development in Xenopus because inhibition of the BMP4 signaling pathway by dominant-negative receptors (lacking the cytoplasmic kinase domain) directs cells to become neural tissue at the expense of epidermis. Similarly, injection of RNA encoding either noggin, chordin, or follistatin into Xenopus animal caps induces them to become neural tissue (reviewed by Hemmati-Brivanlou & Melton, 1997). As discussed above, these proteins bind to BMP4 thereby inhibiting its activity, and transcripts for all three proteins are found in the organizer during gastrulation. These results suggest a model for neural induction in which noggin, chordin, and follistatin secreted by the organizer bind to B MP4 in the ectoderm thereby preventing it from activating its receptors and promoting epidermal differentiation. In the absence of BMP4, ectodermal cells adopt the neural fate. It is still unclear whether inhibiting BMP4 is sufficient for neural induction in vivo, or whether other signals are also required. The neural tissue induced in animal caps by inhibiting BMP4 is invariably anterior in character, expressing genes such as otx2 which is normally expressed in forebrain and midbrain, but not genes such as hoxb9, which is normally expressed in the posterior spinal cord. These results are consistent with a two-signal model for neural induction in which the ectoderm is first induced to become anterior neural tissue by anterior mesoderm. This anterior neural tissue is then transformed to more pos-

terior neural fates by a second signal from the posterior mesoderm. Whereas the first signal is present throughout the dorsal mesoderm, the second signal is proposed to be in an anterior-posterior gradient with the highest concentration in the posterior mesoderm. Recent experiments suggest that this second signal may be a member of the FGF family of PGFs: (1) at least two members of this family, FGF3 (Intl) and FGF4 (eFGF), are expressed in posterior mesoderm during gastrulation in Xenopus, an expression pattern consistent with the proposed posteriorizing signal; (2) expression of the dominant-negative FGF receptor in Xenopus leads to severe defects in posterior neural tissue, a phenotype expected of a posteriorizing signal; (3) FGFs posteriorize anterior neural tissue induced in animal caps by either noggin, follistatin, or a dominant-negative receptor that inhibits BMP4 signaling (reviewed by Doniach, 1995). In these latter experiments, neuralized animal caps express the posterior neural marker, hoxb9, only in the presence of FGE PGFs also appear to play a role in establishing the dorsal-ventral pattern of the neural tube. Grafting experiments in the chick embryo suggest that the notochord is responsible for inducing the ventral floor plate of the neural tube and the motor neurons that differentiate adjacent to the floor plate (reviewed by Kelly & Melton, 1995). Whereas removal of the notochord results in loss of both the floor plate and motor neurons and an expansion of dorsal cell-types, implantation of an additional notochord adjacent to the intermediate neural tube results in the formation of an extra floor plate and motor neurons. Similarly, intermediate segments of the neural plate can be induced to differentiate as floor plate and motors neurons in vitro if they are co-cultured with notochord. Once induced, the floor plate itself can mimic the inductive properties of the notochord. As discussed above, shh is expressed in the notochord--and subsequently the floor plate, from where it helps to pattern the adjacent somites, but could this protein also be involved in patterning the neural tube? The answer appears to be yes because ectopic expression of Shh induces the expression of floor plate markers in vivo, and cells expressing Shh induce floor plate and motor neuron differentiation in neural plate explants in vitro. Consistent with this proposed function for Shh, is the observation that mice lacking the shh gene lack ventral cell-types within the neural tube (Chiang et al., 1996). The dorsal neural tube also differentiates specialized cell types, including the neural crest, which migrates away to form the peripheral nervous system, the roof plate, and commissural neurons. These cell types appear to be negatively regulated by the notochord because notochord grafts repress dorsal markers and notochord ablation results in the expansion of dorsal markers. The activity of the notochord in these assays is presumably mediated by Shh. However, there is also evidence for a dorsal signal, released by the adjacent nonneural ectoderm, that is responsible for inducing the development of dorsal neural cell types. Neural crest cell markers are expressed in intermediate segments of the neural tube that have been grafted beneath the nonneural ectoderm, and the latter tissue is sufficient to dorsalize explants of intermediate neural tube in vitro. The signal released by the dorsal ectoderm appears to be either BMP4 or BMP7; both genes are expressed in the dorsal ectoderm

of chick embryos and both proteins antagonize the ventralizing activity of Shh in vitro (reviewed by Hogan, 1996). Another member of the TGF~ family, dorsalin 1 (dsll), is expressed in the dorsal neural tube, and application of Dsll protein to explants of intermediate neural tube induces the formation of neural crest cells and inhibits the induction of ventral motor neurons by the notochord or floor plate. Expression ofdsll may be a consequence of BMP4 or BMP7 activity and may act as a relay signal for these PGFs in the neural tube. As we can see dorsal-ventral patterning in the neural tube appears to be a consequence of apposing dorsal and ventral signals mediated by BMP4/BMP7/dorsalin 1 and Shh respectively. Parallels with patterning of the somites are quite striking where the apposing activities of BMP4 and Shh may mediate medial-lateral patterning.

The vertebrate limb arises as a small bud from the flank of the embryo (Figure 7), and subsequent outgrowth is characterized by a relatively rapid rate of cell proliferation in the mesodermal layer. As the limb bud grows, it develops asymmetries along the anterior-posterior, dorsal-ventral, and proximal-distal axes (Tickle and Eichele, 1994). The proximal-distal axis is determined in the rapidly dividing mesenchyme at the distal tip, a region known as the progress zone. As the bud grows out, cells leave the progress zone and their proximal-distal fate becomes fixed; the longer they remain in the progress zone the more distal the structures they produce. Overlying the progress zone is a specialized layer of ectoderm known as the apical ectodermal ridge (AER), which runs anterior to posterior along the distal tip of the limb bud (Figure 7). The AER is induced by the underlying mesenchyme to become a morphologically distinct thickening of epithelium. Once induced, the AER is required for bud outgrowth and is responsible for stimulating cell proliferation in the progress zone. Removal of the AER results in a greatly reduced rate of mitosis in the mesenchyme and a truncated limb develops. The initial induction of a limb is probably a consequence of the activity of a member of the FGF family since agarose beads soaked in FGFs induce the formation of additional limbs when placed into the flank mesoderm, between the foreand hind-limb buds, of a chick embryo. FGF8 is the prime candidate for the endogenous signal because, apart from exhibiting the correct biological activity, this gene is expressed in the ectoderm where the fore- and hind limb buds will eventually emerge. FGFs can also substitute for the AER, restoring normal rates of mitosis to limb buds from which the AER has been removed. The FGF-rescued limbs produce a full complement of distal structures, although digits are misshapen suggesting that additional AER signals are required for correct morphogenesis. Thus, an FGF signal from the AER appears to be responsible for promoting outgrowth of the underlying mesenchyme and allowing proximal-distal patterning. Consistent with this is the observation that fg~, fgf4, and f g ~ are all expressed in the AER. B MPs

Figure 7. Development of the chick wing. (A) Diagram of the early limb bud indicating the apical ectodermal ridge (AER), zone of polarizing activity (ZPA), and the progress zone. The dorsal-ventral axis is not shown. (B) Differentiation of the wing bud is clearly visible in a six-day-old embryo. The mesenchymal condensations giving rise to the humerus (H), radius (R), ulna (U), and digits 3 and 4, which can be seen. (C) The wing pattern is fully established after 10 days of embryogenesis.

(bmp2, bmp4, and bmp7) are also expressed in the AER where they may function to control cell proliferation and/or signaling to the progress zone. For example, BMP2 antagonizes the growth stimulatory effects of FGF4 on AER-depleted limbs suggesting that cell proliferation in the progress zone, and as a consequence the normal

outgrowth of the vertebrate limb, are the result of a dynamic equilibrium between the growth stimulatory effect of FGFs and the inhibitory effects of BMPs (reviewed by Hogan, 1996). A second set of interactions in the limb bud involves the zone of polarizing activity (ZPA), a small group of mesenchyme cells at the posterior margin of the bud (Figure 7). The ZPA interacts with cells in the progress zone to control the pattern of structures that develop across the anterior-posterior axis of the limb. This signaling activity can be demonstrated by transplanting the ZPA into the anterior mesenchyme of the developing limb bud, which results in mirror-image duplications along the anterior-posterior axis; in the extreme case, the normal digit pattern of 23-4 is replaced by 4-3-2-2-3-4. ZPA signaling can be understood in terms of a model in which posterior cells are the source of a diffusible signal that becomes distributed in a concentration gradient across the limb bud. Cells at different distances from the ZPA would be exposed to different concentrations of the signal, and this would determine their subsequent development. A candidate for this signal is Shh whose expression in the limb is coincident with the ZPA (Johnson et al., 1994). Cells transfected with shh, when implanted into the chick anterior limb bud, produce the same mirror-image duplications as seen with ZPA grafts, suggesting that Shh may be the signal derived from the ZPA. A possible target for Shh may be bmp2 because transcripts of this PGF are localized to posterior mesenchyme of the limb bud in a pattern that partially overlaps that of shh. ZPA grafts and ectopic expression of Shh induces the expression of bmp2 in anterior mesenchyme, suggesting that BMP2 acts downstream in the polarizing pathway (reviewed by Hogan, 1996). One prediction of this model is that elimination of shh by targeted mutation in the mouse would result in anterior-posterior patterning defects in the limbmyet these are not observed (Chiang et al., 1996). Rather, mice homozygous for a shh loss of function mutation lack distal limb structures, a result consistent with the loss of distal structures following removal of posterior portions of the chick limb bud. One possible explanation for this result is that Shh is required to maintain the expression of FGF4 in the posterior AER and, as discussed above, FGF4 may be required to maintain the distal outgrowth of the developing limb. Similarly, if BMP2 were acting downstream of Shh, it should induce mirror-image digit duplications when expressed in anterior mesenchyme, yet this is not observed (reviewed by Hogan, 1996). Unfortunately, mice homozygous for a loss-of-function bmp2 mutation die before any role in limb development can be ascertained. Signals from the limb bud ectoderm also appear to control dorsal-ventral patterning, and there is now evidence that another PGF, a member of the Wnt family of genes, acts as a signal for dorsal-ventral patterning. Wnt7a is expressed specifically in dorsal ectoderm of vertebrate limb buds, and paws of mice in which wnt7a has been functionally inactivated have a double ventral pattern (Parr and McMahon, 1995). This means that special features of the skin that are normally found on the sole are now found on both sole and upper paw surfaces. Several related Wnt family

members are expressed throughout the limb ectoderm as well as in nonlimb ectoderm and may be involved in less regionalized ectodermal signaling, such as maintaining the undifferentiated state of the sub-apical mesenchyme.

The basic form and pattern of the axial skeleton and the bones of the limbs are first laid down when mesenchyme cells aggregate into regions of high cell density called condensations. Cells within a condensation will first differentiate into cartilage before being replaced by bone, a process known as endochondrial ossification. In contrast, the cranial bones form directly without a cartilaginous phase, a process known as intramembraneous ossification. Bone morphogenetic proteins were first identified in demineralized bone as molecules that could direct endochondrial ossification in vivo (reviewed by Rosen & Theis, 1992), although it is now clear that they have many functions during embryonic development. Definitive evidence that BMPs play a role in bone formation was provided by David Kingsley and his colleagues, who found that the gene encoding BMP5 is defective in mice carrying mutations at the short-ear locus (reviewed by Kingsley, 1994). As the name suggests, short-ear mice have very short external ears, but they also have a wider skull and shorter nose than normal animals and many internal skeletal elements are either missing or reduced in size. These morphological changes have been traced to the initial mesenchymal condensations, which in short-ear mice, are either missing or have altered shapes and sizes. Because bmp5 is expressed in these mesenchymal condensations during normal development it is probably acting as an autocrine or paracrine signaling factor during bone development. Since not all skeletal elements are affected by the short-ear mutation, other genes must be controlling their formation, and although the other BMPs are obvious candidates, there is presently no genetic evidence to support this suggestion. However, molecular analysis of the mouse brachypodism mutations has revealed that they disrupt the gene encoding a member of the TGFI3 family called growth and differentiation factor 5 (GDF5). The limb bones of mice homozygous for brachypodism are severely shortened, while the skull and axial skeleton are largely unaffected. Defects are first detectable around day 12 of gestation, when gdf5 is expressed in the aggregating mesenchyme of the limb bones. These observations for short-ear and brachypodism suggest that specific subsets of skeletal elements require different members of the TGF~ family for normal development (reviewed by Hogan, 1996). The analysis of mutations in both mice and humans has also revealed a role for FGFs in bone morphogenesis (reviewed by Muenke & Schell, 1995). In mammals, FGFs bind with high affinity to a family of four FGF receptors (FGFRs), and in humans several skeletal disorders result from point mutations in the FGFR1, FGFR2, and FGFR3 genes (Table 3). No skeletal disorder has yet been associated with

FGFR4. Mutations in FGFR1 and FGFR2 cause the craniosynostosis syndromes, Apert, Pfeiffer, Jackson-Weiss, and Crouzon, in which intramembraneous ossification of the fiat bones of the skull is affected. Achondroplasia, hypochondroplasia, and thanatophoric dysplasia result from abnormal endochondrial ossification of the long bones and the base of the skull and are caused by mutations in FGFR3. In the mouse,fgfrl,fgfr2, andfgfr3 are co-expressed in prebone cartilage rudiments of the embryo but have distinct patterns of expression during endochondral ossification later in bone development. Targeted disruption offgfrl (see above) andfg[r2 results in early embryonic lethality, whereas mice homozygous for targeted fgfr3 mutations survive embryogenesis and are born with skeletal defects. These skeletal defects include abnormal curvature of the spine and tail and overgrowth of the long bones, fgfr3 is also expressed in tissues other than bone such as the developing cochlea of the ear, and mice lacking fgfr3 also exhibit inner ear defects.

Recent work on vertebrate embryos has demonstrated the important roles that polypeptide growth factors play as cell-cell signaling molecules during embryogenesis. It is now clear that they are the long sought-after inducing factors. This knowledge has greatly aided our understanding of early embryogenesis, but there is much more to be learned; for example, we still know very little about how combinations of these PGFs are used to specify different cell-types. However, progress has been so rapid over the last few years that it is only a matter of time before many of the outstanding questions are answered. Although not discussed in this article, it is quite clear that similar types of molecules are also used by invertebrate embryos. Whilst the embryological circumstances may differ, the ligand, receptor, and signal transduction systems appear to be significantly conserved. This has enabled invertebrate embryos to provide important leads for vertebrate embryologists to follow and will no doubt continue to do so.

The authors are grateful to Dr. Paul Martin and Fiona Wardle for their constructive criticisms of the manuscript. Wherever possible we have cited reviews; these will provide access to the primary references.

Alberts, B., Bray, D., Lewis, J., Raft, M., Roberts, K. and Watson, J.D. (1994). Molecular Biology ofthe Cell (3rd Edition). Garland Publishing Inc., New York. Betsholtz, C. (1995). Role of platelet-derived growth factors in mouse development. Int. J. Dev. Biol. 39, 817-825. Chiang, C., Litingtung, Y., Lee, E., Young, K.E., Corden, J.L., Westphal, H. & Beachy, P.A. (1996). Cyclopia and defective axial patterning in mice lacking sonic hedgehog gene function. Nature 383,407-413. Cui, Y., Brown, J.D., Moon, R.T. and Christian, J.L. (1995). XWnt-8b: a maternally expressed Xenopus Wnt gene with a potential role in establishing the dorsoventral axis. Development 121, 2177-2186. Deng, C.X., Wynshaw-Boris, A., Shen, M.M., Daugherty, C., Ornitz, D.M. & Leder, P. (1994). Murine FGFR-1 is required for early postimplantation growth and axial organization. Genes Devl. 8, 3045-3057. Doniach, T. (1995). Basic FGF as an inducer of anteroposterior neural pattern. Cell 83, 1067-1070. Harland, R.M. (1994). The transforming growth factor 13 family and induction of the vertebrate mesoderm: Bone morphogenetic proteins are ventral inducers. Proc. Natl. Acad. Sci. USA 91, 10243-10246. Hemmati-Brivanlou, A. & Melton, D.A. (1997). Vertebrate embryonic cells will become nerve cells unless told otherwise. Cell 88, 13-17. Hogan, B.L. (1996). Bone Morphogenetic Proteins: Multifunctional regulators of vertebrate development. Genes Devl. 10, 1580-1594. Hoppler, S., Brown, J.D. and Moon, R.T. (1996). Expression of a dominant-negative Wnt blocks induction of MyoD in Xenopus embryos. Genes Devl. 10, 2805-2817. Johnson, R.L., Riddle, R.D., & Tabin, C.J. (1994). Mechanisms of limb patterning. Curr. Op. Genet. Dev. 4, 535-542. Kelly, O.G. & Melton, D.A. (1995). Induction and patterning of the vertebrate nervous system. Trends Genet. 11,273-278. Kessler, D. & Melton, D.A. (1995). Vertebrate embryonic induction: Mesodermal and neural patterning. Science 266, 596--604. Kingsley, D.M. (1994). What do BMPs do in mammals? Clues from the mouse short-ear mutation. Trends Genet. 10, 16-21. Marshall, C.J. (1995). Specificity of receptor tyrosine kinase signaling: Transient versus sustained extracellular signal-regulated kinase activation. Cell 80, 179-185. Moon, R.T., Christian, J.L., Campbell, R.M., McGrew, L.L., DeMarais, A.A., Torres, M., Lai, C.-J., Olson, D.J., & Kelly, G.M. (1993). Dissecting Wnt signalling pathways and Wnt-sensitive developmental processes through transient misexpression analyses in embryos of Xenopus laevis. Development Supplement, 85-94. Muenke, M. & Schell, U. (1995). Fibroblast-growth-factor-receptor mutations in human skeletal disorders. Trends Genet. 11,308-313. Orsulic, S. & Peifer, M. (1996). Cell-cell signaling: Wingless lands at last. Curr. Biol. 6,1363-1367.

Parr, B.A. & McMahon, A.P. (1995). Dorsalizing signal Wnt-7a required for normal polarity of D-V and A-P axes of mouse limb. Nature 374, 350-353. Piccolo, S., Sasai, Y., Lu, B. & De Robertis, E.M. (1996). Dorsoventral patterning in Xenopus: Inhibition of ventral signals by direct binding of Chordin to BMP4. Cell 86, 589-598. Rosen, V., & Theis, R.S., (1992). The BMP proteins in bone formation and repair. Trends Genet. 8, 97-102. Slack, J.M.W. (1991 ). From Egg to Embryo: Regional Specification in Early Development (2nd FAn.). Cambridge University Press, Cambridge. Slack, J.M.W. (1994). Inducing factors in Xenopus early embryos. Curr. Biol. 4, 116-126. Sokol, S.Y. (1996). Analysis of Dishevelled signaling pathways during Xenopus development. Curr. Biol. 6, 1456-1467. Takada, S., Stark, K.L., Shea, M.J., Vassileva, G., McMahon, J.A. & McMahon, A.P. (1994). Wnt-3a regulates somite and tailbud formation in the mouse embryo. Genes Dev. 8, 174-189. Ten Dijke, P., Miyazono, K. & Heldin, C-H. (1996). Signaling via hetero-oligomeric complexes of type I and type II serine/threonine kinase receptors. Curr. Op. Cell Biol. 8, 139-145, Tickle, C. & Eichele, G. (1994). Vertebrate limb development. Ann. Rev. Cell Biol. 10, 121-152. Wrana, J.L. & Attisano, L. (1996). MAD-related proteins in TGFI3 signaling. Trends Genet. 12, 493--496. Yamaguchi, T.P., Harpal, K., Henkemeyer, M. & Rossant, J. (1994).fgfr-1 is required for embryonic growth and mesodermal patterning during mouse gastrulation. Genes Devl. 8, 3032-3044. Zimmerman, L.B., De Jestis-Escobar, J.M. & Harland, R.M. (1996). The Spemann organizer signal noggin binds and inactivates bone morphogenetic protein 4. Cell 86, 599-606.

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Introduction Nerve Growth Factor (NGF) is a Chemoattractant for Regenerating Axons Chemotropic Guidance by Factors Other than NGF Guidance of Spinal Commissural Axons Guidance of Cortical Projection Axons Guidance of Developing Trigeminal Sensory Axons Guidance of Spinal Motor Axons Chernoattractants Might Act in Combination with Other Cues Concluding Remarks Summary

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The functioning of the nervous system is dependent to a large extent on the intricate network of neural connections that is formed during embryonic development. This network becomes established gradually in the embryo as each differentiating neu-

Principles of Medical Biology, Volume 11 Developmental Biology, pages 153-165. Copyright 9 1998 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-816-1

ron extends a thin axon to appropriate target cells with which it forms synaptic connections. The tip of the axon, the growth cone, is a dynamic structure resembling a motile cell, which leads the extending axon along its appropriate trajectory. Axonal trajecto' ries are highly reproducible from embryo to embryo; growth cones rarely make errors of projection, despite projecting over large distances of as much as several centimeters. How do growth cones navigate with such precision? Current evidence suggests that they detect and interpret directional information encoded in specific guidance molecules in their environment to which they respond with directed growth (reviewed in Dodd and Jessell, 1988). The nature of these guidance molecules appears to be quite varied. Firstly, considerable evidence has accumulated for the existence of what may be termed short-range guidance cues. These are molecules that derive from cells along the path that the axons traverse and that are immobilized in the extracellular matrix or on the surface of the pathway cells. Many good candidates for short-range guidance cues have been identified, such as the extracellular matrix molecule laminin, and the cell-surface molecule L 1. The roles of these molecules in guiding axons are reviewed in Dodd and Jessell, 1988; Reichardt et al., 1990, and Hynes and Lander, 1992. In addition, there is evidence that long-range guidance cues may also guide axons. These take the form of soluble factors secreted by the targets of axons. As they diffuse through the cellular environment of the embryo, they set up diffusion gradients that orient axons located some distance from the targets. The idea that axons may be guided by chemotropic molecules is not new: It was proposed a century ago by Ramon y Cajal (Cajal, 1892), soon after his discovery of the growth cone in 1890. His observation came at a time when other studies were establishing that leukocytes can orient in gradients of soluble chemoattractants. Inspired by these findings, he likened growth cones to leukocytes, proposing that they have similar sensory and motile properties and that growth cones may be guided by gradients of target-derived attractants. Despite the appeal of this theory, it lay dormant for the better part of the century for want of supporting evidence. As we shall discuss here, however, evidence has started to accumulate over the past ten years suggesting that chemoattractants play a prominent role in guiding developing axons. A discussion of recent reserch can be found in Tessier-Lavigne and Goodman (1996).

The first clear demonstration that axons can detect gradients of chemoattractants was obtained by Gundersen and Barrett, in their studies of the effects of Nerve Growth Factor (NGF) on regenerating sensory axons in cell culture (Gundersen and Barrett, 1979). NGF is a soluble protein that is secreted by some of the target cells of sensory and sympathetic neurons and is required for the survival of these neurons during development (Barde, 1989). When the sensory axons in cell culture were ex-

posed to a steep gradient of NGF emanating from a capillary tip, all axons turned towards the source within minutes (Gundersen and Barrett, 1979; Gundersen and Barrett, 1980) (Figure 1). These experiments provided the first unambiguous evidence that axons can detect gradients of diffusible factors and turn in response to such a gradient. They also supported the conclusions of earlier studies by LeviMontalcini and colleagues, who found that NGF can act as a chemoattractant in vivo. In those experiments, NGF was injected into the brain stem of neonatal rats. This treatment caused sympathetic axons at all segmental levels (including some over a centimeter away) to take an aberrant trajectory into the spinal cord through the dorsal roots, and then, via the dorsal funiculus, to the injection site (MenesiniChen et al., 1978). NGF is still the only identified molecule that has been shown to act as an axonal chemoattractant; other tissue-derived factors (discussed below) have been shown to act as chemoattractants, but their molecular identity has not yet been established. However, although the observation that NGF can orient regenerating axons is important, there are two reasons for believing that NGF does not actually play a role in guiding developing axons to their targets cells during the initial establishment of neuronal connectivity. First, NGF is only secreted by the targets of developing axons after the first axons have reached them; target-derived NGF could not, there-

fore, be used as a guidance cue by the first developing axons (Davies, 1987). More importantly, developing axons appear to be unresponsive to NGF while they are growing to their targets and only acquire responsiveness after they have reached the target (Davies, 1987). Given these facts, it seems likely that in all experiments that have demonstrated a chemoattractant effect of NGF; responsive axons were regenerating, not developing.

Starting with the studies of Lumsden and Davies about 10 years ago (Lumsden and Davies, 1983), evidence that diffusible molecules can attract several different classes of developing axons has been obtained in tissue culture experiments in which axons and their appropriate target tissues, dissected from the embryo during the relevant stages of development, were cocultured some distance apart in semisolid matrices such as collagen gels or plasma clots (see below). Such matrices provide a favorable experimental preparation to reveal the presence of target-derived chemoattractants because they appear to stabilize gradients that extend over hundreds of microns from the target cells and are sufficiently steep to evoke a directional response (Ebendal and Jacobson, 1977; Lumsden and Davies, 1983). Using this approach, target-derived chemoattractants have been implicated in directing the growth of: (i) spinal commissural axons to midline spinal floor plate cells, (ii) cortical projection neurons to subcortical targets, and (iii) trigeminal sensory axons to their epithelial targets (Figures. 2-5). In addition, a combination of in vivo and in vitro approaches has provided evidence that the axons of spinal motor neurons are also attracted to some of their targets by chemoattractants (see below). These experiments are reviewed in turn.

Studies on the projection of spinal commissural neurons towards an intermediate target, the floor plate at the ventral midline of the embryonic spinal cord (Figure 2), have demonstrated that developing axons within the central nervous system can be guided by chemoattractants. Floor plate explants were shown to influence the direction of growth of commissural axons growing in embryonic day (E) 11 rat dorsal spinal cord explants (Tessier-Lavigne et al., 1988; Placzek et al., 1990a). Within dorsal spinal cord explants cultured alone, commissural axons were found to extend along a characteristic dorso-ventral trajectory (Figure 2). However, when a floor plate explant was positioned to one side of the dorso-ventrally extending axons and ~ 100-200 lam away, the axons were deflected from their trajectory and grew out of the explant, projecting directly towards the floor plate (Figure 2). Other tissues did not deflect the commissural axons. The floor

Figure 2. Chemotropic guidance of spinal commissural axons. (A) In the rat, commissural neurons differentiate between E11 and E13 in the dorsal spinal cord near the roof plate. After extending ventrally close to the edge of the spinal cord, commissural axons change their direction at the developing motor column and project to floor plate cells at the ventral midline. The axons cross the midline at the floor plate, then project to the brain in the ventro-lateral funiculus (not shown). (B) Within dorsal spinal cord explants cultured either alone (not shown) or with control tissue apposed to one side, commissural axons grew dorso-ventrally. In ccmtrast, when a floor plate explant (FP) was placed to one side, all commissural axons within ---250pro from the floor plate reoriented their growth towards it. Similarly, when a floor plate explant was cultured to one side of a dorsal explant, but some distance away, commissural axons projected out of the dorsal explant towards the floor plate (Tessier-Lavigne et al., 1988; Placzek et al., 1990a). (rp): roof plate. (From Tessier-Lavigne and Placzek, 1991).

plate, but not other tissues, can therefore attract commissural axons at a distance, presumably by releasing a diffusible chemoattractant. Experiments in which the floor plate was apposed to one side of a dorsal explant showed that the chemoattractant can diffuse through dorsal neuroepithelium over distances of--250 pm and can reorient the growth of all commissural axons within that range (Placzek et al., 1990a) (Figures. 2 and 3). This range of action would be sufficient to account for the guidance of commissural axons in the developing embryo because they turn towards the ventral midline when they reach the developing motor column, about - 150 lam from the floor plate. The distribution of the floor plate-derived chemoattractant and its specificity of action are consistent with a role in axon guidance (Tessier-Lavigne et al., 1988; Placzek et al., 1990a). During the early period of commissural axon growth, expression of the factor within the spinal cord is restricted to the floor plate. Axons that are within the range of action of the chemoattractant but which do not project to the floor plate (motor, spinal association, and sensory axons) are not attracted to the floor plate in vitro. Finally, the floor plate also appears to be effective in guiding axons in vivo because commissural axons can be deflected from their normal trajectory towards an ectopic floor plate in developing chick embryos (Placzek et al.,

Attraction of spinal commissural axons to the floor plate in vitro. (A) A small E11 floor plate explant (f) was cultured directly apposed to the lateral edge of a strip of E11 dorsal spinal cord (r, roof plate). (B) Phase-contrast micrograph taken after 40 hours, showing that axons have not emerged from the dorsal explant. The dotted line indicates the point of contact between the floor plate (FP) and the dorsal explant. (R) = roof plate. (C) The anterograde diffusion of Dil injected into the roof plate region reveals that axons within about 3001am of the floor plate (indicated by arrowhead) deviate from their initial dorso--ventral trajectory and turn completely towards the floor plate within the dorsal neural epithelium. Axons located at distances greater than 3001am continue to grow along their normal dorso--ventral trajectory. (Scale bar is lO0~tm. From Placzek et al., 1990a).

Figure 3.

1990b; Yaginuma and Oppenheim, 1991). Taken together, these observations suggest that the chemoattractant guides commissural axons to the ventral midline of the spinal cord during development.

Within the central nervous system, chemoattractants have also been implicated in establishing connections between cortical layer-5 neurons and their targets in the brainstem (Figure 4). The axons of layer 5 pyramidal neurons initially grow beyond their brainstem targets (e.g., the superior colliculus and the basilar pons), then sprout collateral branches to innervate them (O'Leary and Terashima, 1988). Evidence that chemoattractants may direct collateral branching was obtained by examining the effect of explants of basilar pons tissue on the growth of cortical axons

Figure 4. Chemotropic guidance of cortical projection axons. (A) Pyramidal neurons in layer 5 ofthe cortex (A) begin to differentiate around E17-20 in the rat. Layer-5 pyramidal neurons in the motor cortex extend axons that reach the spinal cord by birth. Between birth and P3 the axons develop two collateral branches that project to the posterior and anterior basilar pons. Layer-5 pyramidal neurons in the visual cortex (A) likewise project axons that grow past the pons, reaching the spinal cord at P2, and then develop a single collateral branch that projects to the anterior pons (O'Leary and Terashima, 1988). The corticothalamic projection develops through the direct growth of axons of layer-6 pyramidal neurons (o) to the thalamus. (B) In motor or visual cortical explants cultured alone in a collagen gel, axons project from neurons located in all layers of the cortex and emerge out of the explant from the ventral ventricular edge (only axons extending from layer-5 neurons are illustrated). The pattern of growth of layer-5 axons is changed when a pons explant (P) is cultured to one side (axons from other layers appear unaffected). Many layer-5 axons emerging from the ventral edge subsequently reorient their growth within the collagen matrix to project towards the target. In addition, many sprout a collateral branch that projects directly to the pons explant. Control tissues (C) do not produce these effects (Heffner et al., 1990). (From Tessier-Lavigne and Placzek, 1991 ).

(Heffner et al., 1990). In cortical explants cultured alone, axons extended ventrally and projected out of the explant at the ventricular surface. When cortical explants were flanked by pieces of pons tissue and control tissue, however, axons reoriented their growth in one of two ways (Figure 4). Many axons turned towards the pons tissue, but did not turn towards control tissuesl In a second pattern of growth that closely mimics development in vivo, axons grew ventrally but sprouted a collateral that projected straight to the pons tissue. Collateral branching was not observed in the absence of pons tissue. Axon reorientation and directed collateral sprouting were observed not only within the cortical explants but also within the inert matrix provided by the collagen gel, showing that the chemoattractant does not need any accessory factors provided by cortical tissue to direct growth and sprouting (Heftner et al., 1990; O'Leary et al., 1990). Retrograde-labeling studies indicated that only layer-5 pyramidal axons grew into the pons tissue, suggesting that the ponsderived chemoattractant selectively affects these axons. Control tissues (brain re-

gions close to the corticospinal tract that do not receive neocortical input) failed to mimic the effects of the pons (O'Leary et al., 1990). Evidence has also been obtained that the axons of layer-6 pyramidal neurons that project to the thalamus (Figure 4) are guided to this target by a distinct chemoattractant (Bolz et al., 1990). In long-term cultures of cortical explants with pieces of thalamus placed opposite the pial (dorsal) surface, the thalamus was found to be innervated exclusively by the axons of layer-6 neurons, which had grown along inappropriate dorsal trajectories through the cortical explant to reach it; control experiments suggested that the axons of these neurons did not simply grow at random. These observations raise the possibility that chemotropism contributes to the targeting of both layer-5 and layer-6 axons to subcortical targets.

Within the peripheral nervous system, a chemoattractant may operate to guide trigeminal sensory axons to their epithelial targets in the maxillary and submandibular arches (Lumsden and Davies, 1983; Lumsden and Davies, 1986) (Figure 5)

Trigeminal ganglia from El0 mouse embryos were cultured either alone or with an E 10 maxillary arch positioned 300-5001am away (the approximate distance that the axons must traverse in vivo). When ganglia were cultured alone, no axons grew out of them, but when ganglia were cultured with maxillary arches, axonal outgrowth was observed in over 60% of cases, with virtually all axons emerging from the side of the ganglion facing the target tissue. Two observations indicated that this directed growth reflected the detection of a gradient of a chemoattractant, rather than a nondirectional response to an outgrowth-stimulating factor that did not diffuse beyond the proximal face of the ganglion. First, some of the axons growing along trajectories that would not have intercepted the target turned toward it (Lumsden, 1988). Second, when two ganglia were cultured in tandem next to a maxillary arch, more axons grew towards the target from the more distant ganglion than grew away from the closer ganglion (Lumsden and Davies, 1983) (Figure 5). This showed that the factor can diffuse past the closer ganglion and suggested that axons were responding to a gradient (see Figure 5 legend). The maxillary arch-derived chemoattractant, termed Max Factor (MF), is expressed from E9.5 until at least El2 (Lumsden, 1988), the period over which most axons emerge from the trigeminal ganglion. MF has a restricted distribution that is appropriate for a guidance cue (Lumsden and Davies, 1986); activity is detected in the maxillary and mandibular processes (both targets of trigeminal axons) but not in the hyoid process (a target of neighboring geniculate ganglion neurons). Within the maxillary process, MF is secreted by the target epithelium but not the intervening mesenchyme. Furthermore, the response appears to be specific to trigeminal axons inasmuch as MF does not affect the growth of geniculate axons in culture. The timing and pattern of expression of MF and the specificity of its effect are therefore consistent with a role in the long-range guidance of developing trigeminal axons.

In vertebrates, motoneurons innervating limb muscles are restricted to specific segmental levels, while 'epaxial' motoneurons, which grow to the myotome to innervate the axial musculature, are present at all levels. There is now evidence that these two classes of neurons respond to distinct chemotropic cues. Ablation studies in developing chicks provided evidence that the axons of epaxial motoneurons are attracted at a distance by the developing dermomyotome (the structure within the somites that gives rise to axial musculature; Tosney, 1987); consistent with this are the in vitro studies in Xenopus that have shown myoblasts secrete a diffusible factor that can attract the axons of some spinal neurons (perhaps motoneurons; McCaig, 1986). In contrast, muscle tissue is not required for the growth of motor axons to their targets in the limbs, but there is evidence that limb nerves may be attracted to the limb by nonmuscle tissue in the limb itself. The strongest evidence yet for attraction by the limb has come from studies in the medaka fish (Okamoto and Kuwada, 199 la; Okamoto and Kuwada, 1991b). The motoneurons innervating the pectoral fin in this fish

converge from five segmental levels to the base of the fin. If the fin bud is removed by surgical ablation or genetic mutation, the nerves fail to converge; if a fin is grafted to ectopic locations, motoneurons that would normally innervate targets other than the fin are deflected toward the graft. These results extend previous studies in amphibians and birds showing that limb buds can attract motor axons in vivo (Detwiler, 1934; Hamburger, 1934) and the axons of spinal cord neurons (presumably motoneurons) in vitro (Pollack and Liebig, 1977; Pollack et al., 1981).

The studies described above have provided evidence that a number of axonal targets secrete diffusible chemoattractants. In each case, evidence has been obtained that a diffusible target-derived factor does not simply cause the axons to extend but suggests instead that a gradient of the factor actually alters the pattern of axon growth. Indeed, the floor plate-derived chemoattractant has been shown to orient commissural axons without affecting their rate of extension (Placzek et al., 1990a). Consistent with a role in guiding axons in vivo is the observation that different attractants are secreted at appropriate times in development and can selectively reorient the growth of appropriate axons over hundreds of microns in vitro. These experiments do not reveal the exact contribution of diffusible attractants to guidance in the developing embryo. If the chemoattractants do guide axons in vivo, their most important role might be to orient the earliest projecting axons in each system. For their guidance, later-developing axons may rely both on chemoattractants and on cues provided by earlier-developing axons (which may serve as a scaffold for later growth (Davies and Lumsden, 1986)), or on yet other types of cues. For example, laminin is expressed in the extracellular matrix along the pathway taken by trigeminal sensory axons to the mandibular arch in the chick embryo (Riggott and Moody, 1987). Chemoattractants alone may also be insufficient to ensure correct guidance in some cases. For example, in the case of motor corticoponfine projections, axons that travel close to the pons over a distance of--lmm sprout collaterals in only two restricted regions opposite the anterior and posterior pons (O'Leary and Terashima, 1988) (Figure 4), and it is unclear whether the expression profile of the pons-derived chemoattractant can account for this pattern.

The studies described above suggest that chemotropism plays a prominent role in axon guidance during development, but they also emphasize the difficulty of proving that chemotropism contributes to the guidance of axons. Axonal targets secrete factors such as NGF that may act as chemoattractants in vitro without playing a role

in long-range guidance during development if, for example, they are not present at the appropriate time, do not diffuse sufficiently far, or do not affect developing axons. Furthermore, examples of directed axon growth in vivo that are suggestive of chemotropism do not automatically indicate the action of a chemoattractant. The homing of misrouted or experimentally displaced axons to their targets in vivo may be due to chemoattraction, but could equally indicate, for example, the existence of a more global system ofpositional information to which axons can respond. For example, it has been shown that the homing of displaced Xenopus retinal ganglion axons to the tectum (Harris, 1986) does not involve a tectum-derived chemoattractant (Harris, 1989). Finally, with the realization that multiple cues may act in concert to guide axons along a given path comes the likelihood that in some cases chemotropism may "assist, rather than determine, the course of nerve fibers toward their correct destination", as suggested by Ramon y Cajal (1892) and reemphasized by Levi-Montalcini and colleagues (Menesini-Chen et al., 1978). Understanding the precise contribution of different chemoattractants to axon guidance will require selectively altering their expression in vivo and examining how this affects axonal trajectories. This will become possible only as the chemoattractants are characterized biochemically.

Within the developing vertebrate embryo, it appears that axons can be guided to their target cells under the influence of both short-range and long-range guidance cues. Long-range guidance cues take the form of diffusible chemoattractants that emanate from the target cells, establishing a gradient in the surrounding tissue. The growth cones of responsive neurons can sense the gradient some distance from its source, and become oriented, growing ultimately to the distantly located target cells. To date, it has not been established that any known factor can act as a chemoattractant for developing axons, although NGF has been shown to orient the growth of regenerating axons. The strongest evidence in support of long-range chemoattractants for developing axons comes from tissue culture experiments. Specific classes of neurons are cultured in vitro either alone or at a distance from their normal target tissue; turning of the axons towards target tissue (and not towards nontarget tissue) indicates the action of a diffusible chemoattractant. Such experiments have provided evidence that gradients of chemoattractants act to guide the axons of developing spinal commissural neurons and corticopontine neurons in the central nervous system, and developing trigeminal ganglion neurons and spinal motoneurons in the peripheral nervous system. Although particular target tissues that produce chemoattractants have been identified in this manner, the chemoattractants themselves have not yet been isolated from the tissues or characterized biochemically. The isolation and characterization

o f the p u t a t i v e c h e m o a t t r a c t a n t s will m a k e it p o s s i b l e to d e t e r m i n e their c o n t r i b u tion to a x o n a l g u i d a n c e and their m e c h a n i s m o f action, b o t h o f w h i c h c u r r e n t l y rem a i n unclear.

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O'Leary, D. D. M., Bicknese, A. R., De Carlos, J. A., Heffner, C. D., Koester, S. E., Kutka, L. J. and Terashima, T. (1990). Target selection by cortical axons: Alternative mechanisms to establish axonal connections in the developing brain. Cold Spring Harbor Symp. Quant. Biol. 55, 453-468. Okamoto, H. and Kuwada, H. Y. (1991a). Outgrowth by fin motor axons in wildtype and a finless mutant of the Japaense medaka fish. Dev. Biol. 146, 49-61. Okamoto, H. and Kuwada, J. Y. (199 lb). Alteration of pectoral fin nerves following ablation of fin buds and by ectopic fin buds in the Japanese medaka fish. Dev. Biol. 146, 62-71. Placzek, M., Tessier-Lavigne, M., Jessell, T. and Dodd, J. (1990a). Orientation of commissural axons in vitro to a floor plate-derived chemoattractant. Development 110, 19-30. Placzek, M., Tessier-Lavigne, M., Yamada, T., Dodd, J. and Jessell, T. M. (1990b). Guidance of developing axons by diffusible chemoattractants. Cold Spring Harbor Symp. Quant. Biol. 55, 279-290. Pollack, E. D. and Liebig, V. (1977). Differentiating limb tissue affects neurite growth in spinal cord culture. Science 197, 899-900. Pollack, E. D., Muhlach, W. L. and Liebig, V. (1981). Neurotropic influence of mesenchymal limb target tissue on spinal cord neurite growth in vitro. J. Comp. Neurol. 200, 393-405. Purves, D. and Lichtman, J. W. (1985). Principles of Neural Development. Sinauer Associates, Inc, Sunderland, MA. Reichardt, L. F., Bossy, B., Carbonetto, S., De Curtis, I., Emmett, C., Hall, D. E., Ignatius, M. J., Lefcort, F., Napolitano, E., Large, T., Neurgebauer, K. M. and Tomaselli, K. J. (1990). Neuronal receptors that regulate axon growth. Cold Spring Harb. Symp. Quant. Biol. 55, 341-350. Riggott, M. J. and Moody, S. A. (1987). Distribution of laminin and fibronectin along peripheral trigeminal axon pathways in the developing chick. J. Comp. Neurol. 258, 580-596. Tessier-Lavigne, M. and Goodman, C.S. (1996). The molecular biology of axon guidance. Science 274, 1123-1133. Tessier-Lavigne, M. and Placzek, M. (1991). Target attraction: are developing axons guided by chemotropism? TINS 15, 303-310. Tessier-Lavigne, M., Placzek, M., Lumsden, A. G. S., Dodd, J. and Jessell, T. M. (1988). Chemotropic guidance ofdeveloping axons in the mammalian central nervous system. Nature 336, 775-778. Tosney, K. W. (1987). Proximal tissues and patterned neurite outgrowth at the lumbosacral level of the chick embryo: Deletion of the dermamyotome. Dev. Biol. 122, 540-588. Yaginuma, H. and Oppenheim, R. W. (1991). An experimental analysis of in vivo guidance cues used by axons of spinal interneurons in the chick embryo: Evidence for chemotropism and related guidance mechanisms. J. Neurosci. 11, 2598-2613.

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What Are Growth Cones? Growth Cone Behavior The Growth Cone Cytoskeleton Microfilaments Microtubules Organization of Microtubules in Neurites and Growth Cones The Location of Microtubule Formation Elongation of Preformed Microtubules in the Neurite and Growth Cone? Control of Microtubule Assembly in Growth Cones A Linkage between Microtubules and Microfilaments? Summary

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The neuronal growth cone is the highly motile structure located at the tip of growing axons and dendrites (neurites) in developing and regenerating nervous systems (Ram6n y Cajal, 1890). Growth cones fulfill many important functions during

Principles of Medical Biology, Volume 11 Developmental Biology, pages 167-186. Copyright 9 1998 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-816-1

development that are crucial to the formation of a properly connected nervous system. These include navigating an appropriate route to the target cell (pathfinding), target recognition, and synapse formation (reviewed in Dodd & Jessell, 1988). Growth cones are guided to their targets by extrinsic guidance cues, such as extracellular matrix proteins, cell adhesion molecules, and chemotropic factors (see related chapter), to which they possess specific cell surface receptors. In this sense, the growth cone is a sensory structure and has been likened to a leucocyte on a leash (Pfenninger, 1986). Although we have only a meager understanding of molecular events within growth cones, it is likely that, in analogy with other cell types, these receptors are coupled to intracellular second messenger systems that have the ability to modify the cytoskeleton and thus to influence growth cone behavior (reviewed in: Kater & Guthrie, 1990; Letourneau & Cypher, 1991; Strittmatter & Fishman, 1991). For this reason, if we are to understand how the nervous system is properly connected, we need to enlarge our knowledge of the organization of the growth cone cytoskeleton and how its behavior is influenced by extrinsic guidance cues. Specifically, we need an inventory of cytoskeletal proteins in growth cones and an understanding of their interactions with each other and with membrane receptors for guidance cues.

Growth cones have a central (C)-domain, which is relatively thick and filled with various organelles including mitochondria, smooth endoplasmic reticulum, and various kinds of vesicles. The C-domain forms a transition zone between the neurite and the motile, peripheral (P)-domain (Figure 1A).As they advance, growth cones continually extend and retract two characteristic processes from the Pdomain: finger-like processes called filopodia or microspikes, and between adjacent filopodia, sheet-like extensions called lamellipodia or veils (Figure 1A). As the growth cone advances and the neurite elongates the P-domain is converted into C-domain and the C-domain into new neurite. The function of filopodia is thought to be related to the detection of extrinsic guidance cues.The evidence for this includes the obvious fact that they are the first part of the growth cone to encounter new territory as the growth cone advances. Furthermore, when growth cones are in places in the embryo where they must make decisions about which direction to grow, so-called "decision points", they tend to have many, long filopodia whereas when they are growing along established tracts they are more lamellipodial. More direct evidence comes from experiments in which the effects of removing filopodia on the growth cone's abillity to pathfind are observed (Bentley & Toroian-Raymond, 1986; Chien et al., 1993). This was first done in explant cultures of grasshopper limb buds, where sensory neurons pioneering a pathway to the central nervous system (CNS) can be observed directly. Normally, the growth cones of these neurons take stereotypical routes that are identical from ani-

mal to animal, through the limb bud mesenchyme to reach the CNS. However, when the filopodia of these growth cones are removed using the agent cytochalasinnwhich blocks the actin filament formation upon which filopodial extension depends--although the neurite can still extend, the growth cones lose their ability to pathfind (Bentley & Toroian-Raymond, 1986). Consistent with this role in pathfinding, is the fact that filopodia have high concentrations of cell adhesion molecules in their surface membrane (Letourneau & Shattuck, 1989) and autonomous intracellular signaling mechanisms (Davenport et al., 1993). The function of lamellipodia is less clear. They extend and retract between filopodia and become enlarged with organelles in the preliminary stages of conversion of the P-domain into the C-domain, which occurs as the growth cone advances and the neurite is elongated (Goldberg & Burmeister, 1986). The motile behaviour of growth cones, that is the extension and retraction of filopodia and lamellipodia, is actin-based whereas neurite elongation depends on the assembly and stability of microtubules (reviewed in Gordon-Weeks, 1991b). Microtubule assembly and growth cone motility are thought to be coupled together to produce vectorial growth, but how this is achieved is not understood (Mitchison & Kirschner, 1988; Gordon-Weeks, 1989).

The growth cone cytoskeleton is composed principally of microtubules and microfilaments; neurofilaments are rarely present and then usually confined to the Cdomain. The distribution of these two cytoskeletal components is strikingly different (Figure 1B, C). Microtubules are largely found in the C-domain (but see below) whereas there is a considerable concentration of microfilaments in the P-domain, particularly the filopodia.

The cytoskeleton of filopodia consists, characteristically, of bundles of microfilaments containing actin and oriented in parallel with the long axis of the filopodium (Figures 1B, 2). Microfilament bundles are also found in the lamellipodia, but here the number of filaments in each bundle is smaller and filaments also occur individually and are often branched. In lamellipodia, the microfilaments are not oriented in any particular direction. Activation by guidance cues of surface receptors on individual filopodia may lead to selective stabilization of filopodia. Vectorial growth is then thought to occur by directing cytoplasmic organelles into those lamellipodia associated with stabilized filopodia and, eventually, the conversion of that region of the growth cone into new neurite (Goldberg & Burmeister, 1986; A1etta & Green, 1988; Sabry et al., 1991). Directed movement ofmembrane bound organelles in growth cones probably occurs on microtubules and therefore the

Diagrams showing the hypothetical reorganization of microtubules (thick lines), and microfilaments (thin lines), in a growth cone undergoing a turning maneuvere. Time elapses from A to C. It is imagined that the lower filopodia (filo). and lamellipodia (lam). encounter an extrinsic guidance signal that stabilizes these structures. As a consequence, microtubules that are dynamically unstable and randomly probing the peripheral domain are "captured" by filopodial actin filaments and stabilized (arrows in B and C). Dashed lines indicate border between the central (C). and peripheral (P). domains.

Figure 2.

observed selective stabilization of microtubules in growth cones maneuvering under the influence of target cells is significant (Sabry et al., 1991; Lin & Forscher, 1993).

It has been suspected for a long time that microtubules play an important role in elongation and growth of the neurite. Indirect evidence for this idea is that agents that interfere with microtubule dynamics inhibit neurite elongation of neurons in culture and, when substrate adhesion is low, may cause neurite retraction (Seeds et

al., 1970; Yamada et al., 1970; Daniels, 1972; Bray et al., 1978). Furthermore, antisense oligodeoxynucleotides to tubulin mRNA (Teichman-Weinberg et al., 1988) and microinjection of colchicine-tubulin (Keith, 1990), a derivative of tubulin which blocks tubulin polymerization, block neurite extension in culture. Two general roles for microtubules in neurites are recognized. They maintain the structural integrity of neurites and they provide the structural basis of fast axonal and dendritic transport (Bershadsky & Vasiliev, 1988). In addition, microtubules in growth cones may underlie vectorial growth. Microtubules are hollow, unbranched cylindrical filaments (24 nm in diameter) composed of heterodimers of a- and 13-tubulin and a diverse group of accessory proteins known as microtubule-associated proteins (MAPs). MAPs characteristically bind to, stabilize, and co-purify with microtubules and can be grouped into several families according to their molecular structure and function (Wiche et al., 1991). In cells, microtubules are in equilibrium with soluble a-and 13-tubulin dimers which are added to or lost from the filament ends. Microtubules are inherently polar; the ends displaying different kinetics of subunit addition and loss. The so-called "plus" end is that at which subunit addition takes place preferentially and therefore grows faster. Microtubule formation is initiated in cells at microtubuleorganizing centers (MTOC), a well known example of which is the centrosome. Microtubules formed at MTOCs always have their "minus" ends nearest to the MTOC, an arrangement that confers polarity on the microtubules radiating throughout the cell from the MTOC. Individual microtubules in cells are usually undergoing dynamic instability, that is to say the "plus" end is either growing slowly or shrinking rapidly with abrupt transitions between these two states (Schulze & Kirschner, 1988; Mitchison & Kirschner, 1988). It has been suggested that this behavior allows microtubules to continually probe the cell periphery and that this may form the basis for changes in cell morphology as a consequence of the action of extracellular signals (Mitchison & Kirschner, 1988). Such signals, acting locally at the cell surface, may enable the cortical cytoskeleton of the cell to "capture" microtubules probing the cell periphery, and so stabilize them at appropriate sites within the cell. This results in the delivery of material moving along microtubules to particular regions within the cell and therefore to cellular growth at that location. This model could equally well apply, with minor modifications, to growth cones, as discussed below.

In growing neurites, as in most mature axons and dendrites, microtubules are organized into bundles that have an orientation that is parallel to the longitudinal axis of the neurite. Although individual microtubules frequently attain lengths of 100 ~tm, because neurites can be far longer, microtubules are usually entirely contained

within the neurite. At the distal end of a growing neurite where it enlarges into the growth cone, the individual microtubules of the bundle diverge from each other and extend into the C-domain of the growth cone (Figure 1C). (Yamada et al., 1970; Bunge, 1973; Isenberg & Small, 1978; Shaw et al., 1981; Cheng & Reese, 1985; Forscher & Smith, 1988). Individual microtubules frequently extend beyond the C-domain (Figure 1C) (Letourneau, 1983; Tsui et al., 1984; Bridgman & Dailey, 1989; Dailey & Bridgman, 1991; Gordon-Weeks, 1991 a; Sabry et al., 1991; Tanaka & Kirschner, 1991). As they diverge from each other on entering the C-domain, microtubules may have a winding course at their distal ends, whereas in the neurite they are straight (Cheng & Reese, 1985). Furthermore, in vivo there is a marked preference for smooth endoplasmic reticulum and vesicles to be associated with the straight regions but not the winding regions (Cheng & Reese, 1985), although this is not the case in growth cones in vitro (Dailey & Bridgman, 1991). Occasionally, microtubules are also found bundled in the C-domain (Sabry et al., 1991; Tanaka & Kirschner, 1991). In rat cerebral cortical neurons and chick dorsal root ganglion (DRG) neurons in culture, individual microtubules can be seen to extend across the P-domain and run alongside the actin filament bundles of filopodia (Figure 1C; Gordon-Weeks, 1991 a). The occasional insertion of microtubules into filopodia may indicate an interaction between microtubules and filopodia actin-filament bundles (Figure 1C, D). Such an interaction could form the basis of vectorial growth. In this model, microtubules in the growth cone are dynamically unstable, growing slowly forward by polymerization into the P-domain and rapidly shrinking back by depolymerization (Figure 2). Those filopodia that interact with extrinsic guidance cues are stabilized and are able to "capture" individual microtubules and stabilize them by preventing shrinkage. Microtubule stabilization allows the flow of cytoplasmic organelles moving along microtubules (fast axoplasmic transport) to be directed to particular regions of the growth cone and, hence, may underlie vectorial growth (GordonWeeks, 1991 a). What evidence is there that microtubules in growth cones are dynamically unstable? In a study of Aplysia neurons observed in culture with videoenhanced microscopy, microtubules showing dynamic instability at their distal ends were observed in growth cones, although the authors were not able to rule out forward sliding of the microtubules because the proximal ends of the microtubules were not visible (Forscher & Smith, 1988). Similarly, Tanaka & Kirschner (1991) saw growth and shrinkage of fluorescently labeled microtubules in Xenopus neuronal growth cones. The proposal that growth cone microtubules are undergoing dynamic instability is also consistent with the observation that these microtubules are turning over more rapidly than in other regions of the growing neuron (Lim et al., 1989), and that these microtubules are largely composed of tyrosinated a-tubulin which correlates with microtubule instability (see below). This model for vectorial growth predicts a correlation between microtubule capture by filopodia and the direction of growth. This prediction has been tested in the Til pioneer growth cones of grasshopper limb buds growing in explant culture (Sa-

bry et al., 1991). These neurons were microinjected with fluorescent tubulin, which became incorporated into microtubules in the growth cone, which could then be visualized with highly sensitive cameras. Although microtubule invasion into filopodia was not observed, microtubules were seen to selectively invade or be selectively retained in branches that developed from filopodia and became, eventually, new neurite. These authors suggested that differential organization of microtubules across the growth cone is an important component of vectorial growth. A similar conclusion was also reached by Lin & Forscher (1993) studying Aplysia bag cell growth cones interacting in culture and stained by tubulin antibodies. They found that growth cone microtubules became reorganized following changes in the microfilaments consequent upon filopodial contact with another neuron.

So where are microtubules formed within growing neurons? Axons do not contain the biosynthetic machinery, such as ribosomes, for the synthesis of proteins and therefore all proteins must be transported from the cell body, their site of synthesis. Dendrites, however, do have ribosomes along their entire length, a feature that forms one of the distinctions between the two types of neuronal processes. Despite this fact, mRNA for tubulin in neurons is confined to the cell body (Garner et al., 1988). Are microtubules assembled at the cell body, the site of tubulin synthesis, and transported as polymer, or are microtubules assembled in neurites or growth cones from a soluble pool of tubulin? While there is no doubt that neuronal microtubules are formed in the cell body and microtubules in axons and that growth cones can elongate by polymerization, whether or not de novo microtubule formation can occur in neurites or growth cones is controversial. Early biochemical analysis of the incorporation and transport of metabolically radiolabeled proteins in neurons has suggested that tubulin is transported as a polymer in axons after assembly at the cell body (reviewed in Lasek, 1986). In these experiments, 35S-methionine is introduced into the vicinity of the cell bodies of a group of neurons and becomes metabolically incorporated into newly synthesized tubulin and other proteins. After washing out unincorporated radioactivity and allowing a suitable time period to elapse, the movement of radiolabeled tubulin into and along the axons of these neurons is analyzed biochemically by cutting the axons into short segments. Experiments of this kind have established that the radiolabeled tubulin moves out along the axon as a coherent wave that essentially maintains its form as it progresses along the axon. The speed of translocation is similar to that of axon elongation, about 40 lam/hr. On the basis of these experiments, Lasek and his co-workers have proposed that microtubules are assembled in the neuronal cell body and are translocated down the axon as microtubules, rather than as individual tubulin dimers, by an active transport mechanism (Lasek, 1986). The active transport mechanism, which has yet to be identified, was postulated be-

Microtubules in Growth Cones

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cause it was assumed that diffusion of microtubules would be too slow and fails to explain the coherent waves of transported proteins. While this is true of microtubules, it is clear that tubulin dimers can diffuse quite rapidly along axons (e.g., Keith, 1990), although this would be an inefficient method of transport in axons greater than 1 mm (Reinsch et al., 1991). Neuronal microtubules are formed de novo, or nucleated, by the centrosome in the cell body. Recent work from Baas' group (Yu et al., 1993) has shown that sympathetic neurons in culture normally have less than ten microtubules attached to their centrosomes. If, however, microtubules in these cells are extensively depolymerized with nocodazole and then allowed to repolymerize by washing out the drug, hundreds of microtubules form at the centrosome and later become detached and move away from the centrosome. These experiments demonstrate the high efficiency with which the neuronal centrosome can nucleate microtubules and have demonstrated the existence of a mechanism for microtubule detachment from centrosomes that is implicit in Lasek's model, particularly because microtubules are often wholly contained within neurites. But can microtubules nucleate elsewhere in the neuron, for instance in the growing axon or growth cone? At present this question is unresolved, although it is clear that microtubules can elongate by polymerization at both of these locations. Several observations argue against the presence of MTOCs in growing axons or growth cones. Associated with MTOCs is a member of the tubulin superfamily called 3,-tubulin, which seems to be essential for microtubule nucleation (Oakley & Oakley, 1989; Joshi et al., 1992). However, 3,-tubulin is not present in growing axons (Baas & Joshi, 1992). Furthermore, there is no structural evidence for MTOCs in either the axon or the growth cone. Such negative findings do not, however, rule out novel MTOCs. There is a subset of microtubules within axons that are resistant to depolymerization by drugs. After maximal microtubule depolymerization, repolymerization following drug washout takes place only at the distally oriented "plus" ends of those microtubules that resisted drug-induced depolymerization; no new microtubules are formed (Baas & Ahmad, 1992). This finding also suggests that there are no MTOCs within the axon. If de novo assembly of microtubules occurs only in the neuronal cell body, followed by detachment from the MTOC and transport into neurites, then at least a subpopulation of microtubules in neurites should be mobile. In contrast, if microtubules are nucleated at the growth cone and then deposited in the growing neurite, then we might expect microtubules in the neurite to be stationary. Unfortunately, attempts to directly visualize microtubule movement in neurites using fluorescent imaging have produced conflicting results. Two approaches have been used: either fluorescence recovery after photobleaching in which a localized region of the neurite is photobleached after allowing microinjected, fluorescently labeled tubulin to equilibrate with microtubules throughout the cell; or microinjection of cagedfluorescent tubulin that becomes fluorescent on activation with UV light. The first reports used photobleaching and found that the bleached segment did move in

growing PC12 cell neurites (Keith, 1987) or that it did not move in PC12 cell neurites, in chick DRG neurons (Lim et al., 1989, 1990), or in axons of mammalian DRG neurons in culture (Okabe and Hirokawa, 1990). More recently, however, support for both microtubule transport along growing axons and assembly at growth cones has come from experiments using microinjected, caged-fluorescentlabeled tubulin. In Xenopus neurons, microtubules were not stationary, as indicated by the distal movement of fluorescence during neurite elongation (Reinsch et al., 1991; Tanaka & Kirschner, 1991; Okabe & Hirokawa, 1992), whereas in mammalian neurons there was no translocation (Okabe & Hirokawa, 1992). This apparent species difference is not attributable to differences in the two techniques, although the caged-fluorecein tubulin method is more sensitive, because photobleaching in Xenopus neurons also shows segment movement (Okabe & Hirokawa, 1993). At present this entirely unsatisfactory paradox remains unresolved.

Although the question of de novo microtubule formation in neurites and \growth cones remains unanswered, it is clear that pre-formed microtubules can elongate distally by polymerization in neurites and growth cones. For instance, when microinjected into PC 12 cells in culture, biotinylated tubulin, which can be distinguished from endogenous tubulin using antibodies, assembles onto the distal ("plus") ends of existing microtubules in the neurites of these cells (Okabe & Hirokawa, 1988). Additional evidence for distal elongation of existing microtubules in neurites comes from studies of the distribution of post-translationally modified forms of tubulin. There are multiple genes for or- and 13-tubulin (Serrano & Avila, 1990), but in addition to differences in primary sequence, several post-translational modifications of tubulin have been identified including phosphorylation, acetylation, glutamylation and a reversible removal of the C-terminal tyrosine of ot-tubulin by specific enzymes (Serrano & Avila, 1990). Most of the genes for ot-tubulin code for a protein with a C-terminal tyrosine (Serrano & Avila, 1990). This tyrosine can be selectively removed by a specific tubulin tyrosine carboxypeptidase (Argarana et al., 1978) or added by a specific tubulin tyrosine ligase (Barra et al., 1973). These posttranslational modifications are unique to tubulin. In non-neuronal cells, where the existence of biochemically distinct but overlapping subpopulations of microtubules is well established (Gundersen et al., 1984; Piperno et al., 1987), a-tubulin becomes de-tyrosinated and acetylated sometime after assembly into microtubules (Schulze et al., 1987). This phenomenon produces microtubules with variations in ot-tubulin isoforms along their length. There is a correlation between microtubule populations that are relatively stable to depolymerization by cold shock and microtubule depolymerizing agents such as nocodazole, and acetylation and detyrosina-

tion, on the one hand, and labile microtubules and tyrosination on the other (Kreis, 1987; Khawaja et al., 1988). Although these post-translational modifications of tubulin correlate with an increase in the stability of the microtubules to depolymerization, they are not causal to microtubule stability (Piperno et al., 1987; Schulze et al., 1987; Khawaja et al., 1988). The relative age of a microtubule population can be gauged by the distribution within it of the post-translationally modified forms of tubulin. In neurons in culture, immunofluorescence and immuno-electron microscope studies, using antibodies specific for acetylated, tyrosinated, or de-tyrosinated a-tubulin, have also revealed differences in post-translationally modified forms of tubulin in microtubule populations in both neurites and growth cones. Immunofluorecence has shown that in proximal regions of growing axons, detyrosinated and acetylated forms of ~-tubulin predominate over tyrosinated and unacetylated forms, whereas in neurites and particularly in growth cones, the reverse is true (Black et al., 1989; Lim et al., 1989; Robson & Burgoyne, 1989; Baas & Black, 1990; Arregui et al., 1991; Mansfield & Gordon-Weeks, 1991). For instance, in dorsal root ganglion cells in culture, the majority of the axonal growth cones (these cells do not have dendrites) stain for antibodies specific for tyrosinated tx-tubulin but not for de-tyrosinated ct-tubulin (Robson & Burgoyne, 1989). This is also the case with PC12 growth cones (Lim et al., 1989), and with the axonal and dendritic growth cones of cerebral cortical neurons in culture (Mansfield & Gordon-Weeks, 1991). Immunoelectron microscopy has revealed that individual microtubules in neurites have distinct domains ofpost-translationally modified tx-tubulin (Baas & Black, 1990; Baas & Ahmad, 1992; Brown et al., 1993). There is a proximal domain which has predominantly de-tyrosinated and acetylated cz-tubulin, and a distal domain, which is therefore at the "plus" end, of unacetylated, tyrosinated oc-tubulin. The border between the two domains tends to be quite sharp. The events that stabilize microtubules to depolymerization are not known, but strong candidates include structural MAPs because they can stabilize microtubules, probably by cross-linking them. How are these observations of microtubule dynamics relevant to neurite elongation and growth cone behavior? These and several other independent observations support the view that microtubule elongation takes place in the growth cone by the addition of tubulin onto the distal ends of the neurite microtubules that enter the Cdomain of the growth cone, followed by microtubule incorporation into the growing neurite, and that these events are essential for neurite growth (Figure 3; reviewed in Gordon-Weeks, 1991 b; Gordon-Weeks & Mansfield, 1991). Firstly, as in neurites, the microtubules in growth cones are all oriented with their "plus" ends, the end at which tubulin subunit addition takes place preferentially, located distally; an appropriate orientation for elongation distally (Baas et al., 1987). Secondly, the direct application onto the growth cone by micropipette of agents that depolymerize or stabilize microtubules prevents neurite growth (Bamburg et al., 1986). Furthermore, the growth cone is the most sensitive region of the neuron to these agents

Diagrams illustrating the elongation of microtubules in advancing growth cones. De-tyrosinated regions of microtubules are indicated by filled rectangles whereas tyrosinated regions are open. Tubulin dimers are shown as small rectangles. As the growth cone advances some microtubules shrink by depolymerization whereas others elongate at the expense of the soluble tubulin pool (arrows). by orders of magnitude (Bamburg et al., 1986). Thirdly, growth cones contain a large, soluble pool of assembly competent tubulin. This was first shown by observing the effects of the agent taxol on growth cones in culture (Letourneau & Ressler, 1984). Taxol lowers the critical concentration point for tubulin assembly within cells and thus forces the soluble pool of tubulin to form microtubules (reviewed in Horwitz, 1992). The effect of taxol treatment on growth cones is to assemble the soluble pool of tubulin onto the "plus" ends of the microtubules that enter the Cdomain from the neurite shaft (Letourneau & Ressler, 1984; Gordon-Weeks, 1987; Gordon-Weeks et al., 1989; Mansfield & Gordon-Weeks, 1991). At high concentrations of taxol (low JAM),when this effect goes to completion, microtubule loops appear in the C-domain of the growth cone because of the large size of the soluble tubulin pool. One of the consequences of this artificial hyper-polymerization of the tubulin in growth cones is to block neurite elongation, further supporting a role for microtubules in neurite growth (Peterson & Crain, 1982; Letourneau & Ressler, 1984; Mansfield & Gordon-Weeks, 1991). Growth cones can be isolated as a subcellular fraction from developing neural tissue (reviewed in Lockerbie, 1990), and biochemical analysis of such fractions also supports the existence of a soluble tubulin pool (Gordon-Weeks & Lang, 1988; Gordon-Weeks et al., 1989). Finally, microinjection of colchicine-tubulin, a derivatized form of tubulin that is unable to polymerize, blocks neurite elongation in PC12 cells only when it has reached the growth cone and not when restricted to the cell body or proximal neurite (Keith, 1990). Tyrosinated ct-tubulin is the predominant form of t~-tubulin in the growth cone soluble pool (Gordon-Weeks & Lang, 1988; Gordon-Weeks et al., 1989). This soluble pool oftubulin can be visualized in cultured neurons using antibodies specific to

tyrosinated ct-tubulin (Gordon-Weeks et al., 1989; Mansfield & Gordon-Weeks, 1991). If neuronal cultures are fixed with fixatives containing detergent, the soluble pool of tubulin is removed leaving behind the cytoskeleton including the microtubules and actin filament bundles in the filopodia (Gordon-Weeks, 1991 a). Under these circumstances, only the microtubules are seen in growth cones after immunofluorescence staining with tubulin antibodies (Figure 1C). As described above, taxol treatment forces the assembly of tubulin onto the ends of the microtubules in the growth cone, forming microtubule loops in the C-domain. This process exhausts the pool of soluble tubulin and consequently results in a loss of the staining of the P-domain and filopodia with tubulin antibodies (Gordon-Weeks et al., 1989; Mansfield & Gordon-Weeks, 1991). It seems, then, that in neurites and growth cones, microtubules are able to elongate distally by polymerization. How the soluble tubulin in the growth cone is maintained and delivered to the growth cone from the cell body where, at least in axons, it is synthesized, is not known. It may be transported in the so-called "slow component 'a' particles" (described by Weisenberg et al., 1987), which may correspond to the motile varicosities seen in some cultured neurites (Koenig et al., 1985). Alternatively, it may derive by depolymerization from the microtubules that enter the neurite from the cell body (Joshi & Baas, 1993).

What factors might be involved in controlling microtubule elongation and bundling in growth cones and the interaction of microtubules with actin filaments such as those in filopodia? Tyrosinated a-tubulin is no less able to polymerize than de-tyrosinated ct-tubulin. However, if the C-terminal tyrosine is phosphorylated, then assembly is markedly impaired (Wandosell et al., 1987). Even if assembly occurs, the binding of MAPs may be altered by microtubule phosphorylation, and this in turn may lead to less stable microtubules (see below). Experiments with isolated growth cones have shown that tubulin can be phosphorylated on tyrosine residues in the growth cone, but the location of the tyrosine within the molecule is not known (Cheng & Sahyoun, 1988). The tyrosine kinase pp60 c-srcis present in an active form in growth cones (Maness et al., 1988) and phosphorylates tubulin in them, although probably not at the C-terminal tyrosine (Matten et al., 1990). The structural MAPs have been shown to bundle microtubules and to cross-link them to other filamentous proteins and are, therefore, candidates for playing a role in neurite extension by bundling microtubules and cross-linking microtubules and actin filaments (reviewed in Hirokawa, 1991). Interestingly, the ability to bind to microtubules is regulated by phosphorylation. Some of the better characterized structural MAPs are present within growth cones, including MAP1B, MAP2, and

tau, but their distributions within growth cones, particularly whether they are bound to microtubules, and their functions remain unclear. There is considerable circumstantial evidence that MAP 1B may play an important role in neurite outgrowth (reviewed in Matus, 1990; Gordon-Weeks, 1993). MAP1B is present in growth cones, as judged by immunocytochemical and biochemical data (Mansfield et al., 1991). Furthermore, when PC12 cells are induced to form neurites in culture by the action of nerve growth factor, MAP1B is rapidly up-regulated, suggesting that it is required for neurite outgrowth (Greene et al., 1983; Drubin et al., 1985; Brugg & Matus, 1988; Zauner et al., 1992). MAP1B is post-translationally phosphorylated by a casein kinase II-like activity (Diaz-Nido et al., 1988), and there is strong developmental down-regulation of the phosphorylated form (Viereck & Matus, 1990; Fischer & Romano-Clarke, 1990). Interestingly, it is the phosphorylated form of MAP 1B that is most strongly induced during neurite outgrowth (Aletta, et al., 1988; Diaz-Nido et al., 1988). It has been shown that MAP1B is phosphorylated in both isolated growth cones and the growth cones of rat cerebral cortical neurons in culture and in vivo (Mansfield et al., 1991; Gordon-Weeks et al., 1993). In some axons in culture, the phosphorylated form of MAP1B is distributed in a striking gradient that is highest distally and lowest near the cell body, whereas the nonphosphorylated form is distributed throughout the neuron (Mansfield et al., 1991). This suggests that the phosphorylation epitope plays some role in neurite elongation. The phosphorylated form of MAP1B has also been found in high levels in growing axons in vivo (Sato-Yoshitake et al., 1989; Schoenfeld et al., 1989; Viereck & Matus, 1990; Gordon-Weeks et al., 1993). In Alzheimer's disease, in which it is thought there is a massive attempt at axonal and dendritic regeneration, there is an accumulation of hyperphosphorylated MAP 1B (Hasegawa et al., 1990). These results suggest an important role for MAP 1B, particularly the phosphorylated form, in neurite outgrowth. MAP1B probably cross-links microtubules (Sato-Yoshitake et al., 1989) and therefore one possible function for MAP1B in growth cones is to bundle the newly-formed microtubules in the C-domain as it transforms into new neurite (Gordon-Weeks & Mansfield, 1991; Mansfield et al. 1991). The idea that MAP 1B is important in neurite outgrowth can be tested. The obvious approach is to block MAP1B function with antisense probes or antibodies in neurons extending neurites. This has been achieved in PC 12 cells where it was found that antisense oligodeoxynucleotides to MAP1B blocked neurite outgrowth (Brugg et al., 1993). This result supports the view that MAP1B expression is important for neurite elongation. Alternatively, when the binding site(s) of MAP1B on tubulin has been determined it will become possible to compete out MAP1B binding to microtubules with exogenous peptide containing the sequence of the binding site. The function of MAP1B could also be assessed by transfecting cells that do not normally express the protein. In experiments in which cDNA encoding for mouse MAP 1B was transfected into COS cells, which do not usually express MAP 1B, although the expressed MAP1B was localized along the lengths of microtubules in these cells there was no rearrangement of the microtubules, such as bundling, as is

commonly seen when transfecting with MAP 2 or tau cDNA (Takemura et al., 1992). However, transfection with MAP1B cDNA did confer increased stability on the COS cell microtubules, as indicated by their increased resistance to depolymerizing drugs and increased staining for antibodies against acetylated a-tubulin. It is also clear that it is important to determine the kinases that phosphorylate MAP1B during development because this will allow us to explore the control of MAP1B regulation, particularly during neurite outgrowth.

There are several indications that growth cone motility and neurite elongation are linked. For instance, the formation of microtubule loops following taxol treatment is associated with the collapse of the P-domain of the growth cone (Letourneau & Ressler, 1984; Gordon-Weeks et al., 1989; Mansfield & Gordon-Weeks, 1991). When actin filaments are depolymerized following cytochalasin treatment, microtubules advance more distally, suggesting that they are normally constrained from doing so by an interaction with actin filaments (Forscher & Smith, 1988; Lin & Forscher, 1993). These interactions between the two filament systems may, of course, be merely physical and not involve specific factors such as additional proteins. This is also true of the occasional insertion of microtubules into filopodia, which may simply be a consequence of the dynamic behavior of growth cone microtubules. In future studies, it will be interesting to explore these potential functional interactions between microtubules and actin filaments in growth cones.

Neuronal growth cones guide growing axons and dendrites (neurites) through developing embryos by detecting extrinsic guidance cues and transducing the signal into changes in motile behavior. In this chapter, the role of the growth cone cytoskeleton in these events, in particular the microtubules, is discussed. Microtubules in the neurite are mainly bundled into fascicles whereas on entering the growth cone they diverge from each other and traverse the central (C)-domain of the growth cone. Occasionally, individual microtubules extend as far as the peripheral (P)-domain and may even enter filopodia. Microtubules in the growth cone are probably dynamically unstable, exchanging tubulin dimer with a large pool of soluble tubulin. It is proposed that the "capture" of dynamically unstable microtubules by filopodial actin filament bundles is a crucial step underlying directed growth. This may facilitate the delivery of material to specific regions of the growth cone and hence allow vectorial growth. Bundling of microtubules and capture of microtubules by filopodia both imply roles for MAPs. Several MAPs are present within growth

cones, including MAP 1B, MAP 2, and tau. Although the molecular events have yet to be resolved, recent experiments point toward a phosphorylated form of MAP1B as an important component in neurite elongation and in particular in the bundling of microtubules in the growth cone.

I thank Dr. Max Bush for Figure 1A and helpful comments on the manuscript and Rapid Communications of Oxford Ltd. for permission to publish Figure 1B and C, which appeared in Gordon-Weeks, 1991 a.

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Introduction Early Events of Nerve-Muscle Interactions Role of the Muscle during Neuromuscular Junction Development Acetylcholine Receptor Aggregation The MuSK Receptor Accumulation of Acetylcholinesterase Compartmentalization of Other Synaptic Proteins Specializations of Organelles within the Postsynaptic Sarcoplasm Role of the Nerve during Neuromuscular Junction Development The Synaptic Basal Lamina during Neuromuscular Development Maturation of the Neuromuscular Junction Synapse Elimination during Neuromuscular Junction Formation Function of the Synaptic Folds Role of Nerve Impulse Activity in Muscle Fiber Differentiation The Importance of the Pattern of Nerve Impulse Activity Plasticity of the Neuromuscular Junction Hypertrophy and Atrophy of Muscle Effects of Denervation on Skeletal Muscle Fibers Principles of Medical Biology, Volume 11 Developmental Biology, pages 187-207. Copyright 9 1998 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-816-1

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Reinnervation of Skeletal Muscle Regeneration of Damaged Muscle Congenital Disorders of the Neuromuscular Junction Summary and Key Points

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The neuromuscular junction is the highly specialized synapse between motoneurons and skeletal muscle fibers. Nearly 50% of body mass is composed of muscle tissue whose contractile state is directly controlled by nerve impulse activity at neuromuscular junctions by release of the neurotransmitter acetylcholine. This chemical signal is transduced via the nicotinic acetylcholine receptor (AChR), a ligand-gated ion channel. The region of the neuromuscular junction comprises, on the average, less than one tenth of 1% of the total surface area of an individual muscle fiber, yet it is entirely responsible for initiating the subsequent .events of membrane depolarization and excitation-contraction coupling leading to muscle contraction. This relationship between nerve and muscle fiber is relatively permanent in that, once formed during embryonic development, the same neuromuscular junctions appear to persist throughout the lifespan of the organism. In addition to muscle contraction, the physiological importance of the neuromuscular junction in normal regulation of muscle properties becomes readily apparent in such common pathophysiological states as paralysis, muscle wasting disorders, and muscle atrophy and degeneration as a consequence of nerve injury or muscle disuse. Because of its importance in all aspects of motor function, and the relative accessibility of this synapse for physiological and biochemical investigations, the neuromuscular junction is the best characterized of all synapses in the central and peripheral nervous systems. The neuromuscular junction consists of the terminal processes of the motoneurons, the region of specialized basal lamina interposed between the nerve terminals and the invaginated surface of the muscle fiber, and the highly organized plasma membrane and submembrane cytoskeleton of the muscle fiber (Figure 1). In addition, other specialized cells, such as modified Schwann cells and fibroblast-like cells cover the motor nerve terminals and surrounding synaptic basal lamina and most likely play several important roles in the development and maintenance of the motor endplate region of the muscle. To date, more than 50 different proteins have been identified and shown to be concentrated specifically in either the pre- or post-synaptic regions of the neuromuscular junction, or to the specialized basal lamina that separates them (Table 1). Although in a few cases the functions of these molecules are well understood, the role of many junctional proteins remain to be determined. The most studied components from a physiological, biochemical, and molecular standpoint are the nicotinic acetylcholine receptor (AChR) and the enzyme acetylcholinesterase (ACHE), which is responsible

for terminating the actions of acetylcholine once the receptors have been activated. These two proteins were the first to be identified at neuromuscular junctions and have for several decades been used as markers for these specialized sites of nerve-muscle interaction. Together, they have provided a wealth of information regarding the cellular and molecular events involved in synaptogenesis and in the regulation and maintenance of the adult neuromuscular junction. This chapter will focus on the function and regulation of some of the principal molecules that play a role in the development and maintenance of the mature neuromuscular junction.

Skeletal muscle fibers are multinucleated mesodermal derivatives that arise from the fusion of mononucleated myoblasts. Their embryonic origins are in the lateral plate mesoderm which separates into three layers: the dermatome, the myotome, and the sclerotome (Figure 2). In the thoracic and lumbar regions of the embryo, the myotome is more thickened as it gives rise to the presumptive myoblasts, which migrate out into the developing limb buds. Cells from other regions of the myotome can give rise to the skeletal muscle fibers of the back, shoulders, and rib cage. The differentiation of the myotomal ridge proceeds in a rostral-caudal fashion with the more ante-

rior regions, such as those providing myoblasts to the fore limbs, differentiating first. Thus, this period of early muscle development is one of cell commitment, where the cells of the medial most region of the ridge begin transiently expressing musclespecific genes (for example MyoD, AChR a-subunit) and shortly thereafter begin to form the axial musculature along the vertebral column. The cells destined to migrate out and give rise to the limb musculature are present in the lateral aspects and at this stage do not express any of the known muscle-specific genes. The second period is one of cell migration during which myoblasts from the lateral portions of the dermamyotome begin their migration into the developing limb bud. Concurrent with myoblast migration, pioneering nerve processes originating from cell bodies in the medial-lateral region of the neural tube, the motoneurons, begin their projection into the developing limb bud. Shortly after arriving in the limb bud, the myoblasts undergo a period of rapid proliferation and segregation into dorsal and ventral muscle masses that will give rise to the specific groups of flexor and extensor muscles. While this is occurring, the pioneering nerve fibers penetrate the forming individual myoblast groups and await a signal from the developing muscle that they are ready for innervation. When the myoblasts begin to fuse, producing short myotubes, the awaiting nerve terminals form their initial contacts. At this point, the myotubes may consist of several to more than a dozen nuclei derived from fused mononucleated myoblasts (Figure 3).

Once initial contacts are formed, a long series of reciprocal interactions between the nerve and muscle cells are initiated that subsequently give rise to the mature neuromuscular junction. In lower vertebrates, this period can take up to several days whereas during human development the entire process occurs over a period of several months.

The invading nerve initiates aggregation of AChRs and the accumulation of many specific proteins at the presynaptic, postsynaptic, and cleft regions of the neuromuscular junction (Table 1). During the maturation of the junction, specialized folds and invaginations appear on the otherwise smooth muscle fiber surface, and an accumulation of nuclei, often referred to as "fundamental" myonuclei, can be observed beneath the motor endplate. These nuclei are the major contributors of the transcripts that encode the synaptic proteins contributed by the muscle fiber. Studies on developing rat, chicken, and amphibian embryos indicate that surface nicotinic AChRs redistribute and selectively accumulate at sites of nerve-muscle contact at the earliest observable times. Thus, even when the myotubes consist of only a few nuclei, acetylcholine receptor clusters are already apparent. Based on studies using tissue-cultured nerves and muscle cells, it appears that adhesion of the nerve growth cone to the myotube surface membrane is sufficient to induce accumulation of receptors within a period as short as a few minutes after initial contact. Thus AChR aggregation is the first observable marker for the formation of the neuromuscular junction.

Acetylcholine ReceptorAggregation Of all the molecules localized to the neuromuscular junction, the nicotinic AChR is the best studied. The AChR is a heteropentameric protein composed of four homologous subunits (2a,13,5, and either ? or e) that together form a ligandgated ion channel activated by binding of ACh to the two ot-subunits. Developmentally, the ?-subunit of the AChR is replaced by the e-subunit in the transition from an embryonic (~2~y~) to an adult (a213eS) form of the AChR complex after innervation has been established. In developing rat muscle, this occurs shortly after birth whereas the initial nerve-muscle contacts formed over a week earlier. In developing myotubes, AChR subunits are distributed diffusely over the cell surface. Upon contact with the nerve, the AChRs under the nerve become anchored and the AChRs rapidly concentrate at the sites of nerve-muscle contact by a combination of lateral diffusion and preferential synthesis and insertion of AChR under the forming motor endplate. Functional changes intrinsic to the AChR at the developing neuromuscular junction then follow over time. For instance, the half life of

the junctional AChR protein increases from about 24 hours to 10 days, and the mean channel open time decreases concomitantly with the developmental switch from the to the e AChR subunit after birth. In adult muscle, AChR messenger RNAs have been found to be highly concentrated in innervated versus noninnervated regions of muscle. Elegant transgenic mice experiments using reporter gene products regulated by AChR gene-specific promoters demonstrate selective accumulation of these reporter gene products at the motor endplate. Thus, the molecular mechanism of this accumulation of AChR mRNAs and protein is, at least in part, regulated by selective transcription of AChR subunit genes in myonuclei localized to the neuromuscular junction.

MuSK (muscle-specific kinase) is a recently discovered receptor tyrosine kinase specifically expressed by skeletal muscle at the neuromuscular junction. Developmentally, MuSK is expressed after myoblasts fuse to form myotubes that await innervation by motor nerves and appropriately accumulates at sites that become the motor end-plate. Mice genetically deficient in MuSK develop skeletal muscle normally but are lacking in neuromuscular junctions, demonstrating that MuSK is an essential signaling molecule for synapse formation that is necessary during the earliest stages. Absent in MuSK-deficient muscle is aggregation of AChR, ACHE, rapsyn, utrophin, erbB receptors, and other molecules typically found at sites of nerve-muscle contact (Table 1). MuSK may function as a co-receptor for agrin, a nerve-derived signaling molecule important in inducing aggregation of synaptic molecules such as AChR, ACHE, and rapsyn. Binding of agrin to muscle induces tyrosine phosphorylation essential for AChR clustering at the motor endplate, thus the functional agrin receptor is believed to involve a tyrosine kinase. Agrin binds MuSK in association with an as yet undiscovered molecule, and this activated tyrosine kinase receptor complex then rapidly initiates a signaling cascade that includes phosphorylation of MuSK and induces the subsequent accumulation of other synaptic proteins. MuSK also plays a critical role in presynaptic differentiation of the NMJ. Whereas the axon terminal normally splits into short nerve branches that form arborized terminals upon the motor end-plate, the nerve branches in MUSKdeficient mice extend along the length of the muscle fiber as if lost and unable to find an address along its path at which to stop. These wandering nerve terminals are not morphologically differentiated; thus it is clear that MuSK transduces a very early signal in neuromuscular development required for differentiation of the postsynaptic membrane, which in turn generates the signals required for presynaptic differentiation as well. This reciprocal signaling, or "handshaking", is an important feature of the complex cascade of events that regulate formation of the neuromuscular junction.

Acetylcholinesterase (ACHE), which hydrolyzes acetylcholine released by the nerve terminal, is attached to the extracellular matrix in the basal lamina and has long been used as a marker for nerve-muscle interactions because of the availability of specific histochemical reactions that enabled early investigators to easily visualize sites of enzyme accumulation by microscopy. Like the AChR, AChE protein is expressed very early in development, prior to the onset of innervation. However, unlike the clustering of AChRs, the accumulation of AChE at sites of nerve-muscle contact is a later event. This late appearance, which can be up to several days post-innervation in vivo, most likely reflects the requirement for prior accumulation of other extracellular matrix molecules to which AChE enzyme is attached in the basal lamina. The expression of AChE in multinucleated skeletal muscle fibers is also highly compartmentalized, with each individual nucleus responsible for localizing this enzyme to the overlying region of the membrane. In adult muscle fibers, the AChE mRNA has been shown to selectively accumulate at motor endplates. These studies suggest that AChE transcripts are preferentially accumulated by the myonuclei under the neuromuscular junction, do not diffuse very far, and are preferentially translated locally on the rough endoplasmic reticulum surrounding each nucleus. Taken together, studies on the AChR and AChE demonstrate that functional nuclear domains exist at the motor endplate that regulate synapse-specific transcription and translation by the junctional nuclei for at least some of the proteins that are concentrated at the motor end plate.

The compartmentalization of the nicotinic acetylcholine receptors is a harbinger of the multitude of other synaptic proteins that also accumulate selectively at the NMJ to allow for neuronal signaling and maintenance of the motor-end plate as a privileged domain in the muscle fiber. Some proteins, like dystrophin, are found throughout the muscle fibers but are concentrated at the motor endplate. Many other proteins such as utrophin, AChR, and ACHE, are restricted mainly to the neuromuscular junction, especially in the mature muscle fiber. Among the molecules involved in the maintenance of AChR clusters is the 43kDa protein, rapsyn (receptor-associated protein at the synapse), which colocalizes at the NMJ in an apparent 1:1 stoichiometry with AChR. Rapsyn is a peripheral membrane protein associated with the intracellular domain of AChR, co-localizes with AChR, and can induce small clusters of AChR on its own in uninnervated myotubes and transfected cell lines expressing the AChR receptor subunits. Rapsyn is important for development of neuromuscular junctions because mice genetically deficient in rapsyn die shortly after birth and lack AChR clusters. In the absence of rapsyn, AChR cannot be induced to aggregate using known stimuli such as agrin, suggesting that rapsyn functions as an essential

linchpin, cross-linking synaptic proteins organized in the postsynaptic membrane. Surprisingly, rapsyn also appears to play a role in the differentiation of nerve terminals. Nerve axons normally terminate at the motor end-plate with specialized arborizing branches, but in rapsyn-deficient endplates the nerve terminals were simplified and often did not form differentiated arbors. These findings emphasize the importance of reciprocal signaling from the nerve to the muscle, and from the muscle to the nerve, in the development of the motor end-plate. Rapsyn, while participating in a scaffold for synaptic protein accumulation in the muscle, permits signals from the muscle to be sent to the nerve to encourage its further differentiation, thereby cementing local interactions between the presynaptic and postsynaptic cells. Additional molecules that are probably associated in an anchoring complex with AChR and rapsyn include dystrophin, utrophin, actin, and a group of membraneassociated proteins concentrated at the NMJ, collectively referred to as the dystrophin-associated proteins (Table 1 and Figure 1). The dystrophin-associated glycoprotein (DAG) complex consists of at least six different proteins. Dystrophin, the protein defective in the Duchenne and Becker muscular dystrophies, is associated with syntrophin (58 kDa), utrophin (dystrophin-related protein; DRP), a- and 13-dystroglycan, adhalin, and several proteins known only by their molecular weights. Utrophin, a synapse-specific molecule with high homology to Dystrophin, appears to modulate the size of AChR clusters in association with rapsyn, ot-Dystroglycan, which binds the extracellular matrix proteins laminin and merosin, can also act as a receptor for the AChR aggregation factor agrin, but with little or no signal transducing activity. Agrin is believed to bind (x-dystroglycan in a transmembrane complex coupled intracellularly with utrophin and the DAG complex to AChR and rapsyn. Thus, these DAG complex proteins form a structural bridge as well as communication network between the basal lamina (via agrin and other extracellular matrix molecules) and the muscle cell. At the mature neuromuscular junction, the troughs of the junctional folds contain a much higher concentration of voltage-dependent sodium channels, ensuring the efficiency of transmitting the postsynaptic potential from the motor-endplate to the noninnervated portions of the muscle fiber. In the rodent, the voltage-gated sodium channel (NaCh) undergoes a slow maturational change from an embryonic form to an adult form distinguished by their sensitivities to the binding of the sodium channel-blocking neurotoxin called tetrodotoxin (TTX). The transition from an embryonic TTX-resistant to an adult TTX-sensitive form of voltage-gated sodium channel results in a concomitant heterogeneous and possibly nerve-derived agrin-mediated accumulation of embryonic and adult NaCh forms at the motor endplate beginning at about one to two weeks postnatally with completion several weeks after birth in the rodent. One of the first surface proteins shown to be involved directly in nerve-nerve and nerve-,nuscle adhesion and recognition is the neural cell adhesion molecule (NCAM), which also undergoes a postnatal compartmentalization at the motor end-

plate. N-CAM is expressed on the surfaces of both muscles and nerves, and appears as highly sialated embryonic isoforms and less sialated adult isoforms. Several alternative splicing variants of N-CAM exist that give rise to different transmembrane and glycolipid-anchored forms. However, the exact function of the variants is not well understood. In the nervous system, N-CAM mediates, in part, the adhesion of neuronal membranes to form fascicles, or bundles of axons, and is also found on the nerve terminals and nerve terminal-associated Schwann cells at the motor endplate. Postsynaptically, N-CAM isoforms are expressed at all stages of muscle development from myoblasts, myotubes and myofibers, to adult muscle. The observation that incubation of myoblasts with N-CAM antibodies hinders the rate of myoblast fusion also suggests a role for N-CAM in myogenesis. N-CAM is uniformly distributed on the surface of early embryonic primary myotubes and accumulates at pericellular myotube contact sites and, in more mature embryonic myotubes, can form surface clusters. Around the time of birth, N-CAM once again becomes more uniformly distributed on the muscle fiber surface only to begin a process of postnatal compartmentalization that results in a diffuse junctional accumulation in adult muscle. The function and regulation of many of the molecules localized at the neuromuscular junction are still unclear. For example, the neuron-derived agrin protein variant is known to induce AChR clustering, but a functionally distinct muscle-derived agrin isoform, also localized to the neuromuscular junction, may play an important role in the development of the motor endplate, possibly as a retrograde stop signal for neurite outgrowth. Although a defect in dystrophin causes Duchenne muscular dystrophy, the role of dystrophin in the muscle and the structurally related utrophin at the synapse and how the loss of dystrophin results in muscle pathology are unknown.

The postsynaptic sarcoplasm of muscle fibers contains an accumulation ofmyonuclei, referred to as "fundamental nuclei" or "junctional nuclei", based on their distinct flattened morphology contrasted with the elongated sausage shape of the extrajunctional myonuclei. Functionally, these nuclei appear to transcribe genes encoding synaptic proteins, such as AChR, at higher levels than extrajunctional nuclei and are therefore important components in the molecular regulation of neuromuscular junction development. The Golgi apparatus, which usually consists of a series of characteristic flattened stacks enriched in glycoprotein-processing enzymes, plays a key role in the posttranslational processing and sorting of membrane proteins. It is involved in the posttranslational processing of both AChR and ACHE, components of the DAG, as well as all proteins destined for the extracellular matrix. In most cells, the Golgi apparatus is found adjacent to the nucleus in the vicinity of the microtubule organizing center, but in myoblasts is confined to a juxta-nuclear location. Upon fusion of myoblasts into multinucleated myotubes, the Golgi apparatus undergoes a major reorganization from its polarized location to a more diffuse and fragmented perinu-

clear organization surrounding every nucleus of the developing myotube. This reorganization of the Golgi apparatus appears to result from activation of the myogenic program subsequent to myoblast fusion. An accumulation of several Golgi-specific marker proteins within the junctional sarcoplasm of muscle fibers is observed immunocytochemically using antibodies to Golgi-resident proteins. Similar approaches have also shown an accumulation of a differentiated subset of microtubules within the postsynaptic sarcoplasm. These observations suggest that components of the protein secretory pathway also accumulate within the postsynaptic membrane domain. Thus, this reinforces the concept of structural and functional compartmentalization whereby maintenance of the postsynaptic membrane domain in muscle fibers is achieved via local transcription of synaptic protein-encoding genes, and post-translational processing of their protein products with the subsequent focal insertion of the newly synthesized molecules into this specialized region of the muscle fiber membrane. Mitochondria play a crucial role in cellular metabolism and, in muscle, demonstrate remarkable plasticity in meeting the specific requirements associated with chronic alterations in levels of muscle activity. Thus, it is interesting that mitochondria also appear to accumulate within the postsynaptic sarcoplasm. Because mitochondria may perform slightly different functions according to their position in cells, possibly also acting as calcium sequestering organelles, their role at the neuromuscular junction remains an interesting question. How mitochondria accumulate within the postsynaptic sarcoplasm during development and the functional significance of this accumulation is unclear.

When the nerve growth cones make their first contact with the muscle fibers, the morphology of the pioneering nerve ending changes from that of a growth cone to a highly branched terminal that arborizes at the motor end-plate. Later during development, the nerve terminal becomes capped by Schwann cells and their processes. Synaptic vesicles containing the neurotransmitter acetylcholine, which is initially sparse, along with associated synaptic vesicle proteins such as synaptophysin and synapsin, and synaptic enzymes such as choline acetyltransferase, accumulate in the motor nerve terminal. The discharge of these vesicles into the developing synaptic cleft produce small miniature endplate potentials that steadily increase in amplitude and frequency, and that participate in the process of reciprocal signaling between the nerve and muscle. However, the release of acetylcholine and activation of AChR does not appear to be essential for neuromuscular junction formation because chronic pharmacological blockade of AChR signal transduction during early development results in apparently normal nerve-muscle synapse formation.

The first events in nerve-muscle interactions involve formation of cell-cell contacts. In molecular terms, this requires the presence of complementary cell-cell recognition and adhesion molecules on the surface membranes of the cellular partners. Part of this adhesion process between nerve and muscle includes cell adhesion molecules such" as N-CAM and N-cadherin. Only later do the extracellular matrix molecules secreted into the space between the nerve terminal and the muscle fiber membrane become important. Calcium plays an important role in this development through nerve activity-dependent calcium fluxes in the muscle cell and specific calcium channels studded in the nerve terminal membranes. These observations again underscore the importance of precise reciprocal nerve-muscle interactions which are the necessary inductive influences for development of a functional neuromuscular junction. The full complement of signals from the nerve that announce its approach into the muscle and initiate the formation of the NMJ have not yet been elucidated, but several molecules have been implicated as being important for both AChR clustering and continued differentiation of the presynaptic- and postsynaptic elements. In addition to the release of acetylcholine during the early stages of nerve-muscle interactions, several neuronal proteins are known to play an important role in signaling during early development of the neuromuscular junction. Agrin is a complex proteoglycan encoded by several alternatively spliced mRNAs, and is differentially expressed in muscle and neurons. At the developing neuromuscular junction, agrin is expressed, released, and stably anchored in the synaptic basal lamina. Nerve-derived, but not muscle-derived, agrin potently induces AChR, rapsyn, and AChE clustering. Agrin induces clustering of existing diffuse AChR molecules on the muscle membrane and can enforce synapse-specific transcription of AChR mRNA, but it does not actually increase the synthesis of AChR. Motor end-plates from mice genetically deficient in the neuronal agrin isoform do exhibit small AChR aggregates similar to those that form spontaneously on aneural myotubes in tissue culture, but these are very disorganized and reduced in size and number of AChR molecules. These neuronal agrin-deficient mice are stillborn, demonstrating that neuronal agrin is important for survival but is not a crucial molecule for AChR aggregation and is critical for the differentiation and development of the motor end-plate. Similar to what was found with the rapsyn-deficient NMJs, neuronal agrin is important for presynaptic development as well. Nerve terminals from neuronal agrin-deficient mice had decreased numbers of synaptic vesicles, and the nerve terminals branched abnormally along myotubes, again demonstrating that presynaptic development is influenced by postsynaptic development. Agrin also appears to induce junctional expression of extracellular matrix components in the basal lamina such as laminin, heparan sulfate proteoglycan (HSP), and ACHE, but this occurs much later suggesting that these molecules are not involved in AChR aggregation, although they are expressed at the sites of nerve-muscle contact and are important for subsequent development of the NMJ. The accumulation of AChR at the developing neuromuscular junction is subsequently maintained by selective expression of the AChR subunit genes by the post-

synaptic nuclei. One molecular mechanism for this selective synaptic gene expression is mediated by motor neuron-derived ARIA (AChR-Inducing Activity), a member of the neuregulin family of proteins generated by alternative gene splicing. Neuregulins stimulate Schwann cell proliferation and increase transcription of AChR subunit mRNAs as well as the number of sodium channels, but does not appear to affect the expression of synaptic proteins such as ACHE or rapsyn. Like agrin, neuregulins are associated with the basal lamina, and can induce tyrosine phosphorylation of its muscle-bound receptors, the erbB family of receptor tyrosine kinase proteins. Signaling through the erbB receptors induces synapse-specific transcription of proteins such as AChR, which may be part of a more general mechanism for maintaining high levels of synaptic proteins at the neuromuscular junction. The calcitonin gene-related peptide (CGRP), which is expressed in motoneurons and localized in distinct secretory vesicles within the nerve terminals at the motor endplate, also increases expression of AChR in cultured muscle cells. CGRP appears to stimulate muscle cells through CGRP receptors by activating adenylylcyclase, which catalyzes the synthesis of cAMP, which in turn activates cAMP-dependent protein kinases capable of phosphorylating AChR subunit proteins. However, the physiological conditions under which neuronal CGRP is released are unknown. In addition to factors released by the motoneuron, other cell types such as glial cells and fibroblasts are also capable of producing factors that may have activity at the neuromuscular junction. At present, what all these factors are and how they interact among themselves and with the muscle to initiate formation and maturation of the neuromuscular junction in an orderly manner remains an area of active research.

The basal lamina, a specialized dense accumulation of extracellular matrix molecules including type IV collagen, laminin, fibronectin, and heparin sulfate proteoglycans, surrounds each muscle fiber. The synaptic basal lamina is interposed between the nerve terminals and the muscle and specifically accumulates agrin, ACHE, heparan sulfate proteoglycans, and several other associated extracellular matrix proteins including nerve-derived growth factors (see Figure 1). As discussed earlier, anterograde motoneuron-release of agrin onto the synaptic basal lamina can induce and maintain AChR clustering, and the neuregulins can initiate increased AChR synthesis. Moreover, molecules such as s-laminin and heparan sulfate proteoglycan are inducible by agfin where they co-localize in the region of the synaptic basal lamina with AChR, although at much later times. This suggests a role for agrin not only in the induction but also in the maintenance of the neuromuscular junction. Muscle-derived s-laminin, which is deposited into the basal lamina, may also act as a retrograde signal that is important for proper differentiation of the motor nerve terminals. Nerve terminals normally arborize over the endplate, and synaptic vesi-

cles full of neurotransmitters accumulate at the nerve terminal membrane at the active zone juxtaposed over the junctional folds. Interestingly, mice genetically lacking s-laminin have normal synaptic vesicles but are deficient in active zones with accumulated synaptic vesicles, have diminished synapsin levels, and have nerve terminals that are mostly unbranched and covered by abnormally extended Schwann cell processes that have penetrated the basal lamina. Thus, s-laminin appears to be an important retrograde signal for nerve terminal differentiation at the developing neuromuscular junction. Among the molecules anchored in the synaptic basal lamina that may be regulated by release from the extracellular matrix is basic fibroblast growth factor (bFGF), or a closely related molecule in the family of heparin-binding growth factors (HBGFs). These are growth factors that bind to heparan sulfate proteoglycans and are highly enriched in muscle as well as other tissues. In experiments where latex beads were coated with bFGF and locally applied to muscle cells, AChR clustering was observed within 12-24 h suggesting an inductive role for HBGFs, presumably via a growth factor-like receptor. AChR aggregation induced by bFGF can be prevented by tyrosine kinase inhibitors, again suggesting tyrosine phosphorylation as a signaling mechanism for early receptor clustering as demonstrated by agrin.

During the initial stages of synaptogenesis, multiple axon terminals make contact with the same individual perinatal muscle fibers. Subsequent regulatory events eliminate all but one of these motor nerve terminals to leave a single motoneuron branch contacting each muscle fiber. In most muscles, innervation typically occurs near the center of the muscle fiber, because of growth of the muscle fiber by fusion ofmyoblasts at the ends. The accumulated AChR molecules under the supernumerary axons usually disappear within two weeks after birth and concentrate under the single remaining nerve terminal, which matures into the discrete and morphologically distinct neuromuscular junction. While the single nerve innervating the muscle fiber stabilizes its AChR clusters, the remaining nerve terminals that were unsuccessful in maintaining their innervation are retracted. The mechanism of polyneuronal elimination is thought to be controlled by early muscle activity secondary to nerve stimulation. In experiments where a muscle cell is innervated by two neurons, the experimentally stimulated neuron functionally suppresses the activity of the unstimulated neuron, possibly by recruiting AChRs away from the weak sites of nerve-muscle contact. In addition, asynchronously stimulated neurons suppress signaling activity by other neurons and leave one neuron dominant in its signaling pattern whereas synchronous stimulation of neurons does not suppress signaling by other neurons,

thereby demonstrating the importance of electrical activity patterns. This heterosynaptic suppression appears to be regulated by the depolarization of the postsynaptic membrane because buffeting of the calcium influx in the muscle cell inhibits this suppression. Thus, an important element in the maturation of the motor endplate is the elimination of polyneuronal innervation via competition that occurs between the nerve terminals mediated through signals transduced by the muscle cell.

The structure of the synaptic folds is a prominent feature of the mature motor endplate architecture. These synaptic folds begin to form at the sites of nerve-muscle contact after the initial appearance and accumulation of synaptic basal lamina components. With the compartmentalization of synaptic protein expression at the motor end plate, there is additional spatial segregation of several proteins at the level of the synaptic folds. For instance, the AChR, utrophin, and rapsyn are co-localized at the tops of the fold (crests) whereas proteins such as the voltage-dependent sodium channels, ankyrin and N-CAM are localized to the bottoms (troughs) of the folds (see Figure 1). The functional significance of this subcompartmentalization is not clear. Moreover, the function of the synaptic folds are unclear. On the one hand, they may act functionally as a capacitor that could increase the safety margin, or the reserve current in excess of what is necessary to produce an action potential, in conjunction with the current that can be generated by the accumulation of AChRs and NaChs at the neuromuscular junction. Alternatively, the presence of the folds could serve to increase the surface area available for other functionally important receptors and signal transduction systems and to separate these into different functional subcellular regions. Although it has been proposed that, based on the large surface area of the neuromuscular junctions of fast twitch muscle fibers in some species, the main function of the folds is to increase the area available for AChRs and ACHE, a closer examination of cross species differences in the structure of the folds has not borne this out. In fact, some of the fastest contracting muscle fibers, such as the intraocular muscles of higher vertebrates and the flight muscles of some birds, have the shallowest junctional folds of all. Thus the functional significance of the junctional folds remains somewhat of a mystery, and further research is needed to sort out the various possibilities.

The mass of skeletal muscle originates from two waves of cell differentiation occurring during development that are referred to as primary and secondary myogenesis. Primary myotubes form when a population of primary myoblasts fuse together to form multinucleated myotubes whereas secondary myotubes originate from a distinct set of myoblasts that fuse only after the motoneurons penetrate the muscle mass. Similar to adult skeletal muscle fibers, which exist in a variety of fiber types that may be distinguished by several distinct physiological, histochemical,

and biochemical properties, primary and secondary myotubes contain specific isoforms of myosin heavy chain (MHC). Indeed, primary myotubes express mostly slow or embryonic myosin heavy chain, whereas embryonic or fast MHC are present in secondary myotubes. Thus, the heterogeneity in muscle fiber types not only occurs in adult muscles, but may be detected at the earliest stages ofmyogenesis. Experiments using rat and chick muscle indicate that the initial differentiation of primary and secondary myotubes appears independently of innervation, although complete differentiation is not observed in inactive or denervated muscles. In addition, the emergence of fiber type diversity early in development appears to be at least partially independent ofinnervation because fiber type diversity is observed in aneural muscles. These observations have led to the suggestion that muscle fiber types are preprogrammed and derived from intrinsically different populations of myoblasts. Recent experiments however demonstrate that although cell lineage may initially give rise to fiber-type diversity, upon fusion of myoblasts into existing muscle fibers, the innate program can be overridden by local environmental factors such as the pattern of neuronal stimulation and hormonal influences.

A series of experiments carried out in the early 1960's elegantly demonstrated that cross-reinnervation of a fast or slow muscle with a foreign nerve normally supplying a slow or fast muscle, respectively, led to a conversion of the muscle phenotype. Initially, it was assumed that nerve-derived trophic factors were controlling expression of the muscle-specific proteins. Since then, however, there has been increasing evidence indicating that the pattern of nerve impulse activity regulates expression of a distinct set of muscle proteins involved in metabolic and contractile processes conferring muscles the fast- or slow-twitch phenotype. For example, chronic low-frequency electrical stimulation of fast-twitch muscles leads to their conversion into slow-contracting muscles. Changes in the phenotype of stimulated muscles occur at various levels including modifications of the physiological characteristics and biochemical composition of the fibers. Alternatively, the conversion of slow to fast muscle using phasic, high-frequency stimulation has also been reported, although in much less detail. What remains unanswered, however, is the exact mechanism that triggers fibertype conversion. Although persistent changes in the ionic environment or in the levels of expression of specific transcription factors may initiate the cascade of events, recent studies have raised the possibility that rapid and constant reductions in the phosphorylation potential of the ATP system may catalyze fiber-type conversion.

A remarkable feature of adult skeletal muscle fibers is their ability to rapidly respond to changes in the levels of neuromuscular activity. Over the past several dec-

ades, it has become evident that muscle fibers may be altered phenotypically in order to meet the functional demands placed upon the muscle. This plasticity of muscle fibers has in fact been demonstrated using several experimental models that lead to increases or decreases in neuromuscular activation.

In response to high resistance, low-repetition training regimens, human skeletal muscles display significant enlargement. These alterations in muscle mass as a result of this particular type of overload have been studied extensively in a variety of animals models that mimic high-resistance training programs. These studies firmly establish that increases in muscle mass result from hypertrophy of the existing fibers as opposed to the formation of new muscle fibers, a process called hyperplasia. In addition to showing an enhanced cross-sectional area, hypertrophied muscle fibers display several adaptations affecting metabolic and contractile processes. The nature of the initial stimuli that link contractile activity to profound changes in the phenotype of overloaded muscles remains elusive, yet potential candidates include, among others, activation of immediate-early genes such as c-myc and cfos, as well as signals generated by the muscle cytoskeleton and/or extracellular matrix. In contrast, muscle cells subjected to prolonged periods of inactivity such as bed rest, limb immobilization, or exposure to weightlessness show reduced muscle mass, cross-sectional area, total protein content and force. Similar to muscle hypertrophy, the cellular and molecular mechanisms presiding over muscle fiber atrophy have yet to be elucidated, although the product of the Id-I gene, a transcriptional repressor, may be involved in the atrophic response of muscle cells by turning off many muscle-specific genes.

The expression of many muscle proteins is markedly affected by the presence of the motor nerve. This is perhaps best exemplified by the series of prominent changes that occur in muscle fibers as a result of severing or crushing the motor axons that innervate a particular muscle. In addition to changes in the levels of expression of metabolic enzymes and contractile proteins, and to overall atrophy of the muscle fiber, denervation of skeletal muscle leads to significant changes in the levels of synaptic proteins. However, expression of AChR is greatly enhanced shortly after removal of the influence of the nerve. Numerous studies have shown that this increased AChR expression and loss of AChR protein compartmentalization at the junction results from transcriptional activation of the genes encoding AChR in myonuclei located in extrasynaptic regions of muscle fibers. Although the identity of the specific signaling systems involved in this dramatic up-regulation are still being elucidated, converging lines of evidence implicate protein kinase C as well as members of the MyoD family of transcriptional regulatory factors.

Denervation also affects expression of ACHE. In muscles of rodents, expression of AChE is, in general, markedly decreased following removal of the nerve, including reduction in the amount of synaptic (asymmetric) AChE forms. Although in the case of AChE the mechanisms that lead to this reduction in enzyme levels are largely unknown, they likely involve changes at various stages of the biosynthetic pathway including changes at the transcriptional, post-transcriptional levels, and even degradation and/or removal from the synaptic basal lamina.

Following injury, axons of the peripheral nervous system of essentially all species, as well as those from the central nervous system of lower vertebrates show evidence of new nerve outgrowths. This neural regeneration in many cases eventually leads to the successful reinnervation of target cells. Typically, regrowth of severed axons begins after a short delay of one or two days and proceeds at a rate of a few millimeters per day. Nerve regeneration and reinnervation is usually more successful in cases where the nerve was crushed rather than cut. This may be attributed to the fact that under these circumstances, axons can regrow through the basement membrane and sheaths of collagen left intact. Following nerve regrowth, denervated muscle target cells typically become reinnervated at sites along the surface of the sarcolemma where original neuromuscular junctions were found. In these studies, the influence of the muscle on reinnervation was eliminated by causing the existing fibers to degenerate; therefore, cues to guide reinnervation by the motor neurons must have been provided by components of the synaptic basal lamina that survive the muscle degeneration procedure.

In response to a variety of damaging insults, skeletal muscle fibers can undergo degeneration followed by a period of regeneration. This ability of muscle cells is crucial to ensure a relatively stable muscle mass and may in fact be an ongoing remodeling process through which muscle cells can meet the demands resulting from normal physiological activities. Following exposure to stimuli such as periods of ischemia, mechanical or chemical traumas, and even certain types of exercise, muscle fibers can degenerate. This degeneration is characterized typically by necrosis and subsequent phagocytosis of the muscle fibers and cellular debris. Subsequent regeneration will be successful only if the following conditions are met: i) revascularization occurs, ii) muscle satellite cells are activated, and iii) the basal lamina persists following trauma to form a sheath in which new developing muscle fibers can be instructed to differentiate. Although functional innervation is not essential for the regeneration of muscle fibers, the complete recovery of muscle fibers from an insult causing degeneration depends critically on reinnervation and subsequent

muscle activation. Numerous investigations have shown that the sequence of cellular and molecular events that accompanies muscle fiber regeneration loosely resembles that of muscle fiber ontogeny. However, in studies where the nerve, muscle fiber, and nearby cells (e.g., Schwann cells) were selectively destroyed by freezing while the basal lamina was kept intact, the former post-synaptic site ofinnervation showed specific expression and accumulation of AChR protein and mRNA upon regeneration of the muscle fiber beneath the basal lamina without the presence of a nerve. Thus, although the presence of the motor nerve exerts a profound modulatory effect on expression of synaptic proteins such as AChR and AChE, components of the synaptic basal lamina participate in the development, persistence, and maintenance of the neuromuscular synapse and can actually induce the reorganization of the postsynaptic membrane.

Pathologies of the neuromuscular junction tend to be striking clinically because of the basic and important role the neuromuscular junction has in controlling movement. These pathologies can be grouped into two main classes: those that have a strong genetic component and are present at birth, and those which are acquired during the course of life. The etiologies of these diseases vary from genetic, autoimmune, and environmental, to infectious. The acquired disorders of the neuromuscular junction such as myasthenia gravis are discussed in the Neurobiology module. Very few cases of congenital motor-end plate disease are documented in the literature and are typically limited to case studies probably involving partial disorders because a complete deficiency of many of the many motor end plate proteins would probably be lethal to the developing embryo. Of the live births that do occur, most cases probably go undiagnosed because many of these patients probably die early on. Nonetheless, several cases of complete lack of important junctional components such as acetylcholinesterase exist probably because there are complimentary defects that balance or diminish the severity of the pathophysiology. The basis of many of these congenital neuromuscular transmission diseases have been defined more on clinical than on molecular levels. Thus, many of these case studies have strong clinical correlates such as electromyography, clinical symptoms such as muscle weakness, failure to thrive, fatigue, and pharmacological responses that suggest the site of the lesion. In addition, determinations of AChR or AChE levels and microscopic analyses of their motor endplate architecture to suggest possible mechanisms for the disorder have been performed on some patients. Abnormalities of the motor-endplate can be found at nearly every step of neuromuscular signal transmission. Cases of presynaptic defects include patients with decreased size and decreased number of synaptic vesicles. At the synaptic cleft, patients have been found with a complete lack of junctional acetylcholinesterase and

decreased number of junctional folds. Postsynaptically, the acetylcholine receptor can have defects that result in an ion channel that has increased conductance and decreased channel open time, only decreased channel open time, low conductance and increased channel open time, and defects that result in differential binding of known AChR antagonists such as d-tubocurarine. Recently, defects in the voltagedependent sodium channel have also been implicated in congenital forms of periodic paralysis. All of the previously mentioned congenital syndromes are believed to be due to structural defects or deficiencies in components of the motor end-plate. However, newborns can acquire maternal antibodies to the AChR through placental transfer from myasthenic mothers and can develop a transient myasthenic syndrome.

The neuromuscular junction is the complex and highly specialized region of nerve-muscle contact that serves as the conduit for nerve-muscle communication. The precisely choreographed series of cellular and molecular events serve to initiate, maintain, and allow persistence of this specialized synapse throughout the lifespan of the organism. A likely sequence of events in the development of the neuromuscular junction begins with the nerve terminals releasing agrin and neuregulins into the basal lamina. AChRs and other muscle synaptic proteins accumulate at the motor endplate during this initial period of nerve contact, along with reorganization of the cytoskeletal elements in the vicinity of the nuclei beneath the innervated region of muscle fiber membrane. The reorganized cytoskeleton and remodelled architecture of junctional folds in the synaptic muscle fiber membrane then serve as a scaffold for the recruitment of additional membrane proteins including those involved in binding extracellular matrix molecules. a-dystroglycan binds agrin and serves as an agrin receptor suggesting that the basal lamina is intimately associated structurally with the muscle cell membrane through the DAG complex, which is coupled through rapsyn with AChR. Agrin, a-dystroglycan, and the cytoskeletal dystrophin-associated glycoprotein complex might serve to initiate signaling through the agrin receptor MUSK; they might form the scaffold necessary for maintaining the accumulation of the AChR and other synaptic proteins as a structural and functional linchpin from the basal lamina to the muscle membrane to the cytosol and together might constitute part of the special synaptic domain known as the neuromuscular junction. Other molecules from the nerve, such as the neuregulins and CGRP, along with molecules from the muscle, such as s-laminin, HBGFs, and N-CAM, then serve as reciprocal signaling and inductive influences that strengthen the program for neuromuscular development. The biochemical signaling cascades regulating neuromuscular function and the developmental program for junction formation are diverse and include all of the known pathways such as tyrosine phosphorylation,

protein kinase activation, cAMP, and calcium. Secreted extracellular matrix proteins might serve as nucleation sites for the formation of a specialized synaptic basal lamina within which ACHE, agrin, HBGFs, and other specialized motor endplate molecules can accumulate and regulate neuromuscular activity. The highly selective concentration of numerous proteins at the neuromuscular junction, especially at the motor endplate, begs the question of regulation. For instance, how is it that the AChR and many other proteins become so highly concentrated to an area that represents less than 1% of the muscle cell surface? This finding leaves open several possible models for regulation at the junction: (i) the proteins or their transcripts might be preferentially de-stabilized at extrajunctional regions or (ii) they might be transported, anchored, stabilized, and/or expressed at the neuromuscular junction by fundamental myonuclei in nuclear domains. The latter possibility of selective transcription has been demonstrated at least for AChR. Many more synaptic proteins are probably under similar transcriptional control by fundamental nuclei at the motor endplate and along with other nerve-derived factors, the proteins are important for the development of the neuromuscular junction and await discovery. Most importantly, the formation of the neuromuscular junction is mediated by a series of inductive molecular handshakes between the nerve and the muscle that lead to committed differentiation into a mature synapse.

Buckingham, M. (1994). Molecular biology of muscle development. Cell 78, 15-21. Carbonetto, S. & Lindenbaum, M. (1995). The basement membrane at the neuromuscular junction: A synaptic mediatrix. Curr. Opin. Neurob. 5, 596--605. Duclert, A. & Changeux, J.-P. (1995). Acetylcholine receptor gene expression at the developing neuromuscular junction. Physiol. Rev. 75, 339-368. Fischbach, G.D. & Rosen, K.M. (1997). ARIA: A neuromuscular junction neuregulin. Ann. Rev. Neurosci. 20, 429-458. Glass, D.J., DeChiara, T.M., Stitt, T.N., DiStefano, P.S., Valenzuela, D.M., & Yancopoulos, G.D. (1995). The receptor tyrosine kinase MuSK is required for neuromuscular junction formation and is a functional receptor for agrin. Cold Spring Harb. Quant. Biol. 61,435--444. Hall, Z.W. & Sanes, J.R. (1993). Synaptic structure and development: The neuromuscularjunction. Cell 72/Neuron 10 (Suppl.), 99-121. Kleiman, R.J. & Reichardt, L.F. (1996). Testing the agrin hypothesis. Cell 85, 461-464. Nguyen, Q.T. & Lichtman, J.W. (1996). Mechanism of synapse disasembly at the developing neuromuscular junction. Curr. Opin. Neurob. 6, 104-112. Salpeter, M.M. (Ed.) (1987). The Vertebrate Neuromuscular Junction. Alan R. Liss, Inc., New York. Sanes, J.R. (1997). Genetic analysis of postsynaptic differentiation at the vertebrate neuromuscular junction. Curr. Opin. Neurob. 7, 93-100.

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Introduction What Are Somitomeres? The Discovery of Somitomeres How Somitomeres Are Visualized Animals in which Somitomeres Have Been Described Evolutionary Aspects of Somitomeres When Do Somitomeres Form and What Are Their Fates? When Do Somitomeres Form? All Somites Condense from Somitomeres The Fates of Somites Fates of the Cranial Somitomeres that Do not Condense into Somites The Ability of Somitomeres to Regulate When are Regional Differences Determined in Trunk Segments? The Influence of Somitomeres on Other Structures Relationships between Cranial Somitomeres and Brain Divisions and Rhombomeres Do Somitomeres Specify the Segmentation of the Rhombomeres? Somitomeres are Pathways for Neural Crest Migration Somitomeres and Somites Guide the Segmentation of the Peripheral Nervous System Segmental Vessels are Related to Somitomeres

Principles of Medical Biology, Volume 11 Developmental Biology, pages 209-228. Copyright 9 1998 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-816-1 209

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Molecular Expression Patterns Related to Somitomeres

Genes Expressed during Segmentation of Somitomeres and Somites Summary

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Vertebrate animals, including human beings, are segmented. Voluntary muscles, axial bones, and dermatomes, all of which derive from the somites, are arranged in segmental patterns that reflect their ,~rigin from the somites. Somites are now known to condense from earlier segmented units named somitomeres. The segmental patterning of spinal and most cranial nerves, of neuromeres in the central nervous system, and of parts of the vascular system appear to be secondarily related to the somitomeric segmental pattern. Somitomeres are morphological units of paraxial mesoderm, metamerically arranged to flank the prechordal plate and the notochord, and extending laterally precisely to the lateral limits of the prospective neural plate. They are bilaminar discs composed of mesenchymal cells whose cell bodies and processes are arranged in concentric circles, and the entire unit is often concave or convex. When just formed, somitomeres have the appearance of being expansion figures. Somitomeres form in pairs across the long axis, and these pairs are tandemly arranged along the axis. They are found in early embryos of all groups of vertebrates that have been examined. Somitomeres are formed during gastrulation and tail bud elongation, appearing in a cranial to caudal progression beginning at the tip of the head and forming last at the tip of the tail. Somitomeres condense to form epithelial somites in the occipital region of the head, in the trunk, and in the tail. From the head rostral to the ear placode, the somitomeres do not condense and do not form somites; rather they continue to expand as the brain expands. The somitomeres along the entire axis are the earliest visible segmental pattern and their presence in all of the head reveals that the entire head is initially segmented.

Stephen Meier accidentally discovered somitomeres in chick embryos when he examined with stereo scanning electron microscopy the field ofparaxial mesoderm that lies beneath the prospective otic placode. He saw a metameric arrangement of units of mesenchymal cells and their processes (the somitomeres as he named the units), which made him turn his attention to analysis of the entire paraxial head mesoderm and to the presomitic mesoderm of the trunk region (Meier, 1979).

Somitomeres were discovered only recently because their visualization requires special techniques. Somitomeres are best seen in stereo scanning electron micrographs (SEMs). The somitomeric pattern is revealed in the positions of the cells, the array of their cellular processes, and the convex or concave three-dimensional structure of the whole unit, as well as the consistent relationship between the positions of somitomeres and the neuromeric bulges and furrows of the adjacent neural plate or tube. Somitomeres always appear in pairs to each side of the notochord. Specimens must be prepared for SEM in a manner that allows dissection to remove the overlying ectoderm or underlying endoderm, and that washes away the extracellular matrix that would otherwise obscure the pattern ofmesenchymal cells and of their processes that compose the somitomeres. Specimens are first fixed in halfstrength Karnovsky's glutaraldehyde/formaldehyde fixative, then dissected to remove the overlying tissues and washed in cacodylate buffer to remove the extracellular matrix among the somitomeric cells. After postfixation in osmium tetroxide, the specimens are dehydrated, sputtercoated, and examined in stereo (Meier, 1980).

Somitomeres have been described in representatives of six classes of vertebrates, namely cartilagenous fish, bony fishes, amphibia, reptiles, birds, and mammals. The forms chosen to represent these classes were mostly common laboratory animals, namely, they were, for mammals, the common lab mouse (Mus musculus) and rat (Rattus norvegicus); for birds, the chick (Gallus domesticus) and quail (Coturnix coturnixjaponica); for reptiles, a snapping turtle (Chyledra serpentina); for amphibians, a newt (Taricha torosa) and a frog (Xenopus laevis); for bony fish a teleost, the Japanese medaka (Orizias latipes); and for cartilagenous fish, the dogfish shark(Squalus acanthias). References for each species, about 30 papers, are listed in reviews by Jacobson and Meier (1986), Jacobson (1993), and Tam and Trainor (1994).

The somitomeres in amniotes are similar in appearance, but in amphibia, teleosts, and sharks, somitomeres initially appear more condensed. Studies of gastrula-stage embryos of lampreys and myxines are not available, but in postneurula stages, the entire paraxial mesoderm is segmented like somites (reviewed in Jefferies, 1986). Several people have described the segmentation of the the entire head mesoderm in shark embryos using ordinary light microscopy (Balfour, 1878, 1881; de Beer, 1922; Goodrich, 1930). These primitive forms have condensed any somitomeres they have in the head into somites.

One gets the impression that the mesenchymal somitomeres cranial to the otocyst in amniotes have failed to condense as the brain greatly expands in those regions and the somitomeres expand with it. Brains of teleosts and amphibia expand moderately, but less than those of amniotes, and cranial somitomeres are correspondingly less expanded. Somitomeres initially segment the heads of all the forms examined. Additional evolutionary aspects of somitomeres are discussed in Jacobson (1993).

In all the species examined, somitomeres form during gastrulation, and they form in a strict cranial to caudal order. In amniotes that use a primitive streak to gastrulate, somitomeres make their first appearance at the upper end of the primitive streak adjacent to Hensen's node (the primitive node of mammals) while the streak is forming and elongating. Gastrulation continues as the streak regresses, and neurulation occurs at the cranial end of the embryo in the regions already laid down by the streak. Therefore, in amniotes, one end of the embryo may be gastrulating while the other end is neurulating. The cells that form the somitomeres ingress from the adjacent streak, which in turn is supplied with cells converging from the epiblast (Packard, 1986a; Bellairs, 1986). Somitomeres thus arise from epiblast and streak cells. Experimental studies reveal that there is a prepattern for segmentation in the epiblast and primitive streak (Meier and Jacobson, 1982). Triplett and Meier (1982) looked for the first appearance of somitomeres during the earliest period of gastrulation in the chick embryo. Using stereo SEM, they found that the most cranial pair of somitomeres is formed during primitive streak formation while the streak is elongating, before reaching the "definitive streak stage." The prechordal plate, the axial mesoderm at the midline that is rostral to the forming streak and is rostral to the notochord when it forms, is also formed during this early period of primitive streak formation. The prechordal plate is an unpaired circular domain of mesenchymal cells that looks much like a somitomere at this time. Figure 1 (stage 3+) illustrates the locations of these first somitomeres and the prechordal plate. When the first pair of somitomeres has formed, the second pair is in the process of forming at a position just caudal to the first pair. Shortly before the streak reaches its fullest extension and is poised to begin to regress, the second pair of somitomeres has formed (Figure 1, stage 4). The streak and node then begin regression in a caudal direction. As the streak regresses, the third pair of somitomeres form slightly

caudal to Hensen's node at a location Spratt (1955) called the "somite forming center" because somites were disrupted if he disrupted that position. Cells ingressing from the node lay down a trail of cells between the second pair of somitomeres. These cells compose the first unit of the notochord (Figure 1, stage 5). This process is reiterated as the streak regresses, laying down successive somitomere pairs while the node places notochordal units between the penultimate pair of somitomeres. The notochord looks segmental for a time after the node has composed it, but this appearance is soon lost. The underside of the third somitomere is illustrated in Figure 2. Before the first somite condenses from a somitomere, the whole length of the paraxial mesoderm of the embryo is segmented into somitomeres. For example, in a

newt embryo, when the first somite appears, the embryo has a total of 18 somitomeres and the tail bud is actively adding additional ones (Jacobson, 1993). The first somite seen in a newt arises from the seventh somitomere (numbers begin with the most cranial somitomere). Xenopusalso forms first the seventh segment into a somite. The seventh is the most-cranial trunk somite in amphibia. After condensation of the seventh somitomere pair into somites, condensation of somitomeres six and five occur consecutively (forming the occipital somites), followed by somitomeres eight, nine, and so forth on down the trunk. In amniotes, the eighth somitomere condenses to form the first occipital somite, and additional sornites condense caudally in order. Figure 1 (stage 9) illustrates the appearance of segments in a chick embryo after a few somites have formed. Somitomeres lying cranial to the first somite expand as the brain area expands and never normally condense into somites. The somitomeres caudal to the formed somite files are in what has been called the segmental plate in birds, or the presomitic mesoderm in mammals. The regressing primitive streak is at the caudal end of the segmental plate. The streak adds new somitomeres to the caudal end of the segmental plate and these somitomeres progressively condense into somites, so new somites form from the most cranial somitomeres of the segmental plates as new somitomeres are added at the caudal end (Meier, 1979).

Meier (1979) has described in the chick embryo the progressive condensation and epithelialization of segmental plate somitomeres into somites, as have others (Cheney and Lash, 1984; Lash, 1985). Meier measured the changing diameters of the somitomeres as they progressively condense into somites in the segmental plate. They condense from 175-185 ~m in diameter when first formed beside the regressing Hensen's node to 85-95 jam at the cranial end of the segmental plate just before converting into a somite. The newly-formed somites are 95-105 ~m in diameter. During somitomeric epithelialization, the interface between the lamina that compose a somitomere enlarges into a cavity that becomes the myocoel of the somite. Cells of the epithelial walls of the newly-formed somite have their apical ends toward the myocoel and their basal ends toward the surface of the somite.

All somites are condensed from somitomeres. In the amniotes, the most cranial somites and the first to form are condensed from the eighth pair of somitomeres, which are located just caudal to the otocyst (prospective inner ear mechanism). The ninth and all remaining somites condense from somitomeres in tandem cranial--caudal progression. After the first few somites become apparent, regions both cranial and caudal to the somite files remain as somitomeres. Cranially, amniotes and teleosts have seven subotic and preotic somitomeres. Amphibians have just four (Figure 3). Caudally, between the last-formed somite and the anterior end of the regressing primitive streak, somitomeres compose the segmental plate of the chick or the presomitic mesoderm of the mammal. Chick and quail embryos have 10-12 somitomere pairs in their segmental plates, the medaka (a teleost) has 10, the mouse and turtle have six to seven, and the newt and Xenopushave five to seven (Jacobson, 1993, Tam & Trainor, 1994). Segmental plates are dynamic structures from which somites condense at their cranial ends as new somitomeres are added to their caudal ends. The segmental plate of an older chick embryo is more than twice as long as in a young embryo. Despite this considerable variation in length during development, young and old segmental plates nevertheless produce the same numbers of somites (Packard and Jacobson, 1976). Several sorts of experimental evidence confirm that somites arise seriatim from the somitomeres of the segmental plate. Explanted chick segmental plates were cut transversely between the last-formed somite and the segmental plate, and just anterior to the node (so no more somitomeres could be deposited in the explant). These explants formed, after culturing, a series of 11-12 somites (Packard and Jacobson, 1976). Meier (1979) then found that the same number of somitomeres could be visualized in the segmental plate. Packard (1978) cut the segmental plate into rostral and caudal parts, and each part formed the number of somites it would have formed without the cuts. Packard and Meier (1983) prepared two explants from the chick segmen-

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Figure 3. This drawing illustrates the positions of somitomeres, brain parts, and rhombomeres in an amphibian embryo (left) and an amniote embryo (right). The positions of the amniote rhombomeres are taken from Lumsden and Keynes (1989), and the correlated positions of the amniote somitomeres from Jacobson (1993). The positions for the amphibian somitomeres and rhombomeres are from my observations of newt and Xenopus embryos and from Jacobson and Meier (1984). The positions for the prospective otocysts ("ears") are deduced from the positions of the preotic and postotic streams of neural crest illustrated in Jacobson (1993) for both amniotes and amphibians. Amphibian brains are relatively shorter than amniote brains, so the newt side of the diagram has been stretched to bring the brain parts into register. tal plate region by making a transverse cut across the embryo, separating both segmental plates from the last-formed somites, and at the caudal end they separated the segmental plates from the primitive streak and node and then slit down the midline to make two equivalent explants. One explant was immediately examined with stereo SEM to visualize the total number of somitomeres in the segmental plate. The other, equivalent, segmental plate was cultured until several somites were formed, it was then examined with stereo SEM, and the total number of somites plus somitomeres in that half was determined. The number of segmental units in each half was found to match closely. Similar results were obtained with turtle embryos (Packard and Meier, 1984) and with mouse embryos (Tam et al., 1982). The conclusion must be that somites arise from somitomeres, a conclusion also implicit in the observations made by Meier (1979) and others who found that the somitomeres gradually epithelialize and condense into somites.

More somitomeres form in the tail than become condensed into somites. This cessation of condensation of somites appears, in chick embryos, to result from the caudal-most somitomeres succumbing to apoptosis (Bellairs, 1986). The lengths or the absence of the tail in various animals may result from this mechanism.

Newly-formed somites are epithelial balls surrounding a cavity, the myocoel. Many textbooks describe the gradual conversion of the epithelial somite to mesenchyme. The medio-ventral aspect of the epithelial ball converts to a mesenchymal sclerotome, which gives rise to segmentally arranged axial bones. The remaining epithelial dermamyotome subdivides and produces myotomal mesenchymal cells, which become the myoblasts that fuse to compose the muscle fibers of segmental voluntary muscles. The dermatome produces mesenchymal cells that contribute to the dermis.

Rather than condensing into somites, the cranial preotic and subotic somitomeres remain mesenchymal and expand along with the enlarging brain parts. Several studies reveal the eventual fates of these somitomeres. They make many of the same structures that somites form more posteriorly. These somitomeres form all the voluntary muscles of the head (except for three of the extrinsic eye muscles that form from the prechordal plate), as well as some bones (otic capsule, corpus sphenoidalis, and orbitosphenoid). They contribute to periocular mesenchyme, branchial arches one through four, and to mesencephalic and metencephalic meninges. The distribution pattern of the mesenchyme from the cranial somitomeres is closely followed by the associated neural crest. There are conflicting reports as to whether the somitomeres contribute to the dermis in the head. The fates of these somitomeres were determined using transplants from quail embryos to chick embryos, and then by seeing where the cells with the quail nucleolar marker were located (Noden, 1983; Couly et al., 1992). In mouse embryos, the fates were determined by using cell grafting and fluorescent cell labeling techniques (Trainor et al., 1994). Trainor and colleagues present the results of all three studies in a table. These authors found that each somitomere contributes parts mainly to the region in which it is situated, but some mesenchyme cells from each somitomere also contribute to the domains of adjacent somitomeres. The neural crest associated with each somitomere is distributed in a similar pattern. There is experimental evidence that these cranial somitomeres can condense into somites rather than continue expanding if they are denied their association with the expanding brain (Meier and Jacobson, 1982).

Two sorts of experimental evidence indicate that somitomeres may regulate to restore the normal segmental order after disturbance. In bird embryos, the somitomeres in the segmental plate may be disrupted by stirring the cells of the segmental plate with glass needles (Menkes and Sandor, 1977). Within three hours the segmental plates reformed and somites later formed from the segmental plate with only a few abnormalities. Sandor (1972) removed prospective intersomitic borders in segmental plates of explanted chick embryos and found later that somites appeared in their usual order. The somitomeres appear to be able to sort themselves out following such disruption or partial deletion and to condense to form mostly normal somites. A second indication of ability of the segmental plate to regulate comes from experiments by Packard (1986b). He transplanted quail segmental plates in place of segmental plates in chick embryos and found that the number of somites formed approximated the number expected of the host embryo, as seen on the unoperated side. Underlying somitomeres may impose a covert pattern of segmentation in the prospective neural plate, which may in turn facilitate regulation of disturbed files of somitomeres, directing their return to a normal number. Together with the observations in the section above, these experiments suggest that normally each somitomere in the segmental plate eventually transforms into a somite, but the somitomeres in the segmental plate still have some labile properties and can regulate to disturbances.

There are many regional differences among the final differentiated parts of the trunk segments. When is regional character determined in paraxial mesoderm of the trunk? Experimental evidence suggests that some regional differences are already specified in the somitomere stage and others are not. Thoracic segments make ribs whereas cervical segments usually do not. If a portion of cervical segmental plate is removed and replaced with a piece of thoracic segmental plate, then the somitomeres of the implant form somites that differentiate vertebrae with ribs in that inappropriate axial position (Kieny et al., 1972). The axial skeletal parts are thus determined by the somitomere stage. The same is true of parts that form from the dermatome. The plumage pattern of a chick is already specified in the somitomeres (Mauger, 1972). Grafting segmental plate to foreign levels of the axis, however, does not change the nature of muscles that arise from myotomes. Rather, the muscles that form are appropriate for the new location, so myotome cells have their regional fates determined at some later stage (Chevallier et al., 1977).

In all the species examined, there are consistent relationships between somitomeres and forebrain, midbrain, and rhombomeres in the hindbrain. In all the species, the first somitomere is associated with the forebrain. In teleosts and the amniotes, the second somitomere is associated with the first mesomere of the midbrain, the third somitomere with the second mesomere of the midbrain, and the fourth somitomere with the first rhombomere (the metencephalon). The next six rhombomeres appear first as three large rhombomeres in direct apposition to the next three somitomeres (somitomeres five through seven). These large rhombomeres subdivide to form two rhombomeres each (Chick: K~ill6n, 1953; Mouse: Jacobson and Tam, 1982) (Figure 3).

Somitomeres are morphologically visible at gastrulation well before rhombomeres form. Rhombomeres are first visible at neural tube stages in birds and amphibia, but at neural plate stages in mammals. Because just-formed somitomeres extend laterally precisely to the prospective boundary between the future neural plate and the nonneural ectoderm and have consistent relationships with brain parts and rhombomeres, it seems most probable that somitomeres impress their segmental pattern upon the neural plate. A less probable alternative is that a covert segmentation pattern in the neural plate impresses its pattern on the epiblast and primitive streak cells that form the paraxial mesoderm containing the somitomeres. The timing and arrangement of parts seem quite wrong for this possibility. A third possibility is that common or separate segmentation mechanisms determine the segmentation patterns in both tissues. Other alternatives may exist as well. Considering the early timing of morphological appearance of somitomeres and the role of somitomeres and somites in patterning the peripheral nervous system, it seems most probable that somitomeres represent the primordial segmentation, and that they pattern the other tissues including the rhombomeres. Rhombomeres have a complicated history of emergence. When they first form, they are adjacent to somitomeres, but most rhombomeres then divide again, and when they do the boundaries between the daughter rhombomeres coincide in position with the rows of cell processes that divide nearly all somitomeres into rostral and caudal parts. In amniotes, the rhombomeres that subdivide are the ones that form rhombomeres 2-3,4-5, and 6-7. Rhombomere one is next to somitomere four, and rhom-

bomere "eight" is indistinct and is associated with all four occipital somites (Figure 3, fight side). The ear (otocyst) of amniotes lies between rhombomeres five and six. In the mesencephalon, a somitomere is adjacent to each of the mesomeres. In amphibia, such as the newt and Xenopus, both the rhombomeres and the somitomeres are in an order different from those of amniotes. The entire mesencephalon lies adjacent to somitomere two. The first and second rhombomeres are next to somitomere three, and the third and fourth rhombomeres next to somitomere four. The otocyst (ear) lies between rhombomeres three and four. The amphibia have but two occipital somites and they lie next to rhombomeres 5-6 and 7-8 (Figure 3, left side). These relationships were obtained from files of scanning electron micrographs used for the study of somitomeres in the newt, Taricha torosa (Jacobson and Meier, 1984). Observations from unpublished SEM photos and from stained and dissected embryos indicated that the pattern in Xenopus was similar to the pattern in the newt. The brains of amphibia are much foreshortened compared to those of amniotes, and in Figure 3, the amphibian side has been stretched to match levels with the longer amniote brain. Using quail--chick transplants to identify cell lineages, Couly and Le Douarin (1990) defined segmentally arranged domains of epidermal ectoderm that they called "ectomeres" in the heads of chick embryos. These ectodermal units are in register with neuromeres and with somitomeres. Their segmentation is most likely imposed by somitomeres.

Cranial neural crest migrates in sheets and makes large contributions to the head, face, and branchial arches (Hall and H/Srstadius, 1988). The prosencephalon contributes crest cells only from its caudal border. The mesencephalon contributes large amounts of crest cells to the head, and the rhombencephalon contributes somewhat lesser amounts. Amniote embryos, represented by the chick (Anderson and Meier, 1981) and turtle (Meier and Packard, 1984) have similar patterns of migration of the cranial neural crest over the cranial somitomeres. Crest cells from the mesencephalon and caudal prosencephalon migrate laterally, eventually covering somitomeres two and three, while somitomere one becomes extensively overlain by crest cells that migrate rostrally from these same sources. The rhombencephalic crest covers somitomeres four and seven through eleven, but gaps are left over most of somitomere five and over somitomere six and part of seven. These gaps coincide with the future positions of the fifth cranial ganglion and the otocyst. A preotic crest stream moves laterally between somitomeres five and six, and a postotic crest stream migrates over most of somitomere seven. This same pattern of cranial neural crest migration is seen in amphibians, represented by a newt (Jacobson and Meier, 1984), but different somitomeres are the pathways of migration. Amphibians have fewer somitomeres in the head than do

amniotes. The gap associated with the fifth ganglion is over somitomere three, and the otic gap is over somitomere four. The preotic stream of crest moves laterally between somitomeres three and four; the postotic stream migrates between somitomeres four and five. The chick, turtle, and newt patterns are illustrated and compared in Jacobson (1993).

Spinal nerves are segmentally arranged, reflecting the segmentation of the somites. Early in this century, studies by Lehmann (1927) and Detwiler (1934)established that the segmental pattern of spinal nerves was imposed by the somites. Spinal nerves have two components; they are a sensory dorsal root and ganglion and a motor ventral root. The dorsal root ganglia are of neural crest origin, and the nerve cell bodies of the ventral roots are located in the basal plate of the spinal cord. Ventral roots emerge from the spinal cord only into the rostral portion of the sclerotome of the somite and that fact gives them their segmental pattern (Keynes and Stern, 1984). Surgical manipulations reversing somite files or portions of the spinal cord, or that created rows either of caudal halves of somites or of rostral halves, clearly demonstrated that ventral roots may emerge anywhere along the spinal cord, but the normal alternation of caudal and rostral half sclerotomes always restricts the ventral root to emerge in the rostral half unit (reviewed in Keynes and Stern, 1986). The migration of neural crest cells is also restricted to the rostral halves of sclerotomes, accounting for the segmental arrangement of the dorsal root ganglia. The neural crest cells that compose these ganglia arise above both rostral and caudal halves of somites. Neural crest cells adjacent to rostral halves of sclerotomes migrate directly into those halves and compose much of each ganglion, but the neural crest cells above caudal half segments migrate axially until adjacent to rostral halves of the somite; then they turn laterally to enter the rostral half segment and contribute to the dorsal root ganglia (Teillet et al., 1987). The existence of rostral and caudal domains in the sclerotomes of somites was seen morphologically a long time ago. As a somite matures, its sclerotome becomes separated into rostral and caudal parts by a deep fissure called von Ebner's fissure, which is as prominent as the clefts that separate somites. These fissures appear at the same levels as the transverse rows of cell processes that subdivided the progenitor somitomeres into rostral and caudal domains. Von Ebner's fissures are nicely illustrated in Stern and Keynes (1986; 1987). As noted above, some rhombomeres also subdivide along lines in register with the transverse rows of processes in cranial somitomeres. The first rhombomere is the future metencephalon and it is associated with somitomere four in amniotes. The second through eighth rhombomeres are parts of the myelencephalon. The fifth, sixth, and seventh somitomeres are each associated with two rhombomeres

that had subdivided. The even-numbered rhombomeres are opposite the rostral domain of the associated somitomeres, and the odd-numbered rhombomeres are opposite the caudal domains (Figure 3, right side). Motor roots of cranial nerves V, VII, and IX emerge from the even-numbered rhombomeres into the rostral half segments of somitomeres; V into somitomere five, VII into six, and IX into seven. The "eighth rhombomere" is poorly defined, and motor roots of cranial nerves X, XI, and XII appear to emerge near the rostral ends of the four occipital somites associated with rhombomere eight. The ganglia of nerves that have them are also associated with the even-numbered rhombomeres and are thus next to the rostral parts of their associated somitomeres. The disposition of these nerve roots were determined by and diagrammed by Lumsden and Keynes (1989), and the associations with somitomeres was illustrated in Jacobson (1993). It appears that the arrangement of many cranial nerves may be imposed by the associated rostral portions of the somitomeres just like the arrangement of spinal nerves in relationship to trunk somites. This conclusion is reinforced by the observation that the rather different numbers of cranial somitomeres seen in amphibian embryos matches both the different arrangement of rhombomeres (Figure 3, left side) and the different grouping of emerging cranial nerves found in these animals. Segmental Vessels are Related to Somitomeres In chick embryos, the dorsal aortae send intersegmental branches between successive somitomeres. England (1986) used several means to illustrate these precocious segmental vessels in the segmental plate. England viewed the intersegmental vascular branches between the somitomeres with SEM. They were located on the medial-ventral surface of the segmental plate (see her Figure 5). Injections of ink into the vascular system also revealed the presence of intersegmental arteries in the segmental plate (her Figure 6). The paraxial mesodermal cells that lie in the crotches between successive somitomeres are a likely source of the vascular endothelial cells that compose the intersegmental vessels in the segmental plate. Meier (1980) examined vascularization mainly of the dorsal surface of the mesoderm in early chick embryos. On the dorsal surface, one sees the posterior cardinal vein (overlying the intermediate mesoderm) extending branches between the somites, and also into intersegmental grooves that extend laterally across the lateral plate surface (see Meier's Figure 14). In the somitomere region of the anterior segmental plate, Meier (1980) illustrates cords of vascular cells protruding into incipient clefts between adjacent somitomeres. These prospective intersegmental veins protrude at fight angles from a condensing network of vascular tissue on the dorsal surface of the intermediate mesoderm. In the trunk, somitomeres condense into somites before their surfaces become vascularized, but the upper surfaces of some cranial somitomeres of the mouse embryo become covered with an endothelial vascular network the pattern of which

beautifully mimics the cellular pattern of the underlying somitomere (Meier and Tam, 1982; their Figure 22).

Segmentation of the paraxial mesoderm into somitomeres, the conversion of somitomeres to somites, and the transfer of the somitomere and somite segmental plan onto nervous system components must involve a number of molecular patterning events. Portions of this series of molecular events are becoming known. Some mo -~ lecular expression patterns clearly coincide with the pattern of somitomeres previously seen primarily with stereo SEM.

Members of the conserved Notch family of proteins are transmembrane receptors. They are necessary for many cell-fate decisions in Drosophila as well as for boundary decisions and transitions to, and maintenance of, the epithelial state (references in Conlon et al., 1995; Jen et al., 1997). Delta is one of the ligands for the Notch receptor proteins. Suppressor of Hairless (a transcription factor) is a part of the Notch signaling pathway. Expression patterns of vertebrate homologues of these genes appear to have a role in the segmentation of the paraxial mesoderm. The Delta-related mouse gene DLL1 is expressed during gastrulation of the mouse in the presomitic (somitomeric) mesoderm and down-regulates to persist only in the rostral portions of forming somites (Bettenhausen et al., 1995). A murine homolog of the Drosophila Notch gene, Notch1, has been shown, when mutated, to result in delayed and disorganized somite formation (Conlon, et al., 1995). Both Notch- 1 and Notch-2 are expressed in the mouse presomitic mesoderm. Jen et al. (1997) have isolated from Xenopus embryos a new form of Delta called XDelta-2. X-Delta-2 is expressed in the segmental plate but not in the somites. In the segmental plate, X-Delta-2 is expressed in a segmental pattern as seen in somitomeres. In caudal regions, X-Delta-2 is expressed throughout the somitomere with greater expression in the rostral region of each somitomere, but cranial toward the last-formed somite, X-Delta-2 down-regulates to narrow its expression to the rostral border of the morecranial somitomeres within the segmental plate. The expression pattern of X-Delta-2 defines the pattern of somitomeres previously seen primarily with stereo SEM. RNA of an antimorph made to X-Delta-2 was injected into blastomeres on one side of the embryo where it caused segmentation defects on the injected side. X-Delta-2 is likely to be a ligand for Notch-related receptors. Jen et al. found that X-Notch-1 was expressed at the same time as X-Delta-2, but thatX-Notch-I was expressed in both the segmental plate and the somites.

Jen et al. generated a DNA-binding mutant of Xenopus Suppressor of Hairless (XSu(H) DBM)and injected synthetic RNA of the mutant into the marginal zone of two-cell stage embryos. The result was that clear segmental boundaries did not form. Jen et. al. conclude that the Notch signaling pathway (X-Delta-2, X-Notch-I, and XSu(H)) is required for the organization of the presomitic mesoderm into a segmental somite pattern. It is interesting that following heat shock of Xenopus embryos at late gastrula stages, five sornites form normally, then subsequent somites are disrupted (Elsdale et al., 1976; Elsdale and Davidson, 1986). Because Xenopus has five to seven somitomeres in its segmental plate, the heat shock appears to be affecting possibly just the most recently formed somitomere and the process of forming somitomeres from the streak. Similar experiments with heat shock have been done with chick embryos leading to similar conclusions (Veini and Bellairs, 1986). Besides the above noted gene expressions, which probably are rather directly involved in the segmentation processes, there are other gene expression patterns in the axial mesoderm whose roles in patterning the paraxial mesoderm are not yet certain. Tam and Trainor (1994) illustrate the general nature of the expression patterns of 34 genes in the epiblast and primitive streak of a mouse (see their Figure 6). Since somitomeres arise from these tissues (Packard, 1986a; Bellairs, 1986), some or all of these genes may have some role in somitomere origin. Tam and Trainor (1994) also illustrate general expression patterns of 32 genes and gene products in mouse presomitic mesoderm (in somitomeres) and in the recently formed somites cranial to the presomitic mesoderm (see their Figure 12). These genes and products may have roles in the conversion of mesenchymal somitomeres into epithelial somites, or in the specification of somitomeres. While the expression of many candidate genes and the distribution of gene products has been noted, the complete interacting network of gene activities that is responsible for sornitomere specification, segmentation, conversion to sornites, acquisition of regional characteristics, regulative abilities, signals that affect segmentation of spinal nerves and segmental vessels, and so forth will have to emerge from future work. Sets of somites at different cranial-caudal levels are defined by expression patterns of homeobox-containing genes (Hox genes), and gain-of-function experiments show that some Hox genes are involved in segment specification (see pp. 293-294 in Tam and Trainor for a review and references). Tam and Trainor (p.293) state; "Expression of the Hox genes in specific somite groups begins with their somitomeres in the presomitic mesoderm and persists until the stage of chondrification of the prevertebrae."

The initial segmentation of the vertebrate body is into somitomeres. These segments are at first circular expansion figures of mesenchymal cells in the paraxial

mesoderm. They are bilayered with similar dorsal and ventral faces and form during gastrulation in tandem order from the tip of the head to the tip of the tail. In amniotes, somitomeres arise from epiblast and primitive streak cells that may be covertly segmented before being composed into paraxial mesoderm. The first seven cranial somitomeres of amniotes and teleosts extend from the tip of the head to beneath the otic vesicle, and these never condense into somites but rather expand along with the brain. In amphibia and sharks, the first four cranial somitomeres extend from the tip of the head to beneath the otic vesicle. As a rule, after the first somitomere, which is associated with the forebrain in all vertebrates examined, the succeeding somitomeres in the amphibia include only one somitomere where the amniotes and teleosts have two. In all forms, the somitomeres caudal to the otic vesicle eventually condense into somites. The somites are epithelial when first formed, then they sequentially revert to mesenchyme that forms segmental bones from the sclerotome and voluntary muscles from the mesenchymal myotomes, and finally they contribute cells to dermis from the mesenchymal dermatomes. The somitomeres of the more cranial parts of the head that do not condense into somites nevertheless form similar structures in the head that somites form in the caudal parts of the head, the trunk, and the tail. Segmental patterns seen in the vasculature, axial bones, spinal and cranial nerves, and central nervous system in the pattern of brain parts and rhombomeres are a reflection of the original somitomeric pattern. Somitomeres and their descendents, the somites, appear to impress these patterns upon the other segmented parts. Many of the regional differences seen in descendent cells of the somites, for example in bones and dermis, are already established in the progenitor somitomeres. The regional differences in voluntary muscles is an exception. Myoblasts from cranial somitomeres get their regional character from the local connective tissue they associate with, and this connective tissue originates from the neural crest (Noden, 1983). Myoblasts from myotomes of somites also get their regional designation from local connective tissue with which they associate, but this connective tissue arises from various local mesodermal sources. Recently formed somitomeres are distinct segmental units, but they are not firmly fixed in their fates. The mesenchymal cells that compose them exchange to some extent with adjacent somitomeres. Also the pattern may regulate and restore itself if the somitomeric file is disrupted. If somitomeres of a chick are replaced with somitomeres of a quail, the file will regulate to the size and number of host somitomeres. Signals from tissues adjacent to the somitomeres, such as notochord, neural plate, and neural tube may serve to direct this regulative ability. Somitomeres are pathways for neural crest migration. The cranial somitomeres that never condense, together with their associated neural crest, populate many areas of the head and the branchial arches in quite similar patterns. Many gene expression patterns have been seen in somitomeres, somites, and adjacent tissues. So far, the most interesting genes are a receptor gene, X-Notch-1, one of its ligands, X-Delta-2, and a downstream gene related to the Drosophila Suppres-

sor of Hairless, which may be part of the signaling pathway for the receptor-ligand signals. X-Delta-2 is expressed in the Xenopus embryo in the somitomeres of the segmental plate in a way that suggests its product has a role in the establishment of boundaries between future somites. It seems clear that the formation of somitomeres at gastrulation is a prelude to the segmental patterns that appear in much of the embryo at later stages, and these later patterns may be disrupted by events that disrupt the patterning and conversions of the somitomeres.

Anderson, C.L. & Meier, S. (1981). The influence of the metameric pattern in the mesoderm on migration of cranial neural crest cells in the chick embryo. Dev. Biol. 85, 383-402. Balfour, F.M. (1878). A Monograph on the Development of Elasmobranch Fishes. J. Anat. & Physiol., 1876-1877, and 1878. Macmillan, London. Balfour, F.M. (1881). A Treatise on Comparative Embryology, Macmillan, London. Bellairs, R. (1986). The tailbud and cessation of segmentation in the chick embryo. In: Somites in Developing Embryos. (Bellairs, R., Ede, D.A. & Lash, J.W., Eds.) pp. 161-I 78. Plenum Press, New York. Bettenhausen, B., de Angelis, M.H., Simon, D., Gu6net, J.L. & Gossler, A. (1995). Transient and restricted expression during mouse embryogenesis of DLL1, a murine gene closely related to Drosphila Delta. Development 121, 2407-2418. Cheney, C.M. & Lash, J.W. (1984). An increase in cell--cell adhesion in the chick segmental plate results in a meristic pattern. J. Embryol. Exp. Morphol. 79, 1-10. Chevallier, A., Kieny, M. & Mauger, A. (1977). Limb-somite relationship: Origin of the limb musculature. J. Embryol. Exp. Morphol. 41,245-258. Conlon, R.A., Reaume, A.G. & Rossant, J. (1995). Notch1 is required for the coordinate segmentation of somites. Development 121, 1533-1545. Couly, G. & Le Douarin, N.M. (1990). Head morphogenesis in embryonic avian chimeras: Evidence for a segmental pattern in the ectoderm corresponding to the neuromeres. Development 108, 543-558. Couly, G.F., Coltey, P.M. & Le Douarin, N.M. (1992). The developmental fate of the cephalic mesoderm in quail--chick chimeras. Development 114, 1-15. de Beer, G.R. (1922). The segmentation of the head of Squalus acanthias. Quart. J. Micr. Sci. 66, 457-474. Detwiler, S.R. (1934). An experimental study of spinal nerve segmentation in Amblystoma with reference to the pleurisegmental contribution to the brachial plexus. J. Exp. Zool. 67, 395-441. Elsdale, T. & Davidson, D. (1986). Somitogenesis in the frog. In: Somites in Developing Embryos. (Bellairs, R., Ede, D.A. & Lash, J.W., Eds.) pp. 119-134. Plenum Press, New York. Elsdale, T., Pearson, M. & Whitehead, M. (1976). Abnormalities in somite segmentation following heat shock to Xenopus embryos. J. Embryol. Exp. Morphol. 35, 625--635. England, M.A. (1986). Aspects of somite formation in the early chick embryo. In: Somites in Developing Embryos. (Bellairs, R., Ede, D.A. & Lash, J.W., Eds.) pp. 47--60. Plenum Press, New York. Goodrich, E.S. (1930). Studies on the Structure and Development of Vertebrates. Macmillan, London. Reprint Dover, New York, 1958. Hall, B.K. & Htirstadius, S. (1988). The Neural Crest. Oxford Science Publications, London.

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Jacobson, A.G. (1993) Somitomeres: Mesodermal Segments of the Head and Trunk. In: The Skull, Vol. 1, 42-76. (Hanken, J. & Hall, B.K., Eds.) The University of Chicago Press, Chicago. Jacobson, A.G. & Meier, S.P. (1984). Morphogenesis of the head of a newt: Mesodermal segments, neuromeres, and distribution of neural crest. Dev. Biol. 106, 181-193. Jacobson, A.G. & Meier, S.P. (1986). Somitomeres: The primordial body segments. In: Somites in Developing Embryos. (Bellairs, R., Ede, D.A. & Lash, J.W., Eds.) pp. 1-16. Plenum Press, New York. Jacobson, A.G. & Tam, P.P.L. (1982). Cephalic neurulation in the mouse embryo analysed by SEM and morphometry. Anat. Rec. 203, 375-396. Jefferies, R.P.S. (1986). The Ancestry of the Vertebrates. British Museum (Natural History), London. Jen, W-C., Wettstein, D., Turner, D., Chitnis, A. & Kintner, C. (1997). The Notch ligand, X-Delta-2 mediates segmentation of the paraxial mesoderm in Xenopus embryos. Development 124, 1169-1178. Keynes, R.J. & Stem, C.D. (1984). Segmentation in the vertebrate nervous system. Nature 310, 786-789. Keynes, R.J. & Stem, C.D. (1986). Somites and neural development. In: Somites in Developing Embryos. (Bellairs, R., Ede, D.A. & Lash, J.W., Eds.) pp. 289-299. Plenum Press, New York. Kieny, M., Mauger, A. & Sengal, P. (1972). Early regionalization of the somite mesoderm studied by the development of the axial skeleton of the chick embryo. Dev. Biol. 28, 142-162. K/illtn, B. (1953). On the significance of the neuromeres and similar structures in vertebrate enbryos. J. Embryol. Exp. Morphol. 1,387-392. Lash, J.W. (1985). Somitogenesis: Investigations on the mechanisms of compaction in the presomatic mass and a possible role of fibronectin. In: Developmental Mechanisms: Normal and Abnormal. pp. 45-60. Alan R. Liss, New York. Lehmann, F.E. (1927). Further studies on the morphogenetic role of the somites in the development of the nervous system of amphibians. The differentiation and arrangement of the spinal ganglia in Pleurodeles waltli. J. Exp. Zool. 49, 93-129. Lumsden, A. & Keynes, R. (1989). Segmental patterns of neuronal development in the chick hindbrain. Nature 337, 424--428. Mauger, A. (1972). R61e du mtsoderme somitique dans le dtveloppement du plumage dorsal chez l'embryon de poulet. II. Rtgionalisation du mtsoderme plumigtne. J. Embryol. Exp. Morphol. 28, 343-366. Meier, S. (1979). Development of the chick embryo mesoblast: Formation of the embryonic axis and establishment of the metameric pattern. Dev. Biol. 73, 25-45. Meier, S. (1980). Development of the chick embryo mesoblast: Pronephros, lateral plate, and early vasculature. J. Embryol. Exp. Morph. 55, 291-306. Meier, S., and Jacobson, A.G. (1982). Experimental studies of the origin and expression of metameric pattern in the chick embryo. J. Exp. Zool. 219, 217-232. Meier, S. & Packard, D.S., Jr. (1984). Morphogenesis of the cranial segments and distribution of neural crest in the embryos of the snapping turtle, Chyledra serpentina. Dev. Biol. 102, 309-323. Meier, S. & Tam, P.P.L. (1982). Metameric pattern development in the embryonic axis of the mouse. I. Differentiation of the cranial segments. Differentiation 21, 95-108. Menkes, B. & Sandor, S. (1977). Somitogenesis: Regulation potencies, sequence determination and primordial interactions. In: Vertebrate Limb and Somite Morphogenesis. (Ede, D.A., Hinchliffe, J.R. & Balls, M., Eds.) pp. 405-419, Cambridge University Press, Cambridge. Noden, D.M. (1983). The role of the neural crest in patterning of avian cranial, skeletal, connective, and muscle tissues. Dev. Biol. 96, 144-165. Packard, D.S., Jr., (1978). Chick somite determination: The role of factors in young somites and the segmental plate. J. Exp. Zool. 203, 295-306. Packard, D.S. (1986a) The epiblast origin of avian somite cells. In: Somites in Developing Embryos. (Bellairs, R., Ede, D.A. & Lash, J.W., Eds.) pp. 37-45. Plenum Press, New York.

Packard, D.S., Jr., (1986b). Change in somite number following interspecific grafts of avian segmental plate mesoderm. In: Progress in Developmental Biology, Part A, (Slavkin, H.C., Ed.) pp. 67-70. Alan R. Liss, New York. Packard, D.S., Jr., and Jacobson, A.G. (1976). The influence of axial structures on chick somite formation. Dev. Biol. 53, 36-48. Packard, D.S., Jr. & Meier, S. (1983). An experimental study of the somitomeric organization of the avian segmental plate. Dev. Biol. 97, 191-202. Packard, D.S., Jr., and Meier, S. (1984). Morphological and experimental studies of the somitomeric organization of the segmental plate in snapping turtle embryos. J. Embryol. Exp. Morphol. 84, 35-48. Sandor, S. (1972). Researches on the formation of axial organs in the chick embryo. VIII. Some new aspects of regulation potencies during somitogenesis. Revue Roumaine d'Embryologie et de Cytologie, S6rie Embryologie 9, 113-121. Spratt, N.T., Jr. (1955). Analysis of the organizer center in the early chick embryo. I. Localization of prospective notochord and somite cells. J. Exp. Zool. 128, 121-163. Stern, C.D. & Keynes, R.J. (1986). Cell lineage and the formation and maintenance of half somites. In: Somites in Developing Embryos. (Bellairs, R., Ede, D.A., & Lash, J.W., Eds.) pp. 147-159. Plenum Press, New York. Stern, C.D. & Keynes, R.J. (1987). Interactions between somite cells: The formation and maintenance of segment boundaries in the chick embryo. Development 99, 261-272. Tam, P.P.L., Meier, S. & Jacobson, A.G. (1982). Differentiation of the metameric pattern in the embryonic axis of the mouse: Somitomeric organization of the presomitic mesoderm. Differentiation 21,109-122. Tam, P.P.L. & Trainor, P.A. (1994). Specification and segmentation of the paraxial mesoderm. Anat. Embryol. 189, 275-305. Teillet, M.-A., Kalcheim, C. & Le Douarin, N. (1987). Formation of the dorsal root ganglia in the avian embryo: Segmental origin and migratory behavior of neural crest progenitor cells. Dev. Biol. 129, 329-347. Trainor, P.A., Tan, S-S, & Tam, P.P.L. (1994). Cranial paraxial mesoderm: Regionalization of cell fate and impact on craniofacial development in mouse embryos. Development 120, 2397-2408. Triplett, R.L. & Meier, S. (1982). Morphological analysis of the development of the primary organizer in avian embryos. J. Exp. Zool. 220, 191-206. Vieni, M. & Bellairs, R. (1986). Heat shock effects in chick embryos. In: Somites in Developing Embryos. (BeUairs, R., Ede, D.A. & Lash, J.W., Eds.) pp. 135-145. Plenum Press, New York.

Tam, P.P. & Trainor, P.A. (1994). Specification and segmentation of the periaxial mesoderm. Anat. Embry. 189, 275-305. Tannahill, P., Cook, G.M.W. & Keynes, R.J. (1997). Axon guidance in somites. Cell Tiss. Res. 290, 275-283.

Myogenesis

Introduction Muscle Structure and Function

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The Contractile Apparatus Control of Contraction Fiber Type Connective Tissue

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Embryonic Origin of Muscle Migration of Myogenic Cells Cellular Adhesion Gene Regulation of Myogenesis Cell Lineage Commitment Postnatal Growth

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Regeneration

Practical Applications Summary

Principles of Medical Biology, Volume 11 Developmental Biology, pages 229-259. Copyright 9 1998 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938.816-1

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Skeletal muscle is an elaborate and highly organized tissue, both at the level of the whole organ and the cell. For normal activity, skeletal muscle requires not only muscle cells (fibers), which constitute the bulk of the tissue, but also a complex network of nerves and blood vessels, and a specialized extracellular matrix. Muscle fibers are formed by the fusion of precursor cells (myoblasts) in a process known as myogenesis and are unusual, not only because of their multinucleate nature but also because of their composition; in addition to the organelles found in other cell types, muscle fibers contain an extensive system of intracellular membranes and contractile proteins arranged into highly organized contractile units. The study of myogenesis has been greatly aided by the ability to isolate myoblasts by mechanical and enzymatic disaggregation of muscle tissue, and by the capacity of these myoblasts to grow and differentiate in tissue culture. During myogenesis, myoblasts appear to follow an innate, preprogrammed developmental pathway. Details of this pathway may vary between myoblasts of different developmental origins and can be affected by extrinsic and intrinsic factors, giving rise to fibers with different structural and functional properties. This fiber diversity remains apparent and malleable throughout adulthood; muscle fibers can respond to changes in their external environment by altering their physiological and biochemical properties, and they can regenerate following damage or disease. Myogenesis, has gained particular significance partly because of the identification of genes that directly control the developmental progression of myoblasts to muscle fibers, and partly because of the proposition ofmyoblasts as suitable vectors for the transfer of therapeutic genes into diseased muscle. For the purpose of this chapter, a description of the formation of skeletal muscle will be preceded by a brief outline of its structure and function. Other more extensive texts should be consulted for detailed descriptions beyond the scope of this chapter.

As a reflection of the major function of skeletal muscle, to produce force by contraction, the majority of the cytoplasm of muscle fibers contains the contractile apparatus and intracellular membranes, which respectively act as the molecular motor and couple contraction with nerve impulses.

Within muscle fibers, the contractile apparatus runs in organized myofibrils subdivided into individual repeating units of contraction called sarcomeres. Each sarcomere is bordered by Z-discs and comprise an M-line, a dense (A) band and two lighter (I) bands.

The contractile apparatus consists of two major components, the thick and thin filaments. Aggregates of myosin form the thick filaments of the A band. Myosin filaments form spontaneously under physiological conditions by the association of two myosin heavy chains (MHCs) and four myosin light chains (MLCs) with each other. The MHCs coil around each other and are held together by ionic interactions along their tails, leaving their globular heads projecting from the filaments in a regular array. The heads of MHCs associate with MLCs and have an ATPase function that allows them to hydrolyze ATP to ADP and to change shape, flexing back toward the tail. Thin filaments of the light band also form spontaneously under physiological conditions by the association of globular actin subunits to form long chains of filamentous actin (F-actin). As well as actin, the thin filaments contain the regulatory proteins, troponin and tropomyosin. Troponin is found as a complex of troponin T (tropomyosin-binding), I (inhibitory), and C (calcium binding) and is involved in the regulation of contraction. Troponin T forms a tail-like structure in the troponin complex and acts to bind tropomyosin along the length of the thin filament. Troponin T in combination with the actin-binding troponin I binds to thin filaments, and the addition of troponin C to the complexes makes this process Ca 2§ sensitive. Tropomyosin, which is structurally similar to MHC, acts to stabilize and stiffen the thin filament as well as helping to regulate contraction along with troponin by blocking the association of actin with myosin. Within the myofibril, thick filaments interdigitate with thin filaments in a repeating pattern of sarcomeres. Contraction of the muscle is achieved by the association of the protruding myosin heads with the actin (cross-bridge formation); as the myosin heads hydrolyze ATP and change shape, they pull the thin filaments forward over the thick filaments. Association of myosin and actin, ATP hydrolysis, and movement of the thin filaments occurs in cycles, progressively shortening the sarcomeres as the Z-discs at the end of the thin filaments are pulled together.

For muscles to perform their function effectively, muscle contraction has to be controlled and coordinated by nervous stimulation. This is largely done through the regulation of Ca 2§ within the muscle fibers. Surrounding each myofibril is a membranous structure, the sarcoplasmic reticulum, which acts as a reservoir of Ca 2§ The sarcoplasmic reticulum, via calcium release channels known as "feet", is contiguous with another membranous structure, the transverse T-tubule system. These three structures are collectively known as the triads. T-tubules and feet act to connect the sarcoplasmic reticulum both physically and electrically to the plasmalemma. Sarcolemmal depolarization propagates into the T-tubules and, within the feet, causes the opening of voltage-sensitive Ca 2§ channels. Opening of the voltage-sensitive Ca 2§channels triggers the release of calcium from the sarcoplasmic reticulum into the cytoplasm. The rapid increase in cy-

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Skeletal Muscle Myogenesis toplasmic Ca 2§ effects the release of the troponin complex from the thin filaments, revealing myosin binding sites covered previously by the troponin-tropomyosin complex. Exposure of the myosin binding sites results in actin-myosin crossbridge formation and the initiation of contraction. The increase in cytoplasmic Ca 2§is very short lived. The properties of the sarcoplasmic reticulum membrane return to normal and Ca 2§ pumps in the membrane rapidly push Ca 2§back into the sarcoplasmic reticulum, allowing the troponin complex to reassociate with the thin filaments, blocking cross bridge formation and causing relaxation to occur. The speed of transmission of the electrical impulses along the muscle fibers and the T-tubule system is sufficient to ensure synchronous and efficient contraction of the sarcomeres.

The fibers comprising a muscle are not all identical. Classically muscle fibers are differentiated as fast-oxidative-glycolytic (type ILA), fast-glycolytic (type IIB), or slow oxidative (type I). Muscle fiber type is based on the physiology and biochemistry of the fiber as revealed by correlative studies of ATPase staining, metabolic staining, and contractile properties. However, this system of fiber classification is artificial as fibers with a range of properties exist within the three groups. Type I (slow) fibers are relatively slow contracting and are biochemically oxidative, (i.e., they respire aerobically), and are therefore resistant to fatigue. Slow fibers predominate in postural muscles of the body, such as the soleus where as much as 85% of fibers in the mouse are type I. Type IIB (fast-glycolytic) fibers are fastcontracting fibers prone to rapid fatigue due to their highly glycolytic metabolism (i.e. they respire predominantly anaerobically). Type IIA (fast-oxidative-glycolyric) fibers are intermediate fibers having relatively fast-contracting and fatigueresistant properties. Type II fibers are more common in phasic muscles such as the extensor digitorum longus of the leg, which are used primarily for locomotion, and therefore less frequently than postural muscles. As muscle fibers form, they already contain some of the information relating to their adult fiber type (see later); this program, however, can be altered by extrinsic factors resulting in fiber-type switching. Cross innervation and electrical stimulation of muscle has demonstrated the large role innervating nerves play in determining a muscles characteristics. If a slow and a fast muscle are denervated and subsequently reinnervated using each other's nerves, the result is that the crossinnervated muscles acquire the physiological properties appropriate for the new nerve (Buller et al., 1960). Electrical stimulation also may cause adaptation of muscle. Fast muscles, for example, stimulated at frequencies intrinsic to slow neurons become reversibly transformed into slow muscles (Salmons and Vrbov~i, 1969). Maturation also results in a change of muscle fiber type. In the soleus of the rat, for example, the proportion of slow fibers increases with age, while in the extensor digitorum longus (EDL), the proportion of type IIB fibers (fast glycolytic) increases at the ex-

pense of the type nA fibers (fast oxidative-glycolytic). Other factors are also known to affect muscle fiber type (e.g., thyroid hormone levels or overload of the muscles). Excess thyroid hormone (hyperthyroidism) leads to accumulation of fast fibers, whereas hypothyroidism or increasing the load on individual muscles, either by ablation of synergistic muscles or by stretch, leads to the accumulation of slow fibers. Fiber-type regulation occurs as a result of switching of gene expression either within the multigene family, as with the MHC, or by alternate splicing of exons within one gene, such as the fast skeletal troponin T gene, which has the potential to generate as many as 64 different mRNA transcripts, and therefore protein isoforms, from one gene. Control of the switch in gene expression is only beginning to be understood, but the process is physiologically important as it not only allows the generation of a variety of functionally distinct muscles but also allows them to retain their plasticity, even in mature adults.

Surrounding muscle fibers in muscle tissue is a dense extracellular matrix of connective tissue. The connective tissue acts not only to support the blood vessels, nerves, and muscle fibers, but it is also involved in the development of the muscle during the early embryonic stages and in the transmission of force produced by muscle fiber contraction. Individual, mature muscles are surrounded by a connective tissue layer known as the epimysium. This layer is composed of the structural proteins fibronectin and collagen and is continuous with the myotendinous junction. Within the muscle, bundles of fibers are organized into fascicles, which are bounded by a layer of collagenous connective tissue known as the perimysium. Within the epimysium and perimysium run blood vessels and nerves. The perimysium and epimysium are composed mainly of type I collagen. Individual fibers are covered by yet another layer of connective tissue, the endomysium, which is closely associated with and inserts into a basal lamina. The basal lamina is composed mainly of the glycoprotein laminin, type IV collagen, and proteoglycans such as heparin sulphate. Beneath the basal lamina, but outside the plasmalemma of the muscle fiber, are occasional mononucleate satellite cells, so called because of their position at the periphery of the muscle fiber in transverse section (Mauro, 1961). At the ends of individual fibers, the basal lamina is connected to the myotendinous junction by strands of type III collagen embedded directly into the basal lamina. It is this association of the contractile muscle tissue with the structural connective tissue that allows the transmission of muscle fiber contraction to the skeleton and hence movement.

Until relatively recently, the basic cellular events of muscle fiber formation were poorly understood. Despite good evidence to the contrary, the idea that multinucle-

Figure 2. A. hematoxylin- and eosin-stained culture of myoblasts. Maintained under suitable conditions myoblasts remain as proliferative single cells. Cells undergoing mitosis are arrowed. B. A hematoxylin- and eosin-stained culture of myotubes. By switching culture conditions myoblasts can be induced to cease division, align, and fuse to form long straplike multinucleated early muscle fibers or myotubes (arrowed).

ate muscle fibers were plasmodia formed by repeated nuclear division without cell division persisted (Ali, 1979). The first correct description of muscle fibers as syncytia, formed by the fusion ofmany single, mononuclear cells, was made in 1839 by Schwann. However, it was not until the tissue culture experiments of Konigsberg (Koni gsberg, 1961; Ko ni gsberg, 1963), that this view became generally accepted.

The syncytial formation of muscle fibers has been demonstrated by a variety of experiments in tissue culture (Stockdale and Holtzer, 1961). These experiments take advantage of the fact that myoblasts dissociated from embryos and exposed to tritiated thymidine, a radioactively labeled nucleotide, incorporate it into replicating DNA. The labeled DNA can then be detected autoradiographically in the nuclei of cells that have divided. Initially radioactive label is found exclusively in the nuclei of mononucleated cells and only at later stages after fusion of these cells, in the nuclei of myotubes. Conversely, cultures in which myoblasts have been already fused into myotubes and then pulse-labeled with radioactive nucleotides show no labelling in the nuclei of the myotubes. Direct evidence for the formation and growth of myotubes by the fusion of mononucleate muscle precursor cells after they cease dividing has come from time-lapse photography of differentiating myogenic cultures (Cooper and Konigsberg, 1961). In the following sections of this chapter we will deal with some specific details of myogenesis; for example, the origin of myogenic cells, migration of myogenic cells, cellular adhesion and aggregation of myogenic cells, myogenic determination and terminal differentiation. We will comment on myogenic lineages, postnatal growth, and regeneration of damaged muscle, and conclude by describing the practical applications myogenesis may be put to in the treatment of muscle disease.

The embryonic development of muscle has been extensively studied in two species-the mouse and the chick. As a tool for studying the molecular and cellular biology of myogenesis, the mouse is particularly useful as a large amount is already known about its biology and it is inexpensive and quick to breed. Another advantage of the mouse is that the technology exists to introduce or knock out specific genes in mouse embryos, producing transgenic or null mice which can be used to study the effects of the introduced/removed genes. The chick has been used to study the movement of cells within the developing embryo. Because the presumptive myoblasts are indistinguishable from other cells of the embryo, a system of marking cells and following their fates had to be devised. The answer has been to produce chick/quail chimeras (Jacob et al., 1978), transplanting areas thought to contain the presumptive myoblasts from quail embryos into chick embryos and vice versa. Microscopically, quail and chick cells are easily distinguished from one another by the presence of very prominent nucleoli in the quail nuclei. The chick/quail chimera model has allowed detailed study of the formation of myogenic cells within the mesenchyme. In the mouse, gestation takes 21 days. At 7-10 days p.c. (post coitus) the mesoderm of the developing embryo begins to segment in an anterior-posterior gradient along either side of the midline of the embryo to form blocks of mesoderm called somites. Somites give rise to muscle cells, to cells that comprise the vertebrae and ribs, and to cells that form the dermis of the trunk. The dorsal portion of the somite,

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the dermomyotome, gives rise to premyoblast stem cells; the ventral portion, the sclerotome, gives rise to cells that form the connective tissue. As the dermomyotome matures, premyoblast cells migrate from its dorsal region, adjacent to the neural tube, to a medio-ventral position under the dermomyotome, forming the premuscle masses of the myotome. At about the same time, other primordial muscle cells migrate from the ventrolateral edge of the dermomyotome to form the limb

and trunk muscles. The limbs of vertebrates first appear as four buds on the side of the embryo. In the mouse embryo the anterior limb buds appear at 9 days p.c. and the posterior limb buds at 10 days p.c. In the chick, wing buds form adjacent to somites 15-20 and leg buds adjacent to somites 26-32. Limb buds first appear as thickenings in the somatopleure mesoderm and subsequently elongate along the proximal-distal axis of the developing limb. Initially. the cells of the mesoderm are indistinguishable from each other. As the limb develops they differentiate to form the variety of tissues involved in limb morphogenesis: muscle cells; connective tissue of the skeleton, tendons, and muscle; epithelium of the skin; endothelium of the blood vessels; motor and sensory neurons; and hemopoetic cells of the bone marrow. Experiments in birds (Jacob et al., 1978) have shown that the source of myogenic cells for the developing limb buds are the somites. Grafts were performed between quail donors and chick hosts using somites taken from one of four levels; the neck, the wing, the flank, and the leg. The grafted material contained either somites alone or somites and associated somatopleure, and groups of somites were grafted either to the equivalent site within the chick host (homotopic transplantation) or to one of the other three sites (heterotopic transplantation). Histological examination of the chimeric embryos between 4 and 11 days after grafting showed that where somites alone had been transplanted, the muscle fibers of the developing limb bud were of quail origin whereeas the other components were of chick origin. Somites grafted either homo- or heterotopically participated normally in the formation of the musculature of the chimera, indicating that at the time of migration the somites are not regionally determined. When somite and somatopleure mesoderm were grafted into the embryos, both the muscle fibers and the connective tissue of the limb bud were of quail origin. An extension of this work, grafting either the lateral or medial halves of the developing somite from quail to chick, has shown that myoblasts become determined differently according to their position within the somite (Ordahl and LeDouarin, 1992). Myogenic cells from the medial half of the somite, nearest the notochord, remain in the somite and form the myotome, while myogenic cells in the lateral half migrate to form the muscles of the limb bud and ventral body wall muscles.

Myoblasts migrate from the somites into positions within the developing embryo where they eventually form mature muscles. Little is known about the exact molecular mechanism of migration through the matrix of the embryo to specific sites where muscles are required. It appears to depend on a number of factors such as positional cues, chemotaxis, and adhesive interactions with the extracellular matrix and other cells. A cell placed onto an artificial matrix under particular tissue culture conditions will flatten and form areas of close association with the substrate called adhesive

Skeletal Muscle Myogenesis plaques. Adhesive plaques are sites at which elements of the cytoskeleton, such as actin, become concentrated and linked to receptors of the integrin family (see p. 3), which bind components of the extracellular matrix such as fibronectin, collagen, and laminin. The flattened cell also extends small projections into the surrounding environment that can attach to the substrate and stretch the cell further. Where differences in the components of the matrix cause an increase in adhesiveness in a particular direction, the cell becomes polarized and is pulled in that direction by the preferential extension and adhesion of these projections. In the embryo, locomotion of cells is thought to operate in a similar fashion, namely, association of matrix receptors with the matrix leading to polarization and movement of the cell. Integrins, however, not only act to adhere to the matrix but also transmit signals from the extracellular matrix to the inside ofthe cell via the cytoskeleton. This interaction in turn affects the behavior of the cells causing them to move, proliferate, or differentiate, depending on the types of integrins expressed. Involvement of integrins in the migration of myoblasts has been inferred from experiments using the antibody "CSAT", which recognizes and blocks 131 integrin subunit function. When cells producing this antibody are transplanted into a chick embryo, abnormalities result that appear to be caused by the inappropriate migration of muscle cells in the embryo (Jaffredo et al., 1988). Chemotaxis, the movement of cells along chemical gradients, may also play a part in the attraction of cells to specific sites, as it does in lower organisms such as the slime molds. ,The result of the migration of determined myoblasts into sites of the embryo requiting muscle, such as the limb bud, is the formation of aggregates of myoblasts known as premuscle masses, which eventually differentiate to form first the primary and then the secondary myotubes of the musculature.

An important process in migration, formation of the premuscle masses, and fusion of postmitotic myoblasts is the aggregation of fusion-competent myoblasts. This is achieved by cell sorting via differential adhesive interactions of the developing muscle cells with the extracellular matrix (cell-matrix interactions) and with other similar cells (cell-cell interactions). Receptors of the integrin family of transmembrane glycoproteins are thought to be involved in migration of myoblasts to and from their location on the scaffold of extracellular matrix before fusion as well as affecting the organization of the cytoskeleton. Receptors such as the neural cell adhesion molecule (N-CAM) and the cadherins are thought to be important in the recognition of and adhesion to other fusion-competent muscle cells.

Early studies of integrin expression in muscle used the antibody CSAT to localize integrin expression on cultured cells and in developing chick and rat embryos.

Tissue culture experiments demonstrated 131 integrin sub-unit expression on both myoblasts and myotubes. In developing skeletal muscle in vivo, 131integrin subunit expression on early myotubes is random and punctate. Later myotubes have a more uniform staining pattern, while in developed muscle 131integrin subunit expression is limited to the myotendinous junction and to the lateral points of adhesion (costameres) of muscle fibers (Bozyczko et al., 1989). Because of the widespread expression ofintegrins during skeletal muscle development, it has been suggested that they play an important role not only directing migration but also in early fusion events (Menko and Boettiger, 1987). Cell substrate (i.e., the artificial extracellular matrix of cells) in culture significantly affects myoblast behavior. Myoblasts plated onto a laminin quickly cease proliferation rapidly, fuse into myotubes, and express muscle-specific proteins like myosin. Cells plated on fibronectin continue proliferation and lose expression of early myogenic markers such as desmin because of modulation of the myoblast's phenotype (Von der Mark and Ocalan, 1989). When plated onto a laminin substrate, myoblasts have also been shown to have a greater capacity for migration (Von der Mark et al., 1989). As fusion occurs, however, themyoblasts' laminin binding sites are down-regulated and they gradually lose the ability to move (Goodman et al., 1989). Biochemical analysis of the myoblast laminin receptor has shown it to consist of the 131integrin subunit and a novel t~ chain, the tx7 subunit (Song et al., 1992). It has been proposed that the initial formation of myotubes leads to deposition of laminin, promoting recruitment of new myoblasts by stimulating proliferation, migration, and differentiation of ct7 positive cells. Integrins are not only implicated in events prior to fusion, but also in the maintenance of muscle fibers and the transmission of forces from muscle through the extracellular matrix. Myotubes from a mutant Drosophila, "myospheroid", which lacks the 13integrin subunit of the fibronectin receptor, fail to differentiate normally, forming myotubes with defective Z-bands (Volk et al., 1990). Integrins are thought to be involved in linking muscle fibers to the extracellular matrix at the myotendinous junction and the costameres. At these sites, the intracellular domain of integrins directly associates with cytoskeletal proteins such as vinculin, talin, and tx-actinin, which in turn serve to anchor the contractile apparatus (Burridge et al., 1988). The integrin, VLA 4, is unusual in that it is not only involved in cell-matrix interactions by binding fibronectin, but is also in cell--cell interactions. During secondary myogenesis, myoblasts that form the secondary myotubes and express the adhesion molecule, V-CAM (vascular cell adhesion molecule), locate themselves onto VLA 4 expressing primary myotubes via interactions between V-CAM and VLA 4 (Rosen et al., 1992).

Whereas cell-matrix interactions are thought to be involved in locating myoblasts on the extracellular matrix and promoting the transition to a postmitotic state

Skeletal Muscle Myogenesis ready for fusion, cell--cell interactions are thought to be directly involved in fusion of myoblasts into myotubes by mediating recognition of, and association with, other fusion competent cells. These functions are important in terminal differentiation as fusion can only occur between such myoblasts and because the electrostatic repulsion of similarly charged cell membranes has to be overcome before membrane fusion can occur. Cell--cell interactions can be split into two functional systemsuthe Ca2§ and Ca2+-dependent systems.

Caldum-independent system. Neural cell adhesion molecule (N-CAM) was first identified in the retina of chick embryos. The first studies of N-CAM demonstrated its expression on nerve and glial cells, hence its name; however its expression has since been found on other cell types including skeletal muscle. As anti-N-CAM antibodies disrupt muscle cell aggregation in tissue culture, N-CAM is thought to have an important function in muscle ceil adhesion (Knudsen et al., 1990a). The molecule exists as a number of isoforms generated by the alternate splicing of one gene. The isoforms of N-CAM have a common extracellular region that consists of five immunoglobulinlike domains, and they differ in their intracellular and transmembrane regions. N-CAM molecules interact with other N-CAM molecules via their immunoglobulinlike domains. Three main isoforms of N-CAM are found on muscle tissue. Replicating myoblasts mainly express two transmembrane isoforms that cross and are bound into the plasmalemma of the cell. Postmitotic cells express an isoform that has no transmembrane domain and is covalently linked to the outer lipid layer of the plasmalemma. The significance of the distinct isoforms of N-CAM is unknown, however they are closely regulated in embryonic development and regeneration (Lyons et al., 1992). Experiments have shown the importance of the lipid-bound isoform in the final events of fusion. Its removal from the plasmalemma by digestion with the enzyme phospholipase C blocks fusion of the myoblasts (Knudsen et al., 1989). In addition, when this isoform is introduced into muscle cells in tissue culture, cells containing this exogenous gene fuse at a much higher rate than control cells, which only have their endogenous N-CAM gene (Dickson et al., 1990). This and other experiments (Knudsen et al, 1990a) have shown that N-CAM affects the rate of fusion but not the final extent of muscle differentiation. As such, the Ca2§ adhesion system is thought to complement and trigger the essential Ca2§ adhesion system. Calcium-dependent system. The molecules involved in Ca2§ adhesion are known as cadherins. Currently, four members of this family are known: E-cadherin, which was first described on epithelial cells; P-cadherin, which was found on placental and epidermal cells; M-cadherin, which has recently been described on muscle cells; and N-cadherin, which, like N-CAM, was first found on neural cells but has since been described on a variety of cell types including skeletal

muscle cells. Muscle-specific, M-cadherin is believed to be involved in muscle cell fusion but has yet to be fully characterized. It is known however that the fusion of muscle cells in culture can be blocked by inhibiting the expression of M-cadherin (Donalies et al., 1991). Like N-CAM, N-cadherin is a transmembrane protein that self-associates and is expressed transiently during development. Unlike N-CAM, however, N-cadherin does not have the same diversity of isoforms, its three extracellular domains are not immunoglobulin-like and in the absence of Ca 2§ it undergoes conformational changes that lead to its proteolysis. As with N-CAM, the involvement of cadherins in cellular adhesion was demonstrated by the ability of antibodies, in this case raised against N-cadherin, to disrupt aggregation of muscle cells in tissue culture (Knudsen et al., 1990b). N-cadherin expression has been observed in the myotome, within developing limb and axial muscle, and during myogenesis in tissue culture. On cultured muscle cells, its expression is concentrated around opposing faces of fusion-competent cells. Removal of N-cadherin from myoblasts, which can be achieved by removing Ca 2§ from the culture medium, has a similar, but greater effect than that of removal of NCAM, affecting only the rate and not the extent of fusion. Blocking both N-cadherin and N-CAM with antibodies further reduces the rate of fusion but does not completely prevent it. The effect of removing of either N-CAM or N-cadherin implies that both systems are required for efficient fusion.

The identity of a master regulatory gene, if it exists, that switches pluripotent cells of the mesoderm in embryos to committed muscle progenitor cells remains elusive. Recent studies in tissue culture have, however, led to the identification of a family of genes directly implicated in the control of myogenesis.

The first of the developmental control genes to be identified has been named MyoD (Tapscott et al., 1988). MyoD was identified as a myogenic determination factor by virtue of the effect of its forced expression on the mesenchymal fibroblast cell line, 10Tl/2. Fibroblast cells are not somitic in origin and do not develop as myogenic cells under normal conditions; however, forced MyoD expression is sufficient to convert them to myogenic cells in vitro. Since this discovery, other very similar genes have also been identified that have similar functions. These have been named myogenin (Wright et al., 1989), MRF4 (rat; Rhodes and Konieczny, 1989) (which is also called Herculin (mouse; Miner and Wold, 1990), or Myf6 (human; Braun et al., 1990) and Myf5 (Braun et al., 1989). The protein products of these genes all contain a highly conserved protein structure known as a basic helix-loophelix domain (bHLH). The basic region of the bHLH domain is rich in basic amino

acids, which permits binding to specific sequences of DNA, while the helix-loophelix region allows dimerization/oligomerization with other bHLH molecules. Dimerization/oligomerization of this protein family is an important regulatory feature of their cellular functions. Analysis of the pattern of expression of these genes in differentiating myogenic cell lines in vitro (Montarras et al., 1991) has defined a particular pattern that coincides with particular developmental stages during myogenesis. Reversibly determined muscle precursor cells express myf5. Irreversibly determined proliferating myoblasts express MyoD and, in response to local environmental cues such as growth factor concentrations or cell density, myogenin is expressed and cells enter terminal differentiation. After fusion, the expression of MyoD and myogenindecrease as the myotube becomes established. In myotubes, the appearance of MRF4 coincides with the expression of the acetylcholine receptor e subunit (Montarras et al., 1991), a subunit of the adult form of the AChR. A similar hierarchy of gene expression has been demonstrated during embryogenesis. The first appearance of the myogenic determination genes in the mouse embryo occurs as myf5 is expressed within the somites and limb buds as they form (Ott et al., 1991). The pattern of expression of myf5 echoes the anterior-posterior pattern of somite formation and maturation, indicating that myf5 is a determination factor involved in the earliest stages of the myogenic lineage. Myogenin and MyoD expression in the embryo is at a later stage than myf5 expression (Sassoon et al., 1989). Determined muscle cells forming the myotomes express myogenin and then MyoD. In the developing limb muscles, the order of expression of these proteins is slightly different. Myf5 is still expressed first and MRF4 last; however, myogenin is expressed after MyoD, a fact that is thought to reflect the different somitic origins of muscle in the myotome and limbs. After myoblast fusion, in tissue culture, both MyoD and myogenin expression decrease and MRF4, which is transiently expressed in the early myotomes and limb buds, is re-expressed. In adult muscle, MRF4 expression is constitutive. In vitro and in vivo, MRF4 expression appears to have a function in the maturation and maintenance of muscle phenotype following myogenesis. The production of gene-knockout mice, in which the function of MyoD, myf5 and myogenin genes have been destroyed, gives further insight into the role of these factors during muscle development. In mice in which MyoD function has been destroyed (Rudnicki et al., 1992), myogenesis appears to be unaffected: The defect is apparently compensated for by a rise in myf5 expression. Disruption of the myf5 gene (Braun et al., 1992) is lethal as a result of malformation ofthe ribs, possibly attributable to a slight delay in the development of the muscle in these mice. The expression of other members of the bHLH family appears to be slightly reduced in mice lacking myf5; however, the extent of muscle development is little if at all affected. Mutation of the myogenin gene is lethal at birth (Hasty et al., 1993; Nabeshima et al., 1993); myogenesis in these mice is greatly affected. During embryonic

development of myogenin-deficient mice, myoblasts appear unable to enter terminal differentiation and collect as single cells in the normal myogenic sites, producing nonfunctional muscles containing very few muscle fibers. These knock-out studies indicate that within the bHLH family of myogenic regulatory proteins, despite a very clear hierarchy of expression, there is a certain degree of overlap and functional redundancy. Myogenin, however, does appear to occupy a key point in terminal differentiation.

Functional studies of the myogenic regulatory proteins show that homo- or heterodimers are able to recognize and bind a core CAnnTG (n=A, C, G, or T) sequence of DNA, a known constituent of regulatory DNA sequences such as enhancer boxes (E-box) (Weintraub et al., 1990). Binding of myogenic regulators to cis-actingelements such as E-boxes of muscle-specific genes during differentiation is thought to activate transcription of the genes. Multiple bHLH binding sites are found in the promoter or enhancer regions of many muscle-specific genes, indicating that cooperative binding ofbHLH proteins may help to control expression of these muscle genes (Piette et al., 1990; Weintraub et al., 1990; Wentworth et al., 1991). Promoter and enhancer sequences of the muscle regulatory genes MyoD, myogenin, MRF4, and myf5 themselves contain bHLH binding sites within Eboxes. The presence of these sequences in the gene promoters and enhancers of the bHLH proteins implies that these factors positively auto-regulate their own expression and cross-regulate the expression of other members of the family (Thayer et al., 1989). Transcriptional activation by the bHLH proteins therefore leads not only to the expression of muscle-specific genes, but it also leads to the amplification and reinforcement of initiating myogenic signals from outside and inside the cell and allows coordinated passage of muscle cells through the various developmental stages.

Following initiation of myogenesis, myoblasts leave the cell cycle in the G1/Go phase and become permanently postmitotic (Okazaki and Holtzer, 1966). The events directly preceding cell cycle withdrawal are unclear; it still has to be resolved whether it occurs after an innately programmed number of divisions, the deterministic hypothesis (Quinn et al., 1985), or whether it is because of factors that lengthen G Oand so increase the probability of entering growth arrest, the probabilistic hypothesis (Konigsberg et al., 1978). Our expanding knowledge of the molecular events controlling myogenesis may yet provide an answer. However, it is already known, that growth arrest is influenced by environmental factors such as the presence or absence of specific hormones and growth factors and the composition of the extracellular matrix and cell density.

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Growth arrest ofmyoblasts is strongly linked to the cell cycle, occurring only at a specific point within the G0/G1stage; at other time points, treatment with mitogens such as bFGF is sufficient to reinitiate the cell cycle. It therefore seems likely that it is dependent upon proteins present or activated only during this phase of the cell cycle. The decision either to arrest or to continue proliferation is influenced by antagonistic signals. Serum and peptide growth factors, such as fibroblast growth factor (FGF; Clegg et al., 1987), or activated proto-oncogenes of signal transduction pathways such as v-Erb and v-src (Falcone et al., 1985), inhibit myogenesis through a complex, multimechanism system. Currently, compared to the wealth of knowledge about their function in transcriptional activation, relatively little is known about bHLH protein function in growth arrest. Several lines of evidence, however, point to them being critical not only in the determination of muscle cells but also in the timing of growth arrest and therefore terminal differentiation. Firstly, the bHLH proteins are obvious targets for regulation by intracellular signals, controlling a diverse and genetically unconnected series of genes involved in myogenesis. Secondly, the proteins are able to directly inhibit cell proliferation (Crescenzi et al., 1990; Sorrentino et al., 1990). And lastly, activated proto-oncogenes, which cause cell proliferation, are known to interfere with bHLH expression in the case of ras, or as with c-Fos and c-Jun, directly with the bHLH proteins themselves (Konieczny et al., 1989; Bengal et al., 1992; Li et al., 1992). Although expression of muscle-specific genes is not entirely dependent upon fusion it is dependent upon growth arrest. Cells of the myogenic cell line BC3H-1, which have lost the ability to fuse, and other myogenic cells in which fusion has been blocked by treatment with colchicine, can still express a biochemically differentiated phenotype (Olsen et al., 1983). On the other hand, treatment with dimethyl sulphoxide (DMSO) prevents differentiation by blocking growth arrest, but does not affect cells that are already arrested (Blau and Epstein, 1979). During myogenesis a number of antagonistic regulatory networks appear to coordinate myogenesis. Only when circumstances are appropriate are the cells released from the cell cycle and allowed to enter myogenesis under the control ofbHLH proteins. Close regulation of differentiation, involving inhibitory and stimulatory signals, ensures against inappropriate myogenesis during embryonic development.

Following growth arrest, terminal differentiation occurs: myoblasts begin to synthesize a battery of muscle-specific proteins under bHLH control (myosin, actin, acetylcholine receptor, and tropomyosin) as the cells become permanently postmitotic and align and fuse to form characteristic myotubes. Prior to fusion of myoblasts and expression of the differentiated phenotype, they become incapable of reentering the cell cycle. There are two proposed mechanisms by which this may occur: by the loss of components of the signal transduction pathways that activate mitosis (e.g., those involved in growth factor effects) and by

upregulation of inhibitors of mitosis such as tumor suppresson genes Rb and p53. Growth factor receptors, for example, are known to be lost after differentiation (O1win and Hauschka, 1988); however, the kinetics of this downregulation argue against it being the initial event in permanent cell cycle withdrawal. Recent studies have demonstrated an accumulation of the tumor suppressor proteins, p53 (Halevy, 1993) and Rb (Endo and Goto, 1992) in differentiating muscle cells, as well as direct association between MyoD and Rb (Gu et al., 1993). As both Rb and p53 inhibit cell proliferation and bind the simian virus (SV40) large T-antigen, which is known to prevent entry into myogenesis (Endo, 1992), their presence in the nuclei of myoblasts during terminal differentiation is likely to be more than coincidental The sequences of fusion appear to involve firstly, the intimate association of myoblasts with each other or of myoblasts with myotubes, under the influence of adhesion molecules. Vesicles generated by the Golgi apparatus then collect at the internal opposing faces of the cells, apparently destabilizing the membranes either by their insertion into the plasmalemma or by the release of a cell product. This alteration of the properties of the membranes allows them to coalesce and fuse together resulting in the formation of a single lipid bilayer around the cells. Following fusion of the membranes, the cytoplasm of the two original cells is free to diffuse between them, and the cytoarchitecture is rearranged to form the syncytia of the developing myotube (Kalderon and Gilula, 1977; Robertson et al., 1990).

Myoblasts do not appear to be a homogeneous population of cells; several lines of evidence point to a number of distinct populations (or lineages) of myogenic cells within the developing embryo (Kelly and Zacks, 1969; Bonner et al., 1978). The first myotubes or primitive muscle fibers that form in the embryo do so in the somites at 8.5 days p.c. in the mouse. Within the sites of the limb and trunk muscles, myotubes begin to form between 13 and 16 days p.c. Initial formation of these muscles involves the synchronous fusion of primary myoblasts to form early or primary myotubes. Following this first burst of myogenesis at 16 days p.c. in the mouse, a second population of myoblasts, called secondary myoblasts, appear and become intimately associated with the primary myotubes. These myoblasts proliferate, align, and fuse with one another along the length of, but not with, the primary myotubes, which they use as a scaffold. Fusion of the secondary myoblasts with each other occurs in a synchronous way forming rosettes of secondary myotubes around each primary myotube and within its basal lamina. As the secondary myotubes mature they separate from the primary myotubes and develop their own basal laminae. In the adult, primary myotubes constitute 10-20% of the muscle formed. The remaining bulk of the muscle originates from secondary myotubes. Myoblasts taken at these various stages of embryogenesis and grown in culture have been shown to have distinctive patterns of growth and differentiation and, in the mouse, tolerance to the phorbol ester, TPA (Cossu et al., 1983). It is thought that

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myoblasts may be divided into at least four developmental lineages: somitic, embryonic, fetal, and adult lineages. Somitic lineage cells, which do not migrate from the somites, are determined and committed first. Later, embryonic myoblasts, which have migrated into the limb buds, become committed to myogenesis and express MyoD, fuse rapidly to form primary myotubes, and layout the basic plan of the muscles. Finally, fetal (secondary) myoblasts, migrate to the limb bud, become committed to myogenesis, and fuse to steadily build up the bulk of the muscle tissue. The adult lineage describes those cells that do not fuse during myogenesis and remain as quiescent satellite cells in adult tissue. The developmental expression of MHC genes in myoblasts can also be used to define the primary and secondary lineages in the rat (Harris et al., 1989). Following fusion, both types of myotubes initially express an embryonic form of the protein, MHCemb. As primary myoblasts mature the embryonic form is lost and a mix of slow MHC and neonatal MHC, (MHCslowand MHC,,eo)become the predominantly expressed isoforms. Later, secondary myoblasts accumulate a different neonatal form of the gene (MHC,,eo)as they mature. This in turn gives way to expression of fast isoforms of MHC as the myotubes mature further. The pattern of MHC expression of the mature muscle is modulated by the environmental cues until the muscle fibers finish maturation and the muscle environment becomes stable.

Perinatally, the fiber number of the muscles is established (Montgomery, 1962; Chiakulas and Pauly, 1965). Normal postnatal muscle fiber growth occurs by hypertrophy of these existing fibers (by increasing the cross-sectional area of each of the fibers), and not by hyperplasia (by accumulation of further muscle fibers). The mechanism by which fibers hypertrophy involves an increase in myofibril number. In the mouse, the number of myofibrils contained within a single fiber can increase from 50 in the newly formed myotube to greater than 1000 in mature muscle fibers. Accompanying the increase in myofibril number is an increase in the volume of the T-tubule system and sarcoplasmic reticulum. Myofibrils appear to proliferate by splitting longitudinally when they reach a critical size (Goldspink, 1970). It is thought that splitting is caused by the misalignment of the contractile apparatus in oversized myofibrils leading to stress on the Z discs, and is related to not only the size of the myofibril but also the load on it, the speed of its contraction, hormone levels, and activity. Splitting of the myofibrils allows invasion of the newly formed cleft by extension of the sarcoplasmic reticulum and the T-tubule system, ensuring that the contractile apparatus retains its ability to function correctly. Stretching by the growing skeleton also appears to be the stimulus for fiber lengthening during postnatal growth. Using individual fibers teased out from muscle at a variety of ages, it has been shown that muscles grow in length by the addition of sarcomeres at the end of myofibrils (Williams and Goldspink, 1971). At birth,

myofibrils of the mouse soleus muscle contain about 50 sarcomeres, and this increases to around 2200 in the adult. During postnatal growth, the ratio of nuclei (or DNA) to cytoplasm of muscle fibers remains fairly constant. This finding is consistent with the idea of each myonucleus controlling a specific volume of cytoplasm, the concept of the nuclear domain. However it does pose the problem of how the number of myonuclei can increase with fiber growth, as all myonuclei are postmitotic. Moss and Leblond (1970) used tritiated thymidine to radioactively label dividing cells in new-born mice. Initial accumulation of the label occurred only in satellite cells of the muscles, indicating that satellite cells, unlike myonuclei, synthesize DNA and therefore undergo mitosis during muscle fiber growth. Appearance of the label 24 hours later in a few nuclei of the muscle fibers was interpreted as showing fusion of cells that had undergone division (i.e., cells derived from the satellite cells) with the muscle fiber. Other studies have shown a reduction in the number of satellite cells within the muscle during growth, which correlates with the increase in muscle fiber bulk (Schultz, 1974) and is postulated to reflect the incorporation of satellite cells into the growing fibers.

Some tissues of the body, such as the epithelium and hemopoietic system, are continually replacing lost cells by the proliferation of stem cells in the epidermal basal layers or bone marrow. In more stable tissues like skeletal muscle, however, cell death is much rarer and mitosis is a very sporadic event. Cell death resulting from damage to the muscle fibers may have a number of causes. Biopsies taken from athletes show that in normal individuals, muscle fiber necrosis and regeneration may occur quite commonly as a result of strenuous exercise. Medical concern is usually centered on muscle fiber death and regeneration associated with genetic or acquired disease or arising as a consequence of large scale mechanical or toxic trauma. The events leading to muscle cell death are complex and poorly understood. The later events, however, appear to involve loss by damaged fibers of the ability to regulate their intracellular Ca 2§ leading to hypercontraction of the fiber and activation of Ca2+-dependent proteases and phospholipases. The activity of the proteases and phospholipases predisposes the structure of the fiber and in particular its plasmalemma, to irreversibly breakdown. The type of injury to which a muscle is subjected greatly affects the efficiency of regeneration. Whole muscle grafts, a procedure in which the muscle is removed intact from one muscle bed and transplanted to a new site or returned to the original one, regenerate relatively efficiently and in a characteristic, outside-to-center fashion. Muscle regeneration is more complete following damage that leaves the basal lamina intact, such as whole muscle transplantation or infarction, than after damage, such as severe crush injury, which disrupts the basal lamina. The integrity of

the basal lamina is therefore thought to be of great significance as a scaffold. It may also minimize fibrosis caused by invading fibroblasts during regeneration. Regeneration from the outside occurs as the muscle becomes revascularized and reinnervated from external blood vessels and nerves. This allows the endogenous satellite cells, which, unlike the muscle fibers, remain viable, to proliferate and fuse within the scaffolding of the dead muscle. If the tissue damage is sufficiently severe, whole fibers or sections of fibers become necrotic (segmental necrosis). To preserve unaffected segments, the necrotic segment is quickly sealed off by new membrane which forms between the two areas. As persisting necrotic tissue inhibits regeneration, endocytosis by invading macrophages is an important process, not only for clearing debris but also for actively promoting regeneration, perhaps by stimulating secretion of cytokines. The source of cells involved in the regeneration of muscle tissue has long been assumed to be the satellite cells found beneath the basal lamina (Moss and Leblond, 1971). It has been suggested that satellite cells are induced to divide in response to a variety of stimuli, such as growth factors and the loss of fibronectin from the extracellular matrix. These satellite cells undergo asymmetric division to yield committed myoblasts and new satellite cells. The myoblasts undergo a limited number of divisions, cease to divide, align, and fuse within the basal lamina of the original fi-

ber. They may either form new fibers or replace necrosed segments by fusing with the ends of surviving segments of the damaged fibers. It is a striking feature of muscle regeneration that many of the processes involved recapitulate those of myogenesis in the embryo. After muscle fiber damage, necrosis, and phagocytotic removal of the fiber debris, satellite cells become activated. During activation, the satellite cells are stimulated to divide within the area of damage to produce a population of fusion competent myoblasts. These myoblasts follow a very similar developmental pattern to myoblasts in the premuscle masses of the embryo, expressing the myogenic transcriptional regulatory proteins myogenin and MyoD (Fuchtbauer and Westphal, 1992), and cellular adhesion molecules of the integrin (Hurme and Kalimo, 1992), cadherin (Moore and Walsh, 1993), and N-CAM (Cashman et al., 1987) families. Under the influence of these proteins, the activated myoblasts become growth arrested, align, and fuse with one another. MHC expression following regeneration also mirrors the pattern seen during embryogenesis (Marechalet al., 1984). In the chick, the adult MHC isoform expressed following regeneration appears to be determined within the myoblasts before fusion (Di Mario et al., 1993). Fibers as a whole express the same fiber type along their entire length; it therefore seems likely that chick myoblasts fuse preferentially with fibers of the same fiber type as themselves. Following regeneration in the adult mouse, however, MHC expression appears to be dominated by the type of muscle fiber with which a myoblast fuses, with little influence being found of an intrinsic myoblast program (Hughes and Blau, 1992). In contrast, tissue culture studies of myoblasts show a distinct tendency of inate commitment to expression of type I (adult slow) MHC in myotubes formed by myoblasts derived from muscles ofpredominately slow type in the rat (Dtisterhoff & Pette, 1993) or from individual slow muscle fibers of the mouse (Rosenblatt et al. 1996). Where regeneration is efficient, following maturation of the regenerated fibers the muscle appears normal. In cases where regeneration is inefficient, because of the type of damage or, as may occur in some diseases of muscle, because of failure of regeneration, proliferation of fibrotic tissue and fat deposition may occur, resulting in the disturbance of tissue architecture.

As the details of muscle fiber formation, both during embryogenesis and regeneration, have begun to be understood, so it has been proposed that myoblasts may be of use in treating disease. The basis of this proposition is that normal myoblasts can be injected into, and allowed to fuse with, damaged, diseased muscle, Expression of normal gene products by the transferred myoblasts after incorporation into the muscle should complement the genetic defect and improve the pathology of the disease. This has become known as myoblast transfer therapy (MTT). One disease in particular, Duchenne muscular dystrophy (DMD), has been the focus of experimental MTI'. DMD is one of the most common inherited diseases,

causing severe and progressive muscle wasting in young boys. The devastating progression of this disease, its high incidence and identification of the DMD gene and its protein product (Hoffman et al., 1987; Koenig et al., 1987), have made this an ideal disease in which to study the feasibility of using MTT to treat inherited muscle disease. The study of Mq~ in DMD has also been aided by animal models of the disease. Currently, three animal models of DMD have been recognised: the dystrophic cat, the XMD dog, and the mdx mouse. All three models lack dystrophin and show the degenerative/regenerative muscle lesions that characterize DMD. The cat and the mdx mouse do not show the progressive loss of muscle regenerative capacity, fibrosis, and fatty replacement of muscle tissue, which give DMD and the XMD dog such severe clinical phenotypes. The lack of any severe secondary pathology in the mouse does not affect its status as a true model of DMD and moreover simplifies it, as MTT is primarily concerned with replacing dystrophin before the onset of fibrosis and fatty replacement of muscle tissue. It has been extensively shown that normal mouse myoblasts injected into mdx muscles are able to fuse with dystrophic (dystrophin negative) fibers and biochemically rescue them by expression of dystrophin (Partridge et al., 1989). These experiments have shown a relatively high incidence of conversion of muscle fibers from dystrophin negative to dystrophin positive.

Skeletal Muscle Myogenesis Several problems, however, exist in transferring this therapeutic strategy directly to humans. Firstly, although skeletal muscle is the most overtly affected tissue in DMD, patients commonly die of heart failure caused by slower degeneration of the cardiac muscle. As cardiac muscle is incapable of regeneration, MTT is unlikely to be able to treat the DMD cardiomyopathy. Mdx experiments have been carried out in immunotolerant mice in which the effects of rejection of grafted myoblasts are avoided in ways that are not possible in man. Furthermore, as the muscles of the mdx mouse are small, dispersion of myoblasts from the injection site is not critical; in larger human muscles multiple injections of myoblasts at sites very close together may be needed-- possibly at a distance no greater than 1 cm. As each injection requires a large number of cells (105-106) multiple, closely spaced injections in all of the major muscle groups of the body may be restricted by the number of cells it is possible to grow from donor muscle in tissue culture. Finally, although fibers or sections of fibers may become converted to dystrophin positivity, the effects of this upon the muscle as a whole and the stability of the conversion have yet to be tested. Based largely upon the encouraging results in the mdx mouse, licences have been granted in the USA and Canada for pilot human trials of MTT in DMD. The resuits of the initial trials have been hard to assess due to the experimental design. However, expression of dystrophin has been reported in muscles injected with normal myoblasts, and not in sham-injected muscles (Law et al., 1990; Huard et al., 1991;Gussoni et al., 1992). It is uncertain whether dystrophin expression in the treated muscles has a significant affect upon muscle function. One study has reported an improvement (Law et al., 1991), but others have indicated that where injected myoblasts have survived, they have had little effect upon the injected muscle (Huard et al., 1992a; Karpati et al., 1992). In addition rejection has been reported even of myoblasts matched at the major histocompatability locus, and antibodies to dystrophin itself have been found in the serum of patients in this study (Huard et al., 1992b); this may have serious implications for the whole proposition of replacing dystrophin in dystrophic patients without long term immunosuppression. Although the results of human trials have on the whole been discouraging, they have highlighted features that may be more appropriately studied in animal models such as the XMD dog. The dog being larger and possessing a severe secondary pathology may be of use in successfully transferring techniques into humans after their initial development in the mdx mouse.

Muscle fibers form during embryogenesis by the fusion of mononucleate myoblasts to form multinucleate syncytia. Myoblasts are determined somitic mesoderm cells that migrate to sites of myogenesis throughout the body in a biphasic fashion. Migration to the premuscle masses and the location of myoblasts on the extracellular matrix is controlled by a family of extracellular matrix receptors, the integrins.

Fusion of growth-arrested myoblasts is controlled by two complementary adhesion systems--the Ca 2§dependent and Ca 2§independent systems. Progression of myoblasts through the various compartments of myogenesis is controlled by a family of muscle-specific proteins, the bHLH proteins. Following fusion, the newly formed myotubes follow a typical pattern of development based upon their lineage. Mature fibers retain the ability to change their physiological properties in response to environmental cues, which are responsible for building the adult characteristics of the different muscle groups. Postnatal growth occurs in response to a variety of stimuli by the fusion of myoblasts derived from satellite cells associated with adult fibers. In response to muscle damage caused by either mechanical trauma or disease, muscle is capable of regeneration. The process of regeneration mirrors that of myogenesis in the embryo--by fusion ofmyoblasts derived from the satellite cell population with each other and the damaged fibers. Regeneration of muscle fibers using normal myoblasts injected into diseased muscle (myoblasts transfer therapy or MTT) may be of use in treating inherited muscle diseases. Experimental MTT has already been studied in the disease Duchenne muscular dystrophy with disappointing resuits in human trials.

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Introduction

Hox Genes in Drosophila Development The Expression of Homeotic Genes Hox Genes in Vertebrates Vertebrate Hox Genes in Different Embryonic Contexts The Developing Head and Neck The Trunk Paraxial Mesoderm The Developing Limb ConclusionmDo Hox Genes Control Regional Form in Vertebrates Summary

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The vertebrate body is the product of a hierarchy of developmental decisions, the result of which is the production of appropriate types of cells in the right places at the fight times. The earliest parts of this process are thought to involve the establishment of abasic body plan at stages when the cells involved are overtly identical to each other. It is thought that groups of cells acquire a property known as positional

Principles of Medical Biology, Volume 11 Developmental Biology, pages 261-291. Copyright 9 1998 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-816-1

value, which directs their subsequent behavior and allows them to produce cell types and structures appropriate to a particular part of the body. For example, cells are thought to become able to detect their position along the rostro-caudal axis of the body, and thus determine whether they will give rise to structures appropriate for the head, thorax, or abdomen. As a consequence of detecting these early axial signals, a group of cells will retain a positional memory, perhaps in the form of a particular stable pattern of gene expression, that will be inherited by their offspring and determine their response to subsequent developmental events. In this chapter, I will discuss the evidence that a particular family of genes, the Antennapedia class HOX genes, are able to provide positional memory of this type to embryonic development.

HOX genes have been found in most animal groups and are thought to have the same fundamental role in all animals : the establishment and maintenance of positional value along the rostro-caudal body axis. They were first identified in the fruit fly, Drosophila, and their role is best understood in this organism (Akam, 1987). Mutations that either prevent Hox genes from functioning or cause them to be expressed abnormally, result in changes in body form that imply an alteration in its overall organization. The most spectacular changes of this type result in the transformation of one body structure to another, for instance, the conversion of an antenna to a leg in the Antennapedia mutation, from which the group of genes gets its name. Such an alteration in body structure is known as a homeotic transformation, and the genes able to cause it homeotic genes. An important feature of such transformations is that the same types of cells are made in both normal and mutant animals, but their overall organization is different in the mutant. The fact that equivalent types of cells can still be made suggests that the molecular machinery that actually converts position along the rostro-caudal axis into final structures is functioning normally, but in the mutant, some cells acquired a positional value that they would not normally have and thus made a structure appropriate to their altered state. Therefore, evidence from mutants in Drosophila suggests that homeotic genes are involved in the control of regional identity within the body plan.

Analysis of the expression of homeotic genes within the Drosophila embryo has suggested how they might act to specify position. One way to investigate gene expression patterns is to detect the presence of the RNA made by a gene by in situ hybridization. An RNA complementary in sequence to that made by the gene under investigation is manufactured in vitro, and labeled with either a radioisotope or a chemical marker that can be recognized by an antibody. Whole embryos or sections

of an embryo are exposed to this probe under appropriate conditions such that the probe RNA binds specifically to its complementary sequence within the embryo. The probe, and hence the endogenous sequence, can then be detected by autoradiography or an antibody specific to the epitope present within the probe. In some cases a specific antibody is available, allowing the direct detection of the gene's protein product in situ. When these localization techniques are used on the homeotic genes described above, they are found to be expressed within some particular body segments but not others, which is consistent with the parts of the body affected in their mutants. The expression domains of the Drosophila homeotic genes and the segments they correspond to are indicated in Figure 1. For instance, the gene prod-

uct of the bithorax gene is normally expressed in the third thoracic segment of the fly, where a balancing organ known as a haltere is carried. In mutants in which bithorax function is lost, the third thoracic segment is very similar to the second thoracic segment, with a second pair of wings developing instead of a haltere. In this case, the Antennapedia gene, normally expressed in the second, but not the third, thoracic segment, is present in the third segment, which therefore adopts an identity appropriate to its pattern of gene expression. It is possible to artificially express a gene within new expression domains, in which case segments that would not normally express the gene adopt a morphology similar to those segments that do. These experiments suggest that as a result of their expression in particular segments along the rostro-caudal axis, Hox genes are determining the position of their cells and hence the form of the structures they or their descendants are to make. Analysis of the protein sequence of homeotic genes provides further evidence of how they control regional identity. The proteins all contain a 61 amino acid sequence known as a homeo box or homeo domain, hence the name Hox genes. This protein motif has been shown to bind specific DNA sequences in the control regions of other genes; this suggests that Hox genes are able to control the activity of other genes. Differences in DNA binding specificity and their ability to interact with other proteins would allow different Hox genes to control different downstream genes and hence produce characteristic final morphologies. Thus, it is thought that segment identity along the rostro-caudal axis in Drosophilais the result of the expression of specific combinations of Hox genes (reviewed in McGinnis and Krumlauf, 1992). The organization of Hox genes within the genome of Drosophila suggests a mechanism by which their spatial patterns of gene expression and consequent segment identity is maintained. The Antennapedia class Hox genes are organized into two clusters on the same chromosome, and all but one of the genes are transcribed in the same direction. Furthermore, the relative position of a gene within its cluster reflects the relative position of its expression along the body axis and the segments whose identity it controls, as illustrated in Figure 1. One mechanism by which patterns of gene expression are thought to be stabilized is inactivation or activation the region of the chromosome they occupy. A gene in a region of inactive chromatin cannot be expressed even if the appropriate soluble factors for its expression are present within the nucleus. After segment-specific patterns of Hox expression are established early on in development, then the linear order of Hox genes in their clusters may allow chromosome structure, inherited by daughter cells at mitosis, to maintain combinations of Hox gene activity during subsequent development. In support of this idea, mutations affecting chromatin components can cause segment transformations and misregulation of Hox gene expression, even though the early establishment of expression domains occurs normally. The position of a gene within its cluster may also be important in the initial establishment of gene expression, perhaps by a similar chromatin-mediated mechanism (reviewed in Gaunt, 1991).

rhe sequences within the homeo domain of the Antennapedia class Hox genes that encode the DNA-binding motif show considerable homology to each other. Other types of developmentally important genes contain a DNA-binding domain with similar protein structure, but with much less sequence similarity to Antennapedia class Hox genes. For example the Pax genes of mice and the prd gene of Drosophila have both a homeo domain and another DNA-binding motif known as a prd box. These other types of homeo box containing genes are not clustered, and though they may be members of families of genes, they do not show great sequence similarity to the clustered Antennapedia class genes. In particular the two mammalian genes now referred to as msx-1 and msx-2, but originally termed Hox-7 and Hox-8, thought to play an important role in several developmental interactions, are not to be confused with the Antennapedia class genes discussed in this chapter.

Given the important role of Antennapedia class genes in Drosophila development, it was thought that genes with similar sequences may be important in vertebrate development. When vertebrate DNA is probed with Drosophila homeodomaincontaining sequences under conditions in which similar but not identical DNA can be detected, numerous genes with related sequences are found. Further analysis has revealed four clusters of Antennapedia class Hox genes, containing 40 genes in total (Simeone et al., 1991). The organization and sequences of the mammalian genes, illustrated in Figure 2, suggest that they are derived from a single ancestral cluster by a process of cluster duplication. Subsequently, there have been losses of genes or groups of genes, resulting in the structure of the present four Hox clusters. Because of the cluster duplications, each gene may have up to three closely related equivalents in other clusters; such gene groups, which show greater sequence similarity to each other than they do to the other genes of their clusters, are said to be paralagous. In the Hox nomenclature, genes are referred to by an initial letter, referring to the cluster they occupy, and a number which gives their position within their cluster; this number is the same for the paralogous genes located in other clusters. As such, the 3'-endmost genes in Hox-A, Hox-B, and Hox-D, paralogues of one another and thus of all members of group 1, are known as Hox-A1, Hox-B1, and HoxD1, respectively (Scott, 1992). There are other parallels between vertebrate and Drosophila genes apart from their clustered organization. The 5' end of an mRNA is manufactured first, and is the point where ribosomes bind during translation, while the opposite end of the molecule is known as the 3' end; thus a Hox cluster is said to have a 5' end and a 3' end, as all the genes of a Hox cluster are transcribed in the same direction. A gene's relative position within a cluster is, thus, referred to as 3' or 5'. Particular paralogous groups of vertebrate genes show higher homology to a specific Drosophila gene than they do to other Drosophila genes; particularly remarkable is that the most 3' group of

Figure2.

The structure of the human and murine Antennapedia-class homeobox gene complexes, illustrating the relationship between members of a subfamily of vertebrate genes and their Drosophila equivalents. Each box represents a gene, consisting of two exons. The Drosophila gene, Zen, indicated in brackets in HOM-C, does not now function as a homeotic gene, although it is still located in the Antennapedia complex and shows extensive sequence homology with pb. Members of a subfamily or paralogous group of genes are vertically aligned with each other with their group number indicated at the base of the diagram. Note that some subfamilies are not represented in all Hox clusters. The bold brackets above the mouse genes indicate the Drosophila homeotic genes that have clear homologues in mammals. The dashed brackets indicate that homology relationships may exist but are less distinct based on existing data. The bracketed numbers to the left of the figure indicate the chromosomal location of their respective gene clusters. The bracketed number to the left of the slash is their human chromosomal location and the number to the right of the slash the mouse chromosomal location. The arrow at the base of the diagram indicates the colinear relationship between the position of genes in the complexes and their anterior limits of expression. vertebrate paralogues is most similar to the most 3' Drosophila gene, labial, and that the two next-most 3' families of vertebrate paralogues are most similar to the next-most 3' Drosophila gene, proboscipedia. This relationship between a group or groups of paralogous genes in vertebrates and a gene (or group of adjacent genes) in Drosophila holds for the whole cluster, as illustrated in Figure 2 (Graham et al., 1989), although there is not a 1:1 correspondence between members of a single paralogous group of vertebrate genes and a single Drosophila gene. When the expression patterns of the vertebrate genes were determined, they were found to be spatially restricted within the vertebrate embryo. In numerous tissues, most obviously in the central nervous system and somites (precursors of the vertebrae among other structures), Hox genes have a discrete anterior limit of ex-

pression that corresponds to their relative position within their clusters. 3' Hox genes are expressed throughout the spinal cord and, up to a discrete level, in the hindbrain, whereas progressively more 5' Hox genes are expressed in progressively more posterior parts of the posterior spinal cord. 3' Hox genes are expressed with anterior expression limits in the cervical somites, while more 5' genes are expressed up to limits in thoracic, lumbar, or sacral somites. The correspondence between the position of a gene in its cluster and its domain of expression is identical to that of Drosophila: In both organisms 3' genes are expressed at the anterior end of the body, while 5' genes are expressed at the posterior end (Graham et al., 1989). The extent of these similarities implies that not only similar genes are present in both Drosophila and vertebrates, but that they are both descended from a cluster of genes, present in the ancestor of both insects and vertebrates. Furthermore, the genes are likely to be involved in the same developmental processes and specification of position along the body axis of the embryo; it is otherwise hard to imagine why the relationship between position of a gene in its cluster, orientation with respect to origin of transcription, and position of expression algng the body axis have all been conserved to this extent. Vertebrate Hox genes are thus likely to be important tools in understanding vertebrate development.

With the notable exceptions of the anterior parts of the face and forebrain and midbrain, most of the structures of the body are derived from tissues whose development is likely to involve Hox genes. Unlike Drosophila, where the segmented region of the early embryo gives rise to most of the adult body by broadly similar mechanisms, different mechanisms are involved in the development of various parts of the vertebrate body. It is also less clear how axial patterning in vertebrates is used to control the form ofbody structures, and which tissues are axially patterned. I will discuss the role of Hox genes in positional specification in three different parts of the vertebrate body: the developing head and neck, the somites of the trunk, and the limb.

In contrast to the rest of the body, much of the skeletal structures of the head and neck are derived from an embryonic source known as the neural crest (Couly et al., 1993). At an early stage of development, the region of the body that will give rise to the head consists of three layers of cells; most dorsally, the ectoderm; beneath it, the paraxial mesoderm; and most ventrally, the endoderm, shown in Figure 3. The mesoderm will give rise to the musculature and the occipital region of the skull, while the endoderm forms the secretory components of the endocrine glands of the

Figure 3.

B

neck, as well as the pharynx. The ectoderm becomes divided into a central region, known as the neural plate, either side of which is the surface ectoderm, as shown in Figure 3A. The edges of the neural plate become elevated, and the whole structure rolls up to form a tube, that gives rise to the brain and spinal cord. The ectoderm left

outside of the neural tube becomes the epidermis. These events are summarized in Figure 3B. The neural crest consists of two strips of cells either side of the midline that form the boundar-y between the neural plate and the surface ectoderm. At the same time as the edges of the neural plate are elevating, neural crest cells are detaching themselves from the inner edge of the ectoderm and migrating ventrally. The dorsal crest cells form condensations that give rise to a series of cranial ganglia, while more ventral cells become associated with a series of pouches, known as the branchial arches, in what will become the neck, (shown in Figure 3C). These pouches are formed by a combination of an outpocketing of the endoderm and an inpocketing of the surface ectoderm. The two indentations meet at a number of points along the future neck, forming a series of cavities that are filled by mesenchymal cells, principally neural crest, migrating in between the ectoderm and the endoderm. The endodermal parts of these outpockefings will give rise to the endocrine glands of the neck in combination with the mesenchyme overlying them. The branchial arch series is highly conserved in all vertebrates, and gives rise to a characteristic set of homologous structures, shown schematically in Figure 4. In mammals, the first branchial arch will become the upper and lower jaws, the second will form the lesser wing of the hyoid bone, and the third and fourth will form the greater wing of the hyoid and laryngeal cartilages, respectively. The ossicles of the inner ear are also derived from the first and second branchial arches.

Although the head and neck form as a result of a series of interactions between a number of tissues, there is evidence to suggest that a crucial stage in this process involves the neural crest, which controls the spatial identity and hence the final form of other structures of the head. It is possible to remove regions of neural folds from quail embryos and graft them to a chick embryo within the egg, replacing the host's neural folds. The quail tissue is able to integrate into the host and give rise to the structures that neural crest cells would in normal development. If quail neural crest from the region that would normally colonize the first branchial arch is transplanted into a chick to replace its second arch crest, the transplanted crest migrates into the second branchial arch, but there it gives rise to a first arch skeleton (Noden, 1988). This observation suggests that the route of crest migration is controlled by the local environment because the grafted crest placed over the second arch migrated into that arch. Even though the crest derived from the graft was in an abnormal position as a result of the operation, it still gave rise to the structures it would have if left in place: the chicks had a duplicated jaw skeleton derived from quail cells posterior to their normal one. This suggests that crest acquires some intrinsic property while still within the neural plate. This property controls the form of the structures it will give rise to later on in development; in other words, the crest becomes spatially programmed. Furthermore, this spatially programmed crest is also able to control the development of other noncrest tissues in the branchial arches. When the muscula-

ture associated with the posterior ectopic jaw skeleton was examined, it was found to be first arch in structure, even though derived from second arch muscles. Similarly the ectopic jaws in some cases had induced an extra beak in the second arch ectoderm, which would normally form the skin of the neck. These experiments suggest that the spatial programming in facial crest can be communicated to other cephalic tissues suggesting a mechanism controlling the forms of all structures at this axial level (Noden, 1988).

The early structure of the hindbrain from which the facial crest arises suggests that spatial programming in the branchial region is first established within the neural plate and then transmitted to other tissues via neural crest. Examination of the developing hindbrain at the time when crest emerges reveals a repeated series of bulges known as rhombomeres. The cellular boundaries between rhombomeres are similar in their cellular properties to the embryonic segments of the Drosophilaembryo and are thus said to be compartments. When cells at the center of a compartment are marked in such a way that their daughter cells can be de-

tected, a clone of cells is found spread throughout the rhombomere and mixed with unmarked cells. If, at the same time, a cell close to a rhombomere boundary is marked, its daughters will be found not to spread into the adjacent rhombomere, although they are capable of spreading within their rhombomere and mixing with the cells there. When cells are marked earlier in development, there do not seem to be any barriers to cell movement or mixing. If a group of cells within a particular rhombomere become different in some property from cells in adjacent rhombomeres, then preventing cells from crossing boundaries will maintain these differences throughout subsequent development. Each rhombomere gives rise to a characteristic pattern of neurons whose initial organization is segmental suggesting that cells do develop according to a program that is characteristic of their rhombomere of origin (Lumsden, 1990). As the rhombomeres generate neural crest, hindbrain segmentation may be linked to intrinsic crest patterning as well. The crest cells from a particular branchial arch are always derived from the same rhombomeres; the first (mandibular) arch crest is derived from rhombomere 1 (rl) and r2, the second (hyoid) arch from r4, and the third arch from r6 (Lumsden et al., 1991). The differences between the structures formed from the branchial arches could result from cells of a rhombomere becoming programmed at a particular point in development such that they or their descendants will respond to later developmental events in a particular way.

There is evidence that Hox genes are responsible for the programming of hindbrain and crest. The first indication of this came from the analysis of gene expression in the head. Hox expression is detected in the part of the neural plate that will become the hindbrain before crest cells emerge, with discrete anterior limits in expression domain. Subsequently these expression limits correspond to rhombomere boundaries, with all the Hox genes of a group expressed in the same rhombomeres, (Figure 4; Wilkinson et al., 1989; Hunt et al., 199 la). Furthermore the Hox genes reveal a two-rhombomere periodicity in expression domains so that the genes of group 2, Hox-A2 and -B2, are both expressed in rhombomere 3 and all posterior rhombomeres, while the group-3 genes, Hox-A3, -B3 and -D3, are expressed in rhombomere 5 and all posterior rhombomeres. The characteristic fates of cells within rhombomeres 3 and 4 may reflect that they express Hox-A2 and-B2 in contrast to the cells in rhombomeres 5 and 6, whose expression of Hox-A3, -B3 and -D3, in addition to -A2 and-B2, may mean that they adopt a different fate. There is experimental evidence that odd and even rhombomeres differ in their cellular properties in that cells from odd and even rhombomeres will not mix with each other, but cells from two odd rhombomeres or two even rhombomeres will (Guthrie and Lumsden, 1991). Using a combination of groups of Hox genes and an additional process to distinguish odd from even rhombomeres, it would thus be possible to give each rhombomere of the hindbrain a unique positional address.

The characteristic Hox expression pattern of its rhombomere of origin is also maintained by neural crest as it migrates (Hunt et al., 1991a, c). The migration path of crest cells from their position of origin, in particular rhombomeres, to specific branchial arches is highly consistent within individuals of a species and conserved between species. A consequence of the combination of Hox genes expressed by crest cells at their formation and these consistent migration pathways is that each branchial arch is colonized by crest populations expressing a combination of Hox genes unique to that arch. R1 and r2, which contribute crest to the first branchial arch, have no Hox expression, while r3, which expresses Hox-A2 and-B2, does not produce crest. The crest of the second branchial arch is entirely derived from rhombomere 4, and hence expresses Hox-B2 and -A2. Rhombomere 5 does not produce any crest, while the third arch crest, expressing Hox-B3 and-A3, reflects its origin in r6. The expression patterns of Hox genes in the rhombomeres and neural crest are summarized in Figure 5. The most 3' group of Hox genes, Hox-A1, -B1 and -D1, would be expected to continue the two-segment, one-branchial-arch pattern of expression demonstrated

by other groups. Being the most 3', they would be expected to be the most anterior, expressed perhaps in rhombomeres 1 and 2, and in the first branchial arch. In fact, they appear to be exceptions to many of the "rules" describing the expression patterns of other Hox genes. Although members of the same group, they have very different expression domains from each other, one gene, Hox D1, seems not to be expressed in the central nervous system at all. They are not expressed more rostrally than their neighboring 5' genes of group 2, and the only gene whose expression pattern persists in the head at stages when rhombomeres and branchial arches are distinct, Hox B1, is expressed in a single rhombomere, r4. Hox-B1, unlike the genes of other 3' groups, shows no expression in mesenchymal crest, although it is expressed in the vii/viii ganglion complex. The reasons for this exceptional behavior are not clear, although one idea is that these differences reflect modifications to the anterior parts of the head that occurred during its evolution from a more simple form.

The Expression of Hox Genes in Other Tissues Initially, the other tissues of the head do not express Hox genes. However, after they have come into contact with neural crest with a particular pattern of Hox expression, they adopt the same Hox expression as the crest (Hunt et al., 199 lc). Thus the surface ectoderm of the second branchial arch, which can be induced to form an ectopic beak when brought into contact with first arch crest, adopts a characteristic second-arch pattern of Hox expression after contact with its normal second-arch crest. A particular characteristic of the branchial region is that the same relationships are found between hindbrain, peripheral nervous system, and branchial arch derivatives in all vertebrates. This link between structures could be related to the fact that they express the same combinations of Hox genes during their development; the Hox code may be part of a labeling system that tissues use to identify other tissues during development, which allows them to cooperate to form structures. An attractive idea is that this pattern of labeled tissues is established by contact with crest expressing a Hox code. Throughout the stages of facial development in which neural crest-controlled spatial programming is thought to act, the structures concerned maintain a characteristic Hox code. At later stages in development, when tissues and organs with particular morphologies have been produced, the Hox code of the head becomes modulated (Gaunt et al., 1989). Members of a group of genes remain expressed within their original rostro-caudal domains but become restricted to different tissues within those domains. For example, Hox-A4 is expressed in the thyroid gland, the mesenchyme, but not the lining epithelium, of the trachea, and the thymus. Hox-B4, a gene of the same group, is expressed in these structures and in addition, in the mesenchyme at the base of the tongue, while -D4 wasnot expressed in the trachea. These observations suggest that Hox genes may have additional roles at later stages in development, after the decisions needed to produce correctly patterned structures have been made. This is the time in development when the differentiation

of particular cell types is the major developmental event, rather than the formation and patterning of structures de novo. Hox genes, as transcription factors, could also be involved when precursor cells make choices as to their final roles in tissues, or in stabilizing the results of these decisions once made. The tissue-specific differences in expression patterns that emerge at later stages of their development would be consistent with such a role for Hox genes. The apparent importance of the neural crest in controlling the form of facial structures and the involvement of segmentation in early hindbrain development, together with the early expression patterns of Hox genes in the developing head, suggest a model by which the form of the head and neck is controlled (Hunt et al., 1991b). Families of Hox genes become expressed in the developing neural plate with a two-segment periodicity, the limits of expression domains coinciding with the boundaries between rhombomeres. Particular regions of the neural plate give rise to the neural crest, which then migrates into specific branchial arches. During and after migration, the neural crest continues to express the combination of Hox genes or Hox code it began to express while part of the neural plate, and is able to communicate this code of Hox expression to the surrounding tissues of the head. Hox codes in the various tissues of the head, in concert with other events whose nature is obscure, control the spatial organization of the structures formed from these early embryonic tissues. Once the form of structures is established, Hox genes are reused in a different context; controlling cell differentiation.

Analysis of the expression patterns of vertebrate Hox genes in the head has raised a number of intriguing possibilities as to their roles during both the formation and differentiation of head structures. However, to prove that Hox genes do control the form of facial structures, it is necessary to show that manipulation of the patterns ofHox gene expression produces alterations in facial form in a similar way to the alterations in segment patterning already described for Drosophila. If a particular structure fails to develop resulting in a change in facial form, an alteration in patterning has not necessarily occurred; it is possible that a crucial cell type or interaction failed to occur without which the structure could not form. If a structure is still present, but its form is altered such that it resembles another structure not normally found at that position in the body, then the case is stronger that an actual alteration to body patterning has occurred. There are two main approaches to manipulating gene activity in mammals both of which make use of the mouse. One approach, making a transgenic mouse, involves directly injecting DNA into the fertilized ovum and then reintroducing the injected eggs into the fallopian tubes of a host mouse. DNA introduced in this way will integrate at random in the nucleus, often as multiple tandem copies, and be passed on to all the cells of the embryo. If the introduced DNA contains a control region and the coding sequences of a gene, then the introduced gene is likely to be expressed in a pat-

tern appropriate for the control region used, although, in practice, this control region may not be the only factor directing gene expression. By using the control regions of one gene to drive the expression of another, it is possible, in theory, to express a gene anywhere in the body it is desired. Both the temporal and the spatial expression pattern of a gene are controlled by DNA sequences associated with and sometimes within the protein coding regions. There may be several separate regions controlling a particular gene, and they may be dispersed over extensive regions of genome. There is an upper size limit for the fragments of DNA that can be introduced in transgenic mouse experiments, which is considerably less than the distances that can exist between the control regions in the genome. Thus sometimes, of necessity, sequences capable of producing only part of a gene's expression pattern are in fact used, especially when, as is often the case, an exhaustive analysis of regions controlling a gene's expression pattern has not been carried out. In particular, there have been cases where the spatial expression pattern of a gene can be reconstructed, but the temporal onset of expression may not be correct. Given that developmental decisions occur during defined periods of time, it is important when designing gene misexpression experiments that the introduced control regions are not only producing the correct spatial pattern of expression but also producing that pattern at the appropriate times in development. An additional consideration is that in some cases the introduced DNA integrates within or close to a gene, so it is important to exclude the possibility that the insertion site is modifying the expression pattern of the introduced gene. The second approach, known as gene targeting or gene knock out, is able to replace an endogenous gene with an introduced sequence, most frequently in such a way that the endogenous gene is inactivated. This technique makes use of the fact that it is possible to remove cells from the inner cell mass of the mouse blastocyst, culture them in vitro under conditions in which they will not differentiate, and then reintroduce them into a host blastocyst. Such cells known as embryonic stem cells or ES cells are able to contribute to all the tissues of the chimeric animal derived from the blastocyst, including the germ line, with the result that some of the gametes produced by the chimera will be entirely derived from the cultured cells. When ES cells are in culture, it is possible to expose them to DNA constructs derived from a particular gene, and by means of an antibiotic selection, to isolate cells that have exchanged one of their own genes for the introduced DNA. If the construct is a nonfunctional version of a gene, then cells can be produced that will lack one copy of that gene; such cells can then be reintroduced into a host blastocyst. Once a mouse is produced able to transmit one nonfunctional copy of a gene to its offspring, it is possible by conventional breeding techniques to produce mice lacking both copies of a gene. Such double mutants are preferable in analysis as their phenotypes are usually less ambiguous than those where one functional copy of the gene remains. At this time of writing, three anterior Hox genes have been knocked out--HoxA1, Hox-A3 and Hox-B4 (Chisaka and Capecchi, 1991; Lufkin et al., 1991; Chisaka et al., 1992; Ramffez-Solis et al., 1993)mand the alterations seen in all cases are associated with the region of the body where the anterior limit of gene expression is

located. In no case has transformation of the identity of neural crest-derived structures occurred, although mice lacking Hox B4 show alterations in their cervical vertebrae. Neural crest-derived structures are affected, but the defects seen are more consistent with alterations in the differentiation of cell types rather than the reorganization of spatial form. Defects include lack of thymus and parathyroid glands, greatly reduced thyroid tissue, and absence of the ossicles of the middle ear, but there was no evidence of extra structures forming in their place. Thus, there is evidence to support the idea that Hox genes are important in the differentiation of specific cell types but not in the overall patterning of spatial form. In every case of experiments where a particular member of a Hox group has been knocked out, at least two and sometimes three other genes of the same group remain unaffected in the genome. These other genes show extensive homology to the disrupted gene, and are expressed in identical combinations of rhombomeres and branchial arches. This raises the possibility that they are able to compensate for the lost gene at earlier stages of development at the time when the identity of rhombomeres and branchial arches is being established (Hunt and Krumlauf, 1991). These processes would carry on as normal in mice lacking a single Hox gene, hence the absence of alterations in branchial arch patterning. The mutant phenotypes reflect the first function of the disrupted Hox gene for which the other genes cannot compensate, which corresponds to the time in development when cell differentiation is the major process and expression patterns of group members become different from one another. In flies, there is only one cluster of genes, and hence disruption of one gene could not be compensated for by a gene of similar sequence. To demonstrate a transformation of structures in a vertebrate by Hox gene disruption, it may be necessary to simultaneously inactivate all the genes of a Hox group. In theory, a more straightforward experiment is to express a Hox gene beyond its normal domains, as the other genes of its group would not be expected to be expressed in its new domain. At this time of writing, three experiments misexpressing Hox genes in the cranial region have been described (Bailing et al., 1989; Kessel et al., 1990; Lufldn et al., 1992; Pollock et al., 1992). In one case, misexpression of Hox-A7 was obtained using the actin promoter, while in the other two, Hox gene promoters were used; Hox-D4 under the control of the Hox-A1 promoter and HoxC8 under the control of the Hox-D4 promoter. In the experiment involving the actin promoter, the expression pattern of the construct was not described, and although this promoter might be expected to produce a uniform expression throughout the body, it is not clear whether this is in fact the case. It is rare for a promoter to produce truly ubiquitous expression throughout all tissues of the body, even when it might be expected to. In the experiment involving misexpression of Hox-D4, the expression domain of the transgene was described, but because of the peculiarities in expression domain already described for the group-1 genes, did not result in misexpression of Hox-D4 in neural crest-derived structures. Despite using the promoter regions of Hox-D4, a gene normally expressed in the hindbrain and fourth branchial arch, ectopic Hox-C8 expression was only achieved in the hindbrain and

not in hindbrain crest derivatives. Thus, a clear-cut experiment in which the Hox code of the neural crest has been directly altered has not yet been performed. In experiments involving misexpression of Hox-A7 and Hox-D4, there were alterations to the palate, which forms from the neural crest-derived maxillary process. However, this is likely to be due to secondary effects, as a wide range of teratogens also produce this kind of defect. No effects on hindbrain crest-derived structures were described in the experiments involving ectopic Hox-C8 expression. Alterations in neural crest patterning would be expected to produce more extensive defects in the full range of derivatives of a particular axial level, and given the uncertainty as to whether Hox expression is in fact altered in the crest, the defects in palate development may be a consequence of the alterations to skull form described in the next paragraph. Both Hox-A7 and Hox-D4 misexpression experiments resulted in alterations to the atlas such that it adopted a morphology similar to more posterior vertebrae. In addition, the transgenic misexpressing Hox-D4 showed alterations the skull with a series of structures similar to neural arches in place of the occipital bone. These alterations suggest that the paraxial mesoderm of the skull base and the rostral parts of the neck are adopting the morphology of more caudal vertebrae. This would be the expected result of rostral tissues expressing a Hox gene combination appropriate to more caudal parts of the body with the result that they adopt a morphology dictated by their new Hox code. Artificial and natural mutants in Drosophila that result in transformations of body segments to a more posterior identity result from similar perturbations of Hox expression, and genes are expressed in segments more rostral than they would normally. It is interesting that in the two cases using Hox promoters, misexpression of Hox expression in the hindbrain was observed at mid-gestation, and yet no alterations to hindbrain development were detected. Mice misexpressing Hox-C8 were born alive and able to breathe, which suggests that their hindbrain respiratory centers were functioning normally, arguing against disruption of these hindbrain structures at least. This may reflect that the neuroectoderm is more refractory to Hox gene misexpression, and as with the gene deletion experiments, a single gene is unable alone to perturb development. Thus, while a conclusive experiment has still to be performed for the neural crest, there is evidence from the axial skeleton that alterations in Hox gene expression patterns can alter cranial morphology in a way consistent with alteration in the control of spatial form.

In the early development of the trunk, the neural tube and its derivatives are not thought to control the form of mesenchymal structures. Instead, the patterning influences are thought to be intrinsic to the mesenchyme itself. After the cell rearrangements of gastrulation, the mesodermal tissues of the trunk become organized into a number of regions extending from the midline to the lateral edge of the embryo. Beneath what will become the neural plate lies the notochord, which will eventually become incorporated into the vertebral centrum. On either side of this

are the somites, paired blocks of tissue that will form the dermis of the skin, the skeleton, and skeletal muscle. Lateral of the somites is a segmented region of socalled intermediate mesoderm, which will give rise to the kidney, and most lateral of all is the unsegmented lateral plate mesoderm, which will give rise to the limb buds and visceral musculature. These structures are shown diagrammatically in Figure 6. The skeletal structures of the limb will form from the limb buds, while the limb musculature is derived from the somites that lie opposite the limb bud. There appear to be at least two mesoderm-based patterning systems operating in the trunk, one based on the somites giving rise to the vertebral column, and another independent system patterning the lateral plate mesoderm that gives rise to the limbs. The pattern-forming ability of trunk paraxial mesoderm appears to be intrinsic as somites will produce vertebrae and ribs appropriate to their site of origin when grafted to ectopic sites (Chevallier, 1975). This pattern-forming ability also is important in the development of the spinal cord where, in contrast to the head, the somites control the pattern of nerve outgrowth from the central nervous system. In normal development, spinal motor nerves grow through the anterior halves of their

adjacent somites. This process is under the influence of positional values within the somites, as reversal of the A-P polarity of a group of somites causes corresponding changes in the position of nerve outgrowth, although the neural tube has remained in its original orientation (Keynes and Stern, 1985). In contrast, after neural tube reversal, outgrowth still occurs opposite the rostral halves of somites. Replacement of head paraxial mesoderm with either segmental plate or somites results in a normal head skeleton with normal patterns of neural development (Noden, 1986). This is consistent with the greater importance of neural plate derivatives in head development described in the previous sections. The overtly repetitive nature of the somites raises the possibility that they, like the rhombomeres of the hindbrain, are segmental structures. However, clonal analysis of the type performed in the hindbrain does not suggest the presence of compartments in the somitic mesoderm. Thus, while there is evidence for positional specification of somites, it does not appear to be mediated by the same sort of lineage restriction processes that occur in the hindbrain.

Hox genes are likely to be involved in specification of positional identity in the trunk, but the way in which paralogous genes behave is different from the head, reflecting differences between the molecular processes underlying development in head and trunk. One of the earliest observations of Hox gene expression patterns was that they showed spatial restrictions that corresponded to somite boundaries. In contrast to the situation in the head, there appear to be differences between paralogous genes in their expression limits in somites. The expression patterns of Hox genes in the prevertebrae, derivatives of the somites, are shown in Figure 7. These differences are small in anterior gene groups; thus Hox-A4 shows a prevertebral expression limit in pv3 (prevertebra 3) this is different from Hox-B4 and Hox-D4 in pv2, all of which are expressed in identical rhombomeres and branchial arches. More posterior paralogous genes show greater differences in anterior expression limits, with similar differences also being apparent in the spinal cord. This has led to the suggestion that somite identity is specified by a Hox code (Kessel and Gruss, 1991; Kessel, 1992) and that in contrast to the situation in the branchial region, paralogous genes have independent roles in somite specification from the very outset. It is clear from Figure 7 that not every somite has a unique Hox code and that some members of paralogous groups are expressed with identical somitic cutoffs. Clearly, other genetic systems must also be acting to produce distinctions between the final forms of somite derivatives just as some alternative system in the hindbrain must be specifying the difference between odd and even rhombomeres. It is interesting to note that groups of vertebrae that are similar in their morphology, such as some of those in the cervical region, express identical Hox codes.

Figure 7. Diagrammatic representation of the expression of Hox genes in the prevertebrae, one of the derivatives of the somites. The colored bars represent the expression domains of different groups of Hox genes within the types of prevertebrae indicated on the left. Exnerimental Evidence of thp Rolp of Hox Genes in the Trunk One group of experiments has centerd on the ability of retinoic acid in vivo to produce alterations in vertebral morphology of mouse embryos consistent, in some cases, with transformations of somite identity (Kessel and Gruss, 1991; Kessel, 1992). By feeding the mother with retinoic acid from 7 dpc. (dayspostcoitus) to 8.5 dpc. (i.e., when gastrulation is occurring), animals are produced in which anterior vertebrae could be interpreted to have taken on an identity appropriate for more posterior parts of the body, such as cervical ribs. A number of different developmental abnormalities have been identified in mice consistent with the conversion of anterior structures to more posterior ones (Kessel and Gruss, 1991), while treatment at stages later than 8.5 dpc produces different types of alteration (Kessel, 1992). It is particularly interesting that retinoic acid should produce these kinds of effects on development, as it is known to be able to modulate the expression of Hox genes in cultured mouse (F-9) and human (NTERA-2) teratocarcinoma cells (Papalopulu et al., 1991; Simeone et al., 1991). It is therefore possible that the defects observed are a result of alterations in the domains of Hox gene expression that go on to produce somites with altered morphologies. Consistent with this is the fact that animals treated with retinoic acid at 7.4 dpc. show anterior extension in the domains of expression of particular Hox genes at 12.5 dpc. (Kessel and Gruss, 1991). If this is occurring with

other Hox genes and at the stages when somite specification is occurring, then the result would be an activation of extra Hox genes in particular somites. A somite would thus be expressing a combination or code of Hox genes appropriate to a more posterior axial level and would adopt an appropriate morphology as a result. If the structures a somite forms are controlled by the code of Hox genes that a somite expresses, then it may be possible to change somite identity by direct alteration of the expression of Hox genes. This can be attempted by either inactivating a gene by homologous recombination to produce a null mutant or by expressing a gene inappropriately to produce a gain-of-function alteration. The phenotypes of mice lacking a functional copy of the Hox-C8 gene have been described (Le Mouellic et al., 1992). In contrast to the effects of mutations in the branchial Hox genes, analysis of these mice has revealed several skeletal alterations affecting different axial levels consistent with their transformation to a more anterior identity, for example, ribs on the first lumbar vertebra, extra sternabrae, and additional ribs fused to the sternum. In each case, the transformation was to the morphology of the element immediately anterior. The affected region of the body axis was more extensive than in mice lacking 3' genes, although entirely contained within the normal expression domains of the gene. The described morphological abnormalities were confined to the skeleton, although there was some evidence of abnormal motor activity in the mutants that survived to adulthood. The transformation of vertebrae to the form of those more anterior as a result of a gene deletion is consistent with the idea that vertebral fate is a direct consequence of the combination of the Hox genes they express. However, when the same gene was misexpressed using the Hox-D4 promoter such that its expression domain was moved three prevertebrae to the anterior, a very similar phenotype to the null mutant was found. Ribs were found on the first lumbar vertebra and an extra pair of ribs was attached to the sternum. There was no evidence for transformations of structures to a more caudal identity, even though three prevertebrae expressed Hox-C8 that would have been anterior of its expression domain in normal development. Despite the fact that alteration of the expression pattern of a Hox gene is able to produce alterations in somite identity, it is not easy to relate these findings to a model in which combinations of Hox gene expression alone confer regional identity upon the developing somites.

In the two systems using Hox genes so far described, their primary role is thought to be positional specification along a single dimension, the rostro-caudal body axis. Hox genes in the developing limb appear to be employed in the establishment of regional identity in two dimensions, in contrast to the other two systems we have described. The cells of the limb bud derive from a number of sources the most important of which appears to be the lateral plate mesoderm. This will give rise to the limb carti-

lages and connective tissues, and seems to be the most important tissue in defining the limb structures. Somite-derived cells also enter the limb, but the form of the muscles they contribute to appears to be controlled by the lateral plate-derived tissues. The establishment of structures along the rostro-caudal axis and the proximo-distal axis of the limb bud appears to involve two systems with different properties (reviewed in Tickle, 1991). The limb bud is first apparent as a bulge in the lateral plate mesoderm overlain by a thickened rim of surface ectoderm (Figure 6B) that will go on to form a structure known as the apical ectodermal ridge (AER). As the limb bud grows out from the flank, its structures are laid down sequentially with the most proximal structures (the upper arm or leg) differentiating first and the most distal (the digits) forming last. Recombinations between the ectoderm and mesenchyme of different-aged limb buds show that the mesenchyme contains the information as to the proximodistal identity of the limb structures still to form. It is thought that the apical ridge is necessary to maintain a "progress zone" of undifferentiated mesenchyme where cells successively leave the proximal side and begin to differentiate. The time a cell spends in the progress zone determines the proximo-distal identity of the structures it will form. The cartilages are the first structures to appear with region-specific morphologies emerging from precartilaginous condensations. Patterning along the rostro-caudal axis is thought to involve a positional signal that is interpreted by the cells to form structures of the appropriate type. The caudal margin of the limb bud contains an area of mesenchyme known as the polarizing region. This is able to produce mirror image duplications of structures in the rostrocaudal axis when grafted to the rostral margin of another limb bud whose tissues then become exposed to two polarizing regions. The fate of grafted cells suggests that they exert their effects by signaling to other limb cells rather than by directly contributing to the supernumerary structures. The response of cells to grafts of different amounts of polarizing region tissue and grafts to different locations within the limb bud is consistent with a graded distribution of a signaling substance, the local concentration of which determines the character of a particular region of limb mesenchyme. Implantation of beads soaked in retinoic acid on the rostral margin of the limb bud produces duplication effects similar to a polarizing region graft, suggesting that it is able to affect the normal signalling events that occur during limb development. A reciprocal interaction is thought to occur between limb mesenchyme and AER (Tickle, 1991). Duplication of elements along the rostro-caudal axis of the limb is known to be dependent upon the continued presence of the apical ectodermal ridge and is preceded by an extension in length of the AER that is dependent upon the mesenchyme (Tickle, 1991). Complete removal of the AER results in cessation of limb bud outgrowth, while partial removal results in loss of pattern elements at the corresponding rostro-caudal level (Tickle, 1991). There is no evidence for compartment formation during the early events of vertebrate limb morphogenesis.

At an early stage before limb bud outgrowth, the lateral plate mesoderm of the presumptive limb bud expresses Hox genes of classes 1-9 appropriate to its level along the rostro-caudal axis. During limb bud outgrowth, new patterns of Hox expression involving genes of classes 10-13 are established in limb mesenchyme. The pattern of Hox expression in the limb bud is shown in Figure 8. It is notable that these genes and those of class 9 all show greatest similarity to a single Drosophila gene, Abd-B, and that one vertebrate Hox cluster, Hox-B, does not possess any genes in classes 10-13. This suggests that either the genes of classes 10-13 have arisen by tandem duplication of an ancestral Abd-B-related gene since the divergence of the lineages that lead to vertebrates and arthropods, or they are the product of an earlier duplication in an organism ancestral to both lineages and have been lost in the Hox-B cluster and Drosophila (Izpisua-Belmonte et al., 1991a). The Hox-D genes in both chicken and mouse show progressive spatial restrictions along the A-P axis of the limb bud, an observation consistent with their position within the Hox-D cluster (Dolle et al., 1989; Izpisua'Belmonte et al., 1991b; Nohno et al., 1991). Hox-D9 is expressed throughout the limb bud, while HoxD l l is expressed in a more restricted domain that does not extend to the rostral

surface of the bud. The domains of Hox-Dll and Hox-D13 are contained within the Hox-D9 domain, and are progressively more caudally restricted. The expression domains of Hox genes in the limb are shown in Figure 7. Although these spatial restrictions correspond to regions of the limb bud with consistent fates, the boundaries of gene expression do not correspond with the boundaries of particular structures in the way that expression of Hox genes corresponds to particular rhombomere boundaries. The timing of expression is such that the most anterior gene is activated first, and then successively more posterior and 5' genes are activated successively later. Hox-A genes show spatial restrictions along the proximo-distal axis of the limb (Yokouchi et al., 1991), perpendicular to the expression domains of Hox-D. Successively more 5' Hox-A genes show progressively more distal restrictions, and there is a temporal progression in onset of expression correlating with their spatial distribution. The expression domains are not entirely overlapping, as the Hox-AlO gene eventually comes to be expressed in a band across the limb with both a proximal and a distal expression limit. There is no overlap in expression between this and the more distally restricted Hox-A13 gene. The expression boundaries of Hox-A genes correspond to some of the points in the proximo-distal axis where the number of skeletal elements change, but this also occurs in areas with homogenous Hox expression patterns. The limb makes use of Hox genes in axial patterning in a way independent of the main body axis. Both fore and hind limbs use 5' genes that are also used in the patterning of very posterior parts of the body such as the genitalia (Izpisua-Belmonte et al., 1991 a). Their onset of expression is a function of the age of the limb bud and in the case of the forelimb involves the use of "posterior" genes that are not expressed in any of the other tissues at the axial level of the forelimb. Furthermore, the limb is unique so far in using paralogous Hox genes from two clusters in separate axial patterning events that show distinct developmental properties from each other. These observations suggest that the roles of the Hox-A and Hox-D genes are distinct in limb and trunk. It is, therefore, conceivable that the position of cells within a limb bud, and hence the form of the structures they are to make, is partially determined by reference to the overlapping expression domains of Hox-A and Hox-D genes (illustrated in Figure 7). The expression domain of only one 5' Hox-C gene, Hox-ClO, has been characterized at the time of writing. Unlike the 5' Hox-A and Hox-D genes, which show similar spatial restrictions in both fore- and hindlimbs, Hox-ClO is not expressed in the forelimb (Peterson et al., 1992). The expression domain in the hind limb shows an anterior-proximal restriction suggesting that it could provide additional positional cues in either antero-posterior or proximo-distal axes within the limb. As of the time of writing, it is not clear whether the other 5' Hox-C genes show different expression restrictions within the limb, and it is important to note that the more 3' Hox-C6 gene shows a similar proximo-distal restriction within limb buds (Oliver et al., 1990). If all Hox-C genes show a similar spatial restriction within the limb bud,

they are unlikely to be involved in the types of regional specification within limb buds that employ Hox-A and Hox-D.

The Response of Hox Expression to Manipulation of Limb Development The accessibility of limb buds to experimental manipulation and the extent to which the timing of processes during the development of the limb is understood have provided powerful indirect evidence for their role here. Implantation of retinoic acid-soaked beads or grafts of polarizing regions have been shown to induce ectopic domains of 5' Hox-D gene expression in chicken limb buds, correlating with the duplication of distal limb elements. Izpisua-Belmonte and colleagues (1991 b) were also able to investigate the timing of onset of the ectopic domains of expression. It is known that implantation of retinoic acid-soaked beads must occur for a minimum of 16 hours to produce consistent mirror-image digit duplications, shorter exposure times being without effect. Limb buds exposed to retinoic acid for up to 10 hours showed no alterations in Hox-D gene expression immediately after bead removal; however, after 16 hours, an ectopic domain of Hox-D9 expression was visible at the dorsal margin of treated wings. At successively longer times of exposure to retinoic acid more 5' Hox-D genes were activated, and the fact that a 5' Hox-D gene was in no case activated before its more 3' neighbors suggests that the temporal and spatial sequences of gene activation are interdependent. Retinoic acid, as has been described above, is able in vitro to activate Hox gene expression. However, the in vitro characteristics of 5' Hox-D genes are not consistent with their behavior in retinoic acid-treated limb buds. In vitro, progressively more 5' and posteriorly expressed Hox-D genes are increasingly repressed by retinoic acid; yet in limb buds where a retinoic acid bead is implanted anteriorly, Hox-D genes become activated. This implies that the signaling pathways by which Hox-D genes are repressed by retinoic acid in vitro differ from those that set up the spatial domains of expression in limb buds. A naturally occurring genetic perturbation of limb development, talpid, has been described. Proximo-distal patterning of the limb appears to occur normally, but instead of the normal pattern of digits a single, large plate of cartilage is produced. The expression of Hox-D13 in talpid embryos has been found to be homogenous across the distal parts of the limb bud instead of the normal posterior restriction in expression (Izpisua-Belmonte et al., 1992). This suggests that talpid results in the failure of the rostro-caudal patterning mechanism within the limb bud, resulting in the entire limb adopting a posterior identity reflected in its pattern of Hox expression. Therefore, the continuing correlation between Hox-D gene expression and particular morphologies in limb buds whose development has been manipulated by retinoic acid or genetically suggests strongly that the establishment of particular patterns of Hox gene expression is required for specific types of final morphology

to be produced. The expression patterns are consistent with an involvement in the specification of regional identity by a coordinate system along the proximo-distal and anterior-posterior axes that involve Hox genes of two clusters expressed orthogonally to each other.

Direct Manipulation of Limb Development with Hox Genes The development of retroviral vectors able to introduce genetic material into chick embryos has enabled the direct manipulation of Hox expression in the limb bud (Morgan et al., 1992). A replication-competent retrovirus able to express the murine Hox-DI1 gene was injected into the region of flank that gives rise to the limb bud, such that by the stage when limb mesenchyme cells were still relatively plastic in their development, all cells within the bud had been infected by virus containing mouse Hox-D1 I . Because of the use of mouse Hox-DI 1 within a chick limb bud, it is possible to distinguish between the expression of endogenous chicken genes and introduced mouse genes on the basis of their slight differences in sequence. The expression pattern of the endogenous chicken genes, including HoxD 1 I , is unchanged, while mouse Hox-DI I is expressed throughout the limb bud. This ubiquitous expression of Hox-DI I produces two effects on the limb Hox code. The first is to expand the region of limb bud corresponding to digits 11,111, and IV that expresses Hox-D11 and Hox-DIO. The second is to generate a novel Hox code in the anterior parts of the limb that never occurs in normal development, that is, the combination of Hox-D9 and Hox-D11 without Hox-D10 expression. The effects of this alteration differ between the fore- and hindlimbs. In the forelimb, an extra digit is produced in the anterior portion of the wing with a similar morphology to wing digit 11. This anterior region of the wing bud normally undergoes programmed cell death. In the hind limb bud the number of digits is unchanged, but digit I and, in some cases, the metatarsal from which it arises show a morphology appropriate to digit 11. The manipulation of the expression pattern of a single Hox gene within the limb gives unambiguous evidence that Hox genes are involved in the control of digit form, and that they are likely to be doing so by specifying the spatial identity of the various parts of the limb bud. However, given that both the overexpression and the deletion of Hox-C8 produce similar results in the trunk, it is best to be cautious about the precise mechanism by which Hox codes are "read" in order to produce the final pattern of the limb.

In this chapter, I have discussed the evidence from a number of parts of the body that Antennapedia-class Hox genes are involved in the control of regional charac-

ter in development. When morphology is altered by experimental means, such as a ZPA graft in the limb or retinoic acid treatment in the trunk, the Hox code reflects the new morphology that emerges. The tight link between the two strongly suggests that the Hox code is a prerequisite for morphology to emerge, and given the expression patterns I have described and the extensive conservation with Drosophila, it seems most likely that Hox genes are involved in setting up spatial patterns within different parts of the body, which are then used to produce final morphology. At the moment, however, it is completely unclear how the Hox gene patterns in practice produce the final morphology. As transcription factors, Hox genes can only act by controlling the expression of other genes. It is these other genes that will actually produce the spatial pattern, but for the moment their nature is largely obscure, although this is a very active area of current research. Furthermore, it is not clear how a particular pattern of Hox expression in the vertebrate embryo relates to the structures that are finally formed. A simple model for Hox gene function was originally proposed on the basis of the overlapping patterns of gene expression in Drosophila. In this model, the most 5' gene Hox gene or gene group expressed in a tissue determines the morphology of a structure. If applied to the branchial region, for example, because the third branchial arch expresses the genes of groups 2 and 3, you would predict that the characteristic third arch morphology is a result of the group 3 genes, and the fact that the characteristic group 2 genes of the second arch are expressed there is irrelevant. By this model, misexpression of a third arch gene in the second arch would result in a switch to a third arch morphology, as the single third arch gene would take priority over the genes already there. Thus, misexpression of a gene should result in posteriorization of structures. This model does seem to explain some of the developmental mutants of Drosophila, but in vertebrates when transformations to a posterior phenotype do occur, for example in the vertebral column, in no case has the posterior phenotype appropriate to the gene misexpressed been produced. An alternative, less extreme model suggests that it is the combination of Hox genes expressed at an axial level that controls positional value, but even this cannot explain the results of manipulation of Hox-C8 expression in the trunk, where both gene knockout and overexpression produce similar transformations of vertebral phenotype that do not directly relate to the final Hox code that is produced. The simple models where there is a direct concordance between gene expression and final regional form were based on the Drosophila system. In Drosophila, the adult ectodermal tissues are derived from small disks of cells within the larval body cavity that are isolated from those of other segments during much of the time when interpretation of positional value occurs. Thus, there is considerable autonomy in the development of segments such that perturbations within one do not necessarily have an effect on the derivatives of adjacent segments. In this sort of situation, alteration of the Hox code will lead to clear-cut transformations of an entire segment, as the final form of the structures produced by an imaginal disk is inevitably the re-

sult of the programming of the disk alone, which in turn is a consequence of its pattern of Hox expression. Even though there is evidence for segmental processes in vertebrate development, in particular in the hindbrain and the neural crest derived from it, the subsequent development of the branchial region and other parts of the body involves groups of structures in close contact with one another. To a far greater extent than in the Drosophila ectoderm, the final structures of the vertebrate body form from primordia derived from a number of sources expressing different Hox codes. For example, in the branchial region of the head, the hyoid bone is derived from fusion of parts of both the second and the third arches, each of which has a distinct Hox code. If a number of primordia must interact before a recognizable structure appears, alteration of the programming of one component may prevent or alter its interaction with the rest, the result of which may be that the final structure cannot appear in any recognizable form. In other words, Hox genes may be involved in exactly the same processes of establishing early body regions as in Drosophila, but because of the differences in how these regions go on to produce the final body plan of the organism, vertebrate alterations in Hox function do not produce alterations to the body plan easily interpreted in terms of alterations to the Hox code. In conjunction with this, because of the four vertebrate Hox clusters to the Drosophila one, alteration of a single vertebrate Hox gene produces a much less significant change in the vertebrate Hox code than do Hox gene alterations in Drosophila. To demonstrate a transformation of structures in a vertebrate by Hox gene disruption, for instance, it may be necessary to simultaneously inactivate all the genes of a Hox group. This interpretation may explain why both overexpression and inactivation of Hox-C8 result in the same type of transformations in vertebral phenotype. The actual final phenotype of vertebrae would not be a direct consequence of the combinations of Hox genes the cells express. Instead, Hox genes would act in combination with other gene products to set up the axial character of particular regions of the A-P axis. This would result in the appearance of vertebrae with specific phenotypes but not necessarily entirely as the result of influences intrinsic to each somite in isolation. The Hox genes might act to define the boundaries of action of other developmental influences, with these targets of Hox gene function actually determining which vertebrae carry ribs and so on. Certainly the increase in the number of ribs fused to the sternum and the production of lumbar ribs would be consistent with this type of indirect mode of Hox action.

In this chapter, I have discussed the evidence, both direct and indirect, for the function of Hox genes in vertebrate development. While the evidence for a role of Hox genes in the determination of regional form is strong, the mechanisms by which they act are largely obscure, although this is an active area ofresearch. In addition to

their role in early development, Hox genes are reused at later stages of development in a new role, the control of the differentiation of specific cell types.

I would like to thank Drs. Robb Krumlauf, Cheryll Tickle, and Peter Thorogood for advice and discussions.

Akam, M. (1987). The molecular basis for metameric pattern in the Drosophila embryo. Development 101, 1-22. Balling, R., Mutter, G., Gruss, P. & Kessel, M. (1989). Craniofacial abnormalities induced by ectopic expression of the homeobox gene Hox-l.1 in transgenic mice. Cell 58, 337-347. Chevallier, A. (1975). Role du mesoderme somitique dans le developpement de la cage thoracique de l'embryon d'oiseau. I. origine du segment sternal et mecanismes de la differenciation des cotes. JEEM 33, 291-311. Chisaka, O. & Capecchi, M. (1991). Regionally restricted developmental defects resulting from targeted disruption of the mouse homeobox gene hoxl.5. Nature 350, 473-479. Chisaka, O., Musci, T. & Capecchi, M. (1992). Developmental defects of the ear, cranial nerves and hindbrain resulting from targetted disruption o f the mouse homeobox gene Hox-1.6. Nature 355, 516--520. Couly, G., Coltey, P. & Le Douarin, N. (1993). The triple origin of the skull in higher vertebrates: A study in quail-chick chimeras. Development 117, 409-429. Dolle, P., Izpisua-Belmonte, J.-C., Falkenstein, H., Renucci, A. & Duboule, D. (1989). Coordinate expression of the murine Hox-5 complex homoeobox-containing genes during limb pattern formation. Nature 342, 767-772. Gaunt, S. J. (1991). Expression Patterns of Mouse Hox Genes--Clues to an understanding of Developmental and Evolutionary Strategies. Bioessays. 13,505-513. Gaunt, S. J., Krumlauf, R. & Duboule, D. (1989). Mouse borneo-genes within asubfamily, Hox-l.4, -2.6 and -5.1, display similar anteroposterior domains of expression in the embryo, but show stageand tissue-dependent differences in their regulation. Development 107, 131-141. Graham, A., Papalopulu, N. & Krumlauf, R. (1989). The murine and Drosophila homeobox clusters have common features of organization and expression. Cell 57, 367-378. Guthrie, S. & Lumsden, A. (1991). Formation and regeneration of rhombomere boundaries in the developing chick hindbrain. Development 112, 221-229. Hunt, P. & Krumlauf, R. (1991). Deciphering the Hox Code: Clues to patterning the branchial region of the head. Cell 66, 1075-1078. Hunt, P., Gulisano, M., Cook, M., Sham, M., Faiella, A., Wilkinson, D., Boncinelli, E. & Krumlauf, R. (1991a). A distinct Hox code for the branchial region of the head. Nature 353,861-864. Hunt, P., Whiting, J., Muchamore, I., Marshall, H. & Krumlauf, R. (1991b). Homeobox genes and models for patterning the hindbrain and branchial arches. Development 112 (Supplement: Molecular and cellular basis of pattern formation), 187-196. Hunt, P., Wilkinson, D. & Krumlauf, R. (1991c). Patterning the vertebrate head: Murine Hox-2 genes mark distinct subpopulations of premigratory and migrating neural crest. Development 112, 43-51.

Izpisua-Belmonte, J.-C., Falkenstein, H., Dolle, P., Renucci, A. & Duboule, D. (1991 a). Murine genes related to the Drosophila AbdB homeotic gene are sequentially expressed during development of the posterior part of the body. EMBO 10, 2279-2289. Izpisua-Belmonte, J.-C., Tickle, C., Dolle, P., Wolpert, L. & Duboule, D. (1991b). Expression of homeobox Hox-4 genes and the specification of position in chick wing development. Nature 350, 585-589. Izpisua-Belmonte, J.-C., Ede, D. A., Tickle, C. & Duboule, D. (1992). The misexpression of posterior Hox-4 genes in talpid (ta3) mutant wings correlates with the absence of anteroposterior polarity. Development 114, 959-963. Kessel, M. (1992). Respecification of vertebral identities by retinoic acid. Development 115, 487-501. Kessel, M. & Gruss, P. (1991). Homeotic transformations of murine prevertebrae and concomitant alteration of Hox codes induced by retinoic acid. Cell 67, 89-104. Kessel, M., Balling, R. & Gruss, P. (1990). Variations of cervical vertebrae after expression of aHox 1.1 transgene in mice. Cell 61,301-308. Keynes, R. & Stem, C. (1985). Segmentation and neural development in vertebrates. TINS 8,220-223. Le Mouellic, H., Lallemand, Y. & Brulet, P. (1992). Homeosis in the mouse induced by a null mutation in the homeogene Hox-3.1. Cell 69, 251-264. Lufkin, T., Dierich, A., LeMeur, M., Mark, M. & Chambon, P. (1991). Disruption of the Hox-l.6 homeobox gene results in defects in a region corresponding to its rostral domain of expression. Cell 66, 1105-1119. Lufkin, T., Mark, M., Hart, C., Doll6, P., LeMeur, M. & Chambon, P. (1992). Homeotic transformation of the occipital bones of the skull by ectopic expression of a homeobox gene. Nature 359, 835-841. Lumsden, A. (1990). The cellular basis of segmentation in the developing hindbrain. TINS 13, 329-335. Lumsden, A., Sprawson, N. & Graham, A. (1991 ). Segmental origin and migration of neural crest cells in the hindbrain region of the chick embryo. Development 113, 1281-1291. McGinnis, W. & Krumlauf, R. (1992). Homeobox genes and axial patterning. Cell 68, 283-302. Morgan, B., Izpisua-Belmonte, J.-C., Duboule, D. & Tabin, C. (1992). Targeted expression of Hox-4.6 in the avian limb bud causes apparent homeotic transformations. Nature 358, 236-239. Noden, D. (1986). Patterning of avian craniofacial muscles. Dev. Biol. 116, 347-356. Noden, D. (1988). Interactions and fates of avian craniofacial mesenchyme. Development 103 (Supplement: Craniofacial Development), 121-140. Nohno, T., Noji, S., Koyama, E., Ohyama, K., Myokai, F., Kuroiwa, A., Saito, T. & Tanaguchi, S. (1991). Involvement of the Chox-4 chicken homeobox genes in determination of anteroposterior axial polarity during limb development. Cell 64, 1197-1205. Oliver, G., De Robertis, E. M., Wolpert, L. & Tickle, C. (1990). Expression of a homeobox gene in the chick wing bud following application of retinoic acid and grafts of polarizing region tissue. EMBO 9, 3093-3099. Papalopulu, N., Lovell-Badge, R. & Krumlauf, R. (1991). The expression of murine Hox-2 genes is dependent on the differentiation pathway and displays collinear sensitivity to retinoic acid in F9 cells and Xenopus embryos. N.A.R. 19, 5497-5506. Peterson, R. L., Jacobs, D. F. & Awgulewitsch, A. (1992). Hox-3.6- Isolation and characterization of a new murine homeobox gene located in the 5' region of the Hox-3 cluster. Mech. Develop. 37, 151-166. Pollock, R., Jay, G. & Bieberich, C. (1992). Altering the boundaries of Hox3.1 expression: Evidence for antipodal gene regulation. Cell 71, 911-923. Rarnirez-Solis, R., Zheng, H., Whiting, J., Krumlauf, R. & Bradley, A. (1993). Hox-B4 (Hox-2.6) mutant mice show homeotic transformation of cervical vertebra and defective closure of the sternal rudiments. Cell 73, 279-294. Scott, M. (1992). Vertebrate homeobox gene nomenclature (letter). Cell 71,551-553.

Simeone, A., Acampora, D., Nigro, V., Faiella, A., D'Esposito, M., Stornaiuolo, A., Mavilio, F. & Boncinelli, E. (1991 ). Differential regulation by retinoic acid of the homeobox genes of the four HOX loci in human embryonal carcinoma cells. Mech. Develop. 33, 215-227. Tickle, C. (1991). Retinoic acid and chick limb bud development. Dev. Suppl. 1, 113-121. Wilkinson, D., Bhatt, S., Cook, M., Boncinelli, E. & Krumlauf, R. (1989). Segmental expression ofhox 2 homeobox'containing genes in the developing mouse hindbrain. Nature 341,405-409. Yokouchi, Y., Sasaki, H. & Kuroiwa, A. (1991). Homeobox gene expression correlated with the bifurcation process of limb cartilage development. Nature 353, 443-445.

About similarities between vertebrate and Drosophila Hox genes: Akam, M. (1989). Hox and HOM: Homologous gene clusters in insects and vertebrates. Cell 57, 347-349. Mann, R.S. (1997). Why are Hox genes clustered? Bio Essays 19, 661--664.

About embryogenesis and pathogenesis: Mark, M., Rijli, F.M., & Chambon, P. (1997). Homeobox genes in embryogenesis and pathogenesis. Ped. Res. 42(4), 421-429.

About craniofacial development: Noden, D. (1988). Interactions and fates of avian craniofacial mesenchyme. Development 103 (Supplement: Craniofacial Development), 121-140. Lumsden, A. (1990). The cellular basis of segmentation in the developing hindbrain. TINS 13,329-335. About retinoids and limb development

Tickle, C. (1991). Retinoic acid and chick limb bud development. Development Suppl. 1 (Molecular Basis of Pattern Formation), 113-121.

About Hox gene knockouts: Hunt, P. & Krumlauf, R. (1991). Deciphering the Hox code: Clues to patterning the branchial region of the head. Cell 66, 1075-1078.

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Introduction Teratogenic Mechanisms: Fetal Alcohol Syndrome Physiological Mechanisms: Amniotic Bands Multifactorial Mechanisms: Cleft Lip/Cleft Palate Chromosomal Mechanisms

Aneuploidy: Down Syndrome Contiguous Gene Deletion Syndromes Genomic Imprinting: Prader-Willi and Angelman Syndromes Triplet Repeat Amplification: Fragile X Syndrome Mendelian Mechanisms

Cell Adhesion Molecules: MASA X-Linked Retardation Extracellular Matrix Molecules: Osteogenesis Imperfecta, Marfan Syndrome, and Williams Syndrome Metabolic Errors: Peroxisomal Diseases Signal Transduction Molecules: Miller-Dieker Syndrome Proto-oncogenes: Piebald Trait and Grieg Syndrome Developmental Genes Defined in Lower Organisms: Waardenburg Syndrome Conclusion and Speculation

Principles of Medical Biology, Volume. 11 Developmental Biology, pages 293-325. Copyright 9 1998 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-816-1

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Congenital anomalies rank as the leading cause of infant mortality and sixth among all causes of death in the United States (Schardein 1985). The study of human congenital anomalies is called dysmorphology, literally meaning painful or abnormal shape. Teratology (study of monsters) is a synonym that often connotes environmental etiology. Despite the fact that birth defects impact more than 15 million Americans at an annual cost of more than $13 dollars (Schardein, 1985), the medical approach to congenital anomalies is distastefully empirical. Because few theoretical principles are available, Rutherford's disapproval of biologists as "postage stamp collectors" (Scriver, 1987) is especially applicable to dysmorphology/teratology. However, modern developmental biology and the human genome initiative promise dramatic advances in the understanding of congenital malformations. Until more comprehensive understanding of human embryology is possible, informed classification of congenital anomalies is vital. The several thousand types of congenital anomalies that occur in humans can be divided into two major categories-isolated defects and syndromes (Figure 1). Patients with isolated congenital anomalies exhibit dysmorphogenesis of a localized embryologic region. Examples include cleft palate, congenital heart defects, and neural tube defects. Patients with multiple congenital anomalies have syndromes (from the Greek root meaning "concurrence"). Examples include the Down, Marfan, or Zellweger syndromes (note that the possessive is not employed). Figure 1 displays the contribution of genetics to isolated congenital anomalies versus syndromes. These proportions are rough estimates derived from a literature review (Wilson 1992a), with greater emphasis on genetic factors than is customary (Stevenson 1993). This change in perspective reflects a growing appreciation for

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novel inheritance mechanisms that can cause congenital anomalies without obvious familial transmission. Note that polygenic or multifactorial inheritance predominates for isolated defects, while Mendelian and chromosomal inheritance predominate for syndromes. Distinction between isolated versus multiple defects is the first step towards management, genetic counseling, and scientific investigation of congenital anomalies. In practice, this may be difficult. Some children with syndromes have only one major anomaly (a defect with medical consequences) that is accompanied by several minor anomalies (variants with no medical but considerable diagnostic significance). Thus the infant with Down syndrome may be noticed because of an endocardial cushion defect, with the full syndrome appreciated only after recognizing minor anomalies such as epicanthal folds, upturned nose, single palmar creases, and so forth. The clinician must be alert for minor anomalies in order to recognize the higher genetic and medical risks implied by syndrome diagnoses; the researcher must use care that homogenous patient populations are studied and, for animal models, that the full spectrum of anomalies is defined. The text by Jones (1988) provides an excellent discussion of minor anomalies and their significance. The large contribution of genetics to congenital anomalies (Figure 1) implies that the power of molecular biology will eventually provide an objective science of dysmorphology. A benchmark and tool for progress is the human genome map illustrated in Figure 2. More than 130 isolated anomalies and syndromes have now been mapped to specific chromosome regions; many have been associated with specific gene mutations. The genome map is not sufficiently detailed for easy matching of anomalies with causative genes, but the human genome initiative is making rapid progress. Recent characterization of contiguous yeast artificial chromosome (YAC) segments from whole chromosomes (Vollrath et al., 1992; Mandel et al., 1992), rapid mapping of complementary DNA sequences (putative genes) within YAC or cosmid contigs, construction of genome maps for diverse organisms, and increasing detail of genetic linkage maps (Guyer and Collins, 1993) all suggest that a map containing the location of every human gene will be available within the next decade. Once all genes are localized, technologies for gene amplification (polymerase chain reaction = PCR) or mutation detection (e.g., single-strand conformational polymorphisms = SSCP) promise the rapid identification of genes responsible for human embryogenesis. Matching of most Mendelian syndromes and single defects with their causative genes should soon be accomplished, and these Mendelian loci should provide a starting point for dissecting polygenic phenotypes. Even those anomalies related to environmental agents may be explored through susceptibility genes, genocopies, and animal models. The remainder of this chapter will examine the status of these molecular explorations, proceeding from the least understood teratogenic defects to the best understood genetic syndromes.

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It is quite remarkable that among more than 50,000 drugs and chemicals in common use, only about 20 are proven human teratogens (Schardein, 1985). Considerably more--about 180 of 2800 tested--are teratogenic in two or more animal species (Schardein et al., 1985). The references of Shardein (1985) and Shepard (1993) offer excellent guides to the teratogenic literature, and numerous telephone hotlines have been established at governmental or academic centers. Despite the publicity surrounding the Love Canal or Agent Orange episodes, it is interesting that only one environmental chemical, methyl mercury, is proven as a teratogen. The many lawsuits surrounding agents such as dioxin or Bendectin are truly an American tragedy (Brent, 1983), and these unscrupulous awards provide sad testimony to our inadequate science of congenital anomalies. Although alcohol has long been suspected to cause fetal injury, a specific syndrome resulting from maternal alcohol intake was only documented in the late 1960s (Streissguth et al., 1980). Diagnosis of the fetal alcohol syndrome is based on subjective recognition of minor anomalies combined with typical growth failure and major birth defects. Fetal alcohol syndrome is rarely recognized during the neonatal period, yet it is now estimated to be the most common cause ofmental retardation (1.9 per thousand births-Abel and Sokol, 1986). The characteristic growth pattern of short stature with microcephaly; the facial anomalies including small eyes, shallow nasal bridge, abnormal ears, thin upper lip, and absent philtrum; occasional organ defects affecting the eyes, heart, and joints; and the learning disabilities with poor impulse control and hyperactivity may not be obvious until age 2 or 3. The child pictured in Figure 3A was recognized at age 2 years, with the positive maternal history obtained only in retrospect. The fetal brain is a major target of alcohol, and there are undoubtedly children with learning deficits but without facial changes who encountered alcohol exposure after the first trimester. Limited neuropathological studies in humans have demonstrated smaller brains with abnormal gyri and heterotopias (abnormal cell migration). Brain anomalies and small eyes have been documented in several animal models, including rodents and nonhuman primates (Streissguth et al., 1980). Intraperitoneal injection of mice with ethanol on gestational day 7 produced decreased embryo size, abnormal neuroepithelium, and facial changes that were quite similar to those observed in humans (Sulik et al., 1981). Influence of genetic susceptibility was suggested in these mice because the C57BL/6J strain exhibited a disproportionate incidence of eye anomalies in response to ethanol that correlated with a high baseline rate (Sulik et al., 1981). The mouse thus provides an embryologically and genetically manipulable system for further delineation of alcohol teratogenesis.

The developing embryonic vascular system and the close apposition of embryo and extra-embryonic membranes provide numerous opportunities for morphologic catastrophe. Defects such as porencephaly (cystic lesion in the brain) or hemifacial microsomia (small ear and jaw on one side) have been related to vascular accidents in the fetal or embryonic period. Whether by vascular accidents or other mechanisms, breakdown of the amnion may produce tissue remnants (bands) that encircle fetal parts and cause severe anomalies. These mechanical or physiologic lesions are often called "disruptions" to separate them from true malformations; disruptions almost always occur sporadically with no evidence for genetic transmission in patients with milder defects who survive to reproduce. A particularly high rate of disruptions occurs in monochorionic twins, where vascular connections in their common placenta can produce twin-to-twin shunting. Amniotic bands have an incidence of about 1/10,000 births, with a much higher incidence in abortuses (Gorlin et al., 1990). The fetus illustrated in Figure 3B might first suggest a facial cleft until the amniotic remnant surrounding the fingers is noted. Further inspection reveals that the cleft has a geographic rather than embryologic distribution, more consistent with adhesion and tearing by a band than with a known embryonic cleavage. Depending on its timing and distribution, the amnion rupture sequence of anomalies includes limb, craniofacial, and/or lateral body wall defects. Unfortunately, there are as yet no model systems that can distinguish among possible causes of the amnion rupturew(e.g, amnion hypoplasia, inappropriate cell death, and altered vascular supply).

The majority of common isolated malformations exhibit polygenic inheritance. The baseline 1/1000 incidence of many common birth defects rises to a 2-3% empiric recurrence risk in the sibling or child of an affected individual but returns toward background in second- or third-degree relatives. Such empirical risks can be explained by including a threshold in the polygenic model. Fraser (1976) has given experimental validation to such models by showing interaction of genetic background (strain differences), drug treatment (steroids), and morphologic characteristics (facial width, palatal height, and jaw growth) in producing murine cleft palate. Although rare cases of isolated cleft palate exhibit pure Mendelian inheritance, these may represent extremely severe alleles that override the usual interactions of multiple weak alleles and the intrauterine environment. Despite several centuries of observation, understanding of the pathogenesis of cleft palate is still rudimentary. The embryology is well documented, with failure of the primary palate involved in cleft lip/cleft palate and failure of the lateral palatine process fusion in cleft palate alone. A child with unilateral cleft lip/palate is shown

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in Figure 3C; note the difference from the nonembryologic cleft depicted in Figure 3B. If a parent has cleft palate, then there is an approximate 2-3% recurrence risk for primary relatives to have cleft palate; the same occurs with cleft lip/cleft palate. Both types of clefts breed true and are consistent with polygenic inheritance. As mentioned above, murine strains with genetic susceptibility to cleft palate have allowed definition of morphological mechanisms such as altered palatal height or jaw growth that contribute to palatal fusion. Environmental factors such as hypoxia and anticonvulsant medications including hydantoin, valproic acid, cigarette smoking, and ethanol have all been implicated in the genesis of cleft lip/cleft palate in humans (Lettieri 1993). Although there is considerable variation among studies, from 2-14% of patients with cleft lip and 13-64% of patients with cleft lip/cleft palate have associated anomalies (Cohen, 1991). Conversely, over 300 syndromes have cleft lip or cleft lip/cleft palate as a component defect. This large number of syndromes corroborates polygenic inheritance of the isolated anomalies and implicates many genetic loci in the causation of oral clefts. Three approaches towards defining these genes have been followed. The first was allele association as reported by Ardinger and colleagues (1989). Several growth-factor genes of known importance for epidermal tissue were tested for association in cleft palate families. A higher frequency of certain transforming growth factor-a alleles were found in familial cleft palate patients but not in controls or sporadic patients. The implied involvement of transforming growth factor-a in palatal fusion is now being tested by genetic linkage and embryologic studies. A second strategy was used to examine a Mendelian form of cleft palatemthe X-linked cleft palate/ankyloglossia syndrome. Availability of large families allowed classical linkage studies that assigned this gene to a region on the X chromosome long arm (Williamson 1988). Positional cloning strategies are now underway to isolate this gene and define its role in palatal closure. The third strategy involved defining cleft palate as part of a transgenic mouse phenotype. Increased expression of the mouse homeotic gene Hox 1.1 produced a phenotype that was very similar to the Pierre Robin anomaly in humans--small jaw and cleft soft palate (Bailing et al., 1989). Three different approaches have thus defined genes potentially relevant to polygenic cleft palate, and rapid progress can be anticipated in understanding the molecular embryology of palatal closure.

More than 100 chromosomal diseases have been described, reaching an aggregate frequency of 50% in first trimester abortuses and 0.5% at birth (Jones 1988). Multiple congenital anomalies and mental retardation are hallmarks of this disease category. Entire chromosomes may be duplicated or deleted (e.g., trisomy and monosomy 21), or only portions of the short (p) or long (q) arms (see Figure 2 for depiction of the human karyotype). Partial duplications or deletions of chromosome material may be de-

scribed as trisomy 3q (duplication 3q) or monosomy 7p (deletion 7p) to demarcate the location of extra/missing material. It is remarkable that nearly every complete or partial aneuploidy has a characteristic phenotype that correlates with the aneuploid segment (e.g., dup(3q) syndrome). The more common chromosomal syndromes were recognized prior to the advent of reliable karyotyping in the late 1950s; Down, Edwards, Patau, and Turner syndromes existed before they were correlated with trisomies 21, 18, and 13 and monosomy X, respectively. In addition to providing the explanation for these familiar syndromes, human cytogenetics has led to the discovery of several new inheritance mechanisms (Figure 4).

Down syndrome (Figure 3D) has an incidence of 1 in 600 livebirths that changes little with ethnic background. About 96% of these patients will have trisomy 21, while 4% will have a translocation or mosaicism. Although Langdon Down apparently intended his term of "mongolism" to indicate unity among races, the idea of atavism is unflattering to both the afflicted families and true Mongols. Even worse

is its apposition to the term "idiocy," despite the latter word's origin from the Greek for "special." Down syndrome is the term of choice, best used as the "person with Down syndrome" in consonance with people-first language. Those who consider the semantics unimportant should review the history of institutionalization and forced sterilization of retarded individuals in this country (Gould, 1981), and note the remarkable improvement in outcome that has accompanied home care, anticipatory medical management, and inclusive schooling over the past two decades. A century of phenotypic study has described anomalies of every organ system that occur at higher frequency in the child with Down syndrome. This broad range of anomalies poses a considerable challenge for explanation (Wilson, 1990). One avenue of investigation concerns mice trisomic for a somewhat larger but partially homologous segment of mouse chromosome 16; these animals show some interesting parallels with human Down syndrome, including heart defects and obesity (Epstein 1993). Another approach utilizes partial chromosome 21 duplications to define a "critical region" that is associated with most Down syndrome characteristics. This region lies on the distal long arm of chromosome 21 (Korenberg, 1993). Molecular characterization of overlapping duplications has associated the typical endocardial cushion defects with an even smaller chromosome 21 segment that is now a focus for positional cloning. Other phenotypic correlations such as a high frequency of Alzheimer disease in older individuals with Down syndrome are more puzzling. A genetic form of Alzheimer disease has been linked to a region on chromosome 21, but this lies outside of the critical region (Korenberg 1993). Phenotype/karyotype correlation has been even more difficult in trisomy 18, where at least two separate regions of the chromosome seem instrumental in producing the Edwards syndrome phenotype (Wilson, 1993a).

Contiguous Gene Deletion Syndromes Intermediate between chromosome aneuploidy and Mendelian diseases are the contiguous gene deletion syndromes.(Schmickel, 1986). These deletions involve several genes but are small enough that they require high-resolution (prometaphase) analysis for detection. A prometaphase karyotype resolves more than 1000 bands along the haploid chromosome complement, as opposed to the 300-band routine karyotype; this still translates to more than 1 million base pairs per band based on a haploid genome size of 3 x 109 base pairs. Prometaphase bands will contain enough DNA for more than 20 average-sized genes of 40 kb so that visible prometaphase deletions will usually include several contiguous loci. The contiguous gene deletion syndrome is thus an aggregate phenotype involving alteration of several gene products. Several of the loci listed in Figure 2 were mapped based on their occurrence as a contiguous gene deletion syndrome including the following: Williams syndrome (chromosome 7q), Langer-Giedion syndrome (chromosome 8q), aniridia (chromosome l lp), Prader-Willi/Angelman syndromes (chromosome 15q), RubinsteinTaybi syndrome (chromosome 16p), Miller-Dieker syndrome (chromosome 17p),

Mechanisms of Congenital Malformation Alagille syndrome (chromosome 20p), DiGeorge/Shprintzen syndrome (chromosome 22q), and Duchenne muscular dystrophy with various accessory manifestations (chromosome Xp). Until molecular characterization is completed, it is of course not certain whether a deletion involves one or several contiguous genes. The Duchenne muscular dystrophy gene is sufficiently large enough (>2 megabases) to have been involved in at least tWOcontiguous gene deletions (Francke et al., 1985; Bartley et al., 1986). One of these combined four diseases that were known to exhibit X-linked recessive inheritance: Duchenne muscular dystrophy, chronic granulomatous disease, McLeod red-cell phenotype, and retinitis pigmentosa. Association of these four diseases with the same deletion immediately mapped their causative genes to band Xp21 and provided a route for molecular cloning. A different deletion at Xp21 caused glycerol kinase deficiency and adrenal hypoplasia together with Duchenne muscular dystrophy (see Figure 2C). Molecular analysis has now demonstrated different boundaries for these two contiguous gene deletions and ordered their component loci relative to the X chromosome centromere and telomere. Smaller deletions within the dystrophin gene are common causes of Duchenne or Becker muscular dystrophies, but these are not evident by cytogenetic analysis. This extension from visible prometaphase deletions to submicroscopic deletions has been rewarding in other conditions as well, as illustrated by the "deletion 22" group of disorders (Hall, 1993). Beginning with the DiGeorge anomaly (cardiac defects, abnormal face, thymic hypoplasia, cleft palate, and hypocalcemia, which in this case is due to a branchial arch defect), a prometaphase deletion at band 22ql 1 was noted (see Figure 2C). More recently, seemingly identical deletions were recognized in Shprintzen syndrome, a disorder that can include DiGeorge anomaly as one of its component defects. Completing the story were DNA studies that defined submicroscopic deletions in patients with isolated conotruncal defects, the typical heart lesions associated with Shprintzen syndrome and DiGeorge anomaly (Emmanuel et al., 1993). Thus, a group of clinical disorders that once were considered separate entities now became a phenotypic spectrum that depends on the extent of 22q 11 deletion. It is particularly surprising to find a submicroscopic deletion in patients with isolated heart anomalies, perhaps more than one gene is involved as expected from the general rule of polygenic inheritance for common birth defects.

In a story similar to that described for the 22 deletion group, a microdeletion at chromosome band 15ql 1 was detected in both the Prader-Willi and Angelman syndromes (Figure 5). However, it was difficult to connect the Prader-Willi phenotype of obesity, hypogonadism, and mild mental disability with the very different Angelman phenotype of prominent jaw, seizures, puppetlike jerky motions and more severe mental disability (Wilson et al., 1993b). Molecular analysis resolved this dilemma using DNA restriction polymorphisms to demonstrate that Prader-Willi

Figure 5.

patients always had deletions in the paternally derived chromosome 15, While Angelman patients always had deletions in the maternally derived chromosome 15 (Figure 5). These conclusions jibed with extensive experiments that had identified in mice the phenomenon known as genomic imprinting, named by analogy to the behavioral "imprinting" of young animals on their mothers (Hall, 1990). Several types of experiments have demonstrated that the parental source of genetic material may be as important as its quantity and quality. The first demonstration of imprinting utilized pronuclear transplantation to produce mouse zygotes with two male or two female pronuclei. In contrast to normal zygotes with one female and one male pronucleus, digynous zygotes produced nonviable embryos lacking placentas and diandrous zygotes produced nonviable embryos consisting mainly of placental tissue. An analogous situation occurs in human triploids: molar pregnancies (placental tissue) often have two haploid sets of male-derived chromosomes (plus one female-derived set) while ovarian teratomas (germ layers, but no placental tissue) have the opposite. Systematic construction of mice containing uniparental disomies (e.g., both chromosomes 15 from one parent)

showed that genomic imprinting was important for at least five pairs of chromosomes. In these mice, growth retardation and other congenital anomalies resulted even when the amount, but not the source, of genetic material was normal. These effects are now being related to specific genes on imprinted regions of the chromosomes. For example, the mouse insulinlike growth factor-2 gene is expressed in the fetus only when it is present on the paternally transmitted chromosome 7 (Hall, 1990; Wilson et al., 1993b). As would be expected from the mouse experiments, some patients with the Prader-Willi or Angelman syndromes have no deletions at the chromosomal or DNA level. Rather, they have uniparental disomy for the 15q 11 region when evaluated using DNA polymorphisms. Figure 6A illustrates hypothetical microsatellite patterns that would be typical of each syndrome when caused by uniparental disomy. Microsatellites are stretches of repeating dinucleotides that vary in length among normal individuals (polymorphism). Using PCR, the number of dinucleotide repeats at a given locus can be quantified by sizing the amplified fragment on a polyacrylamide gel. In Figure 6A, DNA amplified from individual II- 1 migrates as a band of double intensity that corresponds to one paternal band. This suggests inheritance of two copies of one paternal chromosome 15 (paternal isodisomy), yielding a phenotype of Angelman syndrome due to loss of maternal alleles. Individual III-1 has inherited two maternal #15 chromosomes, causing Prader-Willi syndrome for lack of paternal alleles in the 15q 11 region (maternal heterodisomy). The human 15q 11 region is homologous to a portion of mouse chromosome 7 that is also known to be imprinted, and a search for the genes whose imprinted expression causes such dramatic anomalies is underway in both organisms. Although genomic imprinting is clearly relevant to human congenital anomalies and particularly to fetal growth retardation, the mechanisms of imprinting have not yet been defined. Late replication and increased DNA methylation are characteristic of genes silenced by imprinting (Wilson et al., 1993b), but the primary events by which a gene is silenced during oogenesis, but not during spermatogenesis (or vice versa), are unknown. Alteration in these primary imprinting mechanisms may be as frequent as chromosome deletions or uniparental disomy in producing imprinting disorders. The mechanism by which uniparental disomy occurs has been suggested through prenatal diagnostic studies. Chorionic villus sampling at 8-10 weeks of pregnancy has been associated with higher rates of chromosomal mosaicism than in later diagnosis by amniocentesis. Sometimes this mosaicism is confined to the placenta, that is, a different karyotype in the trophoblast and future placenta than in the inner cell mass that forms the embryo proper. Kalousek (1993) has reported trisomy 16 mosaicism in placenta as diagnosed by chorionic villus sampling followed by uniparental disomy 16 in the later stillborn fetus. These results imply the conceptus began as trisomy 16 (a frequent karyotype in abortuses) with correction to disomy 16 in the embryo. If the original trisomy 16 conceptus has two maternally derived 16s and one paternally derived 16, then there is a 1/3 chance that the correction will produce maternal uniparental disomy 16. The fact that uniparental disomy 16 fe-

Figure 6. Nucleotide repeats as disease markers or disease etiology. (A) Hypothetical DNA diagnosis of uniparental disomy as the cause for Prader-Willi or Angelman syndromes. Variable numbers of dinucleotide repeats at a locus within the Prader-Willi/Angelman critical regions allows tracking of parental chromosomes after amplification with PCR primers (arrows) and sizing of the products via gel electrophoresis. Individual I1-1 has a single band of double density, implying that he inherited two copies of the same paternal chromosome 15 (paternal isodisomy) and thus has Angelman syndrome (lack of maternal chromosome 15 material). Individual II1-1has two bands that correspond with those of his mother, indicating maternal heterodisomy (both maternal chromosomes 15) associated with Prader-Willi syndrome (lack of paternal chromosome 15 material). D, Angelman syndrome; E~, Prader-Wiili syndrome. (B) DNA diagnosis of Fragile X syndrome by measuring the number of trinucleotide repeats. Ranges of repeat numbers corresponding to particular phenotypes are shown below, while their hypothetical measurement via PCR amplification and gel electrophoresis is shown in the box (middle). Note that males have a single PCR product (one X chromosome), while females have two. m, affected with Fragile X syndrome, | carrier. tuses were growth-retarded suggests that chromosome 16 is imprinted in humans. The growth retardation might also result from the accompanying placental mosaicism or from autozygosity (two identical recessive alleles) for a mutation on chromosome 16 that causes growth retardation. Nevertheless, it is easy to see how study of these and other chromosomal anomalies will soon elucidate those regions of the human genome that are subject to imprinting.

More than 80 forms of X-linked mental retardation (XLMR) have been described that explain the 20-50% excess of males with severe retardation (Tariverdian and Weck, 1982). Nearly half of these XLMR disorders are "nonspecific", meaning that they lack distinctive metabolic or morphologic findings. Many have been mapped to particular regions of the X chromosome, and several are listed in Figure 7. Originally described by Martin and Bell in 1943, patients with fragile X syndrome were separated from other XLMR patients based on their long face, large ears, prominent jaw, megalotestes, lax joints, "autistic" behavior, and unusual speech. Delineation was greatly assisted by chance induction of a fragile site that was eventually related to culture oflymphocytes in low-folate media (Figure 7). Although the cytogenetic marker was extremely useful in targeting molecular studies, it was not always helpful in diagnosing carrier females or unusual normal males who could transmit the disease ("transmitting males"). Cloning of the gene responsible for fragile X syndrome began with linkage studies and culminated with the isolation of a gene segment from band Xq27 that exhibited marked-length variation in fragile X families. The length variation reflected the number of CCG trinucleotide repeats upstream of a newly characterized FMR-1

gene, and the repeat number correlated with clinical severity (Warren and Nelson, 1994). These correlations allow DNA diagnosis of suspect individuals using PCR to measure trinucleotide repeat number (Figure 6B). Normal individuals have between six and 52 repeats, while female carriers or transmitting males have up to 230 repeats. Males with full clinical and cytogenetic expression of fragile X syndrome can have from 230 to 1000 repeats (Warren and Nelson, 1994). When more than 50 repeats are present as a "premutation", the DNA becomes extremely unstable during early embryogenesis and during female meiosis. Fragile X males and affected carrier females are often mosaics of cells containing different numbers of repeats so that a broad band of variably sized DNA fragments is seen after PCR or Southern analysis (Figure 6B). The hypothetical DNA analysis shown in Figure 6B demonstrates an affected male (individual 11-2) whose single X chromosome has numerous triplet repeats upstream of the FMR-1 gene. The broad band representing a range of repeat sizes indicates somatic instability and mosaicism for repeat length in his cells. High degrees of repeat amplification have been associated with preferential nucleosome formation in vitro and increased DNA methylation in vivo; these and possibly other factors combine to shut off the FMR-1 gene. Individual I- 1 is a female carrier with a "premutation," whereas her daughter (11-3) has greater amplification and is probably symptomatic. Note that the females I- 1,11-3, and 1I-4 all exhibit two DNA bands by PCR analysis because they have two X chromosomes. Individual II-1 is a transmitting male with a premutation. He will be asymptomatic and negative for cytogenetic fragile X testing. For unexplained reasons, repeat instability is much greater during female meiosis, conferring a negligible risk for transmitting males (II- 1) to have symptomatic daughters but a higher risk for females I- 1 and especially 11-3 to have affected sons. The mutation rate of one per gamete per generation that occurs for repeat lengths >50 is unprecedented in human genetics where rates of 10-4 are considered high. An incidence of about 1/800 to 1/1000 births qualifies Fragile X syndrome as the most common genetic cause of mental retardation. The implications of DNA testing for this disorder are significant for several reasons. Firstly, as diagrammed below in Figure 7, fragile X is one of many X-linked mental retardation syndromes, and mental retardation is only one of several behavioral traits that exhibit sex bias. X chromosome loci for aggression and homosexuality have been proposed (Brunner et al., 1993; Hamer et al., 1993). Second, there are more than 16 heritable fragile sites distributed around the genome and many more that can be induced by various chemicals. These may also be sites of triplet repeat instability with clinical implications. At the DNA level, more than 40 loci containing triplet repeats have been discovered (Riggins 1992) that are associated with clinical diseases (Martin, 1993) including the following: Steinert myotonic dystrophy (CGG repeats near a muscle protein kinase gene), Huntington chorea (CAG repeats near a gene on the chromosome 4p telomere), spinobulbar muscular atrophy (CAG repeats in the androgen receptor gene), and spinocerebellar ataxia type 1 (CAG repeats in a gene of unknown

Mechanisms of Congenital Malformation

function on chromosome 6). Common to several of these disorders is a phenomenon called anticipation: affected members of successive generations show increasing severity. New scrutiny of inherited isolated defects and syndromes is underway to consider which may be examples of triplet repeat instability.

In addition to the novel genetic mechanisms diagrammed in Figure 4, a large number of isolated congenital anomalies and syndromes exhibit traditional Mendelian inheritance (Wilson, 1992a). Review of a compendium of genetic diseases (McKusick, 1990) revealed that about 1/2 of the >3500 listed disorders involve altered morphogenesis, including 721 isolated defects and 1040 syndromes. The approach to understanding congenital anomalies can thus parallel that of understanding neoplasia, an area where sporadic occurrence and environmental causation also received initial emphasis. The progression from Boveri's chromosomal hypothesis to specific chromosomal rearrangements and oncogenes is an object lesson for dysmorphology. Indeed, the idea of neoplasia as a sequence of genetic change and the fact that many developmentally important genes are proto-oncogenes provide a direct model for frequently sporadic, occasionally Mendelian congenital anomalies. For Mendelian anomalies, the route from phenotype to causative gene follows two main routes. Chance observation of chromosomal rearrangments (deletions, translocations), searching for uniparental disomy, or genetic linkage studies can associate a particular chromosome region with the phenotype. Candidate genes are then proposed based on their location, structural motifs (i.e., transmembrane region), or function in lower organisms. Probes for the candidate gene are used to search for altered restriction fragments by Southern analysis (translocations, deletions) or altered nucleotide sequences by SSCP and related techniques. Even when a particular gene segment has been amplified by PCR and shown to have four bands rather than two on partially denaturing gels (the result for a heterozygote with one normal and one mutant allele examined by SSCP), the mutation must be confirmed by DNA sequencing and examined in populations to diminish the possibility of polymorphism. Characterization of a gene responsible for a particular anomaly or syndrome is the surest route to defining the mechanism of normal or abnormal development. Nevertheless, considerable study of gene expression in cellular or animal model systems may be required to explain pleiotropy and variability of the human phenotype. Several exampies of genotype-phenotype correlations and the emerging molecular approach to human developmental biology will now be considered.

MASA syndrome is another example of the X-linked mental retardation disorders that were discussed together with fragile X syndrome. The disorder is named

as an acronym on the basis of the presence of mental retardation, adducted thumbs, spasticity, and aphasia. In the absence of an obvious candidate gene for the condition, a panel of X chromosome DNA polymorphisms was used to link the MASA gene to band Xq28 (Figure 7). It was intriguing that similar studies of X-linked hydrocephalus also highlighted the Xq28 region, and that individuals with hydrocephalus and/or MASA syndrome were reported in the same family. Focus on the Xq28 region revealed a L 1 cell adhesion molecule (L1CAM) that became a plausible candidate gene for these conditions. Soon deletions and point mutations were observed at the L 1CAM locus in MASA and X-linked hydrocephalus individuals (Figure 7 Rosenthal et al., 1992; Vits et al., 1994). Study of the developmental expression and cerebral distribution of the L1CAM molecule should now give insights into the hydrocephalus and spasticity associated with these mutant alleles. Cell adhesion molecules have been postulated to have central roles in development, and it is interesting that the X-linked Kallman syndrome of mental retardation, anosmia, and hypogenitalism is caused by a KAL-1 gene on the distal Xp region that has a CAM-related motif (Figure 2C). Numerous studies indicate that extracellular matrix, filamentous, and cell-adhesion molecules will be richly informative about human congenital anomalies (Table 1).

The tissue distribution of several extracellular matrix molecules provided link with the symptomatology of certain genetic diseases. Osteogenesis imperfecta is named for the fragile bones that implied a disorder of connective tissue. Severely affected patients had numerous fractures at birth, while mild patients had longer lives complicated by many fractures and bony deformities. The blue scerae (thinning of whites of the eyes to display underlying choroid) and altered cranial bones correlated with the tissue distribution of type-I collagen (Table 1). Soon mutations causing osteogenesis imperfecta were defined in either the otl or or2 type-I collagen chains (Byers, 1993). The nature and position of amino acid substitutions within the collagen chains correlated with symptom severity: Those mutations that disrupted glycine spacing or that occurred near C-terminal regions (the start of triple helix assembly) were extremely severe. Surprisingly, the clinical assumption that mild cases were autosomal dominant and severe cases were autosomal recessive turned out to be false. Molecular studies have indicated dominant inheritance as the rule with de novo mutations or germline mosaicism explaining families where normal parents have affected children. As shown in Table 1, mutations in other collagen molecules have been detected in certain patients with Ehlers-Danlos syndromes, a category of at least eight genetic conditions involving elastic skin, loose joints, and vascular disease. More than 15 different types of collagen exist so that many novel mutations and types of connective tissue await discovery. Other extracelluiar matrix molecules such as

Mechanisms of Congenital Malformation Table 1. Diseases OI types I, II OI type I

fibrillin and elastin also seem important in development and postnatal function, because they have been respectively implicated in the Marfan and Williams syndromes (Table 1). These discoveries contrast two methods for associating anomalies with molecules. Genetic linkage has associated the autosomal dominant Marfan syndrome to a region of chromosome 15 (Figure 2B), focusing attention on fibrillin as a candidate locus (Pyeritz, 1993). The tissue distribution of this molecule correlated with the loose connective tissue (tall stature), lens dislocations, and aortic dilatation that were typical of Marfan syndrome. Williams syndrome was usually a sporadic disorder, and the genetic contribution was highlighted by several patients who had a subtle deletion on chromosome 7q (Figure 2B). Elastin is one of the candidate genes in the deleted region, and a haploid dose of this gene may explain the loose joints, unusual voice, and vascular changes (pulmonic stenosis, renal disease) that occur in Williams syndrome (Ewart et. al., 1993). Involvement of contiguous genes is suggested by the euphoric affect, the selective cognitive defects that spare language skills, and the hypercalcemia that are also seen in Williams syndrome.

Inborn errors of metabolism are usually associated with functional defects such as metabolic acidosis, seizures, neurodegeneration, unusual odors, and so forth. However, a growing category of metabolic diseases includes major and minor congenital anomalies in their phenotype. A striking example is the Zellweger syndrome (Figure 8A) with severe hypotonia, unusual facies, neuronal migration defects, micronodular cirrhosis of the liver, renal cysts, and bone dysplasia (Lazarow and Moser 1989). Numerous metabolic abnormalities were associated with the syndrome including excess of very long-chain fatty acids, pipecolic acid, and phytanic acid and deficiency of plasmalogens, myelin, and certain bile acids (Wilson, 1992b). Multiple enzymes responsible for degradation or synthesis of these chemicals, all located in peroxisomes, are deficient in Zellweger tissues. Like most metabolic disorders, Zellweger syndrome is an autosomal recessive disease. This implies a single gene mutation is responsible for the multiple metabolic and enzymatic alterations. A first clue to pathogenesis occurred when Zellweger patients were shown to lack visible peroxisomes in their livers. Later, antibodies to peroxisomal membrane proteins (PMPs) identified "ghost" structures in affected tissues, implying normal membrane synthesis with aberrant assembly of matrix proteins (PmaPs; Figure 8B). Attention was thus focused on the PMPs as candidates for mutation in Zellweger syndrome, a concept validated when defects in the 70- and 35-kDa PMPs were demon-

strated (Gartner et al., 1992; Shimozawa et al., 1992). A scheme for peroxisome assembly can be hypothesized as illustrated in Figure 8B, showing predominance of PMP synthesis in early embryonic tissues or brain that have scanty matrices with minimal catalase staining (Wilson, 1992). Import of PMaPs leads to easily visualized peroxisomes in liver and kidney, with surprising variation in peroxisome morphology from tissue to tissue (Roels, 1991). Three PMPs of 70-, 35-, and 22-kDa have been characterized, one (PMP35) cloned based on its function in restoring deficient peroxisome assembly in cultured cells (Shimozawa et al., 1992). PMP mutations presumably follow a common pathway in altering peroxisome assembly with consequences for brain, liver, and kidney development. Interesting regions of PMP genes include a putative zinc finger region (PMP35) and ATP-binding sites (PMP70) that will be interesting to examine in transgenic experiments. Other metabolic diseases cause acquired dysmorphology due to stored or deficient molecules. The mucopolysaccharidoses are characterized by coarsening of the face, alterations of the skeleton, and organ dysfunction due to deficiency of lysosomal enzymes. Accumulation of mucopolysaccharides and other lysosomal substrates explains the postnatal dysmorphology. Mitochondrial DNA deletions or point mutations have also been implicated in certain syndromes with optic atrophy, muscle hypoplasia, and brain lesions. Depending on the timing and severity of mitochondrial defects, the anomalies may surface in fetal, juvenile, or senile periods. Metabolic disorders that alter tissue/organ structure offer abundant opportunities for defining mechanisms of altered morphogenesis.

The chromosome deletion associated with Miller-Dieker syndrome led to positional cloning of a gene involved in cerebral differentiation (Dobyns et al. 1993). Ninety per cent of Miller-Dieker patients and 15% of patients with isolated lissencephaly had deletions within a 350 kb region that included the LIS-1 gene. This gene is expressed in brain and shows homology to G proteins that are known to be involved in nerve cell signaling. A 13-transducinlike repeat in the LIS-1 gene is homologous to a cell cycle regulator in yeast and the Drosophila Groucho protein that is involved in neurogenesis. Diverse evidence supports a role for the LIS-1 gene product in the development of cerebral gyri that are absent in lissencephaly. Deletions imply that half-normal LIS-1 gene dosage is sufficient for pathology; altered G protein subunit ratios may be implied. Again, we see that "sporadic" cases of lissencephaly could in fact be due to genetic changes, representing de novo mutation or abnormal imprinting of one LIS-1 gene copy.

Piebald trait refers to a variable region of depigmentation on the ventral surface of the body (Figure 9A). Human piebald trait is remarkably similar to the appear-

ance of mice with the trait dominant spotted. Characterization of mutations in the proto-oncogene c-kit in dominant spotted mice drew attention to the fact that the human homologue had been mapped to chromosome band 4q 12. This was a region that was known to be deleted in several patients with piebald trait (Figure 2A). Scanning of the human c-kit gene for deletions (Southern blotting) or point mutations (SSCP) revealed mutations in several patients with piebald trait (Fleischman et al., 1991; Spritz et al., 1992). Relation of depigmentation to a tyrosine kinase involved in signal transduction implies that normal c-kit signaling is required for melanoblast migration. The ventral, midline patterning of white patches merely reflects the longer distance that dorsally derived melanoblasts must travel to reach these positions. In a similar approach, translocations occurring in patients with Grieg syndrome (wide-spaced eyes, polydactyly) have highlighted a member of the GLI proto-oncogene family as a candidate gene (Vortkamp et al. 1991). The mouse mutant extra-toes also is caused by mutations in the GLI3 gene. Characterization of GLI gene mutations in these vertebrate syndromes has additional significance because the GLI gene family is homologous to the Drosophila kruppel gene involved in insect segmentation. DNA sequence homologies may thus uncover unsuspected similarities in developmental mechanisms, in this case relating aspects of vertebrate midline and limb morphogenesis to fruit fly segmentation.

The intuitive expectation that successful developmental programs would be conserved in evolution is gathering support from molecular genetics. Experimentally tractable organisms have revealed characteristic signatures (motifs) in genes that control their development, allowing searches for these motifs in the human ge-

nome. Numerous examples of sequence conservation exist, and insect segmentation genes are among the most exciting. Genetic investigation has attributed segmentation to the sequential expression of five groups of genes: maternal, gap, pair-rule, segment polarity, and homeotic (Figure 10A). These elegant investigations began with the observation of dysmorphic flies wherein one structure was replaced with a homologous normal structure from another body segment (homeotic mutations; Bender, 1985). After studying a series of homeotic mutations, Lewis (1992) predicted that segment identity required a combination of genes, allowing

stepwise transformation of a featureless abdominal segment (ground state) to a complex antenna. Cloning of these homeotic genes revealed a shared 180-bp, 60 amino acid expressed region, or homeobox = (HOX), that was conserved from insects to mammals. The homeobox and other motifs such as paired boxes = (PAX) were also detected in genes that act early in Drosophila morphogenesis to specify the orientation and number of body segments. Many of these motifs also are found in the human genome (Figure 10A, bottom). Evidence that Drosophila gene homologues actually function in human development has already been mentioned regarding the kruppel-related GLI3 protooncogene that is mutated in Grieg syndrome. Additional evidence concerns the Waardenburg syndrome, an autosomal dominant condition that causes widely spaced and differently colored eyes (heterochromia), deafness, and white streaks in the hair (poliosis-Figure 10B). Mapping of one type of Waardenburg syndrome to chromosome region 2q35 (see Figure 2A) focused attention on a human PAX gene at that location, and mutations in the PAX3 gene were soon described (Tassabehji et al., 1993). As in the Drosophila gene, paired, mammalian PAX genes have a version of the homeobox near the paired box. Tassabehji and colleagues (1993) demonstrated that a mutation deleting only the paired box lacked the widely spaced eyes (Waardenburg type 2), while mutations deleting both motifs had full expression of the syndrome (Waardenburg type 1). This provides a beginning in correlating molecular structure with syndrome phenotype. Other PAX gene mutations have been found in autosomal dominant aniridia (absent iris) at chromosome band 1 lp 13 (see Figure 2B), and in the murine conditions undulated (vertebral anomalies), splotch (spina bifida and neural crest defects), and small eye (absent eyes and nasal structures in homozygotes; Glaser et al., 1992; Stapleton et al., 1993). These mammalian PAX6 mutations (aniridia, small eye) reciprocated in uncovering a similar Pax-6 gene alteration in a Drosophila eyeless mutant (Quiring et al., 1994); similar developmental control of insect compound and vertebrate lens eyes was completely unsuspected. Since human homologues are known for each of the five groups of insect segmentation genes shown in Figure 10A, results with the PAX gene family offer an exciting vista for future exploration.

This chapter emphasizes genetics as the key to understanding congenital anomalies. Although many human anomalies seem sporadic, novel mechanisms for inheritance and DNA instability dramatize the potential frequency and variety of de novo mutations. Based on a survey of genetic catalogs, Mendelian anomalies are not at all exceptional; they can be viewed as extremes that provide scalpels to dissect multifactorial defects in the way that a rare hypercholesterolemia was related to common heart attacks (Brown and Goldstein, 1976). The abundance of developmental regulatory genes being discovered in simpler organisms, plus the ability to identify,

Mechanisms of Congenital Malformation

test, and experimentally manipulate these genes in mammals promise rapid progress in dysmorphology. Accelerating this progress will be the complete map of transcribed regions and motifs to be provided by the human genome initiative. More challenging, perhaps, will be the synthesis of molecular information into a comprehensive understanding of morphogenesis. Not only will such theories be important in making sense of the bewildering variety of human anomalies, especially those of environmental origin, but they can guide selection of candidate genes when there are no clues as to map location. Such concerns justify clinical participation in what will be mainly a technical enterprise, simply because disease phenomena often stimulate new ideas. As an example of this synthetic approach, I would like to conclude with a speculation concerning human homeotic defects. Two human genetic diseases caused by HOX gene mutations (Muragaki et al., 1996; Mortlock and Innis, 1997), together with knock-out experiments in mice (e.g., Bailing et al. 1991), indicate that these genes are important for numerous aspects of vertebrate development. As discussed above for contiguous gene deletions and for the PAX mutations in Waardenburg syndrome, the extent of deletion may determine whether one has an isolated defect or syndrome. Thus mutations in the same developmental gene may be responsible for a broad spectrum of congenital anomalies. It is entirely possible that a large portion of human congenital anomalies relate to alterations in homeotic genes. If so, then we have a repertory theater: the players are known, only their roles remain to be defined. Human situs is an example of handed asymmetry that results in distinctive left and fight sides (situs solitus--heart apex, bilobed lung, spleen, and descending colon on the left). A heterogenous group of situs anomalies includes patients with complete reversal (situs inversus), isomerism (e.g., bilateral bilobed lungs, symmetrical heart, malrotated colon), or situs ambiguus (partial reversal or isomerism where cardiac situs may differ from abdominal situs; Wilson et al., 1991). Brown and Wolpert (1990) have proposed a model for handed asymmetry, whereby oriented asymmetric molecules ("F" molecule in Figure 10A) produce differences between left and fight compartments (imagine a morphogen being transported along the "F"; it will accumulate at the fight midline and the left periphery). In Figure 10A, human situs determination is broken down into steps of embryonic axis, boundary, polarity, and compartment identity that are analogous to those of insect segmentation (Wilson, 1993c, 1996). It is suggested that asymmetric helix-turn-helix motifs such as the homeobox function as "F" molecules to produce transcript/protein gradients and that insect segmentation homologs are involved in the patterning of most human organs. Note that the midline anomalies produced by documented human PAX (Waardenburg syndrome) and GLI (Grieg syndrome) mutations conform to the analogy between genes controlling human side and fly segment boundaries (Figure 10A). Rare inherited situs abnormalities in humans (Casey et al., 1993) or mice (Brueckner et al., 1989; Yokoyama et al., 1993) have not been mapped near known homeotic loci, although one translocation breakpoint at 12q13.1 is at the site of the human HOX3 cluster (Wilson et al., 1991; Wilson, 1996).

Will the above speculations prove useful? Ifhomeosis is defined as normal structure at an abnormal position, then malpositioning of normal spleen or heart would certainly qualify. The sequence of gene families illustrated in Figure 10A also would explain why all such mutations will not map to HOX gene clusters. More concrete mechanisms for tissue development have been disscussed in this chapter, and they involve molecules controlling extracellular matrix, cell adhesion (Edelman 1988), cell signaling, or organelle assembly. The positional information or "address" of cells, however, specified during development (Wolpert, 1969) seems to require a more primary and universal mechanism. Tissue assembly may need direction just as occasional dynein alterations implicate ciliary movement as an effector of situs information (Wilson et al., 1991). The discovery of such mechanisms, whether or not they involve the steps shown in Figure 10A, would initiate a true science of congenital anomalies.

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Achondroplasia, 149 ACHE (nicotinic acetylcholine receptor), 188-189, 192, 193, 197, 206 and denervation of skeletal muscle fibers, 203 as first observable marker for neuromuscular junction, 191 Activin, 133-136 Agrin, 192-194, 197 Alzheimer's disease, 180 genetic form, 306 Amniotic bands, 301 APC gene, 77-78 Apert syndrome, 149 Autonomous differentiation and cellular interaction, 17-18 Basement membranes, 92-93, 94, 99 Becker muscular dystrophy, 194 Bendectin, 300 bHLH proteins, 242-244, 254 and terminal differentiation, 245246 BMP (Bone morphogenetic protein), 137, 148 BP (bullous pemphigoid), 78 Brush border, 74, 75

C-ret, and cadherins, 34 Cadherins, as adhesion molecules, 38, 241-242 B-cadherin, 34 and calcium, 31, 32, 37 and cell signaling events, 49, 54-56, 76 classical, 31, 35-36, 47, 77 and cytoskeletal interactions, 43-46 desmosomal, 78 and disease processes, 56-57 E-cadherin, 32-33, 34, 40, 43, 45, 49-50, 56, 80 EP-cadherin, 34 family tree, 34-35 gene superfamily, 31 glycosylation, 39-40 HAV-containing regions, 39, 45 M-cadherin, 34 and muscle development, 53-54 N-cadherin, 33, 41, 42-43, 50, 51, 54, 197 and nervous, system development, 51-53 P-cadherin, 33, 34 and PCR analysis, 34 phosphorylation, 40-42 and proteolytic processing, 42-43

R-cadherin, 34, 53 T-cadherin, 34, 45, 53 and cell lineage studies, 14, 15-19 Cajal, Radon y, 154, 163 Cancer, and alterations in cadherin expression, 56-57 and cell cycle controls, 8-9 Cardiac development, and N-cadherin, 54 C C mutants, 3-4 Cell adhesion molecules, adhesive plaques, 239 and kinase inhibitors, 41 and MASA syndrome, 313-314 qualitative vs. quantitative factors, 37-39 and regulation of neuronal differentiation, 55, 56 see a l s o Cadherins; Immunoglobulin gene superfamily Cell cycle clock, see Mitotic oscillator Cell death, as an active process in differentiation, 18 of muscle cells, 249 programmed, 11, 18-19 Cell division cycle (CDC), 1 checkpoints, 6, 10 control sites, 2 environmental regulation vs programmed regulation, 25 network of control processes, 3, 5, 10, 14 Cell lineage analysis, 14 Cell migration, 97-98 and local environment, 269 Cell positional value, 261-262, 264 Cell-cell adhesion, 30, 240-242 and specialized junctional complexes, 73 Cell-matrix adhesion, 30, 90, 239-240 Caenorhabditis elegans

Centrosome, see MTOC Charcot-Marie-Tooth disease, 116-117 Chemoattractants as guidance factors, for cortical projection axons, 158160 for developing axons within the central nervous system, 156-158, 163 for motor axons by limb buds, 161162 for regenerating axons, 154-156 for trigeminal sensory axons, 160161 Cleft palate pathogenesis, 301-304 Collagens, 92-93 Connexins, 46-47, 105 Connexon, see Connexins Contiguous gene deletion syndromes, 306-307 Crouzon syndrome, 149 Cyclin-dependent kinases (CDK's), 5 Cytotactin, see Tenascin Darrier's disease, 57 "Determinate" vs. "indeterminate" differentiation, 15 Dioxin, 300 Down syndrome, 305-306 Duchenne muscular dystrophy, 194, 195,251-252, 307 Dysmorphology, 294, 321 chromosomal diseases, 304 importance of parental source of genetic material, 308 isolated effects vs. syndromes, 294 and Mendelian mechanisms, 313320 polygenic model, 301 proxisomal diseases, 316-317 Dystrophin, 24, 194 Edwards syndrome, 305 Endochondrial ossification, 148

Equivalence group cells, 17 Evolution, and cadherin gene superfamily, 31, 35 and conservation of branchial arch series in all vertebrates, 269 and conservation of cell division control mechanisms, 4 and HOX genes common ancestry, 267, 273, 283 and molecular genetics, 318-319 and somitomeres, 211, 219 xylem cell differentiation, 11 Extracellular environment, and cell differentiation, 99 and developmentally regulated cues, 90 and "outside-in" signals, 97 and PGFs, 125-130 Fetal alcohol syndrome, 300 Fibroblast growth factor (FGF), 137138 and bone morphogenesis, 148-149 and limb development, 145-148 and neuromuscular junction, 198 Fibronectin, 93-94, 96 and cell migration, 97-98 Flourescent marking techniques, 22 Fodrin-based membrane-cytoskeletal complex, 79-82 Fragile X syndrome, 310-313 GAG (glycosaminoglycan) chains, 9091 Gangliogenesis, "survival factors", 25 Gap junctions, 103-107, 112 antibody probes vs. dye-coupling, 110, 117 communication, 113 and compaction, 114 in development, 109

gene defects and human developmental disorders, 116-117 and the heart, 115-116, 117 regulatory factors, 107-108 Gated cell division, 7 Genetic manipulation techniques for mammal experiments, gene knock-out, 275 transgenic mouse, 274-275 Glycosphingolipid rafts hypothesis, 72 Golgi apparatus, and membrane proteins processing, 196 G 1 & G 2 (gaps) in cell division cycle, 2 G1 and stationary phase, 9-10 and growth arrest of myoblasts, 244245 Growth cone, 56, 154, 167-168 C-domain, 168 filopodia (microspikes), 168-170, 173, 181 and first contact with muscle fibers, 196-198 microfilaments, 170-171, 181 short- and long-range guidance cues, 154, 168 and taxol treatment, 178 s e e a l s o Microtubules Growth factors, 94 specific to cell type, 2 s e e a l s o Polypeptide growth factors Hailey-Hailey disease, 57 Hemifacial microsomia, 301 Hemmati-Brivanlou, Ali, 135 Histidine-alanine-valine amino acid sequence (HAV), s e e Cadherins Holtfreter's experiment, 31 Homeotic genes, 262-265 HOX genes, 262 expression in the trunk, 279-280 hindbrain and neural crest programming, 271-273

homeo box amino acid sequence, 264, 265 models, 287-288 as part of a labeling system, 273 search for mutation in humans, 321 use in cell differentiation, 274 HSPGs (heparan sulfate proteoglycans), 92 Human genome map, 295-299, 321 Hyaluronan (hyaluronic acid), 91 Hypertrophy and atrophy of muscle fiber, 202-203,248 Hypochondroplasia, 149 Immunoglobulin gene superfamily, ancestral protein, 35 and cell adhesion, 32 and promotion of neurite outgrowth, 54 Induction, criteria for natural signal determination, 135 mesoderm, 132-134 neural, 143-145 and pattern generation in vertebrate embryos, 130 Integrins, 90, 95-97 expression in muscle, 239-240 Intracellular signaling, 54-56, 76, 77 Jackson-Weiss syndrome, 149 Kingsley, David, 148 Konigsberg, tissue culture experiments, 235 Laminin, 94-95, 96 Lateral plate mesoderm, and limb development, 281-282 Mangold, Hilde, 130, 140, 143 MAPKs (mitogen-activated protein kinases), 126

Marfan syndrome, 306, 314-315 MASA syndrome, 313-314 MDCK (Madin-Darby canine kidney), 71, 72, 81 Meier, Stephen, 210 Melton, Doug, 135 Membrane protein sorting sites, 71 hierarchy mechanism, 81 Mesenchymal cells, and development of a polarized cell phenotype, 71,282 and epithelial tissues, 70 Mesodermal patterning, 140-143 Microtubules, 171-172 age estimation, 177 elongation, 176-179, 181 formation location, 174-176 organization, 172-174 phosphorylation, 179 unknown active transport mechanism, 174-175 Miller-Dieker syndrome, 306, 317 Mitochondria, and cell position, 196 Mitosis (M) state of cell division cycle, 1-2 Mitotic oscillator, 6-8 Moon, Randy, 139 MTOC (microtubule organizing centers), 172 absence from growth cones and growing axons, 175 Muscle fiber regeneration, 204, 249250 and stimulation of satellite cells, 250-251,254 vs. cardiomyopathy, 253 Muscle structure and function, 230-234 connective tissue, 234 contraction control via the triads, 231 embryonic origin, 236-238 fiber types, 233-234

MuSK (muscle-specific kinase), 192-

193, 206 Myasthenia gravis, 205 Myoblast cell lineages, 23-24, 246-248 and position within the somite, 238239 Myoblast transfer therapy (MTT), 24, 230, 251-253,254 MyoD, see bHLH proteins Myogenesis, 230, 234-248 myogenic regulatory factors (MRFs), 24 primary vs. secondary, 246-248 use of chick/quail chimera model, 236 see also bHLH proteins N-CAM (Neural cell adhesion molecule), 195-196, 206,239-240, 241 Nematode worm, see C. elegans Nerve growth factor(NGF), as chemoattractant for regenerating axons, 154-156 and disease treatment, 26 Nerve impulse activity pattern, 201-202 Neural crest, cell lineages, 24-26 extracellular matrix molecules and migration pathways, 98, 269 head and neck development, 267-269 and hindbrain segmentation, 270-271 migration patterns and T-cadherin expression, 53 migration and somitomere pathways, 220-221,225 Neural regeneration, 203-204 Neuromuscular junction, 188 congenital disorders, 204-205 "junctional nuclei", 195-196 mechanism of polyneuronal elimination, 200-201 and pathophysiological states, 188

reciprocal signaling, 193 role of junctional proteins, 188-189 Nieuwkoop, Peter, 132 Nodal gene, 136-137 Nuclear domain, 249 Nutrients, requirements for unicellular organism cell division, 2 Oncogenes, 9 Paracellular transport, 70 Patau syndrome, 305 Pfeiffer syndrome, 149 Pierre Robin anomaly, 304 Plasma membrane domains, 72 apical, 70, 72 basal-lateral, 70-71 sorting mechanism questions, 73 Polypeptide growth factors (PGF), 124-125 and differentiation and morphogenesis, 125, 130 as inducing factors, 130, 149 and loss-of function mutation technique, 132 paracine and autocrine action, 125 receptors, 125-130 Wnt gene family, 128, 139, 147 Porencephaly, 301 Postsynaptic sarcoplasm of muscle fibers, 195-196 Prader-Willi/Angelman syndromes, 306-310 Primordial germ cells (PGCs), migration, 98 Proteoglycans, 90-92, 100 Ras genes, 9

Retroviruses, an HOX gene manipulation experiments, 286 as genetic markers, 22 RGDS, 93, 94

Rhombomeres, 219-220, 270-271 odd vs. even, 271,279 Rous sarcoma virus (RSV) studies, 4041 Schwann, and muscle fiber description, 235 Schwann cells, and nerve regeneration, 95 Segmentation, in leeches vs. insects, 19, 21 in pattern of spinal nerves and somites, 221 Skeletal muscle fibers, embryonic origins, 190-191 and primary and secondary mugginess, 201 Sodium pump polarity, E- vs. B-cadherins, 82 Somitomeres, 210, 215-217 and brain divisions, 219 earliest formation, 212-215,224-225 and regulation ability, 218 and segmental vessels, 222-224 "somite forming center", 213 Spemann, Hans, 130, 140, 143 Spemann organizer genes, 139 Synaptic folds, 200-201 Synthesis (S) stage of cell division cycle, 1

Talpid, 285 Teloplasm, and cell lineages, 19-21 Tenascin, 95 Teratogens, human, 300 Terminal web, 74, 75 Thanatophoric dysplasia, 149 Tight junction, 74, 75-76 and "fence and gate" functions, 76, 79 trans-Golgi network, and sorting signals, 71 Trunk paraxial mesoderm, 277-279 Tumorigenesis, 2 Turner syndrome, 305 Ultradian clock, 7-8 Vgl, 136 Viral marking techniques, 22 von Ebner's fissure, 221 Waardenburg syndrome, 318-320, 321 Williams syndrome, 306, 314-315 Zellweger syndrome, 316-317 Zonula occludens, see Tight junction

ZPA (zone of polarizing activity), and signaling in limb bud development, 147-148, 282