Advances in Developmental Biology, Volume 2

ADVANCES IN DEVELOPMENTAL BIOLOGY

Volume 2

1993

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ADVANCES IN DEVELOPMENTAL BIOLOGY Editor:

PAUL M. WASSARMAN Department of Cell and DevelopmentaI Biology Roche lnstitute of Molecular Biology Nutley, New Jersey

~

VOLUME 2

1993

@ Greenwich, Connecticut

JAl PRESS INC. London, England

Copyright 0 1993 by)Al PRESS INC. 55 Old Post Road, No. 2

Greenwich, Connecticut 06836 JAt PRESS LTD. The Courtyard 28 High Street Hampton Hill, Middlesex W l 2 IPD England

All rights reserved. N o 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, without prior permission in writing from the publisher. ISBN: 1-55938-582-0

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CONTENTS

vii

LIST OF CONTRl BUTORS PREFACE PauI Wassarman

ix

THE Sry GENE AND SEX DETERMINATION IN MAMMALS Blanche Cape1 and Robin Lovell-Badge

1

MOLECULAR AND GENETIC STUDIES OF HUMAN X CHROMOSOME INACTIVATION Carolyn 1. Brown and Huntington F. Willard

37

GENOMIC IMPRINTING IN THE REGULATION OF MAMMALIAN DEVELOPMENT Colin L. Stewart

73

CELL INTERACTIONS IN NEURAL CREST CELL MIGRATION M a rianne Bronner-Fraser

119

ENZYMES AND MORPHOGENESIS: ALKALINE PHOSPHATASE AND CONTROL OF CELL MIGRATION Saul L. Zackson

153

INDEX

185

V

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LIST OF CONTRIBUTORS Marianne Bronner-Fraser

Developmental Biology Center University of California Irvine, California

Carolyn 1. Brown

Department of Genetics Case Western Reserve University School of Medicine Cleveland, Ohio

Blanche Cape1

Laboratory of Eukaryotic Molecular Genetics National institute for Medical Research London, England

Robin Lovell-Badge

Laboratory of Eukaryotic Molecular Genetics National Institute for Medical Research London, England

Colin L. Stewart

Department of Cell and Developmental Biology Roche Institute of Molecular Biology Nutley, New Jersey

Huntington F. Willard

Department of Genetics Case Western Reserve University School of Medicine Cleveland, Ohio

Saul L. Zackson

Department of Medical Biochemistry University of Calgary Health Science Center Calgary, Alberta, Canada vii

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PREFACE

Advances in Developmental Biology was launched as a series by JAI Press in 1992 with the appearance of Volume 1. This series is inextricably linked to the companion series,Advances in Developmental Biochemistry, that was launched at the same time. As stated in the Preface to Volume 1: “Together the two series will provide annual reviews of research topics in developmental biologyhiochemistry, written from the perspectives of leading investigators in these fields. It is intended that each review draw heavily from the author’s own research contributions and perspective. Thus, the presentations are not necessarily encyclopedic in coverage, nor do they necessarily reflect all opposing views of the subject.” Volume 2 of the series follows these same guidelines. I am grateful to the authors for their contributions,as well as for their cooperation and patience during the preparation of this volume. I thank Alice O’Connor for excellent editorial assistance throughout the project.

Paul M. Wassarman Series Editor

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THE Sry GENE A N D SEX DETERMINATION IN MAMMALS

Blanche Capel and Robin Lovell-Badge

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 I1. Candidates for the Testis-Determining Gene . . . . . . . . . . . . . . . . . . . 4 A . The H-Y Antigen and Sxr . . . . . . . . . . . . . . . . . . . . . . . . . . 4 B . BkmSequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 C . Deletion Mapping and ZFY . . . . . . . . . . . . . . . . . . . . . . . . . 5 I11. Isolation and Properties of SRY . . . . . . . . . . . . . . . . . . . . . . . . . . 7 IV. Direct Evidence that SrylSRY is the Sex-Determining Gene . . . . . . . . . . . 9 A . Mutationstudies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 B . Transgenic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 V. X and Autosomal Testis-Determining Genes . . . . . . . . . . . . . . . . . . 13 VI . Gonadal Differentiation and Expression of Sry . . . . . . . . . . . . . . . . . 14 VII. Molecular Structure and Biochemistry of SRY/Sry . . . . . . . . . . . . . . . 25 VIII. Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Advances in Developmental Bidogy Volume 2. pages 1-35 Copyright 8 1993 by JAI Press Inc All rights of repduction in any form reserved ISBN: 1-55938-582-0

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1

2

BLANCHE CAPEL and ROBIN LOVELL-BADGE

PREFACE During development,certain genes are thought to act as genetic switchesregulating molecular cascades which control the differentiationof specialized tissues and cell types. The process of sex determination in mammals is believed to depend on the pivotal event during embryogenesiswhich diverts the development of the indifferent gonad along the male or female pathway. All subsequent sex-specificdifferentiation results from secretions of the testis or ovary. The Y chromosome is known to be sexdetermining in mammals. Over the years, a number of candidates for the testis-determining gene have been suggested and tested. In the course of the last two years, a gene has been identified in human (MY) and mouse (Sry) which maps to the portion of the Y chromosomeknown to be associated with sex determination. The structure and expression pattern of this gene are entirely consistent with this central role as a master switch gene in sex determination. Furthermore, genetic and transgenic studies prove its importance in testis determination and show that it is the only gene from the Y chromosome required for male development.We discuss the properties of this gene with respect to cellular events underlying differentiation of the gonad and sex determination.

1. INTRODUCTION The process of sex differentiation involves interacting networks of autocrine, paracrine, and endocrine signals leading to the development of male or female characteristics normally affecting the whole organism. This is preceded by the primary event of sex determination which must include agenetic switch responsible for the decision to become male or female. A wide variety of mechanisms appear to have been adopted for this purpose throughout evolution. In mammals, the two sexes differ in genetic make-up: regardless of the number of X chromosomes, the presence of the Y chromosome acts as a dominant male determinant (Ford et al., 1959; Jacobs and Strong, 1959; Welshons and Russell, 1959). In Drosophila, it is the X:autosome ratio that is critical for the activation of one or other pathway, and although there is a Y chromosome in males, it is required only for fertility (Baker, 1989). This may be contrasted to the situation found in many other species where all the genes responsible for sexual dimorphism are present in both males and females. In many lower orders such as yeast, the switch is the presence of a particular allele at an active mating type locus. In some amphibians, such as alligators, where, again, there are no differences in chromosome constitution, the switch is environmental (Ferguson and Joanen, 1982). It is worth noting that the Y-chromosome sex determination mechanism which has evolved in mammals appears to be very stable since few intersexes occur. It also represents an independence from environmentalinfluences affecting sex ratios (Mintz, 1968).

The Sry Gene and Sex Determination in Mammals

GENITAL RIDGE --+

10.5

TdY

--b

/....-.+

11.5

3

........+......-Germ Celldependent Signal? ..*-

12.5

TIME (dPC)

Figure 7. Gonadal development in the mouse. Tdy is thought to act during a narrow window of development (-1 1.5 dpc in mouse) to initiate a cascade of events which divert the differentiationof the cells of the indifferent gonad along the testis pathway. In the absence of the appropriate expression of Tdy, ovarian'developmentensues.

The primary event in sex determinationin mammals is the differentiationof the indifferent gonads (or genital ridges) into testes rather than ovaries. In eutherian mammals, all of the secondary sexual characteristics are a result of the action of hormones or factors produced by the developing gonads, although in marsupials, some characteristics, such as the development of pouch versus scrotum, may be determined independently from the gonad, perhaps by the X:autosome ratio (0et al., 1988). Jost showed almost 50 years ago that castrated rabbit embryos of either chromosomal sex develop as females, indicating that the presence of a testis is necessary for the development of male characteristics (Jost, 1947). Female development can be considered the normal or default pathway. The Y chromosome acts to divert development along the testicular pathway. The male-determiningactivity of the Y chromosome has therefore been attributed to a gene or genes termed TDF, for testis-determiningfactor in humans, and Tdy, for testisdetermining Y gene in mice (Fig. 1). Over the years, there has been a great deal of interest in isolating and defining the testis-determininggene. In addition to elucidating the process of sex determination itself, it is also possible that this gene may help us to understand the genetic control of other processes in the embryo that involve developmentaldecisions. For example, Tdy could be a member of a family of genes with similar functions in development,andor it could lead to an understanding of a common type of pathway used in cell differentiation and morphogenesis.

4

BLANCHE CAPEL and ROBIN LOVELL-BADGE

II. CANDIDATES FOR THE TESTIS-DETERMINING GENE For a long time after the Y chromosome was observed to be present in male mammals (Welshons and Russell, 1959), it was considered by many to have the single function of testis determination. Over the years, the search for Tdy has led to the isolation of a number of genes with a variety of interesting properties, and both the search and the experimentsdesigned to test candidatesfor Tdy have led to considerable information about the structure and behavior of the Y chromosome. A. The H-Y Antigen and Sxr

The H-Y transplantation antigen was the first assayable Y-linked gene product, and largely on this basis, was proposed as the first candidate for the testis deterZfy- I

Zfy- 1

'Ya

Ubely-I

Zfy-2

;py

zfy-2

SY

rdY

sry

Ubely- 1

sry zfy-211

Zfy-1

Y

j

Y Sxr

Zfy-1

j

X Sxr

X Sxr'

Figure 2. The mouse Y chromosome and the origin of Sxr'. Sxr arose as a duplication and transposition of the short arm of the Y to the pseudoautosomal region. Obligatory crossover within this region transfers Sxr to the X chromosome, leading to XX Sxr males. These are positive for the H-Y transplantation antigen. Sxr' resulted from a deletion event brought about by homologousrecombinationbetween Zfy-2 and Zfy-1. This deletion resulted in a loss of H-Y antigen and a gene involved in spermatogenesis termed Spy. Ubely-7 i s a gene encoding a homologue of the X-linked ubiquitin-activating enzyme, and is proposed as a candidate for Spy (Kay et al., 1991; Mitchell et al., 19911.

The Sry Gene and Sex Determination in Mammals

5

mining gene. This antigen was identified as the cause of female rejection of male skin grafts in otherwise syngeneic transplants (Wachtel et al., 1975). Hya, the structural gene for H-Y (or the gene controlling its expression), was dismissed as a candidate only when it was found to lie outside the sexdetermining region of the mouse Y-chromosome.Much of what is known about the structure of the mouse Y has come from studies of the sex-reversed mutation (Sxr) found by Cattanach (1971). which leads to XX males. This mutation is now thought to have resulted from an event in which most of the very small short arm of the Y chromosome, including Tdy, became duplicated and transposed to the end of the long arm (see Fig. 2). This placed it distal to the region responsible for pairing and exchange in male meiosis, such that it was transferred, by virtue of an obligatory crossover in this region, to the X chromosome at a frequency of about 50%.XX Sxr males were H-Y positive, placing Hyu on the same small fragment as Tdy. However, a further mutational event resulted in a deletion within Sxr. The resulting fragment, termed Sxr’, retained its sex-reversing properties, but XX Sxr’ males had no H-Y antigen (McLaren et al., 1984, 1988; Roberts et al., 1988). Subsequently, the human gene controlling H-Y antigen was mapped to the long arm of the Y chromosome, whereas TDF maps to the short arm (Simpson et al., 1987). Sxr and Sxr’ are now referred to as Sxra and Sxrb. B. Bkm Sequences

Banded krait minor (Bkm) satellite sequences were the first sequences cloned from the Y chromosome. It was thought that these repetitive sequences,because of their conservation across heterogametic species, could have a role in sex determination (Singh et al., 1980, 1984). In the mouse they were found to map to both Sxr and Sxr‘ (Singh et al., 1984). It was found, however, that there is no detectable concentration of these sequences on the human Y chromosome (Kiel-Metzger et al., 1985), and their unrelated organization in different species suggested that they were not important for sex determination (Levinson et al., 1985). C. Deletion Mapping and ZFY

The most promising strategy to locate the testis-determining gene was one of deletion mapping the human Y chromosome in sex-reversed individuals that arise from abnormal X:Y interchange at meiosis (Fig. 3). This would allow thedefinition of the smallest region consistently present in sex-reversed XX males, and absent in XY females. Data from several groups using this approach had suggested that TDF mapped very close to the boundary with the pseudoautosomal region (Guellaen et al., 1984; Vergnaud et al., 1986). Indeed, it was thought possible that TDF could define the boundary itself as transfer to an X chromosome would lead to infertility. However, study of an XX male with just 280 kilobases (kb) of Y-unique sequence, and an XY female deleted for the proximal half of this region, suggested

BLANCHE CAPEL and ROBIN LOVELL-BADGE

6

Normal Exchanae

x

y

Al E) DF

tD

Figure 3. Abnormal X:Y interchange leading to sex-reversal in humans. During male meiosis, pairing and exchange normally occurs within the pseudoautosomal region, represented by the open or filled boxes at the distal ends of the short arms of the X and Y. However, consistent with the model first proposed by Ferguson-Smith (19661, exchange can occasionally occur below the position of TDf within Y unique sequences. Study of resulting XX males, with DNA probes derived from the Y chromosome, allowed a deletion map to be constructed which placed TDFvery close to the pseudoautosomal boundary.

that TDF was at least partly within the deleted portion, 140 to 280 kb from the boundary. The existence of a gene in this region had been predicted from the presence of a CpG rich island found by long-range mapping studies anchored within the pseudoautosomal region (Pritchard et al., 1987). Further characterization of this region led to the isolation of the gene termed ZFY, for zinc finger gene on the Y (Page, 1986; Page et al., 1987, 1990). ZFY encodes a protein with characteristics of a transcription factor, including a potential activating domain and a DNA binding domain with 13 zinc fingers. It was also shown to be conserved on the Y chromosome of all eutherian mammals tested. It was therefore proposed k a candidate for TDF. Evidence gathered over the next few years indicated that ZFY could not be TDFlTdy (see Koopman et al., 1991a). The gene was shown to have two copies on the mouse Y chromosome, Zfy-l and Zfy-2, and puzzling homologous loci on the X and autosomes (Mardon et al., 1989; Ashworth et al., 1990).Marsupials, which have a Y chromosomal sex-determining mechanism, were found to have no detectable homologues on either the Y or X chromosomes, but to have only

The Sry Gene and Sex Determination in Mammals

7

autosomal copies (Sinclair et al., 1988).In the mouse, ZfL-2 was deleted from Sxr', and not expressed significantly at embryonic stages. ZfL-1 was shown to be expressed in the genital ridge and fetal testis; however this expression was dependent on the presence of germ cells in the fetal gonad (Koopman et al., 1989). Since sterile mouse mutants such as W/Wdevelop a normal testis, expression of Tdy must be independent of germ cells. Additionally, both Zfi genes appeared to be normal in structure and expression in an XY mouse strain, XY'@''", in which the Y chromosome had been shown to be mutant for Tdy. These XY mice develop as females despite ZfL-1 expression (Gubbay et al., 1990b;Lovell-Badgeand Robertson, 1990). The final proof that ZFY could not be TDF came from identifying a number of human XX sex-reversed males carrying a smaller region of the Y chromosome which did not include ZFY (Palmer et al., 1989).

111. ISOLATION AND PROPERTIES OF SRY As the evidence accumulated against ZFYs role as the pivotal gene in sex determination, it became important to determine whether primary sex-reversal in humans was absolutely dependent on the presence of ZFY. The majority of human XX males had been found to carry portions of the Y chromosome including ZFY; however, there were a few which were ZFY-negative. One explanation for sexreversal in these cases could be dysfunction somewhere in the sex differentiation pathway downstream of TDF. Alternatively, if these cases were the result of a primary sex-reversal event, then they must cany sequences from the Y, most probably resulting from a transfer of the pseudoautosomal boundary and some portion of the Y distal to ZFY. Palmer et a]. (1989) screened a panel of 14 ZEY-negative XX males, first with boundary and then with flanking Y-unique

Figure 4. SRY was localized on the human Y-chromosome using walking probes spanning the region adjacent to the pseudoautosomal boundary to determine the extent of Y-specific sequences present in a panel of XX sex-reversed males. Z F Y lay outside this region; however, within the 35 kb common to these males, one probe defined a sequence conserved on the Y-chromosome of all mammals tested which corresponded to the SRY gene.

8

BLANCHE CAPEL and ROBIN LOVELL-BADGE

probes. While 10 individuals were negative, data from the other four suggested that testis-determiningactivity was localized within approximately 35 kb of Y-unique sequence adjacent to the boundary (Fig. 4). A search of this region led to the discovery of a conserved sequence that mapped to the Y chromosome of all mammals tested. Importantly, in the mouse this sequence mapped to the SxJ region, the smallest part of the Y known to be testis-determining,and was deleted from the Y chromosome of XYThml sex-reversed females. A conserved open reading frame was found when human, rabbit, and mouse genomic sequences were compared, indicating that the sequence formed part of a gene. This was named SRY (for gene in the sex-determining region of the Y) in humans, and Sry in mice (Gubbay et al., 1990a; Sinclair et al., 1990). It can be seen from Figures 2 4 that the human and mouse Y chromosomes are organized differently. In humans, SRY is located on the short arm about 5 kb from the pseudoautosomal boundary, with the transcription unit running 5‘-3’ towards the boundary. The ribosomal protein subunit4 gene, RPS4-Y, which is implicated in Turner syndrome (Fisheret al., 1990),lies in between SRY andZFY. In the mouse, Sry is also located on the short arm, but the pseudoautosomal region is located at the end of the long arm (Fig. 2). A contiguous map of the short arm is not yet available, and the gene order shown in Figure 2 is only provisional. No RPS4-like gene has been detected on the mouse Y chromosome, and no Ubel-like gene on the human Y. The human and mouse SRYISry genes share considerable homology over a 79-amino acid region (see Fig. 14) corresponding to an HMG box-type of DNAbinding domain (Gubbay et al., 1990a; Sinclair et al., 1990). However, the homology falls off abruptly outside this region. The open reading frame for the human gene appears to be present within a single exon, and encodes a protein of 223 amino acids. The structure of the mouse transcript is not yet clear, but available evidence suggests that the protein it encodes is at least 395 amino acids (unpublished observations). An additional and curious feature of the genomic organization of the mouse gene is that it lies within 2.8 kb of unique sequence at the center of a large inverted repeat extending at least 15.5 kb on either side (Gubbay et al., 1992). The repeat is almost exact, with only seven nucleotide differences seen within about 6 kb sequenced each side. This seems likely to lead to some instability of the locus (see description of Tdf” below). This genomic organization is not present adjacent to the human locus, and it is not known when it first arose in evolution; however, it is present in Y chromosomes of both the Mus musculus domesticus and M.m.musculus type, suggesting that it has been maintained for at least one million years. The close sequence homology on either side is curious, and may imply some function such as duplication of regulatory elements. Alternatively, the close homology may be maintained by gene conversion or homologous recombination events (Gubbay et al., 1992).

The Sry Gene and Sex Determination in Mammals

9

IV. DIRECT EVIDENCE THAT Sry/SRY I S THE SEX- DETERMINING GENE There is now overwhelming evidence that SrylSRY is genetically and functionally equivalent to TdyflDF. We review some of that evidence below. A. Mutation Studies

Evidencethat mutations within SRY/Sry disrupt the function of sex determination has been accumulated from both humans and mice. In a study of sex-reversed humans who carry a Y chromosome, test positive for SRY, but develop as females, approximately 10 to 15% possess mutations specifically within the DNA-binding domain of SRY. Some of these are small deletions, while others involve point mutations leading to amino acid substitutions or frame shifts (Berta et al., 1990; Jager et al., 1990;Jager et al., 1991;Harley et a]., 1992;Hawkins et al., 1992).Most of these are de now mutations which have occurred in the affected individual and which are not present in the SRY gene from the father of the XY female. However, several XY females have now been found whose father andor brothers have an identical amino acid substitution. Since similar substitutions have not been found in many control XY males, these are unlikely simply to be variants. It is quite possible that they are conditional mutations dependent on some aspect of genetic background. Study of such cases may help lead to the identification of autosomal or X-linked genes involved in testis determination. Most of the sequence analysis of these sex-reversed females has included only the conserved box region of SRY. The remaining 85% or so of cases of SRY positive XY females could be due to mutations affecting the structure of the gene product outside the HMG box, to mutations affecting the regulation of the gene, or to mutations in genes elsewhere in the testis-determiningpathway (see McElreary et al., 1992). No point mutations affecting Sry function have been found in the mouse to date. However, Sry appears to be the only gene deleted from the Y chromosomecanying the Tdy”’ mutation. This arose in an experiment designed to generate insertional mutants of Tdy using a retroviral vector (Lovell-Badge and Robertson, 1990). XY embryonic stem cells were multiply infected with the vector, then used to make chimeras. Chimeras were test bred to females homozygous for X-linked gene markers to reveal female offspring that carried a single X chromosome. One chimera was identified that had XY females among his offspring. Unlike most XY female mammals, some of these mice proved to be fertile. The mutation was found to segregate with the Y chromosome; furthermore it could be complemented by Sxr’, thus proving that Tdy had been affected. However, no retroviral vector could be found associated with the mutation. We now know that this mutation is due to the deletion of only 11 kb around Sry, and it seems likely that the peculiar inverted repeat structure flanking the gene contributed to this event (Gubbay et al., 1992).

BLANCHE CAPEL and ROBIN LOVELL-BADGE

10

B. Transgenic Studies

Transgenic experiments have demonstrated that Sry is the only gene from the Y chromosome necessary for testis determination (Koopman et al., 1991b).Fertilized mouse eggs were injected with a 14 kb genomic fragment carrying the mouse Sry gene, and were then reimplanted in pseudopregnant females. After embryos had been allowed to develop in urero for about 14 days, they were sexed by gonad morphology. Chromosomal sex was determined by scoring for sex chromatin in amnion cells of each embryo, indicative of the presence of two X chromosomes. Two embryos were found to be developing testes despite the fact that they were chromosomally female. Southern blot analysis confirmed the absence of a Y chromosome, but indicated that these two embryos carried copies of Sry as a transgene. To determine the frequency with which this 14 kb DNAfragment containing Sry was able to give sex-reversal, all the female embryos were examined for the presence of the transgene. In total, nine embryos were found to be transgenic for Sry sequences. Of these, seven developed as females, while two developed as males (Table 1). There are several reasons why the transgene may have failed to sexreverse in these cases. Four of the seven transgenic females had fewer than one copy of Sry per cell, indicating that they were mosaics. It is known from chimera and mosaic studies that approximately 25% of the somatic cells of the genital ridge must carry Tdy in order for the gonad to develop as a testis (Burgoyne, 1988). It is possible that Sry was present in too few cells in these embryos. Alternatively, the timing or level of expression of Sry in some transgenics may be ineffective. It is known that transgenes often show very different levels of expression from one line to another, probably due to the site of integration. There is good evidence that the regulation of Sry expression is quite precise during gonadal development (see below). Since for the purposes of this analysis it is function that is being assayed,

Table 1. Summary of Transgenic Data Stage of development & Construct

Embryos 74 1 Adult 741 Adults 741mutl Adult 741 Line 32.10

Total Number of X X Transgenics XX Females X X Males XX Intersex

9 5 2

45

7 4 1 37

2 1 1 6

-

2

Nore: In total. 4 out of 16 XX mice which were fwnder transgenics for Sly have been sex-reversed (sum of top 3 lines). Subsequent breeding of non-sex-reversed uansgenics has produced 6 males and 2 intersexes from 45 XX transgenics (bottom line). All tlansgenics. analyzed as 14.5 Qc embryos (top line) or adults (bottom 3 lines), carry a 14 kb genomic fragment containing Sly. 741 or 741mutl. 741mutl has an engineered single base change which simplifies detection of the transgene. but does not change the amino acid encoded

The Sry Gene and Sex Determination in Mammals

11

and not simply tissue specific expression, the timing or level of expression may be critical to produce the effect of sex-reversal. Some of the injected embryos were allowed to develop to term and a number of adult transgenics obtained. One of these had a normal external male phenotype as shown in Figure 5, but was chromosomally female both by karyotypic analysis and by Southern and PCR testing for the Y specific marker Zfy. This animal exhibited apparently normal male reproductive behavior, but was sterile as would be expected for an XX male. When examined internally, he was found to have a normal male reproductive tract, with no signs of hermaphroditism. This indicates that Sertoli cells and Leydig cells must have differentiated and functioned normally during embryonic development of 11133.13, at least in terms of Anti-Mullenan Hormone (AMH) and testosterone production. However, his testes were considerablysmaller than those of control XY litter-mates. Histological examination of the testes revealed a normal structure and the presence of all somatic cell types, Sertoli cells, Leydig cells, and pentubular myoid cells, but with a complete lack of spermatogenesis. It is known that the presence of two X chromosomes leads to adysfunction during the meiotic stages of spermatogenesis (Burgoyne and Baker, 1984). In addition, these transgenics lack the Y chromosomal genes involved in spermatogenesis, at least one of which has been genetically mapped to a region between Zfy-Zand Zfi-2on the mouse Y chromosome (see Fig. 2). The testes of m33.13 in fact looked identical to those of sex-reversed XX Sxr or XX Sxr’ male mice which also carry only part of the Y (Cattanach et al., 1971; Sutcliffeand Burgoyne, 1989). To date, of seven founder adult XX transgenics produced by microinjection, two have developed as males. This result demonstrates that this 14-kb sequence is functionally equivalent to Tdy, and indicates that all other genes required for the development of the somatic male phenotype reside on chromosomesother than the Y. In order to eliminate the possibility that other genes affecting sex determination are present on the 14 kb genomic fragment used in these experiments, detailed sequence analysis has been carried out, and the entire sequence searched for additional exons (Gubbay et al., 1992; and unpublished data). Extensive sequence comparison, cross-hybridization with sequences adjacent to the human gene, and species conservation analysis have failed to reveal the presence of any other genes within the injected DNA. We conclude that Sry alone is able to initiate male development on an otherwise chromosomally female background. A third category of sex-reversed transgenics has been produced by breeding XX Sry transgenic carriers which were not themselves sex-reversed, but developed as normal fertile females. n o of these females generated in the original experiment carried multiple copies of Sry as a transgene. One of these transmitted the transgene to offspring which also did not sex-reverse. This ruled out mosaicism as an explanation for the failure to sex-reversein this line. In contrast, some of the carriers have given rise to sex-reversed offspring in breeding experiments both against normal (1 case) and against other transgenic siblings (5 cases). Two intersex offspring have also been observed in these matings. Preliminary results suggest that

Figure 5. Several XX mice transgenic for Sry developed as normal phenotypic (but sterile) males. 33.13 (the example above) has been shown by Southern and PCR analysis (lower part of the figure) to carry the Sry transgene, but not the Y-specific marker, Zfy-7. 33.1 7, normal male littermate; 33.9, normal female littermate. 12

Tbe Sry Gene and Sex Determination in Mammals

13

the transgene is being expressed but perhaps at a lower level than normal. It is possible that Sry is at a threshold level (Table 1 summarizes our transgenic data at the time of this writing). Careful measurements of the number of transcripts over a time-course of development will be required before a conclusion can be reached. While homozygosity andor copy number appear to increase the incidence of sex-reversal, the correlation is not absolute: not all homozygotes are male. The inconsistenciesin these results suggestthat genetic background effects segregating in these F2(C57BL/6 X CBA) and later generation inter- and back-crosses are affecting sex-reversal (Vivian, Koopman, and Lovell-Badge, in preparation). Transgenic mice have also been generated using a genomic fragment carrying the human SRYgene(Koopman et al., 1991;R. Palmiter, personal communication). However, no evidence of sex-reversal has been seen despite apparently normal expression of the transgene (Koopman et al., 1991).The large number of differences between the mouse and human genes, both at the DNA and protein levels, is presumably responsible for the failure of the human gene to induce testis formation in XX mice. Perhaps the human gene fails to bind to target DNA sequences or fails to interact correctly with other protein factors in the mouse. In fact, recent evidence suggests that there has been a rapid evolution of Sry sequences leading to amino acid substitutions between species inside and outside the conserved HMG box ( S . Whitfield and P. Goodfellow, unpublished observations). The significance of this finding is not yet understood, but it does suggest that there is a great deal of specificity built into Sry’s mode of action.

V. X A N D AUTOSOMAL TESTIS-DETERMINING GENES At least three autosomal loci were identified in the mouse, Tas, Tda-I, and Tda-2, which are thought to interact in a concerted way with Tdy in the sex determination pathway. It has been suggested that all of these loci may have evolved within a species to function in a coordinated manner (Eicher, 1988). In 1982, a Y chromosome from a M.m.domesticus substrain, M.m.poschiavinus, was described which frequently gives sex-reversal when introduced into the genetic background provided specifically by the C57BU6 mouse strain (Eicher et al., 1982; Eicher and Washburn, 1986). In fact all XY embryos arising from these matings develop ovotestes, which sometimes resolve postnatally into either ovary or testis. More recently it has been shown that this YPoschromosome is associated with a delay in testis formation of about 14 hours (Palmer and Burgoyne, 1991b). These results have been interpreted to suggest that Tdy normally acts during a narrow window of time to divert development along the testicular pathway. If the timing of its expression or its interaction with other genes in the pathway is disturbed, ovarian development will be initiated and “locked in” (Eicher et al., 1982; Eicher and Washburn, 1983; Palmer and Burgoyne, 1991b) (Fig. 1). The sex-reversal associated with the Ypos chromosome may result from a late-acting allele of Sry in

14

BLANCHE CAPEL and ROBIN LOVELL-BADGE

combination with early-acting ovariandetermining genes provided by the C57BL/ 6 genetic background. Tdu-1 and Tdu-2 are genes proposed by Eicher (1986) to be involved in this effect. Other domesricus-type Y chromosomes, for example from the AKR strain, can show a similar effect to that of Yp,but only when present on a C57BU6 background together with specific deletions such as the tuil-hairpin deletion on chromosome 17. T-associated sex-reversal (Tas) is the gene proposed to lie within this particular deletion (Eicher and Washburn, 1986; Eicher, 1988). However, it is conceivable that any genetic abnormality that interferes with the finely balanced program of cell interactions required for testis differentiation (see below) will result in operation of the default ovarian pathway. From this type of analysis, it seems that it is possible to rank Y chromosomes with respect to the “strength” of TdyBry, with Yps being weakest, Yakrand some other domesticus type Y chromosomes being intermediate,and other domesticus and all musculusY chromosomes being the strongest (Biddle and Nishioka, 1988; Palmer and Burgoyne, 1991b). One might expect to find differences between the Sry alleles, but so far the only difference noted is a T to C change leading to a threonine instead of an isoleucineat position 63 within the HMG box of all domesticustype Y chromosomes (see Gubbay et al., 1992;Tucker, 1992; and unpublished observations). X-linked genes have also been implicated in sex determination. Mutations have been described which segregate with the X chromosome and lead to XY female phenotypes in the horse (Kent et al., 1986), wood lemming (Fredga, 1988), and humans (Scherer et al., 1989). In the case of the wood lemming, there is a cytologically distinct X chromosome, termed X*. which appears to override Tdy, even if the latter is present in two copies. In humans, X-linked sex-reversal is often associated with duplications of a region of the short arm. Duplications of the X are generally the only way to produce a double dose of an X-linked gene product because of X-inactivation. It is possible that a high level of this product interferes competitively with the normal function of Sry.

VI. GONADAL DIFFERENTIATIONAND EXPRESSION OF Sry The gonad is unique among organs in the developing embryo in that it arises as an indifferent tissue whose developmental course can follow one of two pathways. If the Y-chromosome is present, Sry will be expressed and development along the male pathway will be determined. If Sty is not expressed, the gonad will follow the female pathway of development to form an ovary. The indifferent gonad develops in the context of the larger urogenital system. Three overlapping, sequential kidney systems arise during development: (1) the pronephros;(2) the mesonephros; and the (3) metanephros (Fig. 6). The pronephros appears first as a series of segmental swellingsof the intermediatemesoderm which lies between the somites and the lateral plate in the cervical region of the embryo.

15

The Sry Gene and Sex Determination in Mammals

Pronephric system (segmentedintermediate mesoderm)

Mesonephric system (unsegmented intermediate mesoderm)

Metanephnc system (unsegmented intermediate mesoderm) Ureteric bud

Mesonephnc (Wolffian) duct

Figure 6. The gonad arises within the urogenital system which develops from the

intermediate mesoderm between the somites and the lateral plate. The nephric system is divided into pronephric (cervical, vestigial in mammals), mesonephric (thoracic), and metanephric (definitive kidney) regions. The mesonephric (Wolffian)duct condenses, cavitates, and extends from the pronephros to join the cloaca. The central mesonephros will give rise to the genital ridge. (After Sadler, 1985.) These swellings cavitate and some rudimentary tubules grow toward the dorsal aorta. The pronephros is vestigial in amniotes; however, on the ventral side of these nephroceles, segmental units begin to fuse longitudinally, to form the mesonephric or Wolffian duct, and extend to join the cloaca at the posterior end of the embryo. In the mesonephric region, comprised of the lower thoracic, lumbar, and sacral regions, the extension of the Wolffian duct seems to initiate the organization of S-shaped tubules. These mesonephric tubules are in contact with capillaries from the dorsal aorta, and open at their other ends into the Wolffian duct. This kidney system functions for a limited period in some mammals. Meanwhile, a hollow ureteric bud develops from the Wolffian duct just anterior to its junction with the cloaca and grows back into the sacral region where it induces the elaboration of tubules which results in the development of the definitive metanephric kidney.

BLANCHE CAPEL and ROBIN LOVELL-BADGE

16

Primordial germ cells

Mesonephric tubule

Germinal epithelium

Primitive Mullerian duct

Primitive testis cords

Figure 7. Diagrammatic cross-sectionsof the male urogenital ridge. Mesonephric tubules condense and reach toward the germinal epithelium from the mesonephric duct (top).Primordial germ cells migrate into the region of the gonadal blastema where they are enclosed in primitive testis cords (bottom). A second duct system, the Mullerian duct, invaginates from the coelomic surface of the mesonephros. (After Smith et al., 1969.) The genital system begins to develop within the mesonephros. A second longitudinal duct, the pararnesonephric or Mullerian duct, begins to form from the lateral epithelium of the mesonephros (Figs. 6 and 7). This duct runs parallel to the Wolffian duct for the length of the mesonephros, then turns toward the midline where it fuses with the Mullerian duct from the other side before reaching the cloaca. The medial portion of the mesonephros begins to bulge into the peritoneal cavity by 10.5 dpc and develop as a distinct organ (Figs. 7 and 8). This gonadal blastema is populated by germ cells from day 10.0of gestation in the mouse which arrive via migration from the allantois through the gut mesentery (Mintz and Russell, 1957; Ginsburg et al., 1990) (Fig. 7). While there is some disagreement about the source of the somatic cell population in the gonad, it is likely that there are contributions of cells from both the coelomic epithelium and the tubules and

The Sry Gene and Sex Determination in Mammals

17

Figure 8. Scanning electron micrographs of rnurine urogenital ridge at 10.5 dpc (top) and 11.5 dpc (bottom)as the gonadal portion becomes distinct. m, rnesonephros; g, gonadal blasterna; bar = 100 pm. mesenchyme of the mesonephros (Smith and Mackay, 1991). Scanning electron micrograph studies indicate that cells stream into the gonad from its coelomic surface during this period (Fig. 9). Even though the chromosomal sex of the embryo is, of course, established at fertilization, no sex-specific differences in development of the gonad are recognizable at this stage. within a 24-hour period between 11.5 and 12.5 dpc in the mouse, the initiation of testes development occurs with the alignment of cells into cords in the gonads of male embryos (Fig. 10). Less obvious cellular organization occurs in female gonads (Merchant-Larios and Taketo, 1991). In fact, the first distinct indication of

Figure 9. Scanning electron micrographs of 10.5 dpc murine urogenital ridge. The gonadal blasterna is populated by primordial germ cells, cells fromthe rnesonephros, and probably by cells from the coelomic epithelium. At early time points, cells seem to invaginate from the coelomic surface via pore-like structures. p, pore; m, rnesonephros; g, gonadal blastema; s, gut mesentery; bar = 50 wm. 18

13 I t cipt

Figure 10. Electron micrographs showing cellular organization of male gonads at 11.5 dpc, before primitive testis cords appear (top), and at 14.0 dpc, when cords are organized around primordial germ cells (pgc),recognized by their large round nuclei. Pre-Sertolicells (psc)and Sertoli cellssc (bottom) have polymorphic nuclei and bound the tubule at later stages where they contribute to the basal lamina (bl). Interstitial tissue (i) consists of Leydig and peritubular myoid cells, and forms the structural support for the tubules. 19

BLANCHE CAPEL and ROBIN LOVELL-BADGE

20

9

c?

Indifferent Stage

Mesonephric tubules

form genital duct

$:! \<

Descend / I

Mullerian dwts

Figure 77. In the presence (d, or absence (0) of Sryexpression at the indifferent stage of gonadogenesis, development diverges along male or female pathways. In the male, the rnesonephric (Wolffian) duct is elaborated, due to the action of testosterone produced by Leydig cells, into the male ductal system while the Mullerian duct degenerates due to the action of anti-Mullerian hormone produced by Sertoli cells. In the female the Mullerian duct develops into the female genital tract while the Wolffian duct disappears. (After Smith et al., 1969.)

ovarian development is the entry of germ cells into meiosis which occurs several days later (McLaren, 1988). After 12.5 dpc, a cascade of sex-specific events leads to the rapid divergence of male and female embryos. In males, the pre-Sertoli cells begin to secrete AMH, a growth factor which causes the regression of the Miillerian duct. Under the influence of testosterone and other hormones secreted by the developing testes, the Wolffian duct is elaborated into the male ductal system. The mesonephric tubules become the efferent collecting ducts for the testes which empty into the vas deferens and epididymis. In females, in the absence of AMH, it is the Miillerian duct which is stabilized and which eventually forms the oviduct and uterus while the Wolffian duct regresses (Fig. 11). The adult testes is composed of four basic cell types: (1) germ cells; (2) Sertoli cells, which form the supporting cells of the seminiferous tubules; (3) Leydig cells, which are the steroid secreting cells of the testes; and (4)peritubular myoid cells, which contribute connective tissue surrounding the tubules (Fig. 12). Germ cells are not essential for the organogenesis of the testes. In mutants which lack germ cells, such as W/WorSl/Sl, the testes still forms cords normally (Mintzand Russell, 1957; McLaren, 1985). Ultrastructural studies reveal that the pre-Sertoli cell is the first recognizable cell type to differentiate within the gonadal blastema. Distinguishable by its differential affinity for various cell stains, such as periodic acid Schiff (Kanai et al., 1989) and toluidine blue (Satoh, 1985), the pre-Sertoli cells begin to aggregate in groups surrounding the primordial germ cells. Cell junctions between pre-Sertoli cells, and between germ cells and pre-Sertoli cells, are observed. The pre-Sertoli cell typically contains rod-shaped mitochondria, many lipid droplets, an active Golgi, and well-developed rough endoplasmic reticulum (Fig. 10).

-

The Sry Gene a n d Sex Determination in Mammals

cf

Testis

Prospermatogonia

[ (-+

mitotic arrest)l

4 -

Genital Ridge

primordial

-Q

+

21

Ovary

Oocytes

Germ Cells -+meiotic arrest)

Sertoli Cells Cell Precursors

Leydig Cells

I

- - -

h Testosterond Tunica Peritubular Myoid Cells Blood Vessel

Steroid Cell Precursors

Connective Tissue

Follicle Cells

Interstitial (Them) Cells

Tunics Stromal Cells

Figure 12. The cell lineages of the indifferent genital ridge are thought to give rise to counterparts in testis and ovary.

Some workers have observed a close association between the distal ends of the mesonephric tubules and the location of the earliest pre-Sertoli cells to begin differentiation (Satoh, 1985). This finding has led to the suggestion that the cells of the mesonepbric tubules de-differentiateand re-differentiate as somatic cells of the gonad (Wartenberg, 1982). Smith and MacKay (1991), in a study of the deposition of the basal lamina, have observed no structural barrier partitioning the mesonephric tubules or mesenchyme from the gonadal blastema at early time points; in fact, between 10.5 and 12.0 dpc the mesonephric tubules appear to lose cells to the surroundingmesenchyme and to the developinggonad. Likewise, Smith and MacKay observed no complete basal lamina delineating the coelomic epithelium which is, in some regions, indistinguishable from the somatic cells of the gonad. Either or both of these cell types may be signaling as well as directly contributing to the pre-Sertoli or other cell populations of the gonadal blastema. Leydig cells are identifiable slightly later in the interstitial tissue which remains outside the anastomosing groups of Sertoli cells. Recognizable at the election microscope level by their characteristic smooth endoplasmic reticulum, these are the endocrine cells of the testes which rapidly begin to secrete testosterone.Largely because of its early appearance and location, the pre-Sertoli cell is the most likely candidate for the cell type expressing Sry. Studies of mice mosaic for XX and XY cells have shown that the Sertoli cell population is composed almost entirely of XY

22

BLANCHE CAPEL and ROBIN LOVELL-BADGE

cells as one might expect if the expression of Sry were a requisite for its further differentiation. Other cells of the testes can be either XX or XY with equal probability (Burgoyne et a]., 1988; Palmer and Burgoyne, 1991a). Certainly, the expression of Sry is not germ-cell dependent in the developing testes, since it is still expressed in mutants where germ cells are absent (Koopman et al., 1990). As described earlier in this review, genetic evidence has suggested that there is a window of receptivity in the fetal gonad for the inductive signal which initiates male development. If that signal does not occur, development along the female pathway proceeds (Eicher et al., 1982; Eicher and Washburn, 1983) (Fig. 1). The competence to respond to an inducing signal may involve the transient presence of a cellular state or localization proximal to the inducer, and/or the molecular orchestration of a number of cooperative molecules whose appearance must coincide. Organogenesis is a dynamic process in the mammalian embryo with alternating phases of cellular reorganization and induction. Cell movement and proliferation bring cells into juxtaposition with new cells with which they may communicate.In fact, it has been pointed out that an essential feature of an induction system is that a differenceexists between inducer and responder (Nieuwkoopet al., 1985). Taketo and Koide (1981) showed that gonads explanted with their mesonephroi before 11 dpc did not differentiatefurther, whereas explants after 11 dpc proceeded to develop as testes or ovary. This experiment suggests that the embryonic signals which induce gonadal development converge in a narrow window of time, just as does the competence to respond. Recent experiments in the mouse suggest that the factor which determines testes development is specific to the responding cells. If the 11.5 dpc gonad is explanted and cultured in v i m in the absence of the mesonephros, no cords form in fetal gonads. However, if the 11.5 dpc male gonad is cultured in contact with a male or female mesonephros, cords appear. Cell-markingexperimentsreveal that peritubular myoid cells havemigrated into the gonad in these in vifro experiments. No experiments with female gonad explants were reported (Buehr et al., 1993).This evidence indicates that: (1) contact with the mesonephros is critical to induce further differentiationof gonadal tissue; and (2) the inductive signal is present in male and female surroundingtissue at 11.5 dpc, but only male gonadal tissue has the competence to respond to this signal by organizing into cords at this time. A number of studies have indicated a cooperativitybetween Sertoli and peritubular myoid cells, typical of epithelial-mesenchymeinteraction, in the deposition of the basal lamina surrounding the seminiferous tubules. Laminin has been localized to the Sertoli cell population in vitro, while fibronectin is associated exclusively with myoid cells in vitro and in vivo (Tung and Fritz, 1980; Skinner et al., 1985, 1989).Co-culture of Sertoli and myoid cells prolongs the survival of both cell types in v i m , and affects the migration of Sertoli cells as well as the production of certain molecular markers of Sertoli cell differentiation such as transferrin and androgen binding protein (Tung and Fritz, 1986, 1987). Blocking the direct cell contact between myoid and Sertoli cells inhibits the morphogeneticcascade observed when

The Sry Gene and Sex Determination in Mammals

23

Sertoli cells are plated directly onto existing monolayers ofperitubular myoid cells (Tung and Fritz, 1987). Sertoli cells alone can organize into cord-like structures when provided with an extracellular matrix such as Matrigel (Hadley et al., 1985), but this morphogenesis fails to occur in the presence of an antibody to laminin or its receptor (Hadley et al., 1990). Dym and co-workers have observed an increase in G-protein levels when Sertoli cells are plated on ECM which results in an increase in responsiveness to follicle-stimulating hormone (Dym et al., 1991). These investigators and others have suggested that changes in cytoskeletal structure, which result when matrix receptors are occupied, may favor further differentiation and epithelialization.Changes in the cytoskeletonmay alter gene expression through the associationof polyribosomes,altered messenger stability,or translation efficiency (Bissel et al., 1982). It is particularly apparent in the developmentof the testes that the morphogenetic changes in cell structure and associations are inextricably and reciprocally related to the biochemistry of gene expression. It is possible that Sry transmits a signal to cells of the mesonephros to enter the gonad, or that Sry alters the response of its native cell to these cells or to some other signal from the mesonephros. Northern analyses have failed to detect Sry expression in differentiating genital ridges or embryonic testes from mice. However, with the much more sensitive reverse transcriptase-polymerasechain reaction (RT-PCR) technique, it is possible to demonstrate that Sry transcripts are indeed present for a brief period just prior to overt testis differentiation.Other PCR experimentsand in situ hybridizationstudies provide evidence that the gene is only expressed by cells within the genital ridge (Koopman et al., 1990) (Fig. 13a,b). Furthermore, Sry expression can be seen in genital ridges isolated from 11.5 dpc embryos homozygous for W ,an extreme mutant allele at the W locus, that completely lack germ cells. This shows that the gene is expressed by a somatic cell type within the genital ridge (also an essential condition for Tdy, as discussed above). Experiments in mosaics and with mice carrying the T(X: 16)16H(T16) translocation illustrate the dosage effect seen when Sryexpressing cells fall below a minimal level (thought to be 25%) in the fetal gonad. When Sxr is introduced into mice carrying this translocation, T16/X Sxr mice may develop as males, hermaphrodites, or females. The translocation leads to the normal X being inactivated in all cells, and inactivation is thought to spread into the Sxr region, resulting in a reduced level of expression of Sry (McLaren and Monk, 1982; and see transgenic mosaics above). This expression pattern is therefore, entirely consistent with what might be expected for agene which is conferring on the cells of the gonadal blastema the transient competence to respond to inductive signals. Exactly how Sry is acting to initiate this cascade of events is not yet understood. Sry contains a putative DNA-binding domain (see below). It seems likely that Sry acts as a transcription factor which activates one or more downstream genes. We know from transgenic experiments that the presence of Sry alone on an otherwise XX background leads to the development of testes (Koopman et al., 1991b). This

A

Fetal Stage (days post coifurn) 9.5

10.5

11.5

12.5

B 13.5

Embryo

N

P

Developing Gonad

Figure 13. A, In the mouse, Sry is expressed in the male genital ridge during the window in development when the gonad condenses and testis cord formation is initiated. PCR detects Hprtexpression in the genital ridge at all stages of male development, while Sry expression i s detected at 10.5 dpc, peaks at 11.5 dpc, and disappears by 13.5 dpc. 6, In situ hybridization to a saggital section through a male urogenital ridge at 11.5 dpc shows Sry expression localized to the cells of the gonadal blastema. 0, ducts; CR, genital ridge; M, mesonephros; W ,pre-vertebrae; bar = 50 pm.

The Sry Gene and Sex Determination in Mammals

25

must mean that genes acting immediately upstream and downstream of Sry must be autosomal or X-linked. Furthermore, whatever signals are necessary upstream must be expressed normally on a female background. The Arnh gene has often been considered a likely early downstream target of Sry activation. In sifu experiments on AMH expression in this laboratory have indicated that AMH is first detectable at 12.5 dpc, almost 48 hours after Sry is first expressed (Munsterberg and LovellBadge, 1991). It therefore seems unlikely that Amh is a primary target of Sry. Once the architectural process of testes development is initiated, changes in cell structure will arise from epithelialization and the organization of a basal lamina surrounding the tubules. Changes in cellular neighborhoods will result from cell migration, aggregation, and proliferation. All of these processes are likely to contribute in an interactive way to further differential gene expression and maturation of tubule development. In the adult, Northern analyses have revealed Sry transcripts of about 1.3 kb in mouse testis (Koopman et aI., 1990), and transcripts approximately 900 bp from SRY in human testis (Sinclair et al., 1990). These transcripts appear in mouse at about 20 days afterbirth (Koopman et al., 1990,and Koopman unpublishedresults), and seem to be confined to postmeiotic germ cells (probably round spermatid stages). Sry expression is absent in adult testes of XX Sxr males which are devoid of germ cells. However, it seems that the gene has no cell autonomous function in spermatogenesis, as germ cells deleted for Sry, within a male chimera, were able to give rise to offspring (Gubbay et al., 1990b;Lovell-Badgeand Robertson, 1990; and see section on mutation studies above).

VII. MOLECULAR STRUCTURE AND BIOCHEMISTRY OF SRYISry DNA sequencingof human, rabbit, and mouse SRY/Sry genes revealed the presence of a conserved open reading frame. Within this the region of greatest homology corresponded to a 79-amino acid domain similar to one found in a family of DNA-binding proteins, and referred to as an HMG box. These fall into two classes (Fig. 14). The frst comprises ubiquitous proteins containing several HMG boxes which bind DNA relatively weakly with little or no sequence specificity. Among this class are the high mobility group nonhistone proteins, HMGl and HMG2, in which the domain was first recognized (Einck and Bustin, 1985). These are not known to regulate or bind to specific gene sequences, although they are associated with regions of transcriptionally active chromatin. Also in this first class are genes such as hUBF (Jantzen et al., 1990), an RNA polymerase 1 cofactor that binds to the upstream control element and core of the rRNA gene promoter to activate transcription in a binding site dependent manner, and mtTFl (Parisi and Clayton, 1991), a cofactor of mitochondrial RNA polymerase which binds upstream heterologous promoter elements in order to activate transcription of mitochondrial

IIJI8Sr.y h 4 Y r.bSrT

Lf-2 Sox-1

Sop2 sox-3 11-3

xsorll

Lf-1 sox- 4 MU-13 Lf-4 U - 9

Lf-6 XSox-13

IRE-MIP mi-12

Alli-16 Lf-5

GHVKRPMNAF MVWSRGERHK LAQQNPSMQN TEISKQLGCR WKSLTEAEKR PFFQEAQRLK ILHREKYPNY KYQPHRRAK DRVKRPMNAF IVWSRDQRRK MALENPRMRN SEISKQLGYQ WKMLTEAEKW PFFQEAQKLQ AMHREKYPNY KYRPRRKAK ERVKRPMNAF MVWSQHQRRQ VALQNPKMRN SDISKQLGHQ WlCMLSEAEKW PFFQEAQRLQ AMHKEKYPDY KYRPRRlCVK MVWSRGQRRK MAQENPKMHN SEISKRLGAE WKLLSEAEKR DRVKRPMNAF MVWSRGQRRK MAQENPKMHN SEISKRLGAE WKVMSEAEKR DRVKRFWAF MVWSRGQRRK MAQENPKMHN SEISKRLGAE WKLLSETEKR DRVKRFWAF MVWSRGQRRK MALENPKMHN SEISKRLGAD WKLLTDAEKR MVWSRGQRRK MAQENPKMHN SEISKRLGAD WKLLSDAEKR SRGQRRK MAQENPKMHN SEISKRLGAD WKLLSDSEKR MVCSRGQRRK MAQENPKMHN SEISKRLGAE WKLLSEAEKR

PFIDEAKRLR ALHMKE PFIDEAKRLR ALHMKEHPDY KYRPRRKTK PFIDEAKRLR ALHMKEHPDY KYRPRRKTK PFIDEAKRLR AVHMKEYPDY KYRPRRKTK PFIDEAKRLR AVHMKE PFIDEAKRLR AVHMKDY PYIDEAKRL

GHIKRFMNAF MVWSQIERRK IMEQSPDMHN VKRPMNAF MVWSQIERRK IMEQSPDMHN MVWSQIERRK IMEQSPDMHN VKRPMNAF MVWSQIQRRK IMEQSPDMHN MVWSKIERRK IMEQSPDMHN

PFIQEAERLR PFIREAERLR PFIREAERLR PFIREAERLR PFIREAERLR

AEISKRLGKR AEISKRLGKR AEISKRLGKR AEXSKRRGKR AEISKRLGKR

WKLLKDSDKI WKLLKDSDKI WKLLKDSDKI WKLLKDSDKI WKMLKDSEKI

LKHMADYPDY KYRPRKXVK LKHMMYPDY KYRP LKHMAD LKHMADYPDY KYRP LKHMAD

Percent similarity:

Percent identity :

100

100 71 66

00 81

80 82

63 63 63

77

58

73

54

81

SKIERRK IMEQSPDMHN AEISKRLGKR WKMLNDNEKI PFIREAERLR L K W Y GHIKRPMNAF MVWSQIERRK IMEQSPDMHN PMNAF MVWSQHERRK IElDQWPDMHN VKRPMNAF MVWSQHERRK IMDQWPDMHN MVWSQHERRK IMDQWPDMHN

AEISKRLGKR AEISKRLGRR AEISKRLGRR AEISKRLGRR

WKLLKDSDKI WELLQDSEKI WQLLQDSEKI WQLLHDSEKI

PFIQEADGLR PFVKEADGLL PFVKEAGGLR PFVKEAERLR

LKHMADYPDY KYRpRI(KvK LKHMADYPNY KYRP LKHMADYPDY KYRP LKHMAD

Sox-5

Sox-6

h,

u

SOX-7

EuSox-8 pdm-lk:

Tcp-1 Tcpla mtW1

hUBP h 1

PHIKRMAF MVWAKDERRK ILQAFPDMHN SNISKILGSR ARDERRK ILQAFPDMHN SNISKILGSR AKDERKR LAVQNPDLHN AELSWGKS AKDERKX LAQQNPDLHN AVLSXMLGKA

WKAMTNLEKQ WKSMSNQEKQ WKALTLSQKR WKELNAAEKR

PYYEEQARLS PYYEEQARLS PYVDEAERLR PFVEEAERLR

KQHLEKYPDY KYKPRPKRT KIHLEKY LQHMQDY VQHLRDH

75

54

ERTPRPPNAF ILYRKEKHAT LLKSNPSINN SQVSKLVGEM WRNESKEVRM RYFKMSEFYK AQHQWPGY KYQPRKN

54

31

PTIKKPLNAF MLYMKFMUK VIAECTLKES AAINQILGRR WHALSREEQA KYYELARKER QLHMQLYPGW SARDNYGKK PHIKKPLNAF MLYMKEMRAN WAECTLKES AAINQILGRR WHALSREEQA KYYELARKER QL€W)LYPGW SARDNYGKK

52

29

46

24

PKKPVSSY LRFSKEQLPI FKAQNPDAKT TELIERIAQR WRELPDSKK KIYQDAYEAE WQVYKEEISR FKEQLTPSQ DFPKKPLTPY FRFFMEKRAK YAKLHPEMSN LDLTKILSKK YKELPEKKKM KYIQDFQREK QEFERNLARF REDHPDLIQ NAPKRPPSAF FLFCSEYRPK IKGEHPGLSI GDVAKKGEMW NNTAADDKQ PYEKKAAKLK EKYEKDIAAY RAKGKPD

Figure 14. Sry is a member of a growing family of HMG-relatedproteins which have been found to be highly conserved across species. Percent similarity and percent identity within the 79 amino acid HMG box are given with respect to Sry. Protein sequences are organized in subfamilies depending on the degree of homology and possession of characteristic amino acids at specific positions.

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BLANCHE CAPEL and ROBIN LOVELL-BADGE

DNA. In the second class are proteins containing a single HMG box, which shows considerable sequence specificity and very high affinity in its interaction with DNA. Proteins of this class also appear to be restricted to specific cell types. For example, TCFl (Oosterwegel et al., 1991; van de Wetering et al., 1991) and TCFlcllLEF-1 (Travis et a]., 1991; Waterman et al., 1991) are two transcription factors which participate in the activation of T-cell specific genes upon binding to sequences within their enhancers. Clearly SRY falls into this second class. It has an HMG box related to that of TCFI, showing 29% identity and 52% similarity and is in fact capable of binding the same DNA sequence motif in vitro (Harley et al., 1992; but see also Giese et al., 1992). Recently it has been shown that Sry induces a bend of -60"when it binds target DNA (Ferrari et al., 1992).This finding has given rise to the speculation that Sry may act to juxtapose regulatory elements to repress or activate target genes rather than to directly affect transcription itself. A variety of other genes have also been found recently that contain HMG box domains. The most notable with respect to this review are two genes involved in the mating-type switch of the fission yeast, Schizosaccharomycespombe. Mc is a protein encoded by one of the two alternativealleles of the mating-type locus, matl, of the fission yeast S. pombe (Kelly et a]., 1988). By analogy to the budding yeast, it has been suggested that Mc functions as a transcription factor. Stell is another S. pombe gene cloned more recently which appears to encode a key transcription factor for the regulation of sexual development (Sugimotoet al., 1991).While these two genes involved in sexual development of the fission yeast appear to be molecularlyrelated to Sry, a gene critical for sex determinationin higher organisms, it is likely that this binding domain has simply evolved in a number of proteins which are transcriptional regulators rather than that there is a functional,evolutionary significance in the common pathway of sexual dimorphism. While screening for cDNA clones corresponding to the mouse Sry transcript, a number of clones were obtained from a 8.5 dpc mouse library (Gubbay et al., 1990a). Sequencing revealed that these genes, while distinct from Sry, possess an HMG box showing more than 77% homology to the Sry box. Three of these genes, Sox-I, Sox-2, and Sox-3, have been studied in some detail and show dynamic patterns of expression associated mainly with the developing central nervous system (Collignon and Lovell-Badge, manuscript in preparation). Their structural similarity to Sry combined with their early fetal expression patterns, suggest that they also may be involved in specifying cell fate. This is a rapidly growing family of genes with highly homologous members in all species tested from Drosophila to man. Sry seems to be most closely related to the subfamily including Sox-1, Sox-2, and Sox-3. Sox-.? has recently been mapped to the X chromosome which makes its evolutionary relationship to Sry of considerable interest. The structure of the HMG domain will need to be determined directly, for example, by NMR. Study of the helical potentiality of the HMG box of TCF-la using the Chou-Fasman algorithm predicted that this motif can form two helixes separated by a short non-helical stretch of amino-acids (Waterman et al., 1991).

The Sry Gene and Sex Determination in Mammals

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30Tr

21-

c

Figure 15. D N A binding of protein encoded by the wild-type SRY gene (wt-SRY) i s compared to protein synthesized from the SRY genes in 6 XY females carrying point mutations and deletions within the conserved box domain. (A) Bacteriallysynthesized SRY protein from each of 6 mutant females, recognized for quantitation with rabbit a-SRY sera. (6)Gel retardation assay of the TCF-1 binding site for each protein synthesized above. Of point mutants 1-5, only 190M retains some binding activity. 190M and V60L are thought io be conditional mutations (see text). FS122 is a frameshift mutant for which protein cannot be recognized. )LA is bacterial extract without SRY protein.

Studies of mutations in the HMG box of SRY and LEF-1 in v i m have identified key amino acids necessary for overall structure or DNA binding (Berta et al., 1990; Giese et al., 1991; Harley et al., 1992). Mutations found in human XY females in vivo within the HMG box of SRY (Berta et al., 1990; Iager et al., 1990) have been shown to impair its DNA binding activity in v i m (Harley et al., 1992) (Fig. 15). This demonstrated that the switch in developmental fate controlled by SRY is achieved through specific DNA binding. However, SRY proteins from human and mouse, as well as at least some SOX proteins produced in E. coli, are able to bind

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specifically a TCFl site in an in v i m assay (Harley et al., 1992).It is possible that this may not be the physiologically important target sequence (Giese et al., 1992). Nonetheless, these results raise the question of how distinct proteins with similar DNA-binding specificity achieve precise regulation of cell-type specific target genes? In vitro studies may not reflect in vivo conditions. In particular, there may be other proteins or co-factors which can interact to increase the specificity of DNA-binding. In addition, the time and cell type of in vivo expression of a transcription factor, as well as its concentration and competition with other coincident factors in the cell, are all likely to contribute to the specificity of its DNA binding.

VIII. SUMMARY AND CONCLUSIONS With the isolation and characterization of Sry, a gene with a critical role in development has been defined. Sry can be described as a master regulatory gene in that it has a pivotal role in the sex determination pathway. However, much work must be done to discover how the gene fits into such a pathway, both at the level of molecular cascades and their integration with morphogenic events during organogenesis. First, the precise pattern of expression of Sry suggests that it must be regulated by tissue-specific transcription factors, or a unique combination of factors present within the supporting cell precursor lineage. These factors are clearly not sex-specific. Second, there are likely to be gene products that interact with the Sry protein and contribute to the specificity of its interaction with target genes. Third, the downstream target genes need to be defined. Sry must initiate (or repress) the expression of some critical genes. Sry appears to act only for a very brief period just before testis cord formation; therefore, it is not required for any long-term maintenance of gene activity. How does the expression of Sry lead to the changes in cellular properties which result in organization into testis cords? Determining the nature of the interaction of Sry with DNA, and the definition of target genes will clearly be required before the role of Sry in sex determination can be understood at the cellular and molecular levels.

ACKNOWLEDGMENTS We gratefully acknowledge the Medical Research Council for their generous support. We also thank all members of the groups in this laboratory and Peter Goodfellow’s laboratory at ICRF for their contributions. We would especially like to acknowledge the electron microscopy contributed by Liz Hirst. In addition we thank Marilyn Brennan and others for their work on the manuscript. B.C. is partially supported by the National Institutes of Health, Bethesda.

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MOLECULAR A N D GENETIC STUDIES OF H U M A N X CHROMOSOME INACTIVATION

Carolyn J . Brown and Huntington F. Willard

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Features of the Inactive X Chromosome . . . . . . . . . . . . . . . . . . . . 111. Activity of X-Linked Genes . . . . . . . . . . . . . . . . . . . . . . . . . . A . Direct Analysis of Gene Expression . . . . . . . . . . . . . . . . . . . . B . Indirect Analysis of Gene Expression . . . . . . . . . . . . . . . . . . . IV. Genes that Escape X Inactivation . . . . . . . . . . . . . . . . . . . . . . . . V. The X Inactivation Process . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. The X Inactivation Center and the XISTGene . . . . . . . . . . . . . . . . . VII. Models for X Chromosome Inactivation . . . . . . . . . . . . . . . . . . . . A . Initiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Promulgation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Note Added in Proof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References and Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Advances in Developmental Bidogy Volume 2, pages 37-72 Copyright 8 1993 by JAI Press Inc All rights of reproduction in any form reserved ISBN: 1-55938-582-0

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PREFACE X chromosome inactivation is a unique developmental event which results in the cis-limited inactivation of most genes on one of the pair of X chromosomes in mammalian females. The inactive X also becomes heterochromatic, asynchronously replicating, and differentially methylated. It is unknown if these events are involved in the actual inactivation process or are secondarily acquired traits resulting from the inactivation. Analysis of the evidence for inactivation of human X-linked genes suggests that inactivation is transcriptionally based and does not affect all X-linked genes. The genes known to escape X inactivation are found in multiple locations along the human X chromosome, but more information is required to determine if inactivationoccurs on a regional or a locus-by-locus basis. X inactivation requires the presence, in cis, of a region of the proximal long arm called the X inactivation center (XIC). Both in humans and in mice, the XZST gene is expressed from the XIC region, only from the inactive X chromosome, not the active X chromosome. This suggests that XZST is either involved in or directly affected by the process of X inactivation. The inactivation process is described in terms of three stages-initiation, promulgation, and maintenanceand models are discussed for these stages.

1. INTRODUCTION In somatic cells of mammalian females, one of the two X chromosomes becomes inactivated early in development, compensating for the dosage difference between females and males who have only one X chromosome. X chromosomeinactivation is a unique developmental regulatory event which results in the cis-limited inactivation of most of an entire chromosome-in the case of the human X over 160 million basepairs of DNA and likely over several thousand genes (Mandel et al., 1992). Lyon first hypothesized X inactivation in 1961 on the basis of the observation that X-linked genes show mosaic expression (Lyon, 1961) and on the earlier theory that the sex chromatin or Barr body, which is only observed in the nucleus of female cells (Barr and Bertram, 1949), is derived from a single heteropyknotic X chromosome (Ohno et al., 1959).The tenets of the Lyon hypothesis are: (1) that the heteropyknotic X could be either maternal or paternal in origin: and (2) that it is genetically inactive @,yon, l%l). In the time since the Lyon hypothesis was first proposed, many differences between the active and inactive X chromosomes have been described and the essential features of the hypothesis have been generally proven and widely accepted. Nonetheless the mechanism of X inactivation remains a mystery. Table 1 presents the various components of the phenomenon which remain to be understood in depth. The modest level of our current understanding notwithstanding, Table 1

39

X Chromosome Inactivation TaMe 1. Components of the X Inactivation Process Srep

Potential FactordSires Involved

Initiation

Autosanal product X inactivation center?

Promulgation

X inactivation center? Other X-linked sites? X-linked product?

Maintenance

DNA methylase CpG islands

Other X-linked sites? X inactivation center?

provides a theoretical framework within which to consider the stages of the X inactivation process and the factors potentially involved. The initial mechanism which identifies the chromosome to be inactivated must be able to identify all X chromosomes in excess of one within a cell. Then a signal must extend along the chromosome, only inactivating genes in cis, in the presence of another X chromosome in the same nucleus which is unaffected by inactivation. Once inactivation has occurred, the inactive X must be stably maintained throughout somatic tissues, although reactivation occurs as a normal part of oogenesis. The molecular and genetic dissection of this developmental process has been hindered somewhat by the absence of a tractable experimental system in which to test and examine these components separately. Generally when inactivation is studied in somatic tissues, the net outcome of initiation, promulgation, and maintenance is being observed, but presumably only those processes involved in maintenance of inactivation need still be acting. Thus it may be that many of the features of inactive X chromosomes are events that have occurred to help maintain the inactive state, secondarily to the inactivation process itself. A number of reviews have thoroughly summarized much of the original data on X inactivation, both in humans and in mice, and have extensively discussed many of the theories proposed to explain various aspects of the inactivation process (Lyon, 1972; Gartler and Andina, 1976; Gartler and Riggs, 1983; Grant and Chapman, 1988; Riggs, 1990). This review will focus largely on the human X chromosome, concentratingon recent developments and advances in the molecular and genetic analysis and understanding of X inactivation.

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CAROLYN 1. BROWN and HUNTINGTON F. WILLARD

II. FEATURES OF THE INACTIVE X CHROMOSOME While the inactiveX was first identifiedcytologicallyby its heterochromatic nature, there are many features distinguishing the active and inactive X. Many of the characteristics appear to be interrelated, and it is as yet unclear which (if any) are, in fact, causal of inactivation. As well as yielding insight into the process of inactivation, these features can be useful for identificationof the inactive X (Table 2). Even before Lyon correlated the heterochromatic sex chromatin body with the inactive X chromosome, Taylor had identified a late-replicatingX chromosome in female Chinese Hamster cells and suggested that it might be heterochromatic (Taylor, 1960). Studies of replication timing, originally by tritiated thymidine incorporation (Gilbert et al., 1962; Morishma et ai., 1962; Muldd et al., 1963; German, 1964), and later by 5-bromodeoxyuridine (BrdU) incorporation followed by fluorescence staining (Latt,1973; Willard and Latt, 1976) or antibody conjugation (Vogel et al., 1989), showed that the inactive X starts and finishes replication later than the active X (Reddy et al., 1988), following a specific order of band replication (Willard, 1977; Schwemmle et al., 1989). The basis for chromosomewide differences in replication between active and inactive X chromosomes remains unknown, but suggests an intimate relationship between the regulation of gene expression and the temporal and spatial control of chromosomal DNA replication (Willard, 1983; Holmquist, 1987; Riggs, 1990). Methylation was first proposed as a potential means of maintaining X chromosome inactivation in 1975 (Holliday and Pugh, 1975; Riggs, 1975; Sager and Kichen, 1975).It was subsequentlyshown that DNAfrom the active X chromosome is more efficient in transforming cultured cells than is DNA from the inactive X (Chapman et al., 1982; Venolia and Gartler, 1983), indicating that there is some difference in the DNA between the active and inactive X. Rodentlhuman somatic cell hybrid lines retaining the human inactive X chromosome can be treated with

Tabre 2. Properties of the Inactive X Chromosome Heterochromatinization Barr bcdy formation AsynchronousDNA replication Generally late in S phase Regional differences in timing DNA methylation differences CpG islands hypermethylated Inactive in DNA transformation assay Gene inactivity Transcriptionally repressed

X Chromosome lnactivation

41

5-azacytidine, an inhibitor of DNA methylation, to reactivate genes on the inactive X(Mohandasetal., 1981;Graves, 1982;Lesteretal., 1982;Goss, 1984)andrestore the transforming competence (Venolia et al., 1982), suggesting that DNA methylation is responsible for the epigenetic change. Methylation at specific sites has been analyzed by methylation-sensitiverestriction enzymes (Waalwijk and Flavell, 1978) and genomic sequencing (Church and Gilbert, 1984; Pfeifer et al., 1989; Pfeifer et al., 1990). These data indicated that the CG-rich (CpG) islands (Bird, 1987) associated with numerous X-linked housekeeping genes subject to X inactivation are methylated on the inactive but not the active X chromosome (Wolf et a]., 1984; Yen et al., 1984; Keith et al., 1986; Yen et al., 1986; Hansen et al., 1988; Toniolo et al., 1988; Pfeifer et al., 1989). In contrast, the CpG island associated with a gene known to escape X inactivation, MIC2, is equally unmethylated on the active and inactive X (Goodfellow et al., 1988). In the body of genes and in anonymous DNA fragments, there are usually no consistent differences in methylation (Wolf and Migeon, 1982), although at specific sites the active X can be more methylated than the inactive X (Lindsay et al., 1985; Boyd and Fraser, 1990). Methylation can also be detected at the chromosome level (Miller et al., 1982; Jagiello et al., 1987; Viegas-Pequignot et al., 1988; Prantera and Ferraro, 1990), but such analyses have yielded conflicting results. There are proteins which specifically bind methylated DNA (Zhang et al., 1986; Meehan et al., 1989) and inhibit transcription (Boyes and Bird, 1991), and which may therefore have a role in X inactivation. It has been suggested that the binding of these proteins may also be responsible for the differential accessibility of the active and inactive X chromosomes to nucleases (Antequera et al., 1989). The inactive X chromosome is generally more resistant to the action of DNase than is the active X chromosome (Kerem eta]., 1983; Sperling et al., 1985),and specific sites hypersensitive to nucleases have been identified at the CpG islands of a number of X-linked genes on the active, but not the inactive X chromosome (Riley et al., 1984; Wolf and Migeon, 1985; Yang and Caskey, 1987; Lin and Chinault, 1988). The sex chromatin body or Barr body is often seen flattened against the inner surface of the nuclear membrane (Barr and Bertram, 1949),and in sifu hybridization has shown that sequences from the inactive X tend to be compact and peripheral, while those from the active X are generally decondensed and found equally at central and peripheral locations (Dyer et al., 1989). No differences were observed between active and inactive X chromosomes in the location of scaffold attachment sites which have been associated with transcriptional regulatory sites (Beggs et a]., 1986).However, the telomeres of the inactive X chromosometend to be much more closely associated with each other than do those of the active X chromosome, suggesting that the inactive X chromosome may be a loop formed by telomere association and attachment to the nuclear membrane (Walker et al., 1991). Such a structure could explain the characteristic bends, or “inactivation-associated folds”

CAROLYN I. BROWN and HUNTINGTON F. WILLARD

42

observed in the proximal long arm of the inactive X chromosome (Flejter et al., 1984;Van Dyke et al., 1986).

111. ACTIVITY OF X-LINKED GENES There are well over 100 genes currently identified on the human X chromosome (McKusick, 1990),and there could be several thousand or more X-linked genes in total. While it is generally assumed that most X-linked genes are subject to inactivation, and that this inactivation occurs at the transcriptional level (Lyon, 1968), evidence for specific genes being subject to X inactivation is limited, and direct evidence for the transcriptional basis of X inactivation has been presented for only a very small number of genes (Graves and Gartler, 1986; Nadon et al., 1988; Brown et al., 1990). Initial autoradiographicstudies (Comings, 1966;Fujita et al., 1966)suggested a general transcriptional basis for inactivation by demonstrating that one X is primarily transcriptionally inert. However there are a growing number of genes which are being demonstratedto “escape” X inactivation and to be expressed from both active and (otherwise)inactive X chromosomes.The existence of genes which are not subject to X inactivation was first hypothesized by Lyon for genes in common between the X and Y chromosomesin the pseudoautosomalpairing region to explain the phenotypic effect of X chromosome aneuploidies (Lyon, 1962). Relatively early replicating regions on the inactive X chromosome have also been hypothesizedto contain genes being expressed from the inactive X (Schemppand Meer, 1983; Therman and Sarto, 1983). The originalhypothesisof X inactivationwas based on the observationof “patchy expression” of X-linked genes, and mosaic expression remains one of the best identifiers of genes that are subject to X inactivation.However, as outlined in Table 3, there are a number of different criteria (some stronger than others) that can be used as evidence for X-linked genes being subject to X inactivation. The criteria encompass both the direct assay of expression (of either RNA or protein) and the

Table 3. Evidence for Genes Being Subject to X Inactivation DIRECT

INDIRECT

Mosaic expression of gene product (patches) Dosage of gene product Expression in somatic cell hybrids Mosaic appearance of clinical phenotype (patches) Nonrandom inactivation from clonal selection Nonrandom inactivation in translocation carriers Variable phenotype in carriers

X Chromosome Inactivation

43

indirect monitoring of phenotype. The direct analysis of expression is less circumstantial, but requires knowledge of the gene product. For analysis at the transcrip tional level, the gene must be cloned and an appropriate tissue must be available. As any single line of evidence could be artifactual andor misleading, those genes with multiple demonstrations of inactivity are most likely to be truly subject to inactivation. A. Direct Analysis of Gene Expression

The direct analysis of gene expression by analyzing the presence of FWA transcripts from the active and inactive X chromosomes is the most compelling evidence for a gene being subject to transcriptional inactivation. Similar analyses using the protein product of a gene introduce one more level of complexity (translation),but nonetheless seem conclusive.On the other hand, conclusions from indirect analyses must be considered only tentative since they rely on phenotype, and are therefore subject to additional variabilities such as variable penetrance, expressivity, and environmental influences. Direct analyses require knowledge of the gene function and/or structure, and therefore are limited to genes which have been cloned and/or whose products are well-characterized and readily analyzed. Mosaic Expression of Gene Product

X inactivation is a stable event which is clonally heritable, such that all progeny of a given cell will have the same X chromosomes active and inactive as in the progenitorcell. Therefore,females are a mosaic of two populations of cells in which different X chromosomes are active, as outlined in Figure 1. By culturing cells in vitro,it is possible to isolate homogeneous clonal lineages in which one X is always active and the other X is always inactive. The definitive proof that a gene is subject to inactivation is to identify such clonal populations with differentX chromosomes active and show that one allele (but not both) of the gene in question (i.e., Aor a in Figure 1) is being expressed in each population.To unequivocably demonstratethat a clonal population has a specific X chromosome active or inactive requires the presence of an independentheterozygous marker whose expression is known to be subject to X inactivation. The original characterizable alleles were null alleles or electrophoretic variants (Davidson et al.. 1963); however, molecular biological studies are revealing expressed sequence variants as well (Edwards et al., 1991; Verkerk et al., 1991). Double heterozygotes have been analyzed for hypoxanthine phosphoribosyl transferase (HPRT) and glucose 6-phosphate dehydrogenase (G6PD) (Migeon, 1972), showing that both genes are subject to X inactivation. Different clonal populations of cells were isolated from females heterozygous for steroid sulphatase (STS)deficiency (X-linked icthyosis). and also heterozygous for a polymorphism of the G6PD enzyme. Both G6PD positive and negative clones (that is, clones with either X chromosome active) were found to express STS

Figurn 1. Schematic drawing of the mosaic populations that result from random X inactivation in a heterozygous female. The mosaic populations can be selected or cloned to yield nonmosaic populations for the analysis of X inactivation. 44

X Chromosome Inactivation

45

activity, despite the fact that the mutant STS allele was nonfunctional. These data demonstrated that the STS gene was expressed even when present on the inactive X chromosome (Shapiroet al., 1979). It is also possible to use null alleles of HPRT to select for HPRT and HPRT populations. Such populations have been used to show that HPRT is inactivated at the transcriptional level (Graves and Gartler, 1986). More commonly, it is not possible to identify doubly heterozygous disease carriers. In such cases, tissues or cultured cells from females heterozygous for an X-linked disease have been shown to be mosaic for the gene product, providing evidence that the gene is subject to X inactivation. These genes include pyruvate dehydrogenase (PDHA) (Brown et al., 1989); dystrophin (DMD) (Arahata et al., 1989; Hurko et al., 1989); androgen receptor (AR) (Meyer et al., 1975); alpha galactosidase (GLA) (Romeo and Migeon, 1970); phosphoribosylpyrophosphate transferase synthetase (PRPS) (Zoref et al., 1977; Yen et al., 1978);HPRT(Rosenbloom et al., 1967; Salzmann et al., 1968); G6PD (Davidson et al., 1963; Linder and Gartler, 1965);iduronate sulfatase (IDS) (Danes and Bearn, 1967;Capobianchi and Romeo, 1976;Migeon et al., 1977); cytochrome b-245 (CYBB) (Windhorst et al., 1967; Beuscher et al., 1985); ornithine transcarbamylase (OTC) (Ricciuti et al., 1976); phosphoglycerate kinase (PGKI) (Gartler et al., 1972); and phosphorylase kinase (PHK) (Migeon and Huijing, 1974). Dosage of Gene Product

Without X inactivation, females would be expected to have twice as much product from X-linked genes as do males. Early studies, which demonstrated that the products of X-linked genes such as Factor IX and G6PD were not twice as abundant in females as males, were used to support hypotheses of X inactivation (Beutler et al., 1962). Morerecently, dosage at the RNAlevel has been used to argue that the X-linked zinc finger (ZFX)and ribosomal protein S4 (RPS4X) genes escape X inactivation (Schneider-Gadicke et al., 1989; Fisher et al., 1990). Such studies must be rigidly controlled for the quantity and quality of RNA being analyzed, and are complicated by the fact that expression from the inactive X may be incomplete (Migeon et al., 1982a; Migeon et al., 1982b). For proteins which form dimers of like subunits, the lack of heterodimers in a heterozygous individual is compelling evidence that both alleles are not being expressed in the same cell. For example, no G6PD heterodimers are observed in somatic tissues of G6PD heterozygotes (Migeon, 1972),a findingthat provides additional evidence for G6PD being subject to inactivation. Gene Expression in Somatic Cell Hybrids

The isolation of rodenthuman somatic cell hybrids which retain the human inactiveX without the active X permits the assessment of inactivation for any gene

CAROLYN J. BROWN and HUNTINGTON F. WILLARD

46

expressed in the hybrid system. By using similar hybrids retaining the active X chromosome, the normal level ofexpression for the hybrid system can be identified. This method does not require allelic variants of the gene to be studied,but is limited to those genes which are expressed in the hybrids (which are generally of fibroblastoid origin). The active and inactive human X chromosome in somatic cell hybrids have been shown to retain their characteristic replication patterns (Willard, 1983) and gene methylation patterns (Wolf et al., 1984a;Wolf et al., 1984b; Heifer et al., 1990), and only rarely spontaneously reactivate inactivated genes (Kahan and DeMars, 1975;Migeon et al., 1982b).Protein product has been identified in active X- but not inactive X-containing hybrids for a number of X-linked genes, including DNA polymerase alpha (POLA) (Wang et al., 1985), PGKI, HPRT, and G 6 P D (Kahan and DeMars, 1975), thus providing evidence that these genes are subject to inactivation. Transcriptional inactivation has been shown for TIMP (Brown et al., 1990), POLA (Brown et al., 1991b), PGKI (Franco et al., 1991), and for an unidentified cDNA(Nadon et al., 1988). Protein product has been observed in both active X-and inactive X-containing hybrids for STS (Mohandas et al., 1980) and the MIC2 cell surface antigen gene (MIC2) (Goodfellow et al., 1984), and transcription in both active X- and inactive X-containing hybrids has been shown for ZFX and STS (Schneider-Gadickeet al., 1989), RPS4X (Fisher et al., 1990),MZC2 (Brown et al., 1990), and the Kallman syndrome gene (KALIG) (Franco et al., 1991). The ability of active andor inactive human X chromosomes to complement temperature-sensitive defects in mutant rodent cell lines in somatic cell hybrids has been used as evidence for the complementing gene being subject to X inactivation [in the case of multiple undefined genes (Schwartz et al., 1979)], or escaping from X inactivation [in the case of A1S9T (Brown and Willard, 1989)l.

6. Indirect Analysis of Gene Expression There are many genes on the X chromosome which have been identified because of their distinctiveX-linked inheritancepattern, but for which thegene has not been cloned and the protein is not known. There is indirect evidence that many of these genes are subject to X inactivation, although definitive proof will require better knowledge of each gene and its product. Mosaic Expression

If a gene subject to inactivation has “localized expression” (Lyon, 1962), then patches of expressing and nonexpressing tissue may be observed. The Lyon hypothesis of X inactivation was based in part on the coat color patches observed for X-linked coat color genes in mice. Macroscopically visible patches are identifiable in only a few heterozygous female carriers for X-linked human diseases such as chondrodysplasia punctata (Happle, 1979), ocular albinism (Maguire and Maumenee, 1989),retinitis pigmentosa (Falls and Cotterman, 1984),amelogenesis

X Chromosome Inactivation

47

imperfecta (Witkop, 1967), and anhidrotic ectodermal dysplasia (Passerge and Fries, 1973). ' h o populations of cells have been identified in other diseases including: hypochromic or sideroblasticanemia (Pinkerton, 1967); agammaglobulinemia (Fudenburgand Hirschilome, 1964);adrenoleukodystrophy(Migeon et al., 1981);and Alport syndrome (Kashtan et al., 1986). These areas of expressing and nonexpressing tissue are assumed to be the result of expression and nonexpression of the disease allele, therefore implying that the gene is subject to X inactivation. However, in none of these cases has it been demonstrated that the patches are, in fact, related to presence or absence of the X-linked gene product. It is possible that, in some cases, such patterns reflect phenomena unrelated to X inactivation. Nonrandom lnactivation Patterns Clonal selection in vivo. In females heterozygous for some X-linked diseases, in vivo selection apparently occurs against those cells which have the normal allele

inactivated in tissues where the gene product is required. This results in selection for a population which has only one X chromosomeactive, suggesting that the gene is not expressed (or is very poorly expressed) from the inactive X chromosome. For such selection to occur the defect must be severe and expressed while the affected tissue is still growing. For the nonrandom inactivation patterns to be detectable, sufficientamounts of the tissue must be available,and another X-linked locus must be both heterozygous and be analyzable for its inactivation status. Such nonrandom inactivation has been observed for Lesch-Nyhan syndrome (Nyhan et al., 1970), Fabry's disease (Ropers et al., 1977), incontinentiapigmenti (Wieacker et al., 1985), Wiskott-Aldrich syndrome (Gealy et al., 1980), severe combined immunodeficiencydisease (Puck et al., 1987),agammaglobulinemia(Fearon et al., 1987), and adrenoleukodystrophy[in which cells expressing the mutant allele are apparently selected for (Migeon et al., 1981)l. The presence of nonrandom inactivation can be used for carrier detection (Puck et al., 1987). Clonal selection in translocation carriers. Natural clonal selection is also proposed to account for the nonrandom X inactivation pattern observed for translocation carriers. In balanced carriers of Wautosome translocations the normal X is generally inactivated (Therman and Patau, 1974). This is attributed to selection against the imbalances that would occur if inactivation of the translocation chromosome spread to the autosome or was unable to inactivate the entire X chromosome (Mattei et al., 1983). If a translocation disrupts a gene, and the nondisrupted gene on the intact X chromosome is subject to X inactivation,then such translocation carriers will lack any expression of the normal gene and be affected by the disease. This has been observed for DMD (Jacobs et al., 1981), Hunter syndrome (Mossman et al., 1983), Factor IX and hypomanesemia [cited in (McKusick, 1990)], Aicardi syndrome (Ropers et al., 1982), Norrie disease (Ohba and Yamashita, 1986), incontinentia pigmenti (Hodgson et al., 1985), anhidrotic ec-

48

CAROLYN 1. BROWN and HUNTINGTON F. WILLARD

todermal dysplasia (Tbrleau et al., 1989), Menkes disease (Kapur et al., 1987), AarskogScott syndrome (Bawle et al., 1984). hypochromic or sideroblastic anemia (Sessaregoet al., 1983), choroideremia (Cremers et al., 1989;Siu et al., 1990). Lowe syndrome (Hodgson et al., 1986), and Goeminne syndrome (Zuffardi and Fraccaro, 1982). Thus, the presence of a balanced X autosome translocation in a female affected with an X-linked disease can be taken as indirect evidence that the disease gene is normally subject to X inactivation. Variable Phenotype in Heterozygotes

Female carriers of an X-linked disease allele may show variable or partial expression resulting from different proportions of the affected tissue having the mutant allele inactivated. Such variable expression occasionally results in a completely expressing carrier female (termed “unfortunate lyonization”) or a completely normal carrier female. This criterion for X inactivation has been observed for as many X-linked diseases as all other the criteria combined. However, the demonstration of a variable or intermediate phenotype in heterozygotes is unreliable by itself as a criterion for assessing X inactivation, as expression can be variable due to other genetic and environmental interactions. A review of the X-linked diseases listed in McKusick’s catalog (McKusick, 1990) shows 45 of the over 100 wellestablished X-linked disorders having partial expression or occasional complete expression in heterozygous females. Whether these genes are, in fact, subject to inactivation must await confirmation from direct biochemical or molecular studies following firm identification of the gene in question.

IV. GENES THAT ESCAPE X INACTIVATION As discussed in the previous sections, there is strong direct evidence for only a limited number of genes being subject to X inactivation (see Fig. 2), although there is suggestive evidence for many more. Nonetheless, at least seven genes on the humanX chromosomehave been reported toescapeX inactivation, and this number is likely to grow as more genes are identified and studied. The current definition for a gene “escaping” X inactivation is that there is detectable expression from the inactive X, although there may not be as much expression as from the active X. Expression from the inactive X may result either from the gene being refractory to the inactivation event, or from the inactivation of the gene being unstable (see Section VII on Models below). In either case, knowledge of the features common to genes escaping X inactivation will add to our understanding of the process of X inactivation. As shown in Figure 2, the genes currently known to escape X inactivation are scattered along the X chromosome. Most, but not all, are located on the X chromosome short arm, which may reflect an ascertainment bias (as more short

49

X Chromosome Inactivation Genes known to be subject to X lnacttvcrtlon

Gem% k m r n

-xk@=w@

Figure 2. Diagram of the human X chromosome, showing genes which escape inactivation (right), and genes demonstrated to be subject to inactivation (left).Only those genes for which there is direct evidence concerning their X inactivation status are shown. See text for gene names and references. DXS423 is an anonymous cDNA.

arm genes have been analyzed) or the short arm’s evolutionary history, as genes on the short arm are not X-linked in marsupials (Spencer et al., 1991; Watson et al., 1991). Many,but again not all, of the genes that escape X inactivation have a Y-linked homologous locus (MIC2, STS, KALIG, ZFX, and RPS4X). If the Y homologue is expressed, then dosage equivalence between males and females would be maintained without X inactivation (Lyon, 1962). However, for those genes where the Y homologue is not expressed, or where there is not a Y homologue, there must either be an alternative mechanism of dosage compensation, or the dosage imbalance between the sexes must be tolerated or even required. Many of the regions containing genes escaping X inactivation have been described as being “early replicating” on the inactive X chromosome (Therman et al., 1976; Schempp

50

CAROLYN 1. BROWN and HUNTINGTON F. WILLARD

and Meer, 1983), suggesting that there may be large, cytologically visible “ d e mains” of genes which escape X inactivation. Certainly the distal tip of the X short arm,including the pseudoautosomal and adjacent regions, has a clustering of genes escaping X inactivation, while the other genes described appear to be interspersed with genes subject to inactivation. Extrapolating the data to date on human genes escaping X inactivation suggests that there may, in fact, be hundreds of genes that are expressed from both active and inactive X chromosomes. Interestingly, however, the same situation does not exist in mouse. At least three genes known to escape inactivation on the human X (ZFX,RPS4X, and AZS9T) have mouse homologues that are subject to X inactivation (Adler et al., 1991; Ashworth et a]., 1991; Kay et al., 1991; Zinn et al., 1991). These data suggest that there may be significant differences in the inactivation process between humans and mouse, although it can’t be ruled out at present that at least some of these data reflect differences between the experimental systems rather than differences between the X inactivation process per se in the two species. In any case, detailed analysis of both the promoter regions and the chromosomal contexts of these genes on the mouse and human X chromosomes should be revealing.

V. THE X INACTIVATION PROCESS X inactivationis not a single irreversibleevent, but rather may be viewed as a cycle of inactivation and reactivation (see Fig. 3) during which the inactive X acquires the different features described earlier. In female germ cells, cytogenetic and biochemical analyses have shown that one X is inactive until the onset of meiosis (Epstein, 1%9; Gartler et al., 1975;Gartler et al., 1980) at which time reactivation occurs to yield two active X chromosomes (Mangiaet al., 1975). As X inactivation occurs early in development after fertilization, more detailed studies of the timing and progress of X inactivation have been carried out in the mouse (see review by Grant and Chapman, 1988). By following gene expression and X chromosome replication patterns, it has been demonstrated that both X chromosomes are active early in development (Kozak and Quinn, 1975; Chapman et al., 1978; Epstein et al., 1978; Kratzer and Gartler, 1978;Monk, 1978; Monk and Harper, 1978;Takagi, 1983). Inactivation is apparently coincident with the onset of differentiation (Sugawara et al., 1985), occurring earlier in the trophectoderm and primitive endoderm than in the primitive ectoderm (Takagi, 1974; Rastan, 1982; Takagi, 1983). Estimates of the time of inactivation based on the composition of various tissues (Nesbitt, 1971), and experimental cell disruption (Hoppe and Whitten, 1972),oradmixing(GardnerandLyon, 1971;Gardneretal.,1985)agree with these estimates. While the mouse is the best studied, similar analyses have been performed in other mammals including humans, and in general inactivation occurs sometime between the morula and late blastocyst stage (Epstein, 1983). Whereas

Single X Is: heterochrwnatic

Single X remainsactive In romatlc tlguBa

SPERM Both X s are active inactlvatlonoccurs in Reactivationof paternal X

preferentianyinacttvated

EARLY

asynchronously replicating active In DNA trarrrformatkm unmethylatedat HlF Islands

oocws

'\

FEMALE RandomX inactivation In somatic tlssues

I

LATE BLASTOCYST Reoctivatlonof Inactive X

MEIOSIS

PRIMORDIAL GERM CELLS

inactlvatlon potfern Is clonoiiy heritable in somatic tissues and subject to selection

lote replicating inactiv6 In DNA transformation methyloted ot HTF Uands

Figurp3. Cycles of X chromosome inactivationand reactivation occurring in human development. Different features of the inactive X are acquired by the X (and outlined in the rectangles) as it progresses through the different developmental stages shown in capital letters.

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CAROLYN J. BROWN and HUNTINGTON F. WILLARD

X inactivation is random in mammalian embryos, in marsupials it is generally the paternal X which is inactivated (Cooper et al., 1971; Richardson et al., 1971; Samollow et al., 1987; Riggs, 1990). Preferential inactivation of the paternal X chromosome has also been observed in rodent extraembryonic tissue (Takagi and Sasaki, 1975;Wake et al., 1976; West et al., 1977; Kratzer et al., 1983),but this is not conclusively established in human material. The “two active X ’ state and the onset of X inactivation have been mimicked in tissue culture, representing an important (if technically challenging) advance for those interested in early development.Culturesofpluripotent cells (called EK cells) can be established from mouse embryos (Evans and Kaufman, 1981), as have cultures from the undifferentiated embryonal carcinoma (EC)cells of teratocarcinomas (McBurney and Adamson, 1976). The pluripotency of these cells has been demonstrated by their ability to form chimaeras upon injection into blastocysts (Mintz and Illmensee, 1975). These cells resemble early mouse embryonic cells and can have both X chromosomesactive (Martinet al., 1978),although not all XX EC cell lines have two active X chromosomes before differentiation (McBurney and Adamson, 1976).Cultures with two active X chromosomes can be induced to differentiate in v i m , often inactivating one of the X chromosomes (Martin et al., 1978)apparently randomly (Paterno and McBurney, 1985).In this system, activity has been analyzed by ma1e:female ratios of X-linked enzymes (McBurney and Adamson, 1976;Martinet al., 1978;McBurney and Strutt, 1980),ability to mutate an X-linked allele, and DNAreplication patterns (McBurney and Adamson, 1976). Inactivation occurs only when cells differentiate, but not whenever the cells differentiate (Takagi and Martin, 1984).Such cell lines may provide an important in v i m system to study X inactivation. In diploid mammals, all X chromosomes in excess of one are subject to inactivation. However, in tetraploids there are two X chromosomes active in 92,XXXX and 92, XXYY lines, suggesting that the number of autosomal sets may influence the number of X chromosomeskept active (Carr, 1971).In triploids, the case is less clear, with 69,XXX and 69,XXY triploids having one or two (or a mosaic population of both) active X chromosomes (Jacobs et al., 1979; Willard and Breg, 1980; Maraschio et al., 1984).The activity of the X chromosomes present in triploids is stable (Migeon et al., 1979), suggesting that the variability lies in the initial inactivation event. In an attempt to define the existence of a putative autosomal locus whose presence in greater than two copies leads to the variable numbers of active X chromosomes observed in triploids, Migeon and Jacobs looked at diploid females trisomic for 18 of the 22 autosomes, but never observed variable X inactivation (Migeon and Jacobs, 1989). This study suggests either that several autosomal loci are functioningin combination,or that one of the remaining untested chromosomes (1,5, 11, or 19) is responsible. Despite these negative findings, the triploid data seem to provide an important clue for identifying the locus or loci involved in initiation of X inactivation. However, because the initiation event occurs so early in development, its analysis will likely require examination of

X Chromosome Inactivation

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preimplantation embryos, rather than study of already differentiated somatic cultures. The inactivation of an X chromosome in human and mouse somatic cells is extremely stable. In studies of clonal descendantsof single cells only one reactivant has ever been identified (Migeon et a]., 1982b). Cell lines have been treated with a number of agents to induce reactivation of the inactiveX, including hormones, cold shock, dimethyl sulfoxide,bromodeoxyuridine(Migeon et al., 1978), strong selective pressure Wigeon, 1972), infection with SV40 (Beggs et al., 1986), and treatment with 5-azacytidine (Grant and Worton, 1989). Only the latter two had any effect, apparently by disrupting DNA methylation patterns. Reactivants have also been identified after aging (Cattanach, 1974; Wareham et al., 1987; Brown and Rastan, 1988; Migeon eta]., 1988). An inactive human X chromosomein rodenthuman somatic cell hybrids retains many of the characteristics of inactive X chromosomes described earlier (Kahan and DeMars, 1975; Kahan, 1978; Migeon et al., 1978; Mohandas et al., 1980). Identification of the human inactive X in hybrids without the active X (Kahan, 1978), and, in fact, without any other human chromosomes (Willard, unpublished data), demonstratesthat maintenanceof inactivity does not require any human DNA in excess of the inactive X. Local reactivation of individual loci on the inactive X in hybrids has been observed spontaneouslyonly rarely (Kahan and DeMars, 1979, but the frequency of reactivation can be increased by treatment with 5-azacytidine (Mohandas et al., 1981; Graves, 1982; Jones et al., 1982; Schmidt et a]., 1985; Beggs et al., 1986).

VI. THE X INACTIVATION CENTER A N D THE XISTGENE The X inactivation center (XIC) is a region of the X which is required in cis for inactivation to occur (Russell, 1963). On the human X chromosome, this region has been definedcytogenetically (Therman and Patau, 1974;Therman et al., 1974; Therman et al., 1979; Mattei et al., 1981; Mattei et a]., 1982; Couturier and Dutrillaux, 1983; Mattei et al., 1983) and molecularly (Brown et a]., 1991b), to within band Xq13, by analyzing X chromosome rearrangements which are subject to inactivation. There is no convincing or substantiated evidence for more than a single XIC. There is also no evidence for a single region of the X which is required in order for the chromosome to be active, supporting models of an inactivation center rather than an activation center. The region is currently defined by two X chromosome rearrangements (Brown et al., 1991b). Thecentromeric side of the region is limited by an W14 translocation in which the der(l4) is known to be inactivated (Fig. 4A). The telomeric side of the region is defined by the breakpoint in an isodicentric chromosome which is also subject to inactivation (Fig. 4B). The human Xq13 region is also the site of an inactive X-specific fold (VanDyke et al., 1987), and is the site of Ban body

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CAROLYN J. BROWN and HUNTINGTON F. WILLARD

A

PR PHKA

X;14

Inactivated

B

t(X;14)

xw

idic(Xp)

xpi i

xqi x

DXS128

PGKI

tel

8

idic(Xp) Inactivated

figurn 4. Mappingof the human X inactivation center (XIO. (A) The X chromosome complement from an individual with an X;14 translocation is shown. One of the X;14 translocation chromosomes is inactivated, showing that the portion of the X retained (Xql3 - Xqter) must contain the XIC. (B) Similarly, the isodicentric X chromosome is subject to inactivation, so the XIC must be located within the Xpter - Xql3 material. These Xq13 breakpoints then form the proximal and distal boundaries for the XIC region shown on the right with adjacent genes. XlST is the only gene known to date to map within the XIC region, along with DXS128 an anonymous DNA marker.

condensation (Therman et al., 1974; Therman and Sarto, 1983; Therman and Susman, 1990). There is a homologous locus on the mouse X chromosome, Xic, which is necessary in cis for inactivation to occur in that species (Rastan and Robertson, 1985). This locus maps to a region homologous to Xq13 in human, containing the Pgk and A r genes (Keer et al., 1990). This is also the site in mouse of the Xce locus, alleles of which affect the choice of X chromosome to be inactivated (Cattanach et al., 1969; Cattanach, 1970; Ohno et al., 1973; Drews et al., 1974; Cattanach and Johnston, 1981;Johnston and Cattanach, 1981).It is likely, but unproven, that Xce and Xic are, in fact, the same locus.

55

X Chromosome Inactivation

PGKl

RPS4X

425 bp

XfST f 30bp

Figure 5. RT-PCR analysis of cDNA from a male and female human cell line; mouse cell line which was used to create the active and inactive X hybrids; and two independent mouse/human somatic cell hybrids retaining either the human active X chromosome or the human inactive X chromosome. The cDNAs were amplified with human specific primers for the PGKl gene (5‘-TCGGCTCCCTCGTTGACCGA-3’; 5’ AGCTGGGTTGGCACAGCTT-3’); RPS4X gene (5’-AGCGGATGGTGCGGGCATCA3’; 5’-AGCATCTGAAGCGGGTGGCA-3‘) and XIST gene (5’-lTGGGTCCTCTATCCATCTAGGTAG-3‘; 5’-GAAGTCTCAAGGCTTGAGTTAGAAG-3’)

We have recently described a gene, X I S T (Xi Specific Transcripts), which maps to theXIC region. As its acronym suggests, XZSTis only expressed from the inactive X, not the active X chromosome (Brown et al., 1991a). In karyotypically normal individuals this results in female-specific expression (as only females have an inactive X). However, in males with an inactive X (47,XXY or 49,XXXXY),XIST expression is observed, while expression is not detectable in 45,XO females. The location and unique expression pattern of the XIST gene suggest that it is either involved in, o r directly affected by, the process of X inactivation. Therefore an

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CAROLYN J.BROWN and HUNTINGTON F. WILLARD

understanding of the function and regulation of the XIST gene should elucidate events involved in X inactivation. Transcripts from the XZST gene show a high degree of alternative splicing and have been observed to be well expressed in all female tissues examined. XZST is only expressed in male testis (Salido et al., 1992), perhaps due to (or reflective of) the inactivationof the X observed during spermatogenesis(Lifschytz and Lindsley, 1972). Studies in mouse have shown the existence of a similar gene, Xisr, which is also inactive X-specific in expression, and which maps to the Xic/Xce interval (Borsani et al., 1991; Brockdorff et al., 1991). No protein product has yet been described for the XZST gene, despite isolation and analysis of over 9 kb of cDNAs (Brownet al.,unpublished data). Figure 5 shows anRT-PCR analysisin whichRNA from male, female, or mouse-human somatic cell hybrids was reverse-transcribed (RT) into cDNA which was then amplified in a polymerase chain reaction (PCR) (Saiki et al., 1988) using human specific primers for the XZSK RPS4X, and PGKl genes. The PGKl gene is subject to X inactivation, and therefore product is observed only in the human (both male and female) and active X-containing hybrid lanes. The RPS4X gene primers amplify both male and female cDNA as well as both active- and inactive-X containing hybrid cDNAs, indicating that the gene is expressed from both the active X- and inactive X-chromosomes. In contrast to both of these situations, XIST product is only observed in the female and inactive X containing hybrid lanes, demonstratingthe inactiveX-specific nature of its expression. Continuing efforts to characterize the XZS7’gene in both humans and mouse should provide understandingof a possible role for the XZSTtranscripts (or protein product) in X inactivation. However, since the XIC region (as currently defined in humans) spans over 2 million basepairs of DNA, it is conceivable that additional genes in the region are also relevant to X inactivation.

VII. MODELS FOR X CHROMOSOME INACTIVATION A complete explanation of X inactivation would include: how all X chromosomes in excess of one are inactivated with no trans inactivation of the active X chromosome; the requirement for a region in cis on the X to be inactivated; the extreme stability yet reversibility of inactivation; and the non-inactivation of certain genes on the (otherwise) inactive X chromosome. As described earlier, the inactivation process can be visualized as consistingof initiation,promulgation,and maintenance steps (Table 1). While this is a convenient way to subdivide the events, it may not be biologically precise, and the steps are so far not experimentally separable. In Figure 6 the f i s t line diagrams the steps involved in initiation of X inactivation, the second line shows the promulgation of inactivation along the length of the X, and the third line represents the maintenance of the inactive X. These steps are described below along with models for how they could be accomplished.

me autosomes produce a limited factor ' A which binds to only one X at XIC

I. INITIATION

XIC

0 2 Active X's In early embryo

A developmentally triggered signal ' acts at all unblocked XIC's

x,c[ ...

0

This results in a cis-limited spread Of Inactlvation to most, but not a11 regions of the X chromosome

illt MAINTENANCE

Loss of expression is accompanied by the secondary acquisition of the other features of X inactivation

Figure 6. Schematic representation of the processes involved in X inactivation. The eventS are divided into stages of initiation, promulgation and maintenance as described in the text.

57

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CAROLYN 1. BROWN and HUNTINGTON F. WILLARD

A. Initiation

The data from studies of X chromosome aneuploidies show that all X chrome somes in excess of one are inactivated, suggesting that initiation of inactivation entails two events, the first of which renders one X unreceptive to the second event, which would then inactivate all unmarked X chromosomes (see Fig. 6i). The presence of two active X chromosomes in triploids and tetraploids suggests that the “marking” of the active X involves some effect of the autosomal complement. This marking could be by physical changes in the DNA, such as the integration of an episomal element (Grumbach et al., 1963) or an “inversion switch” (Gartler and Riggs, 1983), or involve epigenetic changes, such as protein binding (McBurney, 1988), membrane attachment(Comings, 1967),orDNAmethylation (Riggs, 1975). In order to only bind one X chromosomeper nucleus it has been suggested that this factor “A” is either a single entity (e.g., membrane attachment site), or requires a slow initial step and a fast second “feedback” step (Gartler and Riggs, 1983; McBurney, 1988), such that only one X chromosomeis marked. It is likely that this marking of the X occurs at the XIC, the only region of the X required in cis for inactivation (Rastan and Robertson, 1985), and differential X inactivation may be due to “imprinting”at this site (Lyon andRastan, 1984). Once the single X to remain active has been marked, another factor or inactivation signal acts on any unmarked X chromosomes. XZS7’ expression could either be the result of action of the inactivation signal, or could, in fact, be the inactivation signal, or a component of it. Further characterizationof the expression and function of the XZST gene should clarify its potential role in the process of X inactivation. B. Promulgation

One of the most difficult features of X inactivation to explain is how the inactivation “signal” can be promulgated along a single X chromosome in cis without affectingthe activeX chromosome present in the same nucleus. In addition, the genes which “escape” X inactivation are scattered along the X and may represent individual loci, or regions of genes that are not subject to (or responsive to) the inactivation signal. Alternatively, these genes may be originally inactivated, and then be unable to be maintained in the inactive state. In translocations between the X and autosomes, X inactivation has been observed to spread into the translocated material (discussed in Keitges and Palmer, 1986), suggesting that there are not unique X-linked factors associated with each gene subject to inactivation. Inactivation could proceed by a locus by locus mechanism (McBurney, 1988), although the more commonly proposed mechanisms for a cis-limited spread of inactivation are chromosomal in nature. In the “way-station’’model of Gartler and Riggs (1983) there are sequences along the X chromosome which act to propagate in cis the inactivation event. Other proposed mechanisms include heterochromatization as the result of a specific compartment or nuclear envelope attachment site

X Chromosome Inactivation

59

(Dyer et al., 1985), and a “DNA reeling” concept resulting in cis-limited chrome some folding (Riggs, 1990). C. Maintenance

All the properties of the inactive X described earlier (Table 2) could aid in the maintenance of the inactive state, either singly or cooperatively (Gartler and Riggs, 1983). The increased reactivation rates observed in marsupials, extraembryonic tissues, somatic cell hybrids, and aging cells may be indicative of loss or decreased fidelity of one or more of the maintenance factors (Migeon et al., 1989). Methylation is most commonly proposed as a secondary mechanism being used to “lock in” the inactive state (Shapiro and Mohandas, 1982; Gartler et al., 1985; Migeon et al., 1989). Transcriptional activity associated with the binding of transcription factors could result in the exclusion of methyltransferases from the active X, thereby maintaining the differences between the active and inactive X (Pfeifer and Riggs, 1991). As the XZST gene is constitutively expressed, it is also possible that it is in some way required for the maintenance of the inactive state of the X chromosome.

VIII. CONCLUSIONS The inactivation of nearly an entire chromosome is a remarkable developmental event, and presents many interesting questions, including: how can one chrome some be selected to remain active, despite the presence of similar X chromosomes in the same nucleus?; how can inactivation be promulgated in cis along the entire length of the chromosome?;and why are some genes not subject to the inactivation process? The process shares features (such as heterochromatinization, methylation, and delayed replication) in common with the inactivation of other inactive genes or regions, but whether any of these is integral to the inactivation process, or are secondary events, remains to be elucidated.The same is true for the role of the XZST gene. This gene has many intriguingcharacteristics,including being expressedonly from the inactive X chromosome. However, further characterizationis required to determine if XZST is uniquely involved in, or just affected by the events of X inactivation. Another approach to understanding the mechanism of X inactivationis to identify the reason for genes escaping X inactivation. There are a growing number of genes that are expressed from the inactive X, which prompts a reassessment of the evidence for genes being subject to X inactivation. As we have described, direct evidence for genes being subject to X inactivation exists for only a limited number of well characterizedgenes. Any new genes should be thoroughly assessed for their inactivation status, as one can no longer presume that all X-linked genes are subject to inactivation. The analysis of the differences between the regulatory regions of

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CAROLYN J. BROWN and HUNTINGTON F. WILLARD

genes subject to, and escaping from, X inactivation may aid in the elucidation of the mechanisms of X inactivation. The Lyon hypothesis of X inactivation is now 30 years old. In the time since the hypothesis was first made, much evidence has been accumulated supporting the hypothesis, and many features distinguishing the active and inactive X chromosomes have been described. 'Ihe studies of X inactivation have now been taken to the molecular level with the molecular definition of the XIC region, description of the XIST gene, and cloning of genes subject to and escaping from inactivation. Hopefully the combination of molecular and cytogenetic analysis of the active and inactive X chromosomesand their genes, in combinationwith advances in the study of early development, will yield answers to the questions of X chromosome inactivation in something less than another 30 years.

NOTE ADDED IN PROOF The rapidity with which the study of X chromosome inactivation is advancing is reflected in the number of developments made in the year since this review was written. We present a brief synopsis of the advances concerning genes that escape X chromosome inactivation and the XIST gene. The number of genes described to escape X inactivation has continued to increase. Aduected approach to isolating genes escaping X inactivationby identifying human transcripts in a somatic cell hybrid retaining only the human inactive X chromosome has identified a new pseudoautosomal gene (XE7) and two genes near, but proximal to, the pseudoautosomal region ( X U 9 and XEZ13) (Ellison et al., 1992;Yen et al., 1992). The G S 1 gene is also located in this cluster of genes in Xp22.3 that escape X chromosome inactivation (Yen et al., 1992).ANT3, one of a family of ADP/ATPtranslocase genes, has also been shown to be pseudoautosomal and to escape X inactivation (Scheibel et al., 1993). This brings the total number of genes escaping X inactivationto over 10,with the largest number being clustered in and around the pseudoautosomal region. The complete human (Brown et al., 1992) and mouse (Brockdorff et al., 1992) XlSTKist genes have now been isolated and sequenced.No conservedopen reading frame was identified, suggesting that XIST does not encode a protein product. The genes contain a number of repeat regions, the most 5' of which is conserved between humans and mice. RNA fluorescence in situ hybridization localizes the XIST transcripts to a site coincident with the Barr body (Brown et al., 1992). A role for XlST in X inactivation is further strengthened by the demonstration that Xist is expressedjust prior to inactivation from the chromosomewhich is to be inactivated (Kay et al., 1993). Additional evidence supports XIST expression during spermatogenesis (McCarrey and Dilworth, 1992;Richler et al., 1992; Salido et al., 1992) and cessation of XIST expression precedes or is concurrent with X chromosome reactivation during oogenesis (McCarrey and Dilworth, 1992).

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Brown, R. M., Dahl. H. H. M., and Brown, G.K. (1989). X-chromosome localization of the functional gene for the E l a subunit of the human pyruvate dehydrogenase complex. Genomics 4174-181. Brown, S., and Rastan, S. (1988). Age-related reactivation of an X-linked gene close to the inactivation centre in the mouse. Genet. Res. 52:151-154. Capobianchi. M. R., and Romeo, G.(1976). Mosaicism forsulfoiduronatesulfatasedeficiency in carriers of Hunter’s syndrome. Experientia 3 2 4 5 W O . Cam, D. H. (197 I). Chromosome studies in selected spontaneous abortions: Polyploidy in man. J. Med. Genet. 8164-174. Cattanach, B. M. (1970). Controlling elements in the mouse X-chromosome 111. Influence upon both parts of an X divided by rearrangement. Genet. Res. J6:293-301. Cattanach, B. M. (1974). Position effect variegation in the mouse. Genet. Res. 23:291-306. Cattanach, B. M.,and Johnston, P. (1981). Evidence of nonrandom X-inactivation in the mouse. Hereditas 9 4 5 . Cattanach, B. M., Pollard, C. E., and Perez, J. N. (1969). Controlling elements in the mouse Xchromosome I . Interaction with the X-linked genes. Genet. Res. 14223-235. Chapman, V. M., Kratzer, P. G., Siracusa, L. D., Quarantillo, B. A., Evans, R., and Liskay, R. M. (1982). Evidence for DNArnodification in the maintenance of X-chromosome inactivation of adult mouse tissues. Proc. Natl. Acad. Sci. USA 795357-5361. Chapman, V. M., West, J. D., and Adler, D. A. (1978). Bimodal distribution of alpha-galactosidase activities in mouse embryos. In: GeneticMosaics and Chimaeras in Mammals (Russell, L. B., ed.), Vol. 12, pp. 227-237. Plenum Press, New York. Church, G.M., and Gilbert, W. (1984). Genomic sequencing. Proc.Natl. Acad. Sci. USA81: 199 1-1995. Comings, D. E. (1966). Uridine-5-H3 radioautography of the human sex chromatin body. J. Cell Biol. 28:437-441. Comings, D. E. (1967). The rationale for an ordered arrangement of chromatin in the interphase nucleus. Am. J. Hum. Genet. 19:44&460. Cooper, D. W., VandeBerg, J. L., Sharman. G.B., and Poole, W.E. (1971). Phosphoglycerate kinase polymorphism in kangaroos provides further evidence for paternal X inactivation. Nature New Biol. 230:155-157. Couturier, J., and Dutrillaux, B. (1983). Replication studies and demonstration of position effect in rearrangements involving the human X chromosome. In: Cytogenetics of the Mammalian X Chromosome, Part A Basic Mechanisms of X Chromosome Behavior (Sandberg, A. A., ed.), pp. 375403. Alan R. Liss, New York. Cremers, F. P.M.,van de Pol, T. J. R., Wieringa. B., Collins, F. S., Sankila, E.-M., Siu, V. M., Flintoff, W. F., Bmsman, F.,Blonden, L. A. J., and Ropers, H.-H. (1989). Chromosome jumping from the DXS165 locus allows molecular characterization of four microdeletions and a de nova chromosome W l 3 translocation associated with choroideremia. Proc. Natl. Acad. Sci. USA 86:75107514. Danes, B. S., and Beam, A. G.(1967). Hurler’s syndrome: A genetic study of clones in cell culture with particular reference to the Lyon hypothesis. J. Exp. Med. 126:50%523. Davidson, R. G., Nitowsky, H. M., and Childs, B. (1963). Demonstration of two populations of cells in the human female heterozygous for glucose 6-phosphate dehydrogenase variants. Proc. Natl. Acad. Sci. USA50:481485. Drews, U., Blecher, S. R., Owen, D. A.. and Ohno, S. (1974). Genetically directed prefeiential X-inactivation seen in mice. Cell 1:3-8. Dyer, K. A., Canfield, T. K., and Gartler, S. M. (1989). Molecular cytological differentiation of active from inactive X domains in interphase: implications for X chromosome inactivation. Cytogenet. Cell Genet. 50:116-120. Dyer, K. A., Riley, D., and Gartler, S. M. (1985). Analysis of inactive X chromosome structure by in situ nick translation. Chromosoma 92209-2 13.

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Ropers, H. H.. Zuffardi, 0..Bianchi, E., and Xepolo, L. (1982).Agenesis of corpus callosum ocular, and skeletal anomalies (X-linked dominant Aicardi’s syndrome) in a girl with balanced X/3 translocation. Hum. Genet. 61:364-368. Rosenblom, F. M., Kelley, W. N., Henderson, J. F.. and Seegmiller, J. E. (1967).Lyon hypothesis and X-linked disease. Lancet ii:305-306. Russell, L. B. (1%3). Mammalian X-chromosome action: inactivation limited in spread and in region of origin. Science 140.976-978. Sager, R., and Kichen (1975).Selective silencing of eukaryotic DNA. Science 18942&433. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn. G. T., Mullis, K. B., and Erlich, H. A. (1988).Primer-directed enzymatic amplification of DNA with thermostable DNA polymerase. Science 239487491. Salido, E. C., Yen, P. H.. Mohandas. T. K., and Shapiro, L. J. (1992).Expression of the X-inactivationassociated gene XIST during spermatogenesis. Nature Genet. 2:196199. Salzmann, J., DeMars, R., and Benke, P. (1968).Single-allele expression at an X-linked hyperuricemia locus in heterozygous human cells. Roc. Natl. Acad. Sci. USA 60:545-552. Samollow, P. B.. Ford, A. L., and VandeBerg, J. L. (1987).X-linked gene expression in the Virginia1 Opossum: Differences between the paternally derived Gpdand Pgk-Aloci. Genetics 115:185-1 95. Scheibel, K., Weiss, B., Wohrle, D., and Rappold, G.(1993).Ahuman pseudoautosomal gene, ADP/ATP translocase, escapes X-ionactivation whereas a homologue on Xq is subject to X-inactivation. Nature Genetics 3:82-87. Schempp, W., and Meer, B. (1983).Cytologic evidence for three human X chromosomal segments escaping inactivation. Hum. Genet. 63:171-174. Schmidt, M., Wolf, S. F., and Migeon, B. R. (1985). Evidence for a relationship between DNA methylation and DNA replication from studies of the 5-azacytidine-reactivatedallocyclic X chromosome. Exp. Cell Res. 258:301-310. Schneider-Gadicke, A., Beer-Romero, P.,Brown, L. G.,Nussbaum, R., and Page, D. C. (1989).The ZFX gene on the human X chromosome escapes X inactivation and is closely related to ZFY, the putative sex determinant on the Y chromosome. Cell 571247-1258. SchwartG H. E., Moser, G.C., Holmes, S., and Meiss, H. K. (1979).Assignment oftemperature-sensitive mutations of BHK cells to the X chromsome. Somat. Cell Genet. 5:217-224. Schwemmle, S., Mehnert, K., and Vogel. W. (1989).How does inactivation change timing of replication in the human X chromosome? Hum. Genet. 83:26-32. Sessarego, M., Scarra, G.B., Giuntini, P., and Ajmar, F. (1983).On the Xq13 breakpoint: clinical and cytogenetic observations in a patient with acute myelogenous leukemia. Acta Haemat. 7 0 134136. Shapiro, L. J., and Mohandas, T. (1982).DNA methylation and the control of gene expression on the human X chromosome. Cold Spring Harbor Symp. Quant. Biol. 47631637. Siu, V. M., Gonder, J. R., Jung, J. H., Sergovich, F. R., and Flintoff, W. F. (1990). Choroideremia associated with an X-autosomal translocation. Hum. Genet. 84:459464. Spencer, J. A., Sinclair, A. H., Watson, J. M., and Marshall Graves, J. A. (1991).Genes on the short arm of the human X chromosome are not shared with the marsupial X. Genomics 11:339-345. Sperling. K., Kerem B.-S., Goiten, R.. Kottusch, V., Cedar, H.,and Marcus, M. (1985). DNase I sensitivity in facultative and constitutive heterochromatin. Chromosoma 93:38-42. Sugawara, O., Takagi, N., and Sasaki, M. (1985).Correlationbetween X-chromosome inactivation and cell differentiation in female preimplantation mouse embryos. Cytogenet. Cell Genet. 3 9 2 10-2 19. Takagi, N. (1974).Differentiation of X chromosomes in early female mouse embryos. Exp. Cell Res. 86127-135. Takagi. N. (1983).Cytogenetic aspects of X-chromosome inactivation in mouse embryogenesis. In: Cytogenetics of the Mammalian X Chromosome (Sandberg, A. E., ed.), Vol. 3A, pp. 21-50. Alan R. Liss, New York,

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Wang, T. S. F., Pearson, B. E., Suomalainen, H. S., Mohandas. T., Shapiro, L. J., Schroder, J., and K m , D. (1985). Assignment of the gene for human DNA polymerase a to the X chromosome. Proc. Natl. Acad. Sci. USA82:5270-5274. Wareham, K. A., Lyon, M. F., Glenister, P. H., and Williams, E. D. (1987). Age related reactivation of an X-linked gene. Nature 327725-727. Watson, J. M., Spencer, J. A., Riggs, A. D., and Marshall Graves, J. A. (1991). Sex chromosome evolution: platypus gene mapping suggests that part of the human X chromosome was originally autosomal. Proc. Natl. Acad. Sci. USA 88:11256-11260. West, J. D., Freis, W. I., and Chapman, V. (1977). Preferential expression of the maternally derived X chromosome in the mouse yolk sac. Cell 12:87>882. Wieacker, P., Zimmer, J., and Ropers, H. H. (1985). X inactivation patterns in two syndromes with probable X-linked dominant, male lethal inheritance. Clin. Genet. 28:238-242. Willard, H. F. (1977). Tissue-specific heterogeneity in DNA replication patterns of human X chrome somes. Chromosoma 615-73. Willard, H. F. (1983). Replication of human X chromosomes in fibroblasts and somatic cell hybrids: cytogenetic analyses and a molecular perspective. In: Cytogenetics of the Mammalian X Chrome some, Part A: Basic Mechanisms of X chromosome Behavior (Sandberg, A. A,, ed.), pp. 427447. Alan R. Liss, New York. Willard, H. F.,andBreg, W. R. (1980). Human Xchromosomes: synchrony ofDNAreplicationindiploid and triploid fibroblasts with multiple active or inactive X chromosomes. Somat. Cell Mol. Genet. 6:187-198. Willard, H. F., and Latt, S. A. (1976). Analysis of deoxyribonucleic acid replication in human X chromosomes by fluorescence microscopy. Am. J. Hum. Genet. 28:213-227. Windhorst, D. B., Holmes, B., and Good, R. A. (1967). A newly defined X-linked trait in man with demonstration of the Lyon effect in carrier females. Lancet t723-729. Witkop, C. J. (1967). Partial expression of sex-linked recessive amelogenesis imperfecta in females compatible with the Lyon hypothesis. Oral Surg., Oral Meth. and Oral Path. 23:174-182. Wolf, S. F., Dintzis, S., Toniolo, D., Persico, G.,Lunnen, K. D., Axelman, J., and Migeon, B. R. (1984). Complete concordance between glucose-6-phosphate dehydrogenase activity and hypomethylation of 3’CpG clusters: implications for X chromosome dosage compensation. Nucleic Acids Res. 1219333-9348. Wolf, S. F., Jolly, D. J., Lunnen, K. D., Friedmann, T., and Migeon, B. R. (1984b). Methylation of the hypoxanthine phosphoribosyltransferase locus on the human X chromosome: implications for X-chromosome inactivation. Proc. Natl. Acad. Sci. USA 81:28062810. Wolf, S . F., and Migeon, B. R. (1982). Studies of X chromosome DNA methylation in normal human cells. Nature 292667-671. wolf, S. F., and Migeon, B. R. (1985). Cluster of CpG dinucleotides implicated by nuclease hypersensitivity as control elements of housekeeping genes. Nature 314467469. Yang, T.P., and Caskey, T. (1987). Nuclease sensitivity of the mouse HPRT gene promoter region: differential sensitivity on the active and inadive X chromosomes. Mol. Cell. Biol. 7:2994-2998. Yen, P.H., Ellison, J.. Salido, E. C., Mohandas, T., and Shapiro, L. (1992). Isolation of a new gene from the distal short arm ofthe human X chromosome that escapes X-inactivation. Hum. Molec. Genet. 1:47-52. Yen, P. H., Mohandas, T., and Shapiro, L. J. (1986). Stability of DNA methylation of the human hypoxanthine phosphoribosyltransferase gene. Somat. Cell Mol. Genet. 12:153-161. Yen, P. H..Patel, P., Chinault, A. C.. Mohandas. T., and Shapiro, L. J. (1984). Differential methylation of hypoxanthine phosphoribosyltransferasegenes on active and inactive human X chromosomes. Proc. Natl. Acad. Sci. USA81:175%1763. Yen, R. C. K., Adams, W. B., Lazar, C., and Becker, M. A. (1978). Evidence for X-linkage of human phosphoribosylpyrophosphatesynthetase. Proc. Natl. Acad. Sci. USA 75:482-485.

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Zhang, X.-Y.. Ehrlich, K. C., Wang, R. Y.-H., and Ehrlich, M. (1986). Effect of site-specific DNA methylation and mutagenesis on recognition by methylat4 DNA-binding protein from human placenta. Nucleic Acids Res. 14:8387-8397. Zinn, A. R.,Bressler, S. L., Beer-Romro, P., Adler, D. A., Chapnan, V. M..Page, D. C., and Disteche, C. M.(1991). Inactivation of the Rps4 gene on the mouse X chromosome. Genomics 11:10971101. Zoref, E.. deVries, A., and Sperling, 0.(1977). Evidence for X-linkage of phosphoribosylpyrophosphate synthetase in man. Hum. Hered. 27:73-80. Zuffardi, 0..and Fraccaro, M.(1982). Gene mapping and serendipity. The locus for torticollis, keloids, cryptorchidism and renal dysplasia (31430 McKusick) is at Xq28, distal to the G6PD locus. Hum. Genet. 62:280-281.

GENOMIC IMPRINTING IN THE REGULATION OF MAMMALIAN DEVELOPMENT

Colin L. Stewart

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Derivation of Diploid Embryos with Different Chromosomal Combinations . 111. Developmental Consequences of Eggs Containing Altered Diploid Chromosomal Combinations . . . . . . . . . . . . . . . . . IV. Analysis of Chimeric Embryos . . . . . . . . . . . . . . . . . . . . . . . . . A . Chimeras betweenParthenogeneticandWild-QpeEmbryos . . . . . . . B . Chimeras between Androgenetic and Wild-Qpe Embryos . . . . . . . . V. ES Cells in the Analysis of Imprinting . . . . . . . . . . . . . . . . . . . . . A . Parthenogenetic ES Cells . . . . . . . . . . . . . . . . . . . . . . . . . B . Androgenetic ES Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Genetic Basis of Imprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . A . X Chromosome Imprinting . . . . . . . . . . . . . . . . . . . . . . . . B. Autosomal Imprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . Insulin-Like Growth Factor I1 Gene . . . . . . . . . . . . . . . . . . . . . . VIII . Type 2 IGF Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Advance in Developmental Bidogy Volume 2. p a g e 73-118 Copyright 8 1993 by JAI Press Inc All rights of reproduction in any form reserved ISBN: 1-55938-582-0

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IX. H19Gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X. Abnormal Development of Androgenetic and Parthenogenetic Embryos and Expression of Imprinted Genes . . . . . . . . . . . . . . . . . A. Parthenogenetic Development . . . . . . . . . . . . . . . . . . . . . . . B. Androgenetic Development . . . . . . . . . . . . . . . . . . . . . . . . C. Muscle Formation in Androgenetic Teratocarcinomas . . . . . . . . . . XI. Mechanisms of Imprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Imprinting and Transgenes . . . . . . . . . . . . . . . . . . . . . . . . . B. Imprinting and Endogenous Genes . . . . . . . . . . . . . . . . . . . . XII. Imprinting in Other Species . . . . . . . . . . . . . . . . . . . . . . . . . . . XIII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Note Added in Proof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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PREFACE The genetic basis for regulating the growth and development of mammalian embryos is beginning to be understood. One remarkable aspect is that for normal development,the mammalian embryo must, as a diploid organism,receive both the maternal and paternal alleles of certain genes in order for their correct expression to occur during embryogenesis.This dependenceon the parental origins of particular genes is referred to as genomic imprinting. Diploid mouse embryos that lack either a maternal set of chromosomes (androgenotes) or paternal set (parthenotes/gynogenotes),fail to develop following implantation.Analysis of these embryos,either alone or in combination with wild-type embryos as chimeras, has provided insights into the underlying reasons for this failure. In parthenotes/gynogenotes, abnormal development is associated with loss of cells, initially from the trophoblast and subsequently from somatic lineages in the embryo proper. Thus, there is an apparent requirement for paternal chromosomes to sustain the growth of cells in certain lineages. For androgeneticembryos, lack of maternal chromosomes results in abnormal development of the embryo proper with excessive growth occurring in some tissues. This may be due to the over production of certain factors necessary for development. The molecular basis of these abnormal forms of development is only just becoming apparent. The elegant use of mice carrying chromosome translocations has been instrumental in the identification of chromosomal regions where uniparental disomy results in growth and developmental abnormalities. This, together with classical mapping, positional cloning, and mutagenesis of specific genes, has resulted in the identificationof three imprinted genes: (1) insulin-likegrowth factor 11; (2) the cation-independent mannose &phosphate receptor; and (3) an RNA molecule, H19. Their identification,together with the isolation of embryonic stem cell lines from androgenetic and parthenogenetic embryos, should lead to a better

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understanding of the mechanisms of imprinting; that is, how one allele of a gene remains silent while the other is expressed. With the identificationof other imprinted genes and characterizationof their role in embryogenesis, it should be eventually possible to determine why such a form of gene regulation evolved, and why it is essential for proper development of the mammalian embryo.

1. INTRODUCTION In sexually reproducing organisms, it has long been assumed that the genetic contribution of both parents is equal at fertilization and during subsequent development. Over the last decade this assumption has been challenged. Although the genetic contributions are equal in terms of the numbers of chromosomes and genes (with the obvious exception of the sex chromosomes), the function or expression of certain genes is not. Thus, in the same cell, while one allele may be expressed, its homolog is silent. This difference in expression of genes, depending on their parental origin, has been called imprinting. The term “imprinting” was initially used to describe the selectiveloss of paternal chromosomes from the cells of certain insects (Crouse, 1960; Crouse et al., 1971; Nur, 1990). In female mammals, it has also been used to describe the preferential inactivation of the paternal X chromosome in certain embryonic (Takagi and Sasaki, 1975) and adult tissues (Cooper et al., 1971; Richardson et al., 1971). However, it has become apparent that imprinting also occurs at some autosomal loci and that this is essential for normal embryonic development. Among vertebrates, it may be unique to mammals. This review (previous reviews are by Surani, 1986; Aronson and Solter, 1987; Solter, 1988; Shire, 1989), will cover some of the recent progress made in the analysis of imprinting in mammalian embryos. It will deal with the abnormal embryonic phenotypes in mice that arise as a consequenceof imprinting, since these may offer clues as to why imprinting occurs. The past year has witnessed the discovery of three imprinted genes. Thus, the molecular mechanisms of imprinting are now amenable to analysis, more so because embryonic stem cells have been established from androgenetic and parthenogenetic embryos. In addition, an attempt will be made to determine whether the three imprinted genes alone can account for the embryonic phenotypes associated with imprinting. Imprinting may also be of wider consequence since there is now evidence that in humans it is implicated in causing certain inherited diseases. Indeed, it has become apparent that imprinting is no longer a bizarre exception to the rules of Mendelian inheritance, but that it has a fundamental role in regulating the expression of genes necessary for the normal growth and development of mammalian embryos.

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II. DERIVATION OF DIPLOID EMBRYOS WITH DIFFERENT CHROMOSOMAL COMB1NATIONS Following certain experimental manipulations in mice, it is possible to derive eggs that have four different diploid chromosomal combinations, as illustrated in Figure 1. There are normal biparental diploids (resulting from fertilization) containing a maternal and paternal haploid set of chromosomes. Androgenetic eggs (andre genotes) are diploid, but both sets of chromosomes are paternal in origin. These androgenotescan either be male, containingX and Y sex chromosomes,or female, containing two X chromosomes. Gynogenetic (gynogenotes)and parthenogenetic (parthenotes)eggs are also diploid, with chromosomes that are maternal in origin. These are always females containing at least one and usually two X chromosomes. The difference between parthenotes and gynogenotes is that the former have never been exposed to sperm and thus, do not contain any sperm components such as cell membranes, mitochondria, flagellar proteins, or protamines. Gynogenotes are fertilized eggs which lack paternal/sperm-derived chromosomes. Derivation of eggs containing these chromosomal combinations is either by microsurgical transplantation of pronuclei and/or the activation of unfertilized ovulated eggs. In the mouse egg, approximately 10 to 12 hours after fertilization, the haploid pronuclei become visible as distinct and separate entities. The female or egg pronucleus is usually located near the subcortical zone close to the extruded second polar body, whereas the male (sperm)derived pronucleus is usually larger and can be located anywhere in the egg cytoplasm. By exposing the eggs to drugs that depolymerize the internal cytoskeleton, either (or both) pronuclei can be isolated as karyoplasts using a fine pipette and micromanipulator. This technique does not result in rupture of the cell membrane, and the pronucleus as a karyoplast is introduced back into the egg’s cytoplasm by fusion to the recipient using inactivated Sendai virus or a brief electrical pulse (elecb-ofusion)(McGrath and Solter, 1983; Barton et al., 1987; Barra and Renard, 1988). The pronuclei fuse to form a diploid zygote nucleus (such a technique is essential for producing both androgenetic and gynogenetic embryos, and is shown in Fig. 2). The current method for producing parthenotes (for a description of alternative methods, see Graham, 1974; Kaufman, 1983) in ovulated eggs is by briefly exposing them to a dilute solution of ethanol (Cuthbertson, 1983). This results in the egg completing meiosis I1 and extruding the second polar body. The majority of activated eggs will proceed through cleavage and will have a haploid chromosomal complement. Diploid parthenotes are usually derived by suppressing polar body extrusion immediately following activation so that the two pronuclei fuse. They can also be derived by suppressing karyokinesis at the first meiotic division, resulting in an egg that is tetraploid.Parthenogenetic activation is then induced and extrusion of the second polar body is allowed. The resulting egg is diploid (Kubiak et al., 1991). Both of these methods result in embryos that can be heterozygous. Homozygous diploid gynogeneticembryos are derived by enucleation of the sperm

ACTIVATION

DEVELOPMENT

l Post implantation embryos fail to develop properly. Loss of cells from the trophectoderm, subsequently, from certain somatic lineages in chimeras with wild type embryos.

PARTHENOGENETICEGG

FERTILIZED EGGS

d

E

As

with parthenogenetic embryos

P w r preimplantation development. Post implantation development abnormal with poor embryo development and, perhaps, hypertrophyfplasia of trophectoderm. Chimeras usually die during mid-late gestation. Postnatal chimeras have abnormal rib development.

?

4

Normal development.

CONTROL EGG

Figure 1. The derivation of parthenogenetic, gynogenetic, and androgenetic eggs is shown. Parthenogenetic eggs are derived by activating an unfertilized egg. Diploidy is achieved by suppressing the second polar body formation. Gynogenetic and androgenetic eggs are produced by the removal and transplantation of pronuclei so deriving eggs that either contain maternally derived pronuclei (gynogenetic) or paternally (sperm) derived pronuclei (androgenetic). Control eggs are produced by removing either pronucleus and replacing it with an identical pronucleusof the same parental sex. The developmentalfate of these reconstitutedeggs are X =development aborts; d = development to birth and viable progeny; Q = maternal pronucleus; d = paternal pronucleus.

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Figure 2. This illustrates the actual removal (a-c), transfer (d-f), and fusion (g and h) of both haploid pronuclei as an example of how nuclear transplantation is accomplished. Using inactivatedSendai virus, the last two pictures show fusion ofa binuclear karyoplast to an enucleated egg. A more detailed description can be found in Barton et al., 1987. Photos, courtesy of Jeff Mann.

pronucleusafter fertilization,followed by suppressionof the first cleavagedivision. This results in diploidization of the remaining egg pronucleus (Anderegg and Markert, 1986).

111. DEVELOPMENTAL CONSEQUENCES OF EGGS CONTAINING ALTERED DIPLOID CHROMOSOMAL COMBINATIONS Mammalian embryos, at least those of mouse and man, are acutely sensitive to any alterations in their chromosomal constitution(Epstein, 1986;Gearhart et al., 1987). In the mouse, loss of any one autosome (monosomy) results in failure to develop beyond implantation. Gain of an extra autosome (trisomy) is associated with the

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death of the embryo at various times during development, the time depending on which particular autosome is duplicated. Mice that lose an X or a Y chromosome, and are XO, are viable (Burgoyne and Baker, 1981). Triploid embryos, whether diandric (i.e., containing two paternally derived sets of chromosomes and one maternal set), or digynic (2 maternal sets, 1 paternal, can develop to the 2&25 somite stage, but the digynic embryos are often abnonnal with neural tube defects, whereas the diandric triploids are overtly normal (Kaufman and Speirs, 1987; Kaufman et al., 1989a). Tetraploids develop craniofacial abnormalities around midgestation (Kaufman and Webb, 1990), although some may occasionally d e velop to term (Snow, 1975), but it is unclear if they are viable. Here, however, we are concerned with the development of embryos containinga diploid chromosome complement. The development of both gynogenetic and parthenogeneticembryos is indistinguishable (Surani and Barton, 1983),and this indicates that the haploid pronucleus in the second polar body does not differ from the haploid pronucleus remaining in the egg in its ability to support development. But, because it is easier to derive parthenotes, they have been studied more often. Approximately 60 to 80% of gynogenetic or parthenogenetic eggs can form blastocysts, although the strain of mouse from which the eggs are derived can have a major influence on this development (Iles et al., 1975). Most parthenotes and gynogenotes implant, but post-implantationdevelopment of the embryo is usually poor with failure to develop beyond the early egg cylinder stage (Kaufman et al., 1977; Surani and Barton, 1983; McGrath and Solter, 1984; Surani et al., 1984; Pedersen et al., 1992; Tada and Takagi, 1992). There is some suggestion that delaying the onset of implantation can improve development of the embryos (Kaufman et al., 1977; S. Varmuza, personal communication).To date, there is no example of any parthenote or gynogenote developing to term. Approximately 25% of androgenotesdie within the first two cleavage divisions because they lack an X chromosome (Morris, 1968). Androgenotesthat are either XX or XY also exhibit abnormal preimplantation development (Surani et al., 1986a; Surani et al., 1990a; Mann and Stewart, 1991), and there is evidence that the maternal strain used to provide the eggs can influence the severityof abnormal development (Latham and Solter, 1991). Only about half of the eggs form blast& cysts, and they frequently require an extra day to reach the expanded blastocyst stage. Many of the blastocysts are morphologically abnormal with reduced cell number in the inner cell mass, and appear to have difficulties in hatching from the ~ompellucidu.The frequency of implantation is reduced to between 20 to 35% of transplanted embryos. The postimplantation development of androgenotes and parthenotes/gyn& genotes is dissimilar. Of those gynogenotes and parthenotes that do implant, the trophoblast is frequently abnormal with reduced cell number. Development of the embryo is also usualIy very poor. Occasionally, some individuals develop to the 20-25 somite stage with an overtly normal morphology (some contain an abnormal

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allantois), but they are surrounded by a disproportionately small trophoblast and yolk sac (Kaufman et a]., 1977; Surani et al., 1984;Pedersen et al., 1992;Tada and Takagi, 1992). The postimplantation development of androgenetic embryos is also abnormal. Of the 20 to 35% that implant, less than half the implantation sites contain recognizableembryos at day 8 of gestation. For those that do, the embryo is overtly normal although smaller. Beyond this stage, development of the embryo is abnormal and retarded, with few reaching the 6-somite stage. The development of the trophoblast appears to be normal or is disproportionately increased in size relative to the embryo (Barton et al., 1984; Kaufman et al., 1989b). A similar pattern of development has been observed in human embryos that lack all maternal chromosomes,but contain a diploid complement of paternally derived chromosomes.These are known as hydatidiform moles. The embryo is absent with only the trophoblast remaining. The majority of these are thought to arise by fertilization of an egg, lacking maternal chromosomes, by a single sperm with subsequent duplication of the sperm chromosomes. These cells have a 46XX chromosome constitution. Rarely are 46XY moles found, which presumably arise by fertilization of the egg by two sperm (Kajii and Ohara, 1977; Surti, 1987; Szulman, 1987). Triploid embryos also develop abnormally, frequently with the embryo proper being absent, or retarded and abnormal. Triploids can be either diandric or digynic. In humans, diandric triploids frequently develop as incomplete moles with poor development of the embryo proper but with hyperplasia and hypertrophy of the trophoblast. This abnormal growth of the trophoblast has not been observed in diandric triploid mouse embryos (Kaufman et al., 1989a).

IV. ANALYSIS OF CHIMERIC EMBRYOS A. Chimeras between Parthenogenetic and Wild-Type Embryos

To further understand why these embryos fail, their developmentin combination with wild-type (biparental) embryos as chimeras has been studied. The principal advantage in using chimeras is that they can often give information about the underlying defect(s); that is, whether the defect is intrinsic to the cell (e.g., overproduction or absence of a cell surface receptor, transcription factor, or cytoplasmic enzyme), or whether it is an environmental defect (e.g., the absence or overproduction of some secreted factor such as a hormone or cytokine necessary for growth and differentiation of cells). Chimeras can be made between parthenogenetic and wild-type embryos, either by aggregatingthem when at the 4-8 cell morula stage or by the injection of isolated parthenogenetic inner cell masses (ICMs) into recipient blastocysts. It has been consistently shown that in combination with cells from fertilized biparental embryos, the development and viability of the parthenogenetic cells is substantially

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Figure 3. The contribution of wild-type, androgenetic, and parthenogenetic ES cells, as well as parthenogenetic embryos to chimeras produced by either injecting the ES

cells into recipient wild-type blastocysts, or by aggregating the parthenogenetic B-cell stage embryos with wild-type embryos, is shown. All experiments were between 129/Sv ES lines, parthenogenetic embryos, and C57BL6/J wild-type embryos. The bar lines show the mean contribution of the ES celldparthenogenetic embryos to the chimerictissues, with the number of mice analyzedshown aboveeachgraph.Analysis was done using glucose phosphate isomerase 1 (Gpi 1) alleles as the marker to distinguish between the two strains used. Note the extensive contribution of the wild-type ES cells to all tissues. The contribution of the parthenogenetic embryos i s similar to that reported in previous studies, but parthenogenetic ES cells give a much greater level of contribution to all tissues, although the level of contribution to each tissue is not as high as the wild-type ES cells. The androgenetic embryos exhibited a low level of chimerism to all tissues analyzed with the least being seen in the brain.

improved. Chimeras develop to term, although about half die perinatally. Females that survive as viable adults are often chimeric in the germ line, producing offspring from oocytes derived from the parthenogenetic cells (Stevens et al., 1977; Surani et al., 1977; Stevens, 1978; Nagy et a]., 1989). The chimeras that develop to term, however, differ from those made between fertilized biparentalembryos. They are frequently smaller when born, and as adults attain a lower body weight than their wild-type siblings. Examination of the extent of chimerism in the internal organs shows a nonuniform distribution (Fig. 3). The parthenogenetic derivatives are absent from many tissues such as skeletal muscle, liver, pancreas, and spleen. Other tissues such as the brain, intestine, and heart do

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show a consistent presence of derivatives,although at levels that are not more than about 50% of the tissue and usually in the range of 10 to 25%. (Nagy et al., 1987; Otani et al., 1987; Fundele et al., 1989,1991; Paldi et al., 1989; C. L. Stewart and J. R. Mann, in preparation). By aggregating marked parthenogenetic embryos with biparental 8 cell stage embryos it has been shown that in the chimeric blastocysts, parthenogenetic derivatives were present in the trophectoderm as well as in the ICM. Within 24 to 36 hours after implantation,the parthenogenetic derivatives in the trophectoderm had virtually disappeared, leaving a trophectoderm composed almost entirely of the biparental embryo derivatives.However, the parthenogenetic cells were uniformly present in the embryonic component of the chimera (Clarke et al., 1988; Thomson and Solter, 1988, 1989). In later stages, the parthenogenetic derivatives persisted in the embryonic compartment up to and beyond the onset of organogenesis (approximately days 12-13 of gestation). In the trophectoderm and extraembryonic derivatives of the primitive endoderm, such as the parietal and visceral endoderm of the yolk sac, parthenogenetic derivatives were consistently absent or, at best, present at low levels. In the embryo itself, disappearance of the parthenogenetic cells in certain tissues (see above) appeared to begin after day 13 of gestation and this reduction continued throughout the remainder of gestation (Nagyet a]., 1987;Fundele et al., 1989,1990;Surani et al., 1990a).The conclusions were that the nonuniform distribution in chimerism was not due to the inability of the parthenogenetic cells to differentiate along certain lineages. Rather, there was a consistent selection against the parthenogenetic derivatives (or failure of them to survive), and this was more significant in some tissues than others. However, a problem with using these chimeras, where there is intermingling of cells in all tissues derived from both embryos, is that there is often competition between cells from the two different strains. This results in unbalanced chimeras, with one strain predominating over the other in its contribution (Mullen and Whitten, 1971; Schwartzberg et al., 1989). This is particularly observed with the 129/Sv strain in combination with C57BLWJ where the 129 strain predominates; and between BalWc with a variety of strains where the Balb/c component is in the minority. Thus, certain strain combinations may distort the contribution of the parthenogeneticcells. One way to avoid this problem is to make chimeras between tissues of different lineages that will not intermingle when recombined. This makes it possible to determine whether interactions necessary for normal developmentare occurring between different lineages. Parthenogenetic ICMs have been combined with both wild-type trophoblast and with wild-type primitive endoderm. Both of these extraembryonictissues do not develop properly in parthenogeneticembryos, and this may be a contributing factor to the poor development of the ICM. These combinations did, in a few instances, improve the development of the embryo to a more advanced stage ( 2 5 4 somites), but none developed to term (Barton et al., 1985; Gardner et al., 1990). Developmental arrest appeared to coincide with the time of onset of selection against the parthenogenetic cells in the aggregation chimeras, suggesting that this is a critical stage in development.

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In normal embryogenesis,the proliferation of the trophoblast in the blastocyst is dependenton the presence of an ICM. Thus, failure of the trophectodermto develop properly in the parthenotesmay have been due to the inability of the ICM to provide this stimulus (Gardner, 1971; Copp, 1978). Areciprocal set of transplantations has been performed with the wild-type ICM combined with parthenogenetic trophoblast. This also failed to restore normal development of the parthenogenetic trophoblast (Barton et al., 1985), although wild-type trophoblast did respond to parthenogeneticICM by proliferating.Additionalattemptsto restore normal development by aggregatingparthenogeneticand androgeneticembryos have also failed (Surani et al., 1986b). All of these results indicate that failure of the parthenotes to maintain a trophoblast, and their differentiatedderivatives to sustain themselvesin certain tissues after midgestation, is probably a cell autonomous defect. The fact that the presence of wild-type or androgenetic cells cannot complement the deficiency or does so only to a limited extent, suggests that for normal development to occur; paternal chromosomes must be present in the same cells as maternal chromosomes.

B. Chimeras between Androgenetic and Wild-Type Embryos Chimeras between androgenetic and wild-type embryos have also been produced. The data is less extensive than for the parthenotes, principally because androgenetic embryos have to be made by nuclear transplantation and so are more difficult to derive routinely. In some initial studies it was shown that androgenetic cells were capable of forming both trophoblast and ICM in chimeric blastocysts (Thomson and Solter, 1988, 1989). However, in postimplantationchimeras, by the onset of gastrulation, androgeneticderivatives in most chimeras were restricted to the trophectodermand its derivatives, with some contribution also being made to the visceral yolk sac endoderm (Thomson and Solter, 1988). Thus, the distribution of the androgenetic derivatives was opposite to that seen with the parthenotes and gynogenotes with loss of the androgenetic derivatives occurring in the embryo proper (Surani et al., 1988; Thomson and Solter, 1988). Recently it has been shown that these results may have been influenced by the strain combinations of embryos. By using different inbred strains, it was demonstrated that androgenetic cells can contribute both at a reasonable frequency and quite extensively to the embryo of midgestation chimeras. These chimeras have been made either by aggregating 8cell stage embryos or by the injection of the androgenetic ICM into a recipient wild-type blastocyst (Barton et al., 1991; Mann and Stewart, 1991).In chimeras produced by aggregation, the androgenetic derivatives were most frequently found in the yolk sac and trophoblast. However,about 40% also had a significantcontributionto the embryo. Those made by injection of the ICM cells showed an extensive contribution to the yolk sac and embryo, but no contribution to the trophoblast was reported.

COLIN L. STEWART

84

.+

figure 4. The skeletons from androgenetic wild-type chimeras, 11 days old. The upper skeleton which shows normal development is from a nonchimeric individual. The middle skeleton is from a chimera made between an androgenetic 129/Sv embryo aggregated with a wild-type C57BL6/J embryo. The lower skeleton is from a chimera made by injecting androgenetic 129/Sv ES cells into a C57BL6/J blastocyst. Note the abnormalities to the axial skeleton in the androgenetic chimeras and, in particular, hyperplasiahypertrophy in the costal (rib)cartilage.

In both series, a significant proportion of the chimeras were retarded or abnormal with a higher frequency of the aggregation chimeras being defective. In fact, some of the abnormal chimeras produced by aggregation only contained a detectable androgenetic contribution in the yolk sac andor trophectoderm. This suggested that their presence in these tissues, alone, could be detrimental to the normal develop ment of the embryo. In many of the abnormal embryos, the androgenetic contribution to the chimera was much less than that derived from the wild-type cells. Very few of the chimeras developed to term and the majority of these died either at, or shortly after, birth.

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The surviving chimeras all had very low levels of androgenetic contribution to the coat and internal tissues with no obvious exclusion from any tissue as was seen with the parthenogenetic chimeras (Fig. 3). Within a few days after birth, these chimeras developed severe abnormalities in their axial skeleton with scoliosis (twisting) of the spine, and most noticeably, overgrowth of the ribs due to hyperplasia and hypertrophy of the rib or costal cartillage (Fig. 4). These recent results show that androgenetic cells are pluripotent, since they can differentiate and contribute to a wide variety of tissues in the embryo and newborn mouse. In contrast to the parthenogenetic cells, androgenetic cells appear to have a dominant effect that is detrimental to survival of the wild-type cells, since in the majority of the abnormal chimeras the androgenetic derivativesare the minor component, and only those chimeras with the weakest androgenetic contribution survive to term. What is not understood is whether this is due topreferential allocation of the androgenetic cells to a particular tissue resulting in either abnormal function or development of the tissue, or due to overproduction of some factor(s) that when present in too great an amount, affects wild-type cells. Resolution of this will have to await studies performed with marked cells so that their distribution can be determined in the intact embryo.

V.

ES CELLS IN THE ANALYSIS OF IMPRINTING

Embryonic stem (ES) cells are now established as a routine method for genetically manipulating the germ line of mice (Frohman and Martin, 1989). They have superseded embryonal carcinoma (EC) cells, principally because of their genetic totipotency. They also contribute more extensively and at a higher frequency to the tissues of the chimeras. Since they are derived directly from blastocysts grown in culture, their establishment in v i m is relatively easier than for EC cells, which are derived from tumors induced in adult mice (Graham, 1977, for a review). The derivation of ES cell lines from parthenogenetic and androgenetic embryos may be of substantial use in the study of imprinting. Their main advantage is that, as a cell line, they can be maintained continuouslyin culture. They can be expanded to provide sufficient amounts of material for molecular and biochemical analysis. This is not possible when working with embryonic material of androgenetic or parthenogenetic origin, since these embryos not only die, they usually do so at an early stage of development when the number of cells and amount of available material is very small. Also, in any molecular analysis of these embryos, it would be difficult to determine whether any detectabledifferences were directly involved with imprintingor were a consequenceof the abnormal developmentof the embryo. Both androgenetic and parthenogenetic ES cell lines have been derived from the respective blastocysts (Kaufman et al., 1983;Evans et al., 1985;Mannet al., 1990). These lines are morphologically similar to, and have the same growth requirements as wild-type ES cell lines. However, it is important to determine that their estab

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lishment in culture has not in any way disrupted or altered the imprint, resulting in biological properties different from those of the embryos from which they were derived, as this would nullify their use as a source of material in any imprinting study. A. Parthenogenetic ES Cells

Parthenogenetic ES cells can produce chimeras (Evans et al., 1985). A detailed analysis of these chimeras has been made recently, and a comparison between parthenogneticES cell chimeras and parthenogenetic embryo aggregationchimeras has shown that the two differ (C. L. Stewart and J. F. Mann, in preparation). Adult chimeras derived by either aggregation of parthenogenetic embryos with wild-type (biparental) embryos or by injection of parthenogenetic ICM cells into blastocysts invariably have a low level of parthenogenetic contribution to some somatic tissues with exclusion from others (see Section IV and Fig. 3). In contrast, injection of parthenogenetic ES cells into blastocysts produced adult chimeras with an overall higher level of parthenogenetic contribution to all tissues; that is, there was no exclusion from any somatic tissue with the possible exception of the liver (Fig. 3). Some of the females were also chimeric in the germ line, demonstrating that, like their embryonic counterparts, they were totipotent. However, the somatic distribution of chimerism has shown that the parthenogenetic ES cells do not resemble their embryonic equivalents. Rather, their tissue contribution was more, but not entirely, like that of their wild-type counterparts. It is unlikely that this is due to the method used in making the chimeras, since parthenogenetic aggregation chimeras do not differ from those made by blastocyst injection (Kaufman et al., 1977; Otani et al., 1987). The tissue contribution of the parthenogenetic ES cells did differ from wild-type ES cells in that wild-type ES cells (especially of the 129/Sv strain, as were used in these experiments), when injected into C57BU/J recipient blastocysts, frequently produce unbalanced chimeras that are almost entirely derived from ES lines. No such chimeras have been observed with the parthenogenetic ES lines tested. Instead, they rarely produce chimeras with a contribution to any tissue that exceeds

50%. B. Androgenetic ES Cells

ES cell lines with a diploid karyotype have been established from androgenetic blastocysts (Mann et al., 1990). In culture they differentiate either as embryoid bodies or as monolayers into cells that are morphologicallysimilar to those derived from normal ES cells. However, when injected subcutaneously into adult mice, instead of forming a tumor consisting of many different cell types characteristic of a typical teratocarcinoma, the tumors consist largely of striated muscle and myoblasts with a few other cell types present (Fig. 5). When explanted in vitm. the

f&urp 5. (A) Histological cross section of a typical androgenetic tumor showing that it almost entirely consists of muscle. (6)An electron micrograph from the same tumor showing that most of the muscle is striated, kindly prepared by Steve Mortillo. (C) The tumor when explanted, produces in culture an extensive outgrowth of rnyoblast that fuse to form syncytial myotubes.

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tumors produce myoblasts that can be maintained as activelyproliferatingcultures, and they can be induced to fuse to form myotubes that contract rhythmically.These tumors are unusual for teratocarcinomas since striated muscle has only been observed in about In of all tumors examined and, even then, it was a minor component of the tumor (Stevens, 1982).It is of interest that these cells should form such tumors, since in parthenogenetic embryo chimeras, the skeletal muscle is one of the tissues that is strongly selected against. This may be another example of the reciprocal phenotypes seen between androgenotes and parthenotes. Injection of wild-type blastocysts with androgenetic ES cells results in chimeras that are very similar to androgenetic embryo chimeras. A relatively high frequency (-50%) of midgestational chimeras were derived, most of which were chimeric in the embryo proper. Some of these, and those examined at a later stage in gestation, were abnormal and retarded. As with the embryo chimeras, the androgenetic ES contribution was usually the minor one, again implying that androgenetic ES cell derivatives can have a dominant-acting detrimental effect on the development of biparental embryonic cells. When allowed to develop to term, most of the chimeras died perinatally. A few weak chimeras survived but they also developed the characteristicabnormalitiesin the skeletal system that were seen with androgenetic embryos (Fig. 4). Some of the weakest chimeras survived to adulthood and have been test mated, but no germ line offspring have been produced, possibly because the levels of chimerism were so low that no contributionto the germ line was made (C.L. Stewart, unpublished). Many of these adult chimeras also had one or two abnormal ribs. Regarding the usefulness of androgenetic and parthenogeneticES cell lines as a source of material for molecular analysis of imprinting, it would appear that androgenetic ES cells have retained the imprint. From the above description, androgenetic ES cells closely resemble their embryonic counterparts in their development as chimeras. Whether the entire imprint has been retained or some subtle modification has occurred, remains to be determined. In contrast, it appears that parthenogenetic ES cells have modified the imprint associated with parthenogenetic embryos from which they were derived. To what extent the imprint has been altered is uncertain, but it is sufficient that parthenogenetic ES cells are no longer excluded from certain lineages in chimeras. It is not known whether the derivation of ES cells disrupted an already present imprint or whether it intempted the process of establishing the imprint. Whatever the cause,it would appear that any molecular analysis of imprinting including the use of parthene genetic ES cells, or their derivatives, should proceed with a degree of caution.

VI. GENETIC BASIS OF IMPRINTING The genetic basis of imprinting was initially demonstrated by the developmental consequences of transplanting maternal or paternal pronuclei into fertilized or

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parthenogenetically activated eggs (Mann and Lovell-Badge, 1984; McGrath and Solter, 1984;Surani et al., 1984).These experimentsshowed that the developmental failure of androgenotes,parthenotes, and gynogenoteswas due to factors associated with the pronuclei rather than some extranuclear or cytoplasmic component. Prior to these observations,it was known that an imprintingeffect was associated with preferential inactivation of the paternal X chromosome in female marsupials and rodents. The wider implications, in terms of autosomal imprinting, were not fully appreciated, perhaps because of the association with a sex chromosome. A. X Chromosome Imprinting

In rodents, preferential inactivation of the external X chromosome occurs in the trophectodermand extraembryonic membranes of early postimplantation embryos, whereas in all other tissues, inactivation of the maternal or paternal X is random (reviewed by Lyon, 1989).Preferential inactivation of the paternal X chromosome may also occur in the extra embryonic membranes of human embryos, but the data are not so clear (Harrison, 1989; Migeon, 1990). In marsupials, inactivation of the paternal X chromosome occurs in some adult tissues (Cooper et al., 1971, 1990). With the egg laying monotremes, tissue-specific X-inactivation has been described, but it is not known whether the paternal X is preferentially inactivated (Wrigley and Graves, 1988). The extraembryonicmembranes of parthenogenetic embryos contain two maternally derived X chromosomes, and in hydatidiform moles, both X chromosomes are paternal in origin. In these cases, there is inactivation of an X chromosome (Rastan et al., 1980; Tsukahara and Kejii, 1985). Thus, it appears that preferential inactivation of the paternal X chromosome (or retention of an active maternal X chromosome) is not strictly dependent on the imprint. It is not understood why inactivation of the paternal X chromosome should be preferred in normal develop ment, although there is some evidence suggesting that the presence of an extra maternally derived X chromosome can contribute to the developmental failure of mouse embryos (Mann and Lovell-Badge, 1988; Shao and Tagaki, 1990; Tagaki, 1991). It is possible that the presence of two paternally derived X chromosomes in androgeneticembryos may alsoresult in earlierdeath at certain stages than for those with an XY constitution (Kaufman et al., 1989). B. Autosomal Imprinting

Recently, it has been shown that expression of maternal and paternal copies of some autosomes is not equivalent. These observations arose from phenotypic analysis of the consequences to embryos and adult mice that possessed a normal diploid complement of chromosomes, except that one of the 19 autosomal pairs was either exclusively maternal or paternal in origin. The individuals are referred to as uniparental (paternal or maternal) disomics for a particular chromosome pair.

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They are derived by crossing individuals with Robertsonian translocations;that is, where two acrocentric chromosomes (all mouse chromosomes are acrocentric) have joined at their centomeres to form a metacentric chromosome. In mice heterozygous for these translocations, nondisjunction can occur at meiosis, resulting in some gametes containing both copies of the particular homolog and others lacking both copies. The other 18 pairs of chromosomes segregate normally. When two of these heterozygous mice are crossed, a certain (predictable) percentage of the offspring are derived by fusion between a gamete containing both homologs of a particular chromosome and a gamete lacking both chromosomes. Such offspring will be balanced, therefore diploid, except that for the chromosome in question, both copies will be either exclusively maternal or exclusively paternal in origin. This type of inheritance can be more precisely analyzed by using mice that are known to cany a reciprocal translocation between nonhomologous chromosomes. This leads to the derivation of offspring uniparentally disomic for a particular subchromosomalregion. These mice will have paternallmaternalduplication with the correspondingpaternallmaternaldeficiency for the particular regions. By using such a genetic analysis, Cattanach and his colleagues have been able to screen most of the mouse genomefor phenotypiceffectsarising from the uniparental inheritance of particular chromosomal regions (Cattanach and Kirk, 1985; Cattanach, 1986; Cattanach and Beechey, 1990). Six to seven regions that produce a phenotype have been located to mouse chromosomes 2,7, and 17, and two other regions are found on chromosomes6 and 11. A further four regions may exist on chromosome 1, 5,9, and 14, while other chromosomes (e.g., 12 and parts 7, 10, and 18) remain to be analyzed. The distribution of these regions is illustrated in Figure 6. One of the principal effects associated with uniparental disomy is embryonic lethality at various times during development, which can be caused either by maternal deficiency/paternal duplicationor visa versa (see Figure 6). However, not all duplicatioddeficiencies result in embryonic lethality. The region in the distal part of chromosome 2 results in viable offspring. Those with maternal deficiency are square-bodied, flat-backed, and hyperkinetic, whereas paternal deficiency for this region results in mice that are flat-sided, arch-backed, and hypokinetic. Both usually die shortly after birth. Other regions can affect the growth of the newborn and be nonlethal. These are at the proximal region of chromosome 11 and the distal half of chromosome 17. All the phenotypes appear to be fully penetrant; that is, they occur independently of the strain background on which they are located. Variation in the timing of death is associated with paternal duplicationlmaternal P deficiency of distal 2 (and also with neonatal lethality associated with the P deletion which results in paternal monosomy/maternal deficiency for proximal 17) where some individuals can survive to adulthood (Cattanach and Beechey, 1990; J. Forjet, personal communication). Whether all these regions (shown in Fig. 6) represent the total number of imprinted regions in the mouse genome is not clear since they have been defined

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Figure 6. Map of the mouse autosomal genome showing the chromosomal regions, defined by the translocation breakpoints where defective complementation occurs for maternal duplication/paternaldeficiency and visa versa. These regions (dashed)are the imprinted regions. Solid line = normal complementation. Dotted line = regions of uncertainty where, perhaps, noncomplementation may exist. M = maternal duplication/paternal deficiency; P = paternal duplication/maternal deficiency for the particular chromosome. Thin line = regions still not determined. Figure is from Cattanach and Beechy, 1990.

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Figure 7. The five autosomes that contain imprinted regions which result in overt phenotypes are shown with the locations of the 3 imprinted genes, IGF II, H19 and the IGF2r/M6Pr (type 2 receptor) shown in bold. The other symbols are for genes, those that either affect growth or transcription (for which recombinant probes exist) that are known to map to these regions and, therefore, may also be imprinted. The data used to compile this figure was taken from Mouse Genorneu. Peters, M.F.W. Festing and S.D.M. Brown, Eds.), 1991, VOl. 89.

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by overt (scorable) phenotypes. It is conceivable that other regions may contain imprinted genes, with uniparental inheritancenot resulting in an obvious phenotype (Cattanach and Beechey, 1990). The imprinted regions defined by the translocation breakpoints span relatively large segments of chromosomes and will, therefore, contain a number of genes. It is unlikely that all the genes in those regions would be imprinted, since that would almost certainly result in lethality being associated with all of them. There is evidence that loci closely linked to an imprinted gene, are not themselves imprinted (see below). It is more plausible that each region contains at least one gene, and estimates for the number of imprinted genes in the mouse genome range from 10-100 (Solter, 1988; Surani et al., 1990). Where both maternal and paternal chromosomes are imprinted (e.g., distal 2 and 7, and proximal ll) , it is possible that the phenotypes associated with the regions could be due to over or absence of expression of a particular allele ( e g , the growth effects associated with proximal 11). Or, as with the distal region of chromosome7, the phenotypes (lethalityat early and midstages of gestation) may be due to different imprinted genes located in the same region. A variety of genetic and molecular techniques have been employed in identifying genes that map to these regions and have, by their parent-of-origin patterns of expression, been characterized as imprinted. These have included an, as yet, uncharacterized gene(s) that maps to the proximal region of chromosome 7, which has been disrupted by a transgene insertion, resulting in limb abnormalities. These only occur with paternal inheritance of the transgene (DeLoia and Solter, 1990).Other genes that map to these regions and are, by their pattern of expression, classified as imprinted. These genes are the Insulin-LikeGrowth Factor I1 (IGF-B), the cation-independentmannose 6-phosphate or Insulin Like Growth Factor 2 (type 2) receptor, and the H19 gene. Additional candidate genes that map to imprinted regions, especially those associated with growth control and transcription, and for which probes are available, are shown in Figure 7.

VII. INSULIN-LIKE GROWTH FACTOR II G E N E IGF II is a secreted peptide that belongs to the insulin gene family which consists of IGF I, with which it shares 70% amino acid homology, insulin (50%homology), and the more distantly related, relaxin. Apart from their structural homology, the IGF family also exhibits broadly similar effects on cells in that they are mitogenic, that is, they are required for optimal growth and proliferation in vitm, and they have insulin-like effects on muscle and adipose tissue, resulting in an increased uptake of sugar and amino acids (Humbel, 1990; Sara and Hall, 1990, for reviews). The IGF I1 gene in mice, humans, and rats (as well as other vertebrates),is closely linked to the insulin I1 gene (Bell et al., 1985; Soares et al., 1986; O’Malley and Rotwein, 1988;Rotwein and Hall, 1990). In mice, the IGFII gene maps to the distal region of chromosome 7 (thus it falls within an imprinted region, Fig. 7), in humans

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to chromosome 1lp 15.5, and in the rat to chromosome 1. The murine IGF 1 gene is not linked to IGF II and maps to chromosome 10 (Medrano et al., 1991). In all three species, the IGF I1 gene is expressed as multiple transcripts due to the use of three or four promoters (depending on the species), and different polyadenylation sites. The majority of these transcripts appear to be translated as a single 67 amino acid peptide, although other forms have been detected (Rotwein, 1991). In mice, IGFII is first expressed in the preimplantation embryo (Lee et al., 1990; Rappolee et a]., 1990) and subsequently in extraembryonic membranes of postimplantation embryos. In later stages it is found in the chorion and visceral yolk sac. Only after day 8 of gestation is it detected in the embryo proper, where it is found in the mesenchymal derivatives; for example, the heart and somites, and at later midgestational stages in the placenta, skeletal muscle, and cartilage. Similar patterns of expression have been described for IGF I1 in the rat and human fetus (Beck et al., 1987; Schofield and Tate, 1987; Brice et al., 1980; Ohlsson et al., 1989).All three species have high levels of IGF II during fetal development. However, the levels are different postnatally. In rodents, the levels following birth fall dramatically, although expression continues in the choroid plexus and leptomeninges of the brain (Lee et al., 1990), while humans have high circulating IGF I1 levels persisting into adulthood (Han and Hill, 1992). The function(s) of IGF I1 during embryogenesis is still something of a mystery. There is, however, evidence that one of its principal functions is to stimulate fetal growth, possibly by an autocrine/paracrine mechanism. This was shown by the derivation of mice whose endogenousIGF II gene had been mutated so that it could no longerproduce a functionalprotein. The mutation was effected by gene targeting using homologous recombination in ES cells which were used to introduce the mutated gene into the germ line. Heterozygous offspring inheriting the mutated allele from their fathers grew to a final size that was 60% of the normal siblings (De Chiara et al., 1990). Maternal transmission of the mutant allele had no effect on growth, and the offspring reached normal proportions. However, when normal sized males carrying the maternally inherited mutant allele were crossed with wild-type females, all of the offspring receiving the mutant allele were reduced in size (see Fig. 8 for the pedigree). This showed that IGF 11 is expressed from the paternal allele and can, therefore, be classified as an imprinted gene (De Chiara et al., 1991). The maternal allele is inactive during most of embryogenesis,although expression does eventually occur late in gestation in the choroid plexus and leptomeninges of the brain. Other, more indirect evidence has supported these observations. Embryos carrying a maternal duplicatiodpaternal deficiency for the distal part of chromosome 7 did not express IGF 11, whereas those embryos carrying a paternal duplicatiodmaternal deficiency expressed higher levels and were increased in size relative to their normal siblings (Ferguson-Smith et al., 1991).

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m a l e ch1m.r.

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Figure 8. Pedigree of the IGF I1 mutant offspring. Four generations of offspring are shown, with the phenotype of diminished growth consistently segregating with paternal transmission of the mutant allele and not with maternal transmission. Figure was kindly made available by A. Efstradiatis from De Chiara et al., 1991.

Thus, IGF I1 levels can influence the growth of embryos, although it appears that it is not an absolute requirement. Mice homozygous for the IGF I1 mutation are viable and, therefore, other factors must be regulating growth in addition to IGFII.

VIII. TYPE 2 IGF RECEPTOR ' h o receptors bind IGF 11, the IGF I receptor and the cation-independentmannose 6-phosphate receptor or type 2 IGF receptor (Czech, 1989;Nissley and Lopaczynski, 1991).The IGF 1 receptor is a transmembrane tyrosine kinase protein that is structurally very similar to the insulin receptor. IGF I1 can bind to this receptor, although at a lower affinity than IGF I. It can also bind to the insulin receptor, but with even lower affinity. Thus, the IGF I or type 1 receptor can act as a signaltransducing receptor for IGF II.In the mouse, it maps to the middle of chromosome 7 within in one of the regions which has not been fully tested for an imprinting effect, although there is a suggestion that the IGF I receptor might be maternally imprinted; that is, not expressed from the maternal allele in preimplantation stages (Rappolee et al., 1992). The other IGF I1 receptor, the type 2 receptor, binds IGF I1 with much higher affinity than the type 1 receptor. Structurally, it is very different to the type 1 receptor, consisting of a single amino acid chain with 15 repeated subunits. There is a transmembrane region but no tyrosine kinase domain (Oshima et al., 1988). The principal function of the type 2 receptor is to bind, via mannose &phosphate

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residues, some 50 lysosomal enzymes. In addition to these and IGF II,it can bind other growth factors such as proliferin, the pre-protransforming growth factor p1 or TGF p1 precursor and thyroglobulin.These ligands, when bound to the receptor are targeted to the lysosornes via the biosynthetic or endosyntheticpathways where they are incorporated either for internalization andor degradation.Whether IGF II binding to this receptor results in activation of any signal transduction mechanism in the cell is controversial (Czech, 1989), although recently there has been some indication that it can result in activation of G-proteins (Murayama et a]., 1991). The type 2 receptor gene maps to the proximal region of mouse chromosome 17, where the overlapping T hairpin (I? and ) deletions are located. These deletions result in embryonic lethality in heterozygotes if inherited maternally,with affected embryos dying in late gestation. Inheritance from the father does not result in lethality, but the tails of the progeny carrying the deletion are shortened and kinked. To explain these observations it was postulated that a locus, T-maternal effect (Tme) was located within the deletion and was expressed only from the maternal allele and not the paternal allele (Johnson, 1974, 1975; Winking and Silver, 1984; Barlow et al., 1991). Detailed mapping of this region has shown that at least four genes are located within the deletion, these being T-complex polypeptide-1 (Tcp l), plasminogen ( P l g ) superoxide dismutase-2 (Sod-2),and the IGF type 2 receptor (Barlow et al., 1991). Analysis of the expression of these four genes in heterozygous embryos at midto late gestation, that had inherited the Thp or TU2 deletion from their mothers revealed that the type 2 receptor was expressed from the maternal and not the paternal allele, whereas the other three genes were expressed from both alleles. Thus, the type 2 receptor is imprinted and also appears to be a good candidate for the postulated Tme gene (Barlow et al., 1991) (Fig. 6).

IX. H19GENE The H19 gene codes for a polyadenylated Pol I1 transcribed RNA molecule containing introns and exons. It is thought that this RNA does not code for any proteins because there are multiple.translationa1stops codons in all three reading frames in both the human and mouse transcripts. Its function is unknown, although in cells in which it is expressed, it is associated with a 28s cytoplasmic particle (Pachinis et al., 1988; Brannan et al., 1990). This RNA is one of the most abundantly transcribed RNAs in developing mouse embryos with transcripts ftrst appearing in the trophectodexm of the late blastocyst. The levels increase in abundance in the postimplantation embryo with expression occumng in the extra-embryonic ectoderm and endoderm. In midgestation embryos, transcription occurs in many tissues of mesenchymal origin such as cartillage, muscle, and heart, as well as in liver and intestine. However, it is absent in all cells of neuroectodermal origin; for example, brain, spinal cord, and dorsal root

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ganglia. At birth, the levels decline, although in adults transcription is maintained in the skeletal muscle, thymus, heart, and lung. This pattern of transcription is, except in adults, strikingly similar to that of IGF I1 (Pokier et al., 1991). The murine H19 gene has been mapped to the distal region of chromosome 7, about 75 to 100 kilobases from the IGF 11 gene. In humans, both genes are also closely linked on chromosome 1lpl5. By using interspecific mouse crosses, it has been demonstrated that H19 is only expressed from the maternal allele in adult tissues and so can be classified as an imprinted gene (Bartolomei et al., 1991).

X. ABNORMAL DEVELOPMENT OF ANDROGENETIC AND PARTHENOGENETICEMBRYOS AND EXPRESSION OF IMPRINTED GENES In previous sections, evidence has been presented that parthenogenetic and andre genetic embryos, either alone or in combination with wild-type embryos as chimeras, exhibit characteristicphenotypes associated with their abnormal development. This section will discuss whether the expression of any of the three imprinted genes can account for the abnormal phenotypes. Each imprinted gene will be considered in turn. It is, however, appreciated that some or all of the phenotypes may not necessarily be due to the activity of any one gene, but rather a combination of the genes. Furthermore, other imprinted genes remain to be identified. A. Parthenogenetic Development

Abnormal development of parthenotes is associated with the inability to form normal trophectoderm and the elimination of cells from some somatic lineages starting in midgestation embryos. In mice, maternal disomy for the distal region of chromosome 7, where both H19 and IGFII are located, results in midfetal lethality, which may coincide with the onset of loss of the parthenogenetic cells. It is unlikely that a lack of IGFII expression (the maternal allele is not expressed) is solely responsible for the parthenogenetic phenotypes since mice homozygous for IGF 11deficiency are viable and fertile (De Chiara et al., 1991). Similarly, maternal disomy for the proximal region of chromosome 17, where the type 2 receptor is located, does not result in any overt phenotype (Cattanach and Beechey, 1990). It has been suggested, however, that overexpression of H19 in transgenic embryos may result in lethality (Brunkow and Tilghman, 1991). although the lethality may have been caused by the reported expression of the transgene in tissues where it is not normally expressed. Imprinting of the H19 gene may, therefore, exist to tightly regulate its levels of expression (Bartolomei et al., 1991). However, to demonstrate this, additional work is necessary since viable adult chimeras have been produced between wild-type embryos and embryos maternally disomic for

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the distal region of chromosome 7 (Ferguson-Smith et al., 1991), which may be expressing at least twice the normal H19 levels in the chimeric tissues. B. Androgenetic Development

There are at least three phenotypes associated with the development of the androgeneticembryos: (1) retarded developmentof the embryo proper shortly after implantation with survival or, perhaps, increased growth of the trophectoderm; (2) a high incidence of abnormal development of chimeras from midgestation to birth with only the weakest chimeras surviving birth, usually with abnormal develop

Figure 9. The rib cage of 22-day-old rats, homozygous for the Gruneberg’s lethal (L) mutation showing the increase in growth of the costal rib. A wild-type sibling with normal growth is shown on the left. From Gruneberg, 1938.

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c r

o 3.8 4.1

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Figure 10. The linkage relationship of Criineberg’s lethal (L) to albino (C) and pink eye (p)on what is now called chromosome 1 of the rat. The centromere is to the right of the diagram. The figures are the recombination frequencies or distance between the loci, Gruneberg, 1939.

ment of the axial skeleton and rib cage; and (3) teratocarcinomas derived from androgenetic ES cells consisting almost entirely of skeletal muscle. In mice, there is no known mutation that results in abnormal rib development comparable to that of the chimeras. In the rat, a mutation resulting in a similar phenotype has been described, although the line carrying the mutation is unfortunately extinct (Griineberg, 1938).This mutation was a recessive lethal, which in homozygous newborns resulted in hypertrophyhyperplasia of the costal or rib cartilage, and death at about three weeks of age (Fig. 9).Some tissue and transplantation studies suggested that the defect was cell autonomous (Fell and Griineberg, 1939).The mutation was mapped to a region distal to the rat albino (tyrosinase or C) locus (Fig. 10) on chromosome 1. This chromosome shows a high degree of synteny with mouse chromosome 7,with at least 15 genes in common (Levan et al., 1991).If a locus correspondingto the rat mutation exists on mouse chromosome 7, it could be responsible for the rib phenotype associated with the androgenetic chimeras and it may, by its location, be subjected to imprinting. Uniparental paternal disomy for the proximal region of chromosome 17 results in late gestational/neonatallethality. This results in an absence of type 2 receptor gene expression late in development and is correlated with neonatal lethality, although it is unclear whether this deficiency is directly responsible for lethality. Chimeras made between embryos that have maternally inherited the Thp deletion and wild-type embryos are viable (Bennett, 1978), and viable adult mice can occasionally be derived that apparently lack type 2 receptor expression (Cattanach and Beechey, 1990;J. Forjet, personal communication). It may be that the imprint is “leaky”; for example, subjected to the influence of some “modifier” gene that influences the expression of the paternal allele (Sapienza, 1989),or under certain circumstances another unlinked gene may somehow suppress lethality (J. Forjet, personal communication). Paternal disomy for the distal region of chromosome 7 results in early embryonic lethality as well as affecting the expression of H19 and IGFII. H19 is not expressed from the paternal allele (Bartolomei et al., 1991)and, the consequences of H19 deficiency on embryo development or in adults, are unknown and must await its inactivation by gene targeting in ES cells. Paternal disomy for the distal region of chromosome 7 is thought to result in elevated IGFII levels and, in turn, may result in the increasedgrowthof theembryos

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(Ferguson-Smith, 1991). These levels may be further enhanced by an absence of the type 2 receptor which is associated with paternal disomy for chromosome 17. One of the proposed functions of the receptor is to act as a “sink” to remove excess IGF I1 (Haig and Graham, 1991).Thus, androgenetic embryos would be expressing at least twice the normal amount of IGF 11, and these may be further increased by the absence of the type 2 receptor. This, in turn, could have a number of effects, all of which might be related to the described phenotypes. Elevated IGF I1 levels may induce hypoglycemia (Daughaday, 1992), and this could account for the death of chimeras since the androgenetic derivatives are frequently the minor component in the chimeric tissues and are often only found in the extraembryonic membranes of the chimera. In addition, increased IGF I1 synthesis is associated with stimulating the differentiation of skeletal muscle and cartilage proliferation, and this may be influencing muscle formation in the teratocarcinomasand hypertrophyhyperplasia in the ribs (Vetter et al., 1986;Florini et al., 1991). However, an attempt to explain the role of IGF I1 in these processes has to consider the function of the insulin-like growth factor binding proteins (IGFBP) which are found in mice, rats, and humans (SaraandHall, 1990;Lennonet al., 1991).Theseproteinstightlybindandtransport the IGFs in circulation and can act as a “buffer”, regulating the availability of free IGF II. Their expression during development is not well described, but they might be subjected to imprinting. The human gene for one of the principal members (IGFBPI) is tightly linked to the EGF receptor locus. In the mouse, the EGF receptor gene maps to the proximal region of chromosome 11 (Fig. 7), which is a region that is associated with an imprinting effect that influences postnatal growth. C. Muscle Formation in Androgenetic Teratocarcinomas

During embryogenesis, muscle formation is associated with the expression of at least five muscle-specific regulatory genes. It might be anticipated that some of these genes could be imprinted and, in turn, affect the development of muscle in the androgenetic and parthenogenetic embryos. However, with the exception of MyoD (Davis et al., 1987), none of the genes that either regulate muscle development [e.g., M v f s (Braun et al., 1989),Myfa (Braun et al., 1990),Myd (Pinney et al., 1988), Myogenin (Wright et al., 1989)] or affect its proliferation [e.g., Ski (Sutrare et al., 1990)], are known to map to imprinted regions in the mouse. Myf.5 and 6 map to the distal region of chromosome 10 (H.H. Arnold, personal communication); Ski maps to the distal region of chromosome 4 (Goodwin et al., 1991), and myogenin is located on chromosome 1 (Olson et al., 1990); MyoD maps to the proximal region of chromosome 7, and potentially could be subjected to imprinting. Myd has not yet been fully characterized. It is unlikely, however, that imprinting of MyoD is responsible for the muscle formation in the tumors. Mice carrying chromosomal deletions that span the MyoD locus do not exhibit an abnormal muscle phenotype dependent on parental inheritance (Rinchik and Russell, 1990).In humans, the rhabdomyosarcomalocus (which

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causes malignant pediatric tumors of striated muscle origin) is linked to the MyoD locus, yet there is convincing evidence that disruption of MyoD is not the primary lesion causing these tumors (Scrable et al., 1990). In conclusion, there is, at present, insufficientevidence to directly link any of the known imprinted genes with the abnormal phenotypes, with the possible exception that IGF I1 may cause an increase in the size of chimeras paternally disomic for chromosome 7, and that the type 2 receptor may be associated with the lethal effect of the Tme locus. However, now that techniques are available to manipulate the expression of the genes in vivu,it should be possible to obtain a greater understanding of the roles that these genes play in embryogenesis and to determine whether their aberrant expression contributes to abnormal development.

XI. MECHANISMS OF IMPRINTING For imprinting to occur, certain criteria have to be met regarding the mechanism@) by which it operates. These are: (1) it has to be something that is physically associated with the cell’s nucleus; (2) it has to affect gene expression; (3) it must be stable over many rounds of DNAreplication and during cell differentiation,and (4) it has to be established and erased at some point in the germ line or during gametogenesis (Sapienza et al., 1989b). In addition to these criteria, genes that are imprinted may contain some feature; for example, a DNA sequence or “imprinting box” that specifies that they should be subjected to imprinting (De Chiara et al., 1991). A variety of mechanisms have been proposed to account for imprinting. These include DNA methylation, DNA binding proteins such as histones, variations in chromatin structureflooping, DNA rearrangements, and transvection (Tsai and Silver, 1991). One of the more plausible hypotheses is methylation, which is the additionhemoval of methyl groups to or from cytosine residues in the DNA sequence. It has attracted much interest, principally because it is an epigenetic change to DNA and alterations in levels of gene expression are often associated with changes in methylation (Cedar, 1988). High levels of methylation frequently correlate with absence of gene expression, while low levels are associated with gene expression. Because it can be imposed or erased, it may fulfill some of the requirements of, or at least serve as, an indicator of the type of modification associated with imprinting (Monk, 1990; Surani et al., 1990b). A. Imprinting and Transgenes

Prior to the discovery of endogenously imprintedgenes, a focus for a role of DNA methylation centered on an imprinting or parent-of-origin effect associated with the inheritance of transgenes in mice. The majority of transgenes showed no

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differences in methylation levels that related to parental inheritance. However, for approximately 20% of the transgenes there was a correlation between hypomethylation (also in two instances, expression) and paternal transmission, whereas maternal transmission resulted in hypermethylation (and lack of expression in the same two instances). Reversal of the methylation levels occurred by inheritance of the transgene from a parent of the appropriate sex, with this reversibility being maintained over many generations (Hadchouel et al., 1987; Reik et a]., 1987; Sapienza et al., 1987; Swain et a]., 1987). One study, in particular, has shown that the methylation levels of a transgene changed with its progression through different stages of development in the life cycle of the mouse. In primordial germ cells (PGCs) of either sex, the transgene was unmethylated, but by the time mature gametes had formed, the transgene was methylated and at a higher level in ovulated eggs than sperm. The levels of methylation of both the maternal and paternal allele of the transgene continued to change during pre- and early-postimplantation development, particularly for the paternally inherited allele. By midgestation, the methylation pattern was established such that the paternal allele was always hypomethylated relative to the maternal allele (Chaillet et al., 1991). There are some similarities between the methylation changes of the transgenes during development and with those associated with endogenous genes. Like the transgene, endogenous genes in the PGCs are relatively undermethylated. With formation of mature gametes the levels of methylation of the endogenous genes increases, with differences existing to the genes in the gametes of both sexes. Other more global differences relating to the whole genome have also been noted, with the sperm DNA being relatively more methylated than oocyte DNA (Sanford et al., 1987; Driscoll and Migeon, 1990; Monk, 1990;Howlett and Reik, 1991). The alterationsto both the transgenes and some endogeneousgenes would appear to present an attractive possibility for an association between methylation and imprinting. The relative undermethylation of genes in the PGCs might correspond to the erasure of previously inherited imprints, and preparation for the establishment of the new imprint. By the time mature gametes are formed, differences are seen that relate to the sex of the parent (i.e., the imprint is established). Changes in methylation occur during postfertilization development, but each allele or set of genes retain their parent-of-origin identity. It is, however, still unclear to what extent this model, especially when based on transgenes, really does reflect the processes associated with the imprinting of endogenous genes, since there are some inconsistencies.The number of transgenes (20%) showing methylation differences is higher than expected when compared to estimates of the number of imprinted endogenous genes which are in the range of between 10 to 100 genes or up to 10% of the genome (Solter, 1988; Surani et al., 1990).It is unlikely that the transgenes are imprinted because of their specific DNA sequences, since many different transgenes were analyzed and the majority did not exhibit any parent-of-origin effect on their levels of methylation. An alternative

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possibility is that the transgenes integratedin an imprinted region and consequently were influenced, in a cis-acting fashion, by the region. This appears to be unlikely, since in a few cases the integration sites were mapped (Hadchouel et al., 1987; J. R. Chaillet, personal communication)and, with one exception, all were located in nonimprinted regions as defined genetically (Wu et al., 1989; Sasaki et al., 1991). Furthermore, the methylation levels in the chromosomal sequences flanking the integration sites did not correlate with the changes seen in the transgene, nor did the endogenous sequences exhibit any parent specific methylation pattern in the absence of the transgene. It would appear that the pattern of methylation associated with the transgene is determined by a combination of the transgenic sequences and the genornic site into which it integrated. Yet, it is unclear why the transgenes exhibiting a parent-of-origin effect were almost always hypomethylated following paternal transmission with one exception (Sapienza et al., 1987). If hypomethylation does correlate with gene expression,then some maternally inherited transgenes would be expected to be hypomethylated since maternal inheritance of the imprint can, depending on the gene, result in either expression or repression of an imprinted gene. It is possible that these differences in methylation may be reflecting the response of cells to foreign or transposed DNA, since integrationof foreign DNAat different stages of embryogenesiscan effect its degree of methylation and expression (Jahner et al., 1982; Stewart et al., 1982). The levels of methylation and expression of transgenes can also be influenced by the choice of mouse strain (Stewart et al., 1986).Furthermore, in three independentexamples, breeding of a particular transgene onto a different strain of mice resulted in the transgene being methylated at different levels with expression of the transgene also varying, depending on the particular strain. In one case, the strain specific modification was imposed only after passage through the maternal germ line and not through the paternal germ line (Sapienza et al., 1989; Allen et al., 1990; Engler et al., 1991). Whatever the role methylation plays in imprinting, its function may s m n be determined, because an enzyme responsible for methylation (DNA methyl transferase) has been cloned (Bestor, 1990) and its gene subjected to mutagenesis by homologous recombination in ES cells (Bestor, personal communication). Thus, the effect of complete or partial absence of methylation on gene expression during embryogenesis will become evident. B. Imprinting and Endogenous Genes

The imprinted genes are located in chromosome regions that are defined by translocation breakpoints. In these segments there are a large number of genes, but it is unlikely that they are all imprinted. Instead, it appears that in each region at least one gene (or perhaps no more than two or three) rather than blocks of genes are imprinted. The evidence for this is that the tightly linked H19 and IGF I1 genes (75 kb apart, S. Tilghman,personal communication)are oppositely imprinted (i.e.,

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H19 is expressed from the maternal allele and IGF I1 from the paternal allele), and that the three genes linked to the type I1 IGF receptor gene apparently are not imprinted (Barlow et al., 1991). However, to be certain of a gene’s imprinted status, it is necessary to analyze its expression in all tissues throughout embryogenesis and in adults. Expression of an imprinted gene from either its maternal or paternal allele can vary at different stages of embryogenesis,thus restricting analysis of a gene’s expression to only one stage of development may be misleading. There is evidence that both the maternal and paternal alleles of the type 2 receptor gene are expressed in early developmental stages of the mouse, as well as in androgenetic and parthenogenetic ES cells (M. Lau and C. L. Stewart,in preparation).In later embryonic stages, expression is from the maternal allele alone (Barlow et al., 1991). Similarly, IGF I1 is expressed from both alleles in the choroid plexus of late gestation embryos and adults, whereas in earlier stages, expression is only from the paternal allele (De Chiara et al., 1991). Similaritiesexist between imprinting of autosomal genes and the inactivation in females of an X chromosome. Both processes involve one allele of a pair of genes undergoing cycles of inactivation and reactivation. Both involve a gene remaining silent in the presence of sufficient factors that allow for the expression of the gene’s other allele in the same nucleus (Pfeifer and Riggs, 1991). X chromosome inactivation is believed to occur by some influence originating from a single chromosomal locus, the X inactivation center (Xce),spreading along the chromosome which suppresses gene expression and inactivates most of the X chromosome. The molecular mechanism(s) by which this occurs are not known, although it appears that DNA methylation, while not immediately involved in the inactivation process, acts to maintain the inactivated state (Lock et al., 1987; Bartlett et al., 1991). In humans, at least seven individual genes have been identified that escape inactivation (Davies, 1991). In the mouse, the same genes are inactivated with the possible exception of the steroid sulfatase gene (STS) (Keitges et al., 1985) and, thus, the inactivation of genes is not necessarily conserved between species. In humans, the majority of these genes are expressed from both chromosomal copies, although expression from an allele located on the inactive X is at a lower level than from the allele on the active X chromosome wigeon et al., 1982). One gene in particular, Xist, which is closely linked to Xce, may be involved in X-inactivation process, and is only expressed from an inactiveXchromosome Brown et al., 1991). What prevents these genes from undergoing inactivation is not known. There is some indicationthat genes on the X chromosome are associated with closely linked cis-acting factors that determine their activation status. In one individual, the distal tip of the long arm of the X chromosomewas duplicated and translocated to the tip of the short arm at a point, distal to the steroid sulfatase gene (STS), which is one of the genes that is not inactivated. The translocated duplication from the long arm contained the glucose-fj-phosphate dehydrogenase gene (G6PD) which is normally inactivated. On the inactive X chromosome the STS gene remained active, whereas

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both of the G6PD genes were inactivated. Thus, the inactivation process that spreads from the XCE “skipped” the STS gene and still inactivatedthe translocated G6PD gene (Mohandaset al., 1987).Conceivably, similarprocesses could be acting on autosomal genes to establish their imprint, although whether each imprinted region contains an inactivation center is not known. So far, little has been reported about the molecular differences associated with the known imprinted genes. Preliminary results have indicated that the maternal and paternal alleles of the IGFII gene and type 2 receptor gene have different levels of methylation (Chaillet et al., 1991; I). Barlow, personal communication). Attempts to show that the imprinted genes do retain the imprint when moved to different chromosomal locationsand, thus, have cis-actingfeatures (the “imprinting box”) that determine the imprint, have been inconclusive (Pravtcheva et al., 1991; Lee et al., 1992; S. lilghman, personal communication).

XII. IMPRINTING IN OTHER SPECIES In other organisms, a genetic imprintingeffect has been demonstratedin connection with endosperm formation in the seeds of flowering plants. For this tissue to develop properly, it has to contain maternal and paternal genomes (Kermicle and Alleman, 1990). Among the vertebrates, imprinting appears to be uniquely associated with mammals. In fish, androgenetic and gynogenetic individuals, both of which are fertile, have been experimentally produced (Scheerer et al., 1991). In addition, natural populations of fish exist which reproduce parthenogenetically (Schultz, 1967), as do some species of lizard (Cuellar, 1971). Homozygous frogs may be produced by blocking the first cleavage of parthenogenetically activated haploid eggs (Reinschmidt et al., 1979). Among birds, parthenogenetic reproduction can occur commonly in certain breeds of turkey and, occasionally in chickens (Olsen, 1965; Harada and Buss, 1981). However, in those vertebrates where androgenetic or parthenogenetic development has been experimentally initiated, the frequency of embryos developing into viable adults is often very low. The reasons for this are unclear. It may be that the methods used to stimulate development affectviability. Alternatively,theremay beagenetic element involvedbecause the embryos, initially heterozygous, could have been rendered homozygous for mutant loci. It may, however, be that this is an indication that imprinting exists in these other vertebrates, in which case the imprint is not fully penetrant. Among the eutherian (placental)mammals, imprinting is established in mice and also rabbits (Ozil, 1990). In marsupials, imprinting has so far only been associated with the preferential inactivation of the paternal X chromosome in some female tissues (Cooper et al., 1990),and in the egg-laying monotremes, X-inactivation also occurs in some tissues, but it is not known if the paternal X chromosome(s) are preferentially inactivated.

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In humans, imprinting has been, as already mentioned, associated with the development of complete hydatidiform moles in embryos lacking all maternal chromosomes. However, it has become increasingly apparent that imprinting may be associated with a variety of diseases that have a genetic basis. The best characterized of these are the hder-WilWAngelman syndromes that have been linked to chromosome 15q 11-13. hder-Willi syndrome (PWS) is characterized by lethargy (hypotonia), failure to thrive in infancy, obesity, hyperphagia, mental retardation, endocrine abnormalities, hypogonadism, and short stature. Children with Angelman syndrome (AS) have severe mental retardation, absence of speech, inappropriate laughter, repetitive ataxic movements, large mouth and protruding tongue (Nicholls et al., 1992). Approximately half the individuals having PWS have deletions of the paternal chromosome at 15q 11-13, whereas those with AS have similar deletions, but of the maternal chromosome. Those individuals, with no detectable deletions, were either maternally disomic for the region and had PWS, or paternally disomic with AS. Thus, there is good evidence that parental monosomy or disomy correlateswith PWS or AS. Whether the syndromes arise as a consequence of opposite maternal/paternal imprinting of the same gene, or whether different but closely linked genes are imprinted, has not yet been determined (Nicholls et al., 1989,1992). The gene(s) defective in these syndromes have yet to be identified. However, probes from this region that hybridize to single mRNA transcripts have shown that the maternal and paternal genomic sequences to which they hybridize are differently methylated. These probes have also revealed that a region of synteny exists between 15q 11-13 and the proximal region of mouse chromosome 7 close to the pink-eye locus (Chailletet al.. 1991b),rather than with chromosome2 as previously reported (Reik, 1988). It is of interest that the syntenic region on mouse chromosome 7 falls within a potentially imprinted region. This should make it feasible to determinewhether imprintingof the same gene occurs in different species,although mice with chromosomal deletions in this region exhibit no parent-of-origin phenotypes (Rinchik and Russell, 1990; E. Rinchik, personal communication). Another example where uniparental disomy may be associated with a disease phenotype is Beckwith-Wiedemann syndrome (BWS). This is characterized by gigantism, excess growth of certain tissues, macroglossia (large tongue), and exonphalos (extrusion of the liver and gut). About 10% of affected individuals develop Wilm’s tumor, hepatoblastoma, and rhabdomyosarcomas. BWS is frequently associated with trisomy 1lp, particularly p15.5, the region to which it has been mapped (Koufos et al., 1989). It also correlates with a high incidence of paternal isodisomy; that is, loss of the maternal allele llp15.5 (Henry et al., 1991). This region is syntenic with the distal region of mouse chromosome 7 where IGF I1 and H19 are located. It has been suggested that BWS could be associated with imprinting of both IGF I1 and HI9 in humans, similar to that observed in mice with paternal disomy to chromosome 7. In both humans and mice, this may result in over expression of IGF I1 and absence or under expression of H19 (Little et al.,

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1991),although there is no direct evidence for increased IGFII levels in individuals with BWS (Schofield and Engstrom, 1992). Imprinting may also be associated with the development of other human disease phenotypes. There is a high incidence of loss of heterozygosity with retention of the paternal chromosome associated with Wilm’s tumor, rhabdomyosarcoma,and osteosarcomas (Sapienza, 1991). However, this association may be due to a higher frequency of mutations occurring in the male germ line with a subsequent loss of heterozygosity, resulting in individuals or cells, homozygous for the mutation. Thus, with these tumors, their developmentmay not necessarily be associated with imprinting (Ponder, 1989; Driscoll and Migeon, 1990; Pelletier et al., 1991). For other conditions, there is a correlation between the severity of the disease andor its age of onset and parental transmission.The best example is Huntington’s chorea, which is an autosomal dominant disorder, where juvenile onset is mostly (900/0) associated with paternal inheritance (Reik, 1988; Ridley et al., 1988). Models have been proposed to account for how the disease arises (Solter, 1988; Laird, 1990), yet despite intensive efforts, the identity of the gene(s) responsible is still elusive. Other diseases have exhibited parent-of-origin effects related to the onset and severity of the disease. These include spino cerebellar ataxia (Harding, 1981) and myotonic dystrophy. Myotonic dystrophy maps to human chromosome 19, in a region which shows synteny with the proximal region of mouse chromosome 7 (Saunders and Seldin, 1990). A more extensive list of human diseases that may show a parent-of-origin effect can be found in a review by Hall (1990).

XIII. CONCLUSIONS Genomic imprinting is now a recognized phenomenon having a profound role in the development of mammalian embryos. There is an absolute requirement that paternally and maternally derived chromosomes must be present together in the same cell for embryos to develop properly. To be diploid, with either only maternal or paternal chromosomes, is insufficient. Paternal chromosomes appear to be necessary for normal development of the trophoblast and to sustain certain somatic cell lineages in later stages of postimplantation development. It is not understood what determines the loss of cells in parthenogenetic embryos, but there is a loose correlation with the onset of tissue differentiation. Maternally derived chromosomes are essential since androgenetic embryos also die, although their abnormal development is different to that seen in the parthenogenetic embryos. Analysis of the development of androgenetic embryos or chimeras has suggested that abnormal development may be due to overgrowth of certain tissues such as the trophectoderm, as seen in hydatidiform mole and the costal rib cartilage in chimeras. This may be due to the overproduction of growth factors, such as IGF 11, or a lack of factors required to constrain growth, or both.

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Indeed, it is remarkable, as Cattanach has already commented, that so many of the examples of imprinting appear to involve growth (Cattanach and Beechey, 1990). If imprinting has evolved as a means to regulate embryonic growth, then it is necessary to determinewhy it should apparently be restricted, among vertebrates, to mammals. One suggestion is that, as a consequenceof the evolution of a placenta to support viviparous growth of the embryo, imprinting evolved as a mechanism to conserve maternal resources (Haig and Graham, 1991; Moore and Haig, 1991). In the egg-laying vertebrates,imprinting is not necessary because the developingembryo is dependent on a fixed amount of nutrients (the yolk) that are laid down during egg formation. If this is the principal reason, then imprinting may have evolved independently in those other organisms where embryo development relies on a continuous supply of nutrients from the mother; for example, the viviparous species of cockroach,Diplopterupunctatu, reproduces by secreting nutrients into the uterus that are then absorbed by the embryos (Stay and Coop, 1974), or in the viviparous fish, Anableps anableps, in which development is dependent on a placental structure (nrner, 1947). It would also be of interest to determine whether imprinting may exist in the egg-laying monotremes. Alternative proposals for the function(s) of imprinting have centered on its role in preventing parthenogenesis,thus reinforcing the longer term evolutionarybenefits of sexual reproduction. It is difficult to understand why such a mechanism should have evolved only in mammals and not in other sexually reproducing species. Besides, unisexual “species” are rare among the nonmammalian vertebrates in which there is no evidence for the existence of imprinting and, thus, other powerful means exist to select against parthenogenesis (Vrijenhoeck, 1989). A full understanding of why imprinting occurs, and the biochemical mechanisms by which it functions, will probably only become apparent with the identification and characterization of other imprinted genes. With the experimental techniques currently available, this is a feasible objective for the not too distant future.

NOTE ADDED IN PROOF Recently afourth imprinted gene has been described (Leff et al., 1992; Oqelik et al., 1992). It is a small nuclear ribonucleoprotein (snRNp)-associatedpolypeptide that is not expressed from the maternal chromosome. In the mouse it maps to chromosome 7 in a region syntenic with human chromosome 15q 11-13 and has therefore been proposed as a candidate gene for Prader-Willi syndrome.

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ACKNOWLEDGMENTS In writing this review, I would like to thank all my colleagues and friends for communicating to me their results prior to publication. In particular, I would like to thank Paul Schofield for many discussions, Matt Kaufman for clarification on some points, as well as Chris Graham and David Haig for inspiration. I am also very grateful to Alisoun Carey for her editorial skills in reading successive drafts, Susan Abbondanzo for helping in the preparation of some of the figures, and Sharon Perry for her endless patience in preparing the manuscript. Lastly, I wish to acknowledge my friend and colleague, Jeff Mann, without whom much of what is described here would not have been possible.

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CELL INTERACTIONS IN NEURAL CREST CELL MIGRATION

Marian ne Bronner-Fraser

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Analysis of Neural Crest Migratory Pathways Using Cell Marking Techniques . . . . . . . . . . . . . . . . . . . 111. Extracellular Matrix Molecules Along Neural Crest Pathways . . . . . . . . IV. The Neural Crest Cell Surface . . . . . . . . . . . . . . . . . . . . . . . . . V. Cell Adhesion Molecules on Neural Crest Cells . . . . . . . . . . . . . . . . VI. Cell-Matrix Interactions in Neural Crest Migration . . . . . . . . . . . . . . A . Tissue Culture Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . B . In Kvo Perturbation Analysis . . . . . . . . . . . . . . . . . . . . . . . VII . Role of Surrounding Tissues in Determining the Pattern of Neural Crest Migration . . . . . . . . . . . . . . . . . . . . . . . A . Segmental Information within the Somites . . . . . . . . . . . . . . . . B . Inhibitory Effects of the Notochord . . . . . . . . . . . . . . . . . . . . C . Dorsoventral Patterning of Neural Crest Derivatives by the Neural Tube . . . . . . . . . . . . . . . . . . . . . . VIII. Summary and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Advances in Developmental Bidogy Volume 2, pages 119-152 Copyright 8 1993 by JAI Press Inc All rights of reproduction in any form reserved ISBN:1-55938582-0

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PREFACE During their migration, neural crest cells interact with extracellular matrix components and numerous tissues. Because of the intimate relationship between neural crest cells and the surrounding matrix, it has been proposed that the extracellular matrix plays an important role in the initiation, guidance, and cessation of neural crest cell movement. During migration, neural crest cells themselves synthesize some matrix components and also produce proteolytic enzymes that act on matrix molecules. Thus, not only do the cells interact with the matrix but they also may contribute to and modify the matrix through which they move. In addition, neural crest cells possess cell surface molecules which may be used to contact and/or interact with numerous tissues including the neural tube, somites, and notochord. These tissues also may have an important function in determining the pattern of neural crest migration. We have been examining the importance of interactions between the neural crest cells, the extracellular matrix, and other tissues during their migration. Because it is not yet possible to directly monitor cell-ECM interactions in the embryo, our studies utilize migration and adhesion assays in v i m and transplantation and perturbation analyses in vivo to characterize cell-matrix and cell-tissue interactions. Our results suggest that neural crest cells possess several integrin receptors which are utilized for adhesion to numerous matrix molecules including laminin, fibronectin, and collagens. In addition to integrin-mediated interactions, other cellmatrix or cell-cell interactions may influence the pattern of neural crest migration. For example, inhibitory cues, such as those produced by the notochord and the posterior half of the somite, may play an important role in the patterning of neural crest cells along trunk neural crest migratory pathways. Our results suggest that neural crest cells possess several adhesion mechanisms which are regionally distinct and involved in multivalent adhesive interactions.

1. INTRODUCTION The formation of the embryo involves intricate cell movements, cell proliferation, and differentiation.The neural crest serves as a good model for the study of these processes because neural crest cells undergo extensive migrations and give rise to many diverse derivatives. During development, these cells move along characteristic pathways and form numerous derivatives, ranging from pigment cells and cranial cartilage to adrenal chromaffin cells and the ganglia of the peripheral nervous system. Furthermore, they are accessible to surgical, immunological, and biochemical manipulations during both initial and certain later stages in their development. In the avian embryo, neural crest cells arise in the dorsal neural tube shortly after the neural folds close to form the neural tube. Neural crest cell initiate their

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figurn 1. Diagram illustratingregionsalongthe neural axis which differ in their range of neural crest derivatives and migratory pathways. The cranial neural crest emerges from levels of the neural axis above the otic vesicles. The vagal neural crest arises from the neural tube between somitic levels 1-7. The trunk neural crest emerges from axial levels between somites 8-28, with those cells which contribute to the adrenal gland arising from somitic level 18-24. The lumbosacral neural crest emerges from axial levels beyond the 28th somite. (From Bronner-Fraser and Cohen, 1980).

migration from the dorsal neural tube in a region where the basal lamina is patchy (Martins-Green and Erickson, 1986). Their emigration begins in cranial regions of the embryo and proceeds progressively tailward. Initiation of migration closely follows neural tube closure and somite formation. After emigration, neural crest cells migrate away from the neural tube and follow pathways that are characteristic of their axial level of origin (Fig. 1). In the head region, cranial neural crest cells migrate subjacent to the cranial ectoderm. Whereas some cranial neural crest cells enter the branchial arches to form many of the cartilagenous elements of the facial skeleton, others contribute to the ciliary ganglion of the eye and various cranial sensory ganglia. Vagal neural crest cells migrate under the ectoderm and into gut, where they populate the enteric nervous system, first in anterior and then in

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FigUre2. Schematic diagram illustrating theearly pathways of trunk neural crest cell migration. Neural crest cells emerge from the neural tube (NT) and proceed either ventrally (indicated by large curved arrow) through the somitic sclerotome (Scl) or dorso-laterally (indicated by small curved arrow) between the ectoderm (Ec) and detmomyotome (DM). The ventrally migrating cells only move through the rostra1 (R) half of each sclerotome and are not observed in the caudal (C) half. Neural crest cells avoid the region around the notochord (No).Ao = dorsal aorta.

progressively more posterior regions of the gut. In the trunk region, neural crest cells follow two primary pathways (Weston, 1963):adorsolateral pathway between the ectoderm and somite, and a ventral pathway through the anterior half of the somite (Rickmann et al., 1985; Fig. 2). The cells following the dorsolateral stream give rise to melanocytes, whereas those of the ventral stream localize in the sensory and sympathetic ganglia, as well as in the adrenal medulla (Fig. 3). Sacral neural crest cells, like vagal neural crest cells, contribute to the enteric nervous system. They migrate ventrally similar to truncal neural crest cells, then invade and populate the posterior portion of the gut (Pomeranz and Gershon, 1991; Serbedzija et al., 1991). Neural crest cell emigration from the neural tube occurs for approximately 24 hours, with the cells populatingtheir derivativesin an orderlypattern such that the more ventral derivatives at any given axial level become filled first (Serbedzija et al., 1989, 1990, 1991). There has been some controversy over the exact routes taken by neural crest cells in the trunk region. Weston (1963) originallyproposed that neural crest cells move through the somites whereas Thiery and colleagues (1982) proposed that neural

Figure 3. Trunk neural crest cell migration pathways and derivatives. There are two major pathways: (A) The dorso-lateral pathway between the somite and the ectoderm; and (B) the ventral pathway through the rostral half of each somite. Trunk neural crest give rise to: A’ pigment cells; 6’dorsal root ganglia; C’ sympathetic ganglia; and D’ cells around the dorsal aorta. (Bottom)A schematic representation of the rostral to caudal distribution of Dil in the neural crest derivatives of a single embryo injected at stage 19 and fixed at stage 21 . a-f represents levels along the rostrocaudal axis from which transverse sections were taken. (a) At the level of the 9th somite, Dil-labeled cells were observed along thedorsc-lateralpathway. (b)Atthe level ofthe 15th somite, Dil-labeledcells were observed along the dorso-lateral pathway and in the dorsal root ganglia. (c) At the level of the 22nd somite, Dil-labeled cells were seen along the dorsc-lateral pathway, in the dorsal root ganglia, and in the sympathetic ganglia. (60 From the level of the 38th somite to the caudal end of the embryo, DiClabeledcells were observed in all truncal neural crest derivatives. (From Serbedzija et al., 1989)

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MARIANNE BRONNER-FRASER A 8

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Figure 4. Fluorescence photomicrographsof HNK-I staining in longitudinal sections of somites 19-21 in a 27 somite embryo illustrating the neural crest cell distribution at different levels of the dorsoventral axis. (A) Schematic diagram of transversesection; lines b-e indicate the dorsoventral levels represented by photomicrographs 6-E. (6) A section 20 microns below the dorsal aspect of the dermomyotome. Neural crest cells were only located between the neural tube and somite at this level of the dorsoventral axis. (C)a section 20 microns ventral to (6).(Dl20 micronsventral to C; neural crest cells were visualized in the anterior half ofthe xlerotome. (D)20 microns again, extensive HNK-1 staining i s observed in the anterior half of the ventral to (D); sclerotoy. (348X) NT = neural tube; DM = dennomyotome; A = anterior; P = posterior.

crest cells migrate preferentially in the intersomitic space. The recent availability of monoclonal antibodies that recognize neural crest cells (Tucker et al., 1984; Vincent et al., 1984) has made it possible to better define the migratory pathways followed by this cell type. In the trunk region, neural crest cell migration appears to be intimately linked with development of the somites. Neural crest cells leave

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Figure 5. Fluorescence photomicrographs of HNK-1 staining in transverse sections through a 33 somite embryo. (a) Section through the anterior portion ofthe 21 st somite. Neural crest cells migrate from the neural tube (NT) and are visualized within the anterior sclerotome and around the dorsal aorta (DA); however, they are not seen around the notochord. (265X). (b) A section through the posterior half of the 21st somite, approximately 70 microns below the section illustrated in (a). Neural crest cells appear to migrate for a short distance from the neural tube, but do not enter into the posterior sclerotome (265x1.

the neural tube shortly after the epithelial somites have budded off from the segmental plate. Neural crest cells then migrate both dorsal and medial to the epithelial somite. Once the somites delaminate to form the dermomyotome and sclerotome, neural crest cells migrate through the sclerotomal portion. However, their migratory pattern in segmented. Though they emerge uniformly from the neural tube, neural crest cells move only through the anterior half of each somitic sclerotome.Those across from the posterior half of the sclerotome only migrate for a short distance between the neural tube and somite (Fig. 4; Rickmann et d.,1985;

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Bronner-Fraser, 1986a). This pattern of movement is similar to that observed for motor axons emerging from the ventral neural tube (Keynes and Stern, 1984). Within the anterior half of each sclerotome, neural crest cells are found dorsally between the dermomyotome and neural tube and ventrally around the dorsal aorta. However, there is a cylindrical region around the notochord which contains sclerotomal cells but is free of neural crestcells (Fig. 5). As described below, this crest-free zone may arise because the notochord inhibits neural crest migration. Neural crest cells migrate through or adjacent to numerous tissues including the neural tube, somites, notochord, and gut. Abundant quantities of extracellular matrix (ECM) molecules line their migratory pathways. The neural crest cells themselves possess numerous cell surface molecules including receptors for ECM components and cell adhesion molecules which may mediate self association or attachment to other cell types. Thus, a variety of cellcell and cell-substrate interactions may influence the migration and localization of neural crest cells. Although the pathways followed by neural crest cells have been identified, the mechanisms responsible for the initial emigration, movement, and localization of neural crest cells are not yet understood. This review summarizes recent experiments from our laboratory examining the pathways followed by neural crest cells, the potential ECM ligands available along these pathways, and the tissues with which neural crest cells may interact. The goal of these studies is to understand the mechanisms underlying neural crest cell migration at the tissue, cellular, and molecular levels.

II. ANALYSIS OF NEURAL CREST MIGRATORY PATHWAYS USING CELL MARKING TECHNIQUES An important first step for understanding cell movement in the embryo is to define the precise pathways followedby migrating cells. Because neural crest cells interact with and migrate through numerous tissues, it has been necessary to label the migrating cells in order to follow their trajectories. A variety of cell-marking techniques have been utilized for this purpose. Initially, analyses of neural crest cell migratory pathways in avian embryos involved transplantation of neural tubes marked with either 3H-thymidine (Weston, 1963) or derived from quail embryos (review: LeDouarin, 1982) into unlabeled chick hosts. By examining the distribution of labeled cells in embryo fmed at various stages, it was possible to infer both the routes and derivatives of neural crest cells. These experiments demonstrated that neural crest cells migrate along two predominant pathways in the trunk region: either ventrally towad the dorsal aorta, or dorsolaterally under the ectoderm. Transplantation experiments have established not only the normal derivatives arising from neural crest cells at various axial levels, but also the effects of relocating neural crest cells to new environments.

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Because of the time required for healing of neural tube grafts and the possibility of scarring, other techniques have emerged to follow the early pathways of neural crest cells. For example, monoclonal antibodies NC-1 and HNK-1 antibodies (Tucker et al., 1984) both recognize a carbohydrate epitope that is present on the surface of neural crest cells and some of their derivatives. By examining their immunoreactivity in fixed embryonic sections, it was discovered that neural crest cells that move along the ventral pathway migrate in a segmental fashion, moving through the anterior, but not the posterior half of each somitic sclerotome, and further ventrally toward the dorsal aorta (Rickmann et al., 1985; Bronner-Fraser, 1986a; Fig. 4). Interestingly, their metameric pattern of migration is later reflected in the segmental arrangement of neural crest-derived peripheral ganglia (Teillet et al., 1987; Lallier and Bronner-Fraser, 1988). However, the NC-1 and HNK-1 antibodies are not entirely specific;they recognize several nonneural crest cell types (Kruse et al., 1984) and do not recognize all neural crest cells fleillet et al., 1987). A more direct approach for examining the pathways of neural crest cells migration is to inject the lipophilic dye, DiI, into the neural tube (Serbedzijaet al., 1989). This hydrophobic dye intercalates into cell membranes, thereby labeling all neural tube cells including premigratory neural crest cells. One can monitor directly the pathways followed by neural crest cells by examining embryos either in sections or in whole mounts at various times after injection. The results obtained with DiI-labelingare similar to those observed with HNK-I antibody labeling (Serbedzija et al., 1991). Neural crest cells along the ventral pathway were found to migrate in a segmental fashion through the anterior half of each sclerotome. In addition, DiI-labeled, HNK- 1 negative cells were observed along the dorsolateral pathway, where their migration was unsegmented. Thus, DiI labels neural crest cells along all of their migratory pathways. Although the dye and antibody labeling approaches have different potential pitfalls, they provide complementary and confirmatory results. An additional advantage of DiI-labeling is that, by altering the time of injection, it is possible to establish the order in which neural crest cells contribute to their derivatives. When DiI is introduced into the neural tube at progressively later stages of development, we labeled only those neural crest cells that were premigratory at the time of injection. We found that the later emigrating cells also contributed to progressively more dorsal derivatives (Fig. 3). Thus, the contribution of neural crest cells to their derivatives becomes restricted in a ventral-to-dorsal order such that the last cells to exit the neural tube migrate dorsally under the ectoderm where they eventually form pigment cells (Serbedzija et al., 1989). The conclusion that neural crest cells migrate in an orderly pattern is in agreement with the transplantation results of Weston and Butler (1966), who found that tritiated thymidine-labeled neural tubes from the trunk region of “younger” avian embryos transplanted to trunks of “older” hosts contributed only to dorsal derivatives. An additional advantage of DiI-labeling of neural crest cells is that DiI can be applied at a variety of axial levels and to many species. For example, this approach has been used successfully to map the pathways of sacral neural crest cells

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migration in the chick and mouse (Pomeranz and Gershon, 1991; Serbedzijaet al., 1991)and trunk and cranial neural crest cell migrarion in the mouse (Serbedzijaet al., 1990, 1992). DiI labeling also provides information about the length of time that neural crest cells emigrate from the neural tube, which is approximately 24 hours at most axial levels.

111. EXTRACELLULAR MATRIX MOLECULES ALONG NEURAL CREST PATHWAYS After migrating from the neural tube,neural crest cells migrate into a small cell-free space which contains extracellular matrix ( E M ) molecules synthesizedby neighboring tissues. The most prevalent glycoproteins appear to be fibronectin (Newgreen and Thiery, 1980), laminin (Krotoski et al., 1986), tenascidcytotactin (Tan et al., 1987) and various collagens (Duband and Thiery, 1987;Pems et al., 1991a). Fibronectin in particular has been suggested to play a major role in the adhesion and motility of neural crest cells (Thiery et al., 1982). In tissue culture, neural crest cells migrate avidly on both fibronectin (Rovasio et al., 1983) and laminin (Newgreen, 1984) substrates. Hyaluronic acid is present in high concentrations during the early stages of migration (Pintar, 1978); thus, initiation of neural crest cell movement occurs in a hyaluronate-rich region, analogous to the morphogenesis of some other embryonictissuessuch as thecornealstroma(Too1eandTrelstad,1971). Of the proteoglycans present within the embryo, heparan sulfate proteoglycans (Perris et al., 1991a)appearon neural crest cell pathways, whilechondroitin sulfate proteoglycans are generally present in regions from which neural crest cells are absent (Tan et al., 1987; Perris et al., 1991a), such as the perinotochordal space. Chondroitinsulfate proteoglycanstend to inhibit neural crest cell migration in v i m , consistent with the idea that they may restrict or inhibit migration in the embryo. During embryogenesis,the composition of the extracellular matrix is dynamic. For example, levels of fibronectin and laminin immunoreactivitymodulate during neural crest migration such that they appear reduced during the migratory phase compared to levels both before and after neural crest migration (Krotoski et al., 1986). Hyaluronate concentrations also appear to shift; high levels are present on neural crest pathways at the beginning of migration,whereas the levels in the ECM are reduced following migration and during gangliogenesis (Perris et al., 1991b). It is possible that these changes in ECM composition may be involved in the cessation and/or final localization of neural crest cells. In addition to changing levels of ECM components,dynamic rearrangements in the distribution of some extracellular matrix occur at the time of neural crest migration. Some collagens and proteoglycans, as well as cytotactidtenascin, are initially distributed uniformly within the sclemtome at early stages of migration. By advanced stages, tenascidcytotactin codistributes with migrating neural crest cells in the anterior half of each somitic sclerotome (Tan et al., 1987; Stem et al.,

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1989),whereas collagen types I and 111and a keratan sulfate proteoglycan become restricted to the posterior half (Perris et al., 1991b). Since the reorganization of these molecules occurs relatively late, they may be a consequence, rather than a cause of the neural crest migratory pattern. This appears to be true for tenascin, since ablation of the neural crest results in tenascin in the posterior rather than the anteriorsclerotome (Sternet al., 1989;butseeTanet al., 1987,1991,forthecontrary view regarding cytotactin). Yet another class of molecules remains uniformly distributed within the somites as a function of time; these include fibronectin, laminin, and collagen type IV. Other molecules are selectively distributed within either the anterior or posterior half of the sclerotome over time. For example, a monoclonal antibody discovered by Tanaka et al. (1989)recognizes a molecule in the anterior somite prior to neural crest cell entry into this tissue, whose pattern is unaltered by neural crest cell ablation. Both during and following neural crest cell migration, the posterior half of each somite contains peanut lectin-bindingmolecules (Stern and Keynes, 1987) and a chondroitin sulfate proteoglycan that binds cytotactin (Tan et al., 1987).A functional role in axon guidance for a peanut lectin-binding glycoprotein fraction derived from the posterior sclerotome is suggested by experiments in which liposomescontaining these molecules inhibit sensory growth cones in v i m (Davies et al., 1990). In addition to lectin-binding moieties, T-cadherin (truncatedcadherin), a novel member of the cadherin family of cell adhesion molecules, also is selectively expressed in the posterior half of each sclerotome at all times examined (Ranscht and Bronner-Fraser, 1991).The earliest T-cadherin expression is detected in the posterior portion of the somite concomitant with the initial invasion of neural crest cells into the anterior portion of the same somite. Surgical ablation of the neural crest does not alter the pattern of T-cadherin immunoreactivity, suggesting that neural crest cells are not required for the metameric distribution of molecule. Although its function has not been established, this distribution is consistent with the possibility that T-cadherin plays a role in maintaining somite polarity and in influencing the pattern of neural crest cell migration (Ranscht and Bronner-Fraser, 1991).Hence, differential distribution of permissive and nonpermissive molecules, together with changes in the cells' ability to interact with them, may determine the segmented migratory pattern of neural crest cells in the trunk. Along cranial neural crest migratory pathways, molecules such as fibronectin, laminin, tenascin, and heparan sulfate proteoglycans are abundant (Krotoski et al., 1986;Duband and Thiery, 1987;Bronner-Fraser, 1988).All of these ECM components are found in the basement membrane of the ectoderm, neural tube, and notochord, and within the interstitial matrix of the cranial mesenchyme. Because cranial neural crest cells migrate through the mesenchyme subjacent to the ectoderrn, they encounter and have the opportunity to interact with many of these molecules.Perturbation experiments(see below) suggest that these molecules may play a functional role in cranial neural crest migration.

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Neural crest cells themselves may contribute to the dynamic nature of the ECM by adding to or modifying the matrix through which they move. I n vitro, neural crest cells synthesize glycosaminoglycans (primarily hyaluronate)and glycoproteins (Manasek and Cohen, 1977) which become associated with the neural crest cell surface or their substrate. During their migratory phase, it has been shown that neural crest cells produce proteases such as plasminogen activator (Valinsky and LeDouarin, 1985). which may facilitate breakdown and modification of ECM components such as fibronectin.

IV. THE N E U R A L CREST CELL SURFACE Antibodies that disrupt adhesion of cells to the extracellular matrix have been used to define a class of cell surface receptors that are members of the integrin family. As a class, integrin receptors mediate adhesion of cells to a variety of extracellular matrix molecules including fibronectin,laminin,vitronectin,various collagens,and tenascin (Honvitz et al., 1985; Buck et al., 1986; Tomaselli et al., 1988; Bourdon et al., 1989). An integrin receptor is a heterodimer composed of two noncovalently There are at least 14 a and seven f3 linked transmembrane subunits, a and fi subunits which have been wholly or partially characterized. Different a subunits share a significant amount of sequence identity to each other, as do different fJ subunits, though the alpha and beta subunits are distinct and unrelated (Hynes, 1987). All known integrins are thought to require divalent cations in order to bind to their ligands (but see Section VI). Three major classes of integrin receptors have been defined by their use of a common 8 subunit. The matching of one a subunit with one (J subunit is thought to determine the specificity of the receptor complex. There is evidence that these transmembrane glycoproteins also interact with the cytoskeleton, thereby coupling the extracellular matrix to the cell’s intracellular machinery (Buck and Horwitz, 1987; Bum et a]., 1988). While some integrin receptors appear to have specificity for a single molecule, several have been shown to bind multiple extracellular ligands. Recently, some of the integrins on neural crest cells have been identified. It is well established that neural crest cells possess the subunit of integrin since both CSAT and JG22 antibodies against the chick integrin disrupt their adhesion to fibronectin, laminin, and collagen substrates (Bronner-Fraser, 1985,1986b; Lallier and Bronner-Fraser, 1991; and unpublished). Synthetic RGD peptides, which competitivelyinhibit binding to fibronectin,cause similar disruption of neural crest cell migration in vitro (Boucaut et al., 1984; Pems et al., 1989). However, the a subunits that associate with these fJs on neural crest cells are not understood. The antibody HNK-1 also recognizes a molecule on the surface of neural crest cells. This antibody is directed toward a carbohydrate epitope which is also found on mature neurons, Schwann cells, and a subpopulation of leukocytes (Tucker et al., 1984), and is present on a family of adhesion molecules including N-CAM,

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NgCAM, J1, L1, and MAG (Kruse et al., 1984).In vim, neural crest cells become rounded and detached from laminin substrates in the presence of the HNK-1 antibody (Bronner-Fraser, 1988). Similarly, neurite extension onto laminin is inhibited by this antibody(Ropelle et al., 1986).Neural crest cell surface molecules in addition to integrins may be important for migration. For example, Runyan et al. (1988) have shown that cell surface galactosyltransferaseis involved in interactions between neural crest cells and basal lamina components in v i m . Still other neural crest surface moieties remain to be identified.

V. CELL ADHESION MOLECULES ON NEURAL CREST CELLS Although little is known about the mechanisms underlying emigration of neural crest cells from the neural tube, adhesion molecules have been proposed to play an important role in this process. Thiery and colleagues (1982) suggested that the neural cell adhesion molecules (N-CAM) may be important in neural crest cell dispersion. By immunologicalstainingof avian embryos,N-CAM can be identified throughout the neural tube including the region containing premigratory neural crest cells. The adhesive molecule is then lost from neural crest cells after initiation of migration. Following the formation of neural crest-derived ganglia, N-CAM is again found between the ganglionic cells. Like N-CAM, Ncadherin is expressed throughout the neural plate and neural tube and is lost from the neural crest cell surface during migration (Hatta et al., 1987). Using an in vitm adhesion assay system, Newgreen and Gibbins (1982) report a loss of intercellular adhesiveness in neural crest cells as the cells initially migrate away. The emigration of neural crest cells is Ca2+-dependent(Newgreen and C2+ Gooday, 1985), suggesting that molecules other than Ca*-independent N-CAM (perhaps Ncadherin), may be important in neural crest cell emigration from the neural tube. In the trunk, the dorsal root and sympatheticganglia form aligned with the rostra1 portion of each somite (Teillet et al., 1987; Lallier and Bronner-Fraser, 1988), reflecting the metameric pattern of neural crest cell migration along the ventral pathway. Both dorsal root and sympathetic ganglion cells appear to have N-CAM and Ncadherin immunoreactivity. This has led to the idea that one or both of these molecules may be necessary for gangliogenesis. However, we have found that N-CAM expression in dorsal root ganglia appears several stages after formation of the ganglia (Lallier and Bronner-Fraser, 1988).In contrast, N-cadherin expression occurs concomitant with condensation of the dorsal root ganglia (Akitaya and Bronner-Fraser, 1992). In sum, premigratory neural crest cells and neural crest-derived ganglionic cells have abundant levels of cell adhesion molecules. These appear to be lost during cell migration, when neural crest cells may interact with extracellular matrix

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molecules or other cell types in the surroundings.These observations suggest that the balance between cell-cell and cell-substrate adhesion may play an important role in initiation and cessation of neural crest cell migration.

VI. CELL-MATRIX INTERACTIONS IN NEURAL CREST MIG RAT1 0N A. Tissue Culture Analysis In Vitro Migration and Adhesion Assays

By explanting neural tubes prior to neural crest migration, it is possible to establish cultures of neural crest cells in vifro.The neural crest cells emigrate from the dorsal side of the explanted neural tube, to form a monolayer on a variety of substrates including fibronectin (Rovasio et al., 1983; Perris et al., 1989), laminin (Newgreen, 1984;Lallier andBronner-Fraser, 1991),collagen (Perris et al., 1991b), and other molecules. In order to migrate, cells must attach to a substrate, apply traction, and then detach. Thus, an analysis of cell-matrix interactions must consider not only cell movement but also cell attachment. Therefore, we utilize two assay systems for analyzing neural crest cell-matrix interactions. First, we use a migration assay, which measures the distance traversed by a representative number of neural crest cells over a given time. The migration assay utilizes primary outgrowths of neural crest cells and examines their degree of migration on defined ECM substrates. Analysis is performed by making direct measurements from filmed or videotaped images of the migrating cells. Second, we utilize a quantitative cell adhesion assay, which determines the percentage of cells that rapidly attach to a given substrate. For the adhesion assay, neural crest cells are removed from the substrate and reattached to various coating concentrations of ECM molecules using a centrifugation attachment assay that allows reproducible measurement of the adhesive properties of small populations of cells. One must bear in mind that attachmentand migration, though related, are not equivalent, and that these two assays sometimes give different results. For example, a highly adhesive substrate is often a poor migratory substrate because cells are unable to detach from it and move forward. The quantitative centrifugation adhesion assay is adapted from McClay et al. (1 98 1) and is illustrated in Figure 6. Because this technique is less conventional than some more commonly used assay, it deserves further comment. Roughly lo5 cells per experiment are isolated from primary neural crest cell cultures within 24 hours of explantation from the embryo. This corresponds to a time well before their detectable differentiation,thus providing a population of “migrating” neural crest cells. Radioactively labeled neural crest cells are placed into fluid filled chambers to which antibodies, divalent cations, heparin, synthetic peptides, or oligonu-

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Centrifugal Cell Attachment Assay n Bound

uUnbound

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Advantages:

Re produci biIit y Sensitivity Figure 6. Schematic diagram illustrating the cell adhesion assay.

cleotides can be added. The chambers are sealed and a constant centrifugal force is used to bring the cells into contact with the substrates. Nonadherent cells are removed by inverting the chamber and centrifuging. This assay yields highly reproducibleresults for small numbers of cells. In order to determine the molecular interactions involved in cell migration and adhesion, a variety of perturbing agents including antibodies against cell surface or ECM molecules, peptides that competitively inhibit cell-matrix binding, and anti-sense oligonucleotides that block synthesis of receptors can be added to the assay system. This technique makes it possible to draw meaningful conclusionsfromdiverseassay conditions using small numbers of cells. Migration on Fibronectin, Laminin, and Collagen Substrates In Vitro

Using the migration assay, we have examined the molecular interaction of avian neural crest cells with fibronectin and laminin during their initial migration from the neural tube in vitro (Perris et al., 1989).The extent of neural crest cell migration on a 105-kDa proteolytic fragment of fibronectin encompassing the cell-binding domain was equivalentto that observed on the intact molecule, and neural crest cell migration on both intact fibronectin and the 105-kDa fragment was reversibly inhibited by RGD-containing peptides. In addition to this putative “cell-binding domain,” neural crest cells were able to migrate on a fragment correspondingto the

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isolated heparin-binding I1 region of fibronectin, and were specifically inhibited in their migration by exogenous heparin but not RGD peptides. Heparin potentiated the inhibitory effect of RGD peptides on intact fibronectin,but not on the 105-kDa fragment. These findings suggest that neural crest cell migration on fibronectin occurs primarily through an interaction with the RGD site within the cell-binding domain, whereas other potential attachment sites may act complementarilyfor the stabilization of cell-fibronectin linkages. On substrates of isolated laminin, the extent of avian neural crest cell migration was maximal at relatively low substrate concentrations. Neural crest cell migration on laminin was not affected by RGD peptides. The predominant motility-promoting activity of laminin was localized to the E8 domain, located on the long arm of laminin close to the heparin-binding site. Another set of molecules that may serve as substrates for neural crest cells are collagens. In the embryo, collagens are abundant along neural crest migratory pathways, primarily in the interstitial matrix and along basement membranes of the neural tube and ectoderm. In addition, collagens undergo dynamic reorganizations during the course of neural crest cell migration in viva To examine their functional significance, the ability of purified collagens (Col) to promote neural crest cell migration in vitm was examined using the migration assay. Neural crest cell outgrowth on isolated collagens was most pronounced on Col I and Col VI, whereas Col 11,V, and VII were unable to support cell motility.Furthermore,differentclasses of collagens demonstrate differential abilities to support neural crest cell migration in vitm and their migration-promoting activity can be modulated by association with other matrix components (Perris et al., 1991b). Cell Surface Receptors on Neural Crest Cells

The migration and adhesion assays can be used to characterize the properties of the receptors present on neural crest cells. For example, the CSAT and JG22 antibodies against the subunit of chick integrin can be added to neural crest cells in vim. These antibodies disrupts both neural crest cell migration (Fig. 7; BronnerFraser, 1985) and adhesion (Lallier and Bronner-Fraser, 1991) to fibronectin and laminin substrates. The effects are rapid, causing many cells to detach or aggregate with other cells within 15 minutes, and are readily reversible upon removal of the antibody. These results suggest that binding and dissociation of integrin receptors to matrix molecules occur rapidly. HNK-I is another cell surface epitope which is present on migrating neural crest cells, neural crest-derived neurons, and some other cells (Tucker et al., 1984). Addition of HNK-I antibody to primary cultures of neural crest cells causes detachment and aggregation of the cells grown on laminin but not fibronectin substrates (Bronner-Fraser, 1988). Using the quantitative adhesion assay, we have examined receptors utilized for neural crest cell attachment to laminin substrates in some detail. Our results show that an increasingpercentage of neural crest cells adhere to laminin with increasing substrate concentrations.This contrasts with neural crest cell migration on laminin

Figure 7. Phase contrast photomicrographs illustratingthe effects of JG22 antibodies against the Pi subunit of integrin on neural crest cells in vitro. (a) A culture of neural crest cells grown on fibronectin 24 hr after explantation of the neural tube (nt) onto the culture dish. The neural crest cells have emigrated from the neural tube and have formed a monolayer on the substrate. (b) The same field 15 min after addition of antibody. Many cells have rounded and become phase bright, and often detach from the substrate; other cells form small aggregates. (c)The same field after 60 rnin. More cells have detached or formed aggregates. Note that the neural tube does not appear to be affected. (From Bronner-Fraser, 1985).

MARIANNE BRONNER-FRASER

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Substrate Coating Concentration (pg/ml) Figure 8. Neural crest cell attachment to various concentrations of laminin, ranging from 0.1 to 10 &ml, in the presence of the CSAT and HNK-1 antibodies. CSAT and HNK-1 antibodies (50 pg/mI) were added to test their ability to perturb cell adhesion over a range of substrate/coatingconcentrations. Points represent the mean of at least six experiments and the error bars represent the S.E.M. (From Lallier and Bronner-Fraser, 19911.

which is optimal at relatively low coating concentrations (Perris et al., 1989). Their adhesion to fibronectin and laminin is mediated by integrins, sinceCSAT antibodies completely block this interaction. In contrast, the HNK-1 antibody, which recognizes a carbohydrate epitope, inhibits neural crest cell attachment to laminin at low substrate-coating concentrations (> 1 pg/ml; Low-LM) (Fig. 8), but not at high coating concentration of laminin (10 pgfml; High-LM). Attachment to Low-LM occurs in the absence of divalent cations, whereas attachment to High-LM requires Ca2' or Mn2+. This is surprising since all previously described integrins have required divalent cations for function. These results suggest that neural crest cells have at least two integrins that recognize laminin: one that requires divalent cations for binding; the other that can function without divalent cations. In a preliminary biochemical characterization of the HNK-1 epitope on neural crest cells, we find that it recognizes a 165-kDa protein also found in immunoprecipitates using antibodies against integrin, which precipitates both the and associated a bands. Our preliminary evidence suggests that his band corresponds to the chick a1 subunit of integrin, which is a 165-kDa protein (Syfrig et al., 1991). In addition to antibodies, other reagents can be used to functionally inhibit integrins on cells. For example, short anti-sense oligonucleotides (15- to 30-mers)

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O l i g o c o n c e n t r a t i o n (nM) Figure 9. Graphs showing the affects of antibodies and selected antisense oligonucleotides on neural crest cell attachment to laminin substrates. Left Adhesion of neural crest cells to laminin is sensitive to HNK-1 antibodies, which partially block adhesion, and to JG22 antibodies against the f31 subunit of integrin, which completely block adhesion. Right: Some, but not all, anti-sense oligonucleotides affect binding of neural crest cells to laminin. For example, the 1B1 and 2Aa oligos, anti-sense to the f31 and homologous regions of various alpha subunits, block neural crest cell attachment to laminin in a dose-dependent manner. In contrast, 3A3, another anti-sense oligonucleotide to alpha subunits had no effect.

can be used to knock-out mRNA for proteins in cultured cells. Phosphorothioated oligonucleotides were designed to be anti-sense to a portion of the pi subunit and a region of high homology within the C-terminal domain of a integrin subunits (Lallier and Bronner-Fraser, 1993). Selected oligonucleotides significantly inhibited trunk neural crest cell attachment to laminin and fibronectin (Fig. 9) in a dose-dependent manner, with maximal response at 50 pM. In contrast, other oligonucleotides had no significant effect on cell attachment: for example, sense oligonucleotides, sense plus anti-sense oligonucleotidesas well as some anti-sense sequences against various homologous regions of the a subunit sense. One antisense a oligonucleotide preferentially inhibited neural crest cell attachment to fibronectin but not to laminin (Lallier and Bronner-Fraser, 1993). These results suggest that anti-sense oligonucleotides can be used to selectively inhibit cell surface components on neural crest cells. These techniques promise to be useful for isolating cell type specific integrins.

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B. In Vivo Perturbation Analysis

The particular advantage of tissue culture is that it allows one to examine cell interactions under defined conditions. By examining neural crest cell-ECM interactions in v i m it has been possible to identify some of the receptors present on neural crest cells and some of the matrix components to which they can bind. However, this cannot be taken as evidence that these interactions play a role in the embryo itself. To directly analyze cell-matrix interactionsthat are important for cell movement, one must take the knowledge obtained in tissue culture and return to performing experiments within the embryo. Although chick embryos are easily accessible to experimental manipulations, they are not particularly amenable to genetic manipulations. Therefore, perturbation experiments as described below have been used to analyze the role of cell-matrix interactions in viva Antibody perturbation experiments have been used to functionally “knockout” selected cell-matrix interactions along neural crest migratory pathways. These experiments have been performed primarily along cranial neural crest pathways because numerous extracellular matrix molecules are present along these pathways at times that correlate with initial and active migration of this population. We introduce antibodies by microinjection lateral to the mesencephalic neural tube (Fig. 10).Embryos ranging from the neural fold stage to the nine somite stage are used for injection. Embryos with greater than 10 somites at the time of injection have had no detectable abnormalities, suggesting that they are sensitive to the

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figure 10. Schematic diagram illustrating the procedure for injecting antibodies into the cranial mesenchyme adjacent to the mesencephalon. Neural crest cells in this region migrate through the mesenchyme underneath the surface ectoderm.

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injected antibodies for only a limited time during their development. The uninjected side of the embryo can serve as an internal control, since antibodymolecules diffuse freely on the injected side of the embryo but are barely detectableon the uninjected side. In order to test the role of the integrin receptors in cranial neural crest cell migration in situ, we microinjected antibodies that bind and functionally inactivate the /31 subunit of the integrin. Following injection of blocking integrin antibodies, major defects were observed including: reduced numbers of the neural crest cell on the injected side, neural crest cells within the lumen of the neural tube, ectopic neural crest cells external to the neural tube, and neural tube anomalies (Fig. 11; BronnerFraser, 1985,1986b).Similarresults have been obtained with syntheticRGD peptides containing the fibronectin cell-binding sequence (Boucaut et al., 1984) or antibodies against fibronectin (Poole and Thiery, 1986). In contrast to the antibodies that block cell-ECM interactions,several control monoclonal antibodies which bind to, but do not functionally block integrins, had no detectable effects on cranial neural crest or neural tube development. These findings suggest that integrins are important in the normal development of the cranial neural crest and neural tube. Because both competitively inhibit cell binding to fibronectin, the findings that integrin antibodies and synthetic decapeptides block cranial neural crest migration suggest an important function for fibronectin in cranial neural crest migration. However, these reagents are not specific. For example, the cell-binding sequence of fibronectin is also present in numerous other extracellular matrix molecules. Likewise, integrin antibodies block neural crest cell adhesion to laminin, fibronectin, and collagens. Therefore, we have examined the possible role in neural crest cell migration of other ECM molecules that are ligands for integrins. In particular, we have used an antibody which functionally perturbs cell adhesion to laminin as a first attempt to distinguish between the respectiveroles of these matrix molecules. In its native state, laminin is thought to occur in a complex with heparan sulfate proteoglycan (HSPG). The IN0 (inhibitor of neurite outgrowth) antibody recognizes and functionally blocks cell interactions with this laminin-heparan sulfate proteoglycan complex (Chiu et al., 1986). We have injected I N 0 antibody along neural crest pathways in the mesencephalon in order to examine the possible role of laminin in cranial neural crest migration (Bronner-Fraser and Lallier, 1988).One day after injection, the embryos had severe abnormalities in cranial neural crest migration including: ectopic neural crest cells external to the neural tube; neural crest cells within the lumen of the neural tube; and neural tube deformities. In contrast, embryos injected with antibodies against laminin or heparan sulfate proteoglycan were unaffected. These results indicate that functional blockage of a laminin-heparan sulfate proteoglycan perturbs cranial neural crest migration, providing evidence that laminin/HSPGis involved in aspects of neural crest migration in vivo.

Figure 7 7. (A) Fluorescencephotomicrograph of a transverse cryostat section showing the distribution of the CSAT antibody after injection into the mesencephalon. The embryo was processed 6 hr after injection. The CSAT antibody was obserwd around the neural tube (nt), ectodenn, cranial mesenchyme and surrounding premigratory neural crest cells. Antibodies did not, however, appear to cross the midline. (B) The effects of the CSAT antibody on cranial neural crest migration in an embryo fixed 18 hr after injection. An aggregate of neural crest cells (NC) was observed protruding into the lumen of the neural tube (nt) and a 58% reduction in the neural crest cell volume on the injected side (indicated by arrow) relative to the control side (1 55X). 140

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Other antibodies against extracellular matrix molecules also can inhibit cranial neural crest migration. In addition to IN0 and CSAT antibodies described above, antibodies against tenascin (Bronner-Fraser, 1988), against the HNK-1 epitope (Bronner-Fraser, 1987; perhaps on an u1 integrin subunit), and against fibronectin (Poole and Thiery, 1986), inhibit cranial neural crest cell migration. In addition to antibodies, the galactosyltransferase inhibitor, alpha lactalbumin, inhibits neural crest cell spreading on laminin in v i m indicating a possible role for this enzyme in modulating cell attachment by altering cell surface carbohydrates(Runyan et al., 1986).For the cranial neural crest, it is clear that multiple interactions are necessary for normal migration of cranial neural crest cells. Thus, cell-matrix interactions may involve a number of different molecules and/or surfacereceptors. It is possible that these cell-matrix interactions are multivalent. Alternatively, they may occur as an interrelated sequence of interactions, such that interfering with any one step disrupts the process. This highlights the fact that complicated morphogeneticevents such as neural crest cell migration may be mediated by complex sets of interactions. All of the above-described perturbation experimentsinvolved cranial neural crest cells. The tissue and extracellular matrix environments appear very different in the head and trunk, making it possible that different strategies are important for the migration of cranial versus trunk neural crest cells. In support of this idea, none of the function-blockingantibodies that affect cranial neural crest cell migration have been shown to be functional in the trunk region. Thus, there is no direct evidence for the importance of cell-matrix interactions in guiding the movement of trunk neural crest cells. Described below are experiments examining the possible role of tissuederived cues such as segmental information within the anterior and posterior halves of the somites or inhibitory substances from the notochord.

VII. ROLE OF SURROUNDING TISSUES IN DETERMINING THE PATTERN OF NEURAL CREST MIGRATION During their migration, neural crest cells move in the vicinity of a variety of tissues including the somites, ectoderm, neural tube, notochord, and dorsal aorta. The experimentsdescribed below examine the role of some of these tissues in the pattern of neural crest cell migration. Although these experiments do not discount a role for cell-matrix interactions in the trunk, they suggest that these may play a permissive rather than an instructiverole in the migration of trunk neural crest cells. A. Segmental Information within the Somites

Both neural crest cells (Rickmann et al., 1985;Bronner-Fraser, 1986a;Teillet et al., 1987; Loring and Erickson, 1988) and motor s o n s emerging from the ventral neural tube (Keynes and Stern, 1984) appear to preferentially move through the anterior half of each somite, and fail to move through the posterior half. The

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Figure 12. Schematic diagram summarizing the microsurgical experiment; the segmental plate was rotated 180’ about its rostrocaudal axis.

metameric pattern of migration suggests there may be molecular differences within the sclerotome that influence neural crest cell and motor axon movement. Thus, there may be inhibitory cues in the posterior sclerotome, attractive cues in the anterior sclerotome, or both. For the case of motor axons, Keynes and Stern (1984) have performed a series of grafting operations to determine if the “guiding” information was within the sclerotome itself or was inherent to the axons. Although rotating the neural tube did not affect the pattern of axonal outgrowth, rotating the segmental plate (which gives rise to the somites) 180”about its anterior-posterioraxis causes the axons to traverse the posterior (original anterior) halves of the rotated sclerotomes. One possible explanation for this intriguing observation is that the molecular composition is different in the anterior versus the posterior sclerotome. In support of this, Davies and colleagues (1990) have shown that a molecule recognized by peanut agglutinin is selectively distributed within the posterior, but not the anterior sclerotome, and inhibits axon growth. The molecular nature of anterior/posterior differencesin the sclerotomeare of obvious interest for understandingthe guidance of neural crest migration. To test whether the somites are responsible for guiding neural crest cells, we reversed a length of segmental plate mesoderm about its anterior-posterior axis (Fig. 12;Bronner-Fraserand Stern, 1991).The results are similar to those observed for motor axons. Neural crest cells emerge uniformly from the neural tube, then migrate through the rotated sclerotomes. However, their pattern of migration is inverted such that neural crest cells are now present in the posterior (original anterior) halves of the sclerotomes derived from the rotated mesoderm (Fig. 13). This result suggests that it is the presence of differences between the cells of the

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Figure 73. Fluorescence photomicrographs of a longitudinal section through an operated embryos stained with the HNK-1 antibody. The segmental plate was rotated 180" about its anterior-posterioraxis. By the time of fixation, the segmental plate had differentiated into mature somiteswith dermomyotomesand sclerotomes. Neural crest cells are always observed in the original anterior (A) half of each sclerotcine and are absent from the original posterior (P) halves. The arrow indicates a small sornite which formed at one of the junctions between graft and host tissue. (From Bronner-Fraser and Stern, 1991)

anterior and the posterior halves of the sclerotome that is responsible for the segmental pattern of both neural crest migration and motor axon outgrowth. One candidate molecule for an inhibitory cue is T-cadherin, which occurs in the posterior sclerotome prior to neural crest cell entry into the sclerotome (Ranscht and Bronner-Fraser, 1991; see Section In).

B. Inhibitory Effects of the Notochord The notochord appears to have a variety of functions invoked at different stages of development. Just after neural tube closure, the notochord is known to confer

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ventral properties on the neural tube. The notochord induces the formation of motor neurons and a floorplate, morphologically recognizable by it characteristic wedgeshape (Van Stratten et al., 1988; 1989; Smith and Schoenwolf, 1989), and by the appearance of floorplate specific markers (Yamadaet d.,1991). Any portion of the neural tube, including the dorsal-most region, of approximately stage 10 avian embryos is competent to form floorplate and motor neurons when induced by the neural tube (Yamada et al., 1991). Thus, it is clear that the notochord can induce differentiation of ventral neural tube structures. After emigration of neural crest cells from the neural tube, the notochord appears to exert an inhibitory influence on migrating neural crest cells. Avian neural crest cells migrating along the trunk ventral pathway are distributed throughout the anterior half of the sclerotome with the exception of a neural crest cell-free space of approximately 85-pm width surrounding the notochord. In tissue culture experiments, Newgreen found that neural crest cells avoided the region surrounding notochords with which they were cocultured, suggesting that the notochord produces a substance which inhibits neural crest migration (Newgreen et al., 1986). We have examined the role of the notochord in vivo by implanting a length of quail notochord lateral to the neural tube along the neural crest ventral migratory pathway of 2day chicken embryos. The subsequent distribution of neural crest

A

B A

Figure 74. (A) Schematic diagram illustrating the pattern of neural crest cell migration in normal embryos, and (6)the effects on neural crest cell migration of implanting an extra notochord (No) adjacent to the neural tube.

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Figure 15. Fluorescent photomicrographs of a transverse section through chick embryos implanted with a quail notochord. The section was stained with the HNK-1 antibody, which recognizes a surface epitope on neural crest cells and in the perinotochordal matrix. HNK-1 immunoreactive neural crest cells (arrow) approach but do not contact the implanted notochord (n’). nt = neural tube; n = host notochord (From Pettway et al., 1990).

cells was analyzed in embryos fixed two days after grafting. When the donor notochord was isolated using collagenase, neural crest cells avoided the ectopic notochord and were absent from the area immediately surrounding the implant (mean distance of 43 microns) (Figs. 14, 15). The neural crest cell-free space was significantly less when notochords were isolated using trypsin or chondroitinase digestion and was eliminated by fixation of notochords with paraformaldehydeor

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methanol prior to implantation. These results suggest that the notochord produces a trypsin and chondroitinase labile substance that can inhibit neural crest cell migration (Pettway et al., 1990). A likely candidate for the inhibitory molecule is a chondroitin sulfate proteoglycan which bears the HNK-1 epitope (Henning and Schwartz, 1991).These results suggest that the notochord produces a substancethat inhibits neural crest cell migration in its immediate vicinity. In addition to influencing neural crest cell migration, implanted notochords also can alter the position and size of neural crestderived dorsal root ganglia (Artinger and Bronner-Fraser, 1992). Furthermore, the perinotochordal space appears to be inhibitory for motor axons (Oakley and Tosney, 1990).These experiments suggests that the sculpting of neural crest migratory routes at least partially involves inhibitory regions from which neural crest cells are restricted. C. Dorsoventral Patterning of Neural Crest Derivatives by the Neural Tube

In the dorsoventral direction, neural crest cells display a characteristicpattern of migration and localization.To test whether this pattern is caused by chemoattraction of neural crest cells to their targets, Weston (1963) rotated the neural tube dorsoventrally by 180” to cause neural crest cells to emerge ventrally. He found that the neural crest cells from the rotated neural tube still migrate in two streams away from the neural tube: one dorsally, in reverse direction to that taken normally; the other ventrally towards the aorta. These results suggest that the neural crest cells are probably not directed to their targets by chemoattractants, but rather that they exploit all the spaces available to them. We have reexamined this question using modern cell marking techniques which allow a more detailed analysis of neural crest migratory pathways. Similar to Weston’s experiments, the neural tube, with or without the notochord, was rotated by 180” dorsoventrally to cause the neural crest cells to emerge ventrally. In some embryos, the notochord was ablated and in others a second notochord was implanted. Neural crest cells emerging from an inverted neural tube migrated in a ventral-to-dorsal direction through the sclerotome. As in operated embryos, their migration was segmented, being restricted to the anterior half of each sclerotome. The dorsal root ganglia always formed adjacent to the neural tube and their dorsoventral orientation often followed the orientation of the grafted neural tube. Similarly, the ventral roots emanated from the dorsal portion of the neural tube (originally “ventral” prior to rotation) (Fig. 16). In contrast to the dorsal root ganglia which followed the rotated neural tube in orientation, sympathetic neurons only differentiated near the aorkdrnesonephrosand required the proximity of either the notochord or the ventral neural tube. The results suggest that the dorso-ventral polarity of the neural tube, dorsal root ganglia, and ventral roots appear to follow the orientation of the neural tube. In contrast, the sympathetic ganglia require additional cues for differentiation. These results show that neural tube and notochord exert important effects on neural crest cells; they can influence

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figure 16. Fluorescencephotomicrographsof transverse sections through an embryo in which the neural tube was rotated 180° in the dorsoventral plane in the absence of a notochord. The embryo was fixed at stage 25, which is well after gangliogenesis normally occurs. The neural tube (NT) forms normally but with inverteddorsoventrally polarity. In most sections, the dorsal root ganglia (D) form normally relative to the inverted neural tube. In a few cases, supernumerary dorsal root ganglia form, as illustrated in (c). The ventral roots (VR) project from the dorsal portion of the neural tube and appear to find their targets in the limb, as seen in (a) and (b). HNK-1 immunoreactive nerve roots sometimes appeared to cross the midline, as shown in (d). This i s probably due to the absence of the notochord. G = gut. bar = 100 pm. (From Stern et al., 1991

the direction of migration as well as being required for differentiation of certain phenotypes (Stem eta]., 1991).

VIII. SUMMARY AND FUTURE DIRECTIONS In summary, numerous cell surface and ECM molecules have been identified along neural crest migratory routes. Neural crest cells possess integrin receptors which are importantfor attachmentto a variety of extracellularmatrix molecules including fibronectin, laminin, and collagens. Perturbation studies suggest an important role for some cell-matrix interactions during movement along cranial neural crest

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pathways. It is unlikely that one ECM or cell surface component is uniquely responsible for promotion or inhibition of movement; rather, many macromolecules probably work in concert as cell and substratum adhesion-promoting molecules. There may be regional differences in the mechanisms underlying neural crest migration.Though cell-matrix interactions may dominate in the head region, tissue interactions appear to play a prominent role in trunk neural crest migration. The posterior somites and perinotochordal region appear to represent areas that are nonpermissivefor neural crest migration. If neural crest cells are able to invade all available spaces, such inhibitory cues may define neural crest migratory routes by default. Although this type of scenario does not rule out the possibility that various ECM molecules may represent the substrates on which neural crest cells migrate, it makes a guiding role for ECM molecules less likely in the trunk. Ultimately, we would like to address the role of cell surface and ECM molecules in vivo by performinga combinationof knock-out and over-expression experiments analogous to those elegantly performed in Dmsophilu and mouse embryos. Because aves have poor genetics, we will introduce ectopic genes into neural crest cells and their surroundingsby direct injection of constructs into single cells andor by lipofection of selected tissue regions (Holt et al., 1990). Similarly, it should be possible to knock-out mRNA encoding desired gene products by using anti-sense oligonucleotides;these have worked successfully in our tissue culture experiments. Such a fusion of molecular biology with classical embryologicalexperiments may make it possible to dissect the complicated events underlying neural crest cell migration.

ACKNOWLEDGMENT Parts of the work described in this review were supported by USPHS grant HD-15527.

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Bronner-Fraser, M. (1987). Perturbation of cranial neural crest migration by the HNK-I antibody. Dev. Biol. 123:321-331. Bronner-Fraser, M. ( 1988). Distribution and function of tenascin during cranial neural crest development in the chick. J. Neurosci. Res. 21:135-147. Bronner-Fraser, M., and Lallier, T. (1988). A monoclonal antibody against a lamininheparan sulfate proteoglycan complex perturbs cranial neural crest migration in vivo. J. Cell Biol. 106:1321-1330. Bronner-Fraser, M., and Stem, C. (1991). Effects of mesodermal tissues on avian neural crest cell migration. Dev. Biol. 143:213-217. Boucaut, J. C., Darribere, T., Poole. T.1.. Aoyama, H., Yamada, K. M., and Thiery, J. P. (1984). Biologically active synthetic peptides as probes of embryonic development: Acompetitive peptide inhibitor of fibronectin function inhibits gastrulation in amphibian embryos and neural crest migration in avian embryos. J. Cell Biol. 991822-1830. Bourdon. M. A,, and Ruoslahti, E. (1989). Tenascinmediatescell attachment through an RGD-dependent receptor. J. Cell Biol. 108:114%1155. Buck, C. A.. Shea, E., Duggan, K., and Horwitz, A. F. (1986). Integrin (the CSAT antigen): Functionality requires oligomeric integrity. J. Cell Biol. 103:2421-2428. Buck, C. A., and Horwitz, A. F. (1987). Integrin, a transmembrane glycoprotein complex mediating cell-substrate adhesion. J. Cell Sci. Suppl. 8:231-250. Bum, P., Kupfer, A., and Singer, I. C. (1988). Dynamic membrane-cytoskeletal interactionsspecific association of integrin and talin arises in vivo after phorbol ester treatment of peripheral blood lymphocytes. Proc. Natl. Acad. Sci. USA 85497-501. Chiu, A. Y., Matthew, W. D., and Patterson, P. H. (1986). A monoclonal antibody that blocks the activity of a neurite regeneration-promoting factor:studies on the binding site and its localization in viva J. Cell Biol. 103:1383-1398. Davies, J.. Cook, M., Stem, C. D., and Keynes, R. J. (1990). Isolation from chick somites of a glycoprotein that causes collapse of dorsal root ganglion growth cones. Neuron 4 11-20. Duband, J. L., and Thiery, J. P.(1987). Distribution of laminin and collagens during avian neural crest development. Development 101:461-478. Henning, A. K., and Schwartz, N. B. (1991). Characteristics of a large chondroitin sulfate proteoglycan associated with the notochord in 2- to 4-day old chick embryos. J. Cell Biol. 115731a. Holt, C. E., Garlick, N., and Cornel, E. (1990). Lipofedion of cDNAs in the embryonic vertebrate central nervous system. Neuron 4:203-214. Horwitz, A. F., Dugan, K., Greggs, R., Decker, C., and Buck, C. (1985). The CSATantigen has properties of a receptor for laminin and fibronectin. J. Cell Biol. 101:2134-2144. Hynes, R. 0. (1987). Integrins: a family of cell surface receptors. Cell 48549-554. Keynes, R. J., and Stem, C. D. (1984). Segmentation in the vertebrate nervous system. Nature 31Ct786-789. Krotoski, D., Domingo, C., and Bronner-Fraser, M. (1986). Distribution of a putative cell surface receptor for fibronectin and laminin in the avian embryo. J. Cell Biol. 103:1061-1072. Kruse, J., Mailhammer, R., Wenecke, H., Faissner, A., Sommer, I., Goridis, C., and Schachner, M. (1984). Neural cell adhesion molecules and myelin-associated glycoprotein share a common carbohydrate moiety recognized by monoclonal antibodies L2 and HNK-I. Nature (London) 311:153-155. Lallier, T., and Bronner-Fraser, M. (1988). A spatial and temporal analysis of dorsal root and sympathetic ganglion formation in the avian embryo. Dev. Biol. 12799-112. Lallier, T., and Bronner-Fraser, M. (1991). Avian neural crest cell attachment to laminin: involvement of divalent cation dependent and independent integrins. Development 113:1069-1084. Lallier, T., and Bronner-Fraser, M. (1993). Inhibition of neural crest cell attachment by integrin anti-sense oligonucleotides. Science 259692495. LeDouarin, N. M. (1982). The Neural Crest. Cambridge University Press. Cambridge.

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Loring, J., and Erickson, C. (1987). Neural crest cell migration pathways in the chickembryo. Dev. Biol. 121:230-236. Manasek, F. J., and Cohen, A. M. (1977). Anionic glycopepides and glycosaminoglycans synthesized by embryonic neural tube and neural crest. Roc.Natl. Acad. Sci. USA 7 4 1057-1061. Martins-Green, M.. and Erickson, C. A. (1986). J. Embryol. Exp. Morph. 98219-236. McClay, D. R., Wessel, G.M., and Marchase, R. B. (1981). Intercellular recognition: Quantitation of initial binding events. Roc. Natl. Acad. Sci. USA 784975-4979. Newgreen, D., and Gibbins, 1. (1982). Factors controlling the time and onset of migration of neural crest cells in the fowl embryo. Cell Tiss. Res. 22414S160. Newgreen. D.. and Gooday, D. (1985). Control of the onset of migration of neural crest cells in avian embryos: Role of Ca2+-&pen&nt cell adhesions. Cell Tiss. Res. 239329-336. Newgreen, D. F., and Thiery. J.-P. (1980). Fibronectin in early avian embryos: Synthesis and distribution along the migration pathways of neural crest cells. Cell Tiss. Res. 211:269-291. Newgreen, D. F. (1984). Spreading of explants of embryonic chick mesenchymes and epithelia on fibronectin and laminin. Cell Tiss. Res. 3236265-277. Newgreen, D. F., Scheel, M., and Kastner, V. (1986). Morphogenesis of sclerotome and neural crest in avian embryos: in vivo and in v i m studies on the role of notochordal extracellular matrix. Cell Tiss. Res. 244:299-313. Oakley, R., andTosney, K.W. (1991). Peanut agglutinin and chondroitin-&sulfate are molecular markers for tissues that a d as barriers to axon advance in avian embryos. Dev. Biol. 147 187-206. Pems, R., Paulsson, M., and Bronner-Fraser, M. (1989). Molecular mechanisms of avian neural crest cell migration on fibroneain and laminin. Dev. Biol. 136222-239. Perris, R., Krotoski, D., Domingo, C., Lallier, T., Sorrell, J. M.,andBronner-Fraser, M. (1991a). Spatial and temporal changes in the distribution of proteoglycans during avian neural crest development. Development II1:583-599. Pems, R., Krotoski, D., and Bronner-Fraser. M. (1991b). Collagens in avian neural crest cell development: distribution in vivo and migration-promoting ability in v i m . Development. (In press). Pettway, Z., Guillory, G.,and Bronner-Fraser, M. (1990). Absence of neural crest cells from the region surrounding implanted notochords in siru. Dev. Biol. 142:33>345. Pintar, J. (1978). Distribution and synthesis of glycosaminoglycans during quail neural mest morphe genesis. Dev. Biol. 67:444464. Pomeranz, H. D., and Gershon, M. D. (1991). Colonization of the avian hindgut by cells derived from the sacral neural crest. Dev. Biol. 137378-394. Poole, T., and Thiery, J. P. (1986). Antibodies and a synthetic peptide that block cell-fibronectin adhesion arrest neural crest cell migration in vivo. In: Progress in Developmental Biology, Part B, pp. 235-238. Ranscht, B., and Bronner-Fraser, M. ( ). T-cadherin expression alternates with migrating neural crest cells in the trunk of the avian embryo. Development 111:15-22. Riopelle, R. J. McGarry, R. C., and Roder, J. C. (1986). Adhesion properties of a neuronal epitope recognized by the monoclonal antibody HNK-I. Brain Res. 367:20-25. Rickmann, M., Fawcett, J. W., and Keynes, R. J. (1985). The migration of neural crest cells and the growth of motor axons through the rostra1 half of the chick somite. J. Exp. Morph. Embryol. 90437. Rovasio, R., Delouvee, A,, Timpl, R., Yamada, K., and Thiery. J. P.(1983). In virro avian neural crest cell adhesion and migration: requirement for fibronectin in the extracellular matrix. J. Cell Biol. 96:462-473. Runyan. R. B., Maxwell, G.D., and Shur, B. D. (1986). Evidence for a novel enzymatic mechanism of neural crest cell migration on extracellular glycoconjugate matrices. J. Cell Biol. 102:431-441. Serbedzija, G.,Bronner-Fraser, M., and Fraser, S. E. (1989). Vital dye analysis of the timing and pathways of avian trunk neural crest cell migration. Development 106:80&816.

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ENZYMES AND MORPHOGENESIS: ALKALINE PHOSPHATASE A N D CONTROL OF CELL MIGRATION

Saul L. Zackson

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 I. Introduction: Cell Migration and Cell-Cell Interactions in Development . . . . . . . . . . . . . . . . . . . . . 154 11. The Amphibian Pronephric Duct: A Model System for Studying Directed Cell Migration . . . . . . . . . . . . . . . . . . . . . . 157 111. ExperimentalManipulationoftheAmbystomuPronephricDuct.. . . . . . . 159 A. A Genetic Marker System Facilitates Observation of Cell Migration . . . 159 B. The Lateral Mesoderm Can Support Directed Migration of Pronephric Duct Cells . . . . . . . . . . . . . . . . . . . . 160 C. Chemotaxis Does Not Explain PND Cell Trajectories . . . . . . . . . . 161 D. The PND Guidance Information Can Be Recognized by Other Cells . . 161 IV. A Gradient of Adhesiveness Along the Pronephric Duct Pathway Best Accounts for the Observed Cell Behaviors . . . . . . . . . . . 167 V. Alkaline Phosphatase: A Molecular Marker for the Pronephric Duct Cell Guidance Information . . . . . . . . . . . . . . . . 170 A. Alkaline Phosphatase Displays a Patterned Distribution in the Axolotl Embryo . . . . . . . . . . . . . . . . . . . . 170 Advances in Developmental Biology Volume 2, pages 153-183 Copyright 0 1993 by JAI Press Inc. All rights of reproduction in any form reserved ISBN: 1-55938-582-0

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B. Overview of Alkaline Phosphatase Expression in Development . . . . . 171 C. Is Alkaline Phosphatase Functionally Involved in Guiding Migrating Cells? . . . . . . . . . . . . . . . . . . . 173 VI. Hypothesis: Enzymes that Modify Phosphorylation of Extracellular Pmteins Contribute to the Regulation of Cell-Cell Interactions . . . . . . . . . . . . . . . . . . . . . . 175 A. Cytokines.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 B. Proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 C. Extracellular Glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . 178 VII. Summary: Extracellular Phosphorylation and Modulation of Cell-Cell Interactions . . . . . . . . . . . . . . . . . . . . 179 Notes

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Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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PREFACE An analysis of the mechanism of guidance of a directed embryonic cell migration, that of the elongation of the amphibian pronephricduct, is presented in this chapter. Through the use of transplantation experiments, it is shown that the mechanism providing the guidance cues for the migration of pronephric duct cells has properties of a gradient of adhesiveness. Other potential guidance mechanisms, such as chemotaxis and contact guidance, cannot account for observed cell migration behaviors. Based upon the preferred migration trajectories of grafted cells, a map of the “guidance information” is presented. A molecule whose distribution closely corresponds to this map is identified as the cell-surface enzyme, alkaline phosphatase. Further experimental evidenceconsistentwith afunctional role for alkaline phosphatase in mediating pronephric duct cell migration is presented, and a speculative hypothesis in which enzyme modulation of the phosphorylation state of cell surface and extracellular phosphoproteins contributes to the regulation of cell-cell interactions is discussed.

1. INTRODUCTION: CELL MIGRATION AND CELL-CELL INTERACTIONS IN DEVELOPMENT The morphological transformation of an egg into an organism displaying complex three-dimensional form occurs in large part through the process of directed cell migration: cells receive signals from other cells and/or the extracellular matrix (ECM) that instruct them to start migrating from particular sites, to continue migrating along specific pathways, and finally to stop at appropriate destinations. Morphogenetic cell migrations can occur over distances far greater than the diameter of an individual cell, indicating that specific environmental cues are essential for navigation.

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As an embryonic cell migrates, changes occur along the cell’s surface. Within discrete domains of the plasma membrane, specific adhesions are established or broken with neighboring cells or the ECM. These changes occur continuously and simultaneously at different domains, so that at one point in time, a migrating cell is: (1) establishing new adhesive contacts at some sites; (2) maintaining such contacts at other sites; (3)releasing itself from adhesive contacts at yet other sites; and (4) elsewhere contacting other cells and/or ECM without establishing adhesions. Adhesions can vary in intensity, and can involveany of a number of adhesive receptor-ligand combinations. The localized, dynamic heterogeneity of the cell surface is essential to the motility of a cell, and involves the modulation of the localization and activity of adhesion molecules. Over the last few years, a large number of cell-surface molecules as well as ECM molecules participating in cell adhesion have been identified. Among the former are integrins, which function as cell receptors for ECM molecules as well as counter-receptors for other cell-surface molecules; cudherins, which mediate calcium-dependent adhesion through a homophilic mechanism (i.e., one cadherin molecule is the counter-receptor for another cadherin molecule); members of the immunoglobulin supefmily, which utilize homophilic as well as heterophilic binding mechanisms, and includes molecules such as NCAM and the fasciclins, which have important roles in the morphogenesis of the nervous system; selectins, which recognize specific carbohydrates; some glycosylfruns~eruses,which, in the absence of the nucleotide-sugar appropriate for monosaccharide addition, display lectin-like activity; and cell-sulface proteoglycuns, which interact with many ECM and cell surface molecules. [See Hynes and Lander (1992) for a concise review]. Many adhesion molecules are transmembrane proteins which interact with intracellular proteins via their cytoplasmic domains. For example, an integrin receptor for an ECM molecule will also interact with microfilament cytoskeleton via the proteins talin and vinculin. Immunofluorescence studies on migrating fibroblasts demonstrate that the major cell-substratum adhesion site (the “focal contact”) is also the site for localization of integrin, talin, vinculin, and c-src, as well as serving as points of attachment for F-actin filaments (“stress fibers”) and ECM molecules such as laminin or fibronectin. Thus, cytoskeletal, adhesive, and ECM molecules together form interacting systems with which cells respond to signals from the environment (Burridge et al., 1988). Directed cell migration involves coordinated changes in the adhesive activity of cell surface and cytoskeletal components. Appropriate adhesions are established, maintained, or broken in response to signals emanating from neighboring cells. The changes occurring in the surface domains of a migrating cell must be both rapid and reversible. An adhesion established at the leading edge of a migrating cell becomes located at the cell’s trailing edge as the cell advances; further cell advancement requires the release of the adhesion. For a 10 micron cell migrating at 1 microdminute. adhesions must be established, maintained, and released in an average time of 10

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minutes.Thus, the environmentalsignals (or “guidancecues”) that direct migration directly or indirectly cause local changes in the cell surface molecules that mediate adhesion. Cell responses to guidance cues involve local, rapid, and reversible changes in the cell membrane. Such responses potentially involve direct changes in the activity or distribution of the adhesion molecules themselves. Alternatively, the adhesion molecules can be indirectly affected, as a secondary consequence of a response of the cell to the migration guidance cues. Conceivably, proteins such as membrane ion channels or chemoreceptorsfor soluble extracellular molecules could mediate the primary responses to extrinsic signals, and in turn modulateadhesion molecules. For example, an environmental signal which causes local opening of an ion channel could in turn cause adhesion molecules to migrate within the plane of the plasma membrane and aggregate in the vicinity of the channel. Another class of molecules which could potentially modulate adhesion molecule distribution or activity consists of enzymes localized to the plasma membrane of the migrating cell, or to the migration substratum;’ that is, the extracellular matrix or the cells along which the directed migration occurs. Enzyme activity located on the surface of a migrating cell could be modulated in response to a guidance cue, and could represent an early step in the modulation of the cell’s adhesive behavior. Alternatively, enzyme activity localized to the migration substratumcould itself represent a guidance cue. Enzymes bound to the plasma membrane or to extracellular structural proteins (such as collagen) could locally regulate adhesion either directly or indirectly by modifying the covalent structure of an adhesion molecule, or an associated molecule. For example, an adhesion molecule synthesized in an inactiveprecursor form could be activated by a specific protease, which is itself activated or mobilized by an extrinsic signal. Alternatively, an adhesion-activatingprotease could be localized to the surface of a cell or ECM with which the migrating cell makes contact. The protease would be in a position to activate molecules locally on the surface of the migrating cell, and thus provide “guidance information.” In this connection, it is interestingto note that collagenase,plasminogen activator,and other extracellular proteases have been implicated in mediating cell migration and metastasis (Dan@ et al., 1985). Thus, an important unsolved problem of developmental biology is to identify the environmental signals to which migrating cells respond so that directed migration results. This chapter presents a review of the present state of knowledge regarding one particular embryonic cell migration--that of amphibian pronephric duct cells-and includes evidence that the cell surface enzyme, alkufinephasphutuse, contributes to the control of this migration. A hypothesis is developed that modulation of extracellular phosphorylation could be an important mechanism for regulating morphogenetic cell migrations and other cellcell interactions.

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II. THE AMPHIBIAN PRONEPHRIC DUCT: A MODEL SYSTEM FOR STUDYING DIRECTED CELL MIGRATION Studies on cell behavior in v i m have identified several kinds of mechanisms that could potentially contributeto cell guidance. Among these arechemotaris, in which cells migrate towards a source of diffusible chemoattractant (or away from a source of diffusible chemorepellant); contact guidance, in which cells track along mechanical anisotropies;popula tion pressure (also called contact inhibition),in which cells migrate away from a region of high cell population density; galvanom‘s, in which cells polarize and migrate with respect to an electric field; and haptotaris, in which intrinsic, preexisting differences in substratum adhesiveness per se provide directional migration cues. [SeeTrinkaus (1984) for an extensive review of mechanisms of cell guidance.] In v i m experimental tests of these potential guidance mechanisms primarily consist of creating an environment exhibiting a defined anisotropy, and observing the migration behavior of cells placed in that environment.While these experiments reveal the various sorts of anisotropies to which cells are capable of responding, they do not address the mechanisms actually operating in vivo, in that a single cell type is capable of responding to more than one potential source of guidance information. For example, neural crest cells will exhibit directed migration in response not only to electric fields, but also to population pressure and to artificial adhesive differentials (Nuccitelli and Erickson, 1983; Rovasio et al. 1983; Cooper and Keller, 1984;Erickson, 1985). Thus, in order to understand the guidance cues actually being utilized during morphogenesis, experiments in vivo are necessary. An experimental approach that permits analysis of the mechanisms of cell migration in vivo is that of an “elimination tournament” in which the various potential types of guidance cues all lead to testable predictions regarding the migration behavior of embryonic cells engaged in morphogenetic migrations. Experiments leading to observations that do not conform to one or more predictions demanded by a particular potential guidance mechanism can be used to judge that particular potential mechanism as unlikely to be contributing substantially to the guidance of the migrating cells. One system that is amenable to this experimental approach is the migration of cells of the amphibian pronephric duct (PND). The PND forms part of the embryonic renal system (the pronephros),having the prosaic function of carrying waste products to the cloaca for excretion, and is found as a temporary structure in all vertebrate embryos. The PND primordium first appears as a swelling in the mesoderm, just ventral to the anterior somites. In embryos of the salamanders Ambystoma mexicanum (the axolotl) and A. maculatum, this swelling is first observed shortly after the completion of neurulation and the appearance of the anterior somites at stage 22 (Fig. 1). Theprimordium extends for about five somite widths, and consists of 3000 to 4OOO cells, each cell about 10 microns in diameter. In apparent synchrony with the formation of somite fissures, which appear in an anterior to posterior progression, the cells of the PND primor-

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Figure 1. Scanning electron micrographs of Ambysroma mexicanum embryos fixed before peeling of ectoderm from the right side. The pronephric duct primordium i s visible as a mound, or swelling immediately ventral to the anterior somites. Arrows indicate caudal tip of pronephric duct primordia. Duct rudiment’s extension i s accompanied by the segmentation of additional somites and straightening of the embrtyonic axis. (A) stage 22; (B) stage 24; (C)stage 28; (D) stage 32. Staging i s according to Schreckenberg and Jacobson(1975). After Poole and Steinberg (1 981a).

dium migrate caudad. While migrating, the PND cells maintain contact with one another. Theprimordiumelongates alongthe margin between the somitesand the lateral mesodermso that its posteriortip is located ventral to the antepenultimatesomite, while the anteriorend remains anchored to the pronephros.This elongationof the primordium continues until the posterior tip reaches the target organ, the cloaca During PND cell migration, individual cells do not elongate, nor is there extensive cell division. Instead, the primordium undergoes a cell rearrangemerit: The PND primordium starts out short (approx. 900 microns) and wide (6 to 8 cells in width), and ends up long (approx. 2 mm) and narrow (approx. 2 cells in width). During this journey, which takes about 24 hours, cells at the posterior tip migrate at approximately 1 micron per minute. The rearrangement of the Ambystoma pronephric duct primordium has been demonstrated by vital dye marking experi-

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ments (Poole and Steinberg, 1981a). A dye mark placed across the posterior tip of the PND primordium moved caudally as it became longer and narrower. Because histological examination reveals neither significant shape change nor significant amounts of cell division, this experiment definitively demonstrates that directed cell migation is responsible for the morphogenesis of the Ambysfompronephric duct. As noted above, the pronephric duct is found in all vertebrate embryos. In all species examined, the same general scheme of morphogenesis is observed. A primordium forms from a mesodermal swelling ventral to the anterior somites, followed by its lengthening along a pathway defined by the margin between the somites and the lateral mesoderm (Poole, 1988; Lynch and Fraser, 1990). Differences between species are principally in the dimensions of the primordium, as well in the timing of elongation; the apparent synchrony of PND elongation and somitogenesis in Ambystomu is not observed in Xenopus luevis, chick, or sturgeon embryos (Poole, 1988). Several aspects of the elongation of the amphibian pronephric duct render it a useful system for studying the guidance mechanisms directing embryonic cell migration in vivo. Phenomenologically, it is a fairly simple example of a morphogenetic cell migration; a single primordium, consisting of a single cell type, is located at an easily observed position. Cells migrate in one direction along a single, well-defined pathway, towards a single, well-defined target. The migration is dramatic, on the order of 1 mm. Cell migration is easily observed, and this system is amenable to experimental manipulations.

111. EXPERIMENTAL MANIPULATION OF THE AMBYSTOMA PRONEPHRIC DUCT

A. A Genetic Marker System Facilitates Observation of Cell Migration

Not only is PND cell migration fairly simple phenomenologically, but also it is readily amenable to experimental analysis. Grafts between donor and recipient Ambystom embryos can be analyzed by scanning electron microscopy (SEM) or through the use of a visible marker system which clearly distinguishes donor from recipient tissues. These latter studies have made use of a genetic marker; amphibian embryos normally contain maternally provided melanin granules that are distributed among all the blastomeres. However, embryos spawned from genetically albino axolotl females contain no melanin, and so appear virtually white. Tissue can thus be transplanted between wild-type and albino embryos, with donor and recipient cells unambiguously labelled (Zackson and Steinberg, 1986) (Fig. 2).

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Figure 2. Migration of pronephric duct cells after transplantation to the lateral mesoderm. A pigmented wild-type pronephric duct primordium plus surrounding tissues (boxed),was grafted to the lateralmesodermof an albino recipient at stage 25. Pigmented donor pronephric duct cells that have emigrated from the graft are o n the lateral mesoderm and on the host pronephric duct pathway after fixation and peeling of the embryo at stage 32. Arrow indicates posterior tip of grafted pronephric duct primordiurn.

B. The Lateral Mesoderm Can Support Directed Migration of Pronephric Duct Cells As shown initially by Holtfreter (1944). and later by Poole and Steinberg (1982a,b), PND cells are capable of directional migration not only along their normal pathway, but also along neighboring lateral mesoderm tissue. In Poole and Steinberg’s experiments,pronephric duct primordia were cut out of donor embryos at stage 25 (along with neighboring somites, lateral mesoderm, and overlying epidermis) and grafted onto the lateral mesoderm of recipients of similar stage. PND cells migrated out of the primordia, streaming in a dorsocaudal direction on the host lateral mesoderm, and eventually reaching the host pronephric duct pathway. They then turned caudad, integrated with cells of the host PND, and continued on towards the cloaca. Thus, guidance information for directing PND cells is present not only along the PND pathway, but also on the neighboring lateral mesoderm. These observations have permitted the design of experiments that distinguish between the various potential mechanisms of cell guidance outlined

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above. In addition, these experiments, in demonstratingthat ectopicallyplaced cells will exhibit directed migration, provide an indication that the specificity of guidance cues need not be limited to the cells that comprise the normal migration pathway. C. Chemotaxis Does Not Explain PND Cell Trajectories

If chemotaxis provided key guidance cues, altering the position of the target should cause the migrating cells to divert from their normal trajectory, presumably towards the ectopically located target. With regard to the pronephric duct guidance information, the simplest arrangement involving chemotaxis would be for a chemoattractant molecule to diffuse from the target organ (the cloaca) so that a chemotactic gradient is established that attracts migrating cells towards the source. To test this possibility, a cloaca from a donor embryo was grafted onto the lateral mesoderm of a recipient embryo in which the cloaca (and surrounding tissue) was cut away. The PND cells ignored these changes, and continued to migrate along their normal pathway towards the cut edge. This experiment indicates that the position of the presumptive target does not alter the migration trajectory (Zackson and Steinberg, 1987). When a graft of pathway not containing any putative target tissue was positioned such that the PND cells encountered the grafted pathway after they commenced migrating, the cells chose to migrate along, rather than around, the grafted pathway, even though the latter choice would have enabled them to reach the target in response to a chemoattractant (Fig. 3). Ablations of dorsal, anterior, or ventral tissues that did not remove any PND cells or pathway had no affect on PND cell migration (Poole and Steinberg, 1981ab). None of the experimentalresults described above are consistent with the hypothesis that chemotaxis is the primary mechanism of guidance information. Instead, they are all consistent with mechanisms based primarily upon local cell-cell interactions; namely that changes in direction can only be effected by altering the cells’ migration substratum, and that changes effected by distant cells are not observed. Several possible mechanisms of cell guidance, including galvanotaxis, haptotaxis, population pressure, and contact guidance are consistent with cell responses to cues from the local environment, but make different predictions regarding cell behavior in other grafting experiments. These experiments are discussed below.

D. The PND Guidance information Can Be Recognized By Other Cells Cranial Neural Crest Cells Exhibit Directed Migration O n the Lateral Mesoderm and Pronephric Duct Pathway

As discussed above, PNDcells are not uniquely restricted to migrating only along the PND pathway; presumably the same sorts of guidance cues are also present on

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Figure 3. Pronephric duct pathway ectopically positioned so that it is encountered by migrating host pronephric duct cells. The pronephric duct cells have migrated onto the donor pathway, which does not terminate at a cloaca, rather than continuing towards the host cloaca. Line drawing indicates trajectory of host pronephric duct. Chemotactic mechanisms of cell guidance predict that the duct should migrate around grafted pathway and continue towards cloaca. Scale bar = 0.5 mm. From Zackson and Steinberg (1 987).

the lateral mesodem. A reciprocal question is whether the PND cell guidance information system is recognizable only to PND cells, or if other cell types are also capable of responding to this information. Convenient cells with which to address this question are cranial neural crest cells (CNCCs), which migrate at about the same time as PND cells, but in a different region of the embryo. CNCCs originate at or near the dorsal midline of the developing head, and migrate ventrally along specific pathways, contributing primarily to “mesectoderm” which differentiates into specific head cartilage structures (Horstadius, 1950).CNCCs are ectodermal in origin and do not encounter the pronephric duct or its pathway during normal

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development. CNCCs are readily obtainable; a simple cut through dorsal cranial tissue at stages 22-25 will yield a tissue piece containingpoorly motile neural tube and epidermis, plus hundreds or thousands of highly motile CNCCs. CNCCs are also easily identifiable, particularly when using the wild-type-to-albino marking system. CNCCs, like other neural crest cells, tend to migrate as loosely c o ~ e ~ t e d individuals (LeDouarin, 1982, 1984), thus avoiding the limitation of pathway choice imposed by the intrinsic cohesivenessof PND cells. To address the question of whether CNCCs are able to recognize and follow the PND guidance information, a graft containing CNCCs was transplanted to the lateral mesoderm, and the recipient’s pronephric duct was blocked from migrating by means of a deep incision from the dorsal midline through the presomite mesoderm and presumptive PND pathway (for reasonsto be discussedbelow). The CNCCs emigrated from the graft, migrating dorsccaudally upon the lateral mesoderm, then turned caudad upon reaching the host pronephric duct pathway (Fig. 4), migrating unidirectionallyalong the pathway towards the cloaca. Thus, CNCCs are capable of exhibiting directed migration in response to molecular signals presented by the same cells that are able to direct pronephric duct cell migration. This raises the possibility that the two cell types utilize the same molecular mechanisms of cell guidance in normal development. Cranial Neural Crest Cells Can Serve as Probes for the Distribution of the PND Cell Guidance Information

The tendency of CNCCs to migrate as noncohesive individuals enables the design of experimentsthat test some of the proposed mechanisms of cell guidance. In addition,because the guidance system appears to involve local cellcell interactions, the migration trajectories exhibited by transplanted CNCCs can be used for mapping the distribution of the guidance information.As will be discussed further, one interpretation of the map of guidance information is that it corresponds to a map of the distribution of a molecule involved in directing cell migration. CNCCs migrate bidirectionally along the pronephric duct itself. CNCCs grafted to the lateral mesoderm of otherwise normal embryos exhibited unexpected behavior. The CNCCs initially migrated unidirectionally towards the elongating pronephric duct; that is, dorsocaudally along the lateral mesoderm. Upon encountering the host PND, however, they spread bidirectionally, in contrast to their unidirectional migration along a PND pathway devoid of PND cells (Fig. 4). This bidirectional migration was often quite extensive, with CNCCs spreading over a distance approaching 1 mm (Fig. 5) (Zackson and Steinberg, 1986). These observations are not in concordance with the predictions of at least two of the major proposed mechanisms of cell guidance. The responses of migrating cells to chemotactic or galvanotactic guidance cues would always be unidirectional; gradients of chemoattractant (or chemorepulsive) molecules as well as electric

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Figurre 4. Caudad migration of pronephric duct and cranial neural crest cells along the pronephric duct pathway. (a). A pronephric duct rudiment grafted to the lateral plate mesoderm extends dorsocaudad on the lateral plate mesoderm and turns caudad on the duct pathway. (b)Cranial neural crest cells grafted to the lateral plate mesoderm behave in a similar manner. Narrow arrows indicate regions where grafted cells encounter the pronephric duct pathway and turn caudad (these regions shown at higher magnification in c,d). Wide arrows indicate locations of dorsal incisions blocking host pronephric duct elongation. Scale bar = 0.4 mm in (a, b); 0.1 mm in (c, d). From Zackson and Steinberg (1 986).

fields are, by definition, polarized, and can be considered as vector quantities. A constraint of any vector is that it can point in only one direction from any one place at any one time. The bidirectional migration of CNCCs along the pronephric duct primordium thus renders both chemotaxis and galvanotaxis as unlikely to be major sources of guidance information in this system. CNCCs migrate on the pronephric duct pathway in advance of the host pronephric duct. The apparent synchrony between the craniocaudal progression

of somitogenesis and the migration of PND cells during normal development has led to suggestions that the PND guidance information is expressed in a craniocaudally traveling “wave of change” along the PND pathway and lateral mesoderm,

Figure 5. Bidirectional migration of grafted cranial neural crest cells along the host pronephric duct. (a) Live albino embryo in which wild-type cranial neural crest cells migrate dorsocaudad from a graft and spread bidirectionally on the host pronephric duct. Arrow indicates graft, which includes epidermis, neural tube and neural crest. (b) The same embryo at higher magnification after fixation and local peeling of the ectoderm. Arrows indicatefurthest extents, both cephalad and caudad, of neural crest cell migration. Neural crest cells migrating in intersomitic fissures are also visible in regions of high cell density, particularly at anterior somites. Scale bar = 1 mm in (a), 0.4 mm in (b).From Zackson and Steinberg (1986).

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with the tip of the duct positioned near the “peak” of the putative wave (Poole and Steinberg, 1982b). A test of this hypothesis is to graft cells to the lateral mesoderm of young (stage 20) recipients; that is, recipients just completing neurulation. Such operations present the grafted cells with an opportunity to migrate along the pathway in advance of the host PND cells. If a traveling wave were responsible for directing cell migration, migration of grafted cells would not commence until the tip of the duct “caught up” with the grafted cells, so that both populations would be traveling along with the putative wave. When either CNCCs or PND cells were grafted to young recipients, they migrated along the presumptive PND pathway in the normal direction in advance of the host PND cells, indicating that the lateral mesoderm and pronephric duct pathway were expressing guidance cues prior to the elongation of the duct. These observations indicate that the migrating cells need not be synchronized with host PND cells, implying that it is not necessary to postulate a transient traveling wave of expression or activation of guidance information in order to account for pronephric duct cell migration (Zackson and Steinberg, 1986). An important implication of these observations is that the guidance information is already present before migration commences; that is, the guidance cues are preexisting, and are read or activated by the migrating cells. A “wave” model of cell guidance implies that the guidance information is expressed or is active only very transiently on the substratum, so a map of the guidance information would show a narrow window of expression around the position of the migrating tip on the PND pathway and lateral mesoderm. These observations instead lead to a map of the guidance information as extending along the entire length of the PND pathway (and neighboring lateral mesoderm), from the posterior tip of the PND primordium to the cloaca. CNCCs migrate unidirectionally from a grafted primordium. The potential mechanism of population pressure (or “contact inhibition”) for cell guidance implies that cells will scatter from a source in any directionpresenting a substratum permissible for migration. Grafts of CNCCs (or PND primordia) to the lateral mesoderm show that this is not the case; instead, emigration of CNCCs (and PND cells) from the graft is unidirectional. Thus, population pressure by itself can not account for the guidance of cells in this system. However, it remains possible that population pressure can affect the extent of migration of CNCCs. Although CNCCs migrate as loosely connected individuals, individual CNCCs located are skldom more than a few microns from other CNCCs (unpublishedobservation).This raises the possibility that the extent of CNCC migration is controlled, in part, by contact inhibition of migration by other CNCCs; otherwise, it would be expected that individual CNCCs would be able to migrate extensively without any neighbors present.

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IV. A GRADIENT OF ADHESIVENESS ALONG THE PRONEPHRIC DUCT PATHWAY BEST ACCOUNTS FOR THE OBSERVED CELL BEHAVIORS Many of the proposed potential mechanisms for cell guidance have generated testable predictions regarding how cells should behave in the pronephric duct system. In all cases but one, the prediction(s) of a particular strategy do not agree with the observed cell behaviors (Table 1). These mechanisms include some not considered in detail here, such as tissue-specific ligands (Moscona, 1974, 1980; Moscona and Hausman, 1977), passive movements (Bronner-Fraser, 1982), and “ballistics” (in which the orientation of the cells prior to migration determines their direction). The only strategy consistent with all observed cell migration behaviors is that of haptotaxis (Carter, 1967). In the simplest formulation of this model, migrating cells make and break adhesive connections with cells andor ECM along the migration pathway, translocating so that stronger adhesive connections survive at the expense of weaker ones. This strategy implies that preexisting differences in adhesiveness exist between cells of the migration substratum. In particular, a gradient of adhesiveness is predicted for the pronephric duct pathway, reaching a maximum at the cloaca. A gradient of adhesiveness is also predicted for the lateral mesoderm, increasing towards the pronephric duct pathway.

Table 1 . Summary of Observationson the Pronephric Duct Guidance Information Guidance Mechanism Prediction

Observation

Chemotaxis

Continued migration along pathway

Diversion towards ectopically located target Unidirectional migration of CNCCs on PND primordium Contact guidance Bidirectional migration along pathway Galvanotaxis Unidirectional migration of CNCCs on PND primordium Population pressure Omnidirectional emigration of (Contact inhibition) CNCCs from graft Passive migration Unidirectional caudad migration along primordium Ballistics Orientation of graft determines direction Tissue-specific ligands Migration only upon normal pathways

Bidirectional migration of CNCCs on PND primodium Unidirectional migration along pathway Bidirectional migration of CNCCs on PND primodium Unidirectional emigration of CNCCs from graft Bidirectional, (including “upstream”) migration along PND primodium Substratum determines direction irrespective of graft orientation PND cells migrate on lateral mesoderm. CNCCs migrate on PND primodium PND pathway, and lateral mesoderm

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’Iheabilityofthepronephncductp-imordiumtosupportbi~~CN~migration suggests a uniform distribution of guidance cues along the PND substratum. A fi,uther implication of this model is that cranial neural crest cells adhere better to the ectopic memdemd substrata presentedto them thanto each OdKI., becauseof the tendency of the

CNCCstomigrateasindividuals.Thus,accordingtothismodel,asaPNDcelloraCNCC migrates, i t receives, as a result of contact with preexisting molecules located on the migration substratum, signal(s) to establishadhesions at or near its leadingedge, as well as signal(s) to release adhesions towards its trailing edge. These signals would consist of Merentid strengthsof contactsmade betweensubstratum cells (orsubcellularmembrane domains). A stronger adhesion would lead to its maintenance, while a weaker adhesion would be released Comparativestrengthor weakness could be due to differences in local concentrations, activity, or availability of adhesion molecules. I n the case of the CNCCs migratingalong the pmnephric duct primotdium, one possibility i s that “contact inhibition” behavior, consisting of signals to release adhesions, is generatedby C N E s located behind the CNCCs in the vanguard of migration. One possible (but not exclusive) mechanism for generating the guidance cues implied by the haptotactic model for pronephric duct cell guidance i s that at Ieast one molecule, directly or indirectly involved in modulating adhesiveness, is distributed in the embryo i n a manner corresponding to the preferred migration trajectories o f grafted cells. I n particular, i t i s expected that such a molecule should be distributed: (1) as a gradient on the lateral mesoderm, increasingdormcaudally towards the pronephric duct pathway (due to the unidirectional dorsocaudal migration of cells grafted to the lateral mesoderm); (2) as a gradient on the PND

Figurn 6. (A) Expected distribution of a molecule mediating guidance of pronephric duct (PND) cells. The drawing represents an embryo in which the ectoderm has been peeled off to reveal the underlying mesoderm. The finger-shaped, elongating PND primordium lies immediately ventral to the somites. A molecule putatively involved in directing PND cell migration i s expected to be distributed uniformly on the PND, and on the lateral mesoderm as a gradient field increasing towards the pronephric duct pathway (the margin between the somite mesoderm and lateral mesoderm), and also increasing towards the cloaca. See text for explanation and details. (B-E) Albino axolotl embryos fixed, locally peeled of ectoderm, and stained for alkaline phosphatase activity. (B) Stage 27 embryo stained lightly for ALP activity. A gradient i s discernible along the PND pathway between the tip of the PND primordium and the cloaca. (C)Stage 27 embryo stained more heavily for ALP activity. A gradient is discernibleon the lateral mesoderm. [Other regions of the embryo exposed in peeled whoe mounts but not yet assayed for PND guidance information also exhibit ALP activity. These include the anterior somites, the trunk-level neural tube, the gill primordium, some head cartilage, and the eye primordium.] (D)Stage 3 0 albino embryo which had received a graft of a pigmented wild type PND primodiumat stage 25. The grafted PND has elongated on the lateral plate mesoderm and turned caudad on the pathway, migratingtowards regions of increasingALP activity. Pigmented cells

Figurp 6.(cmt.) on the dorsal and ventral sidesof the graft are donor-derived somite and lateral plate cells, respectively. The somite cells remain segregated from the host, whereas donor and host lateral mesoderm mix locally. Arrow parallels PND cell migration on the lateral mesoderm. (E)Stage 25 embryo which had received a graft of wild type anterodorsal ectoderm at stage 20. Cranial neural crest cell have migrated caudad upon the presumptive PND pathway, following the line of maximal staining. A few cells have also migrated cephalad upon the host PND itself, which also stains intensely for ALP. Arrow parallels migration of CNCCs along presumptive PND pathway. (F) Stage 27 embryo to which wild type anterodorsal ectoderm was grafted across the segmental mesoderm at stage 21. Arrows indicate CNCCs migrating along the trunk level neural tube. [Additional neural crest cells are migratingon the left side of the neural tube, out of view.] CNCCs also migrate extensively along the PND pathway, but not on the sigmental mesoderm. All figures are oriented with anteriorto the right Scale bar in (D) = 1 mm. From Zackson and Steinberg (1 988). 169

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pathway itself, increasing towards the cloaca (due to the unidirectional migration of cells along the pathway); (3) on the pronephric duct primodium, distributed uniformly (due to its ability to support bidirectional CNCCs migration); and (4) reduced or absent from the somitic mesoderm, at least in the vicinity of the tip of the pnd (as this region is not a preferred migration substratum). Such a molecule could itself be an adhesion molecule, or a molecule that modulates the activity, expression, or localization of adhesion molecules, as outlined in Section I. A map of the predicted distribution for such a molecule is presented in Fig. 6A (Section VI). Of course, many more complex mechanisms are possible. It is conceivable that multiple adhesion systems are involved, so that no one molecule maps in a manner corresponding to cell trajectories. Another possibility is that a ubiquitous molecule undergoes a shift in affinity for a counter-receptoror ligand, or undergoes a change in its membrane distribution, so that stronger adhesion sites are generated at sites of the molecule’s aggregation. Also possible is a mechanism involving “antiadhesiveness” (Calof and Lander, 1991);against a background of a ubiquitously present adhesion system, other molecules which interfere with adhesion could exhibit a patterned distribution.

V. ALKALINE PHOSPHATASE A MOLECULAR MARKER FOR THE PRONEPHRIC DUCT CELL GUIDANCE INFORMATION A. Alkaline Phosphatase Displays a Patterned Distribution in the Axolotl Embryo

Working under the assumption that the most likely distribution for a molecule involved in directing pronephric duct migration would be a membrane or ECM protein distributed in a manner corresponding to the map of preferred migration trajectories, an attempt was made to produce monoclonal antibodies against membrane antigens, and screen them through the use of whole-mount immunocytochemistry. Utilizing alkaline phosphatase-conjugated secondary antibodies for visualization of antibody localization, the first hybridoma supernatant tested revealed a staining pattern which corresponded closely to the expected pattern for the pronephric duct migration guidance information (Zackson and Steinberg, 1988). A series of controls led to the conclusion that what was being revealed was, in fact, endogenous alkaline phosphatase (ALP) activity. This activity also proved to be sensitive to the non-competitive,isozyme-specific inhibitor levamisole, indicating that the activity was likely to be the Ambystoma homologue to a particular isozyme, referred to as the “bonelliverkidney” (BLK) or “tissue nonspecific” isozyme in mammalian systems (McComb et al., 1979; Harris, 1982; Millin, 1990). Staining of embryos containing grafted cells further confirmed the correlation between the

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localization of ALP activity and preferred cell migration trajectories (Zackson and Steinberg, 1988).In these experiments,trajectoriesof grafted cells migrating along the pronephric duct pathway coincided with the line of maximal activity staining, delineating the boundary between the lateral mesoderm, which stains positively for ALP activity,and the somitic mesoderm, which stains poorly for ALP activity (Fig. 6B-E). Grafting of CNCCs to the trunk-level neural tube, a region strongly positive for ALP activity, but not previously recognized as a good migration substratum for CNCCs, indicated that, in fact, this tissue was preferred as a migration substratum over neighboring presomite mesoderm (Fig. 6F). It should be pointed out that the correspondencebetween ALP distribution and preferred migration trajectories is not absolute. For example, one region that does not support pronephric duct cell migration (Poole and Steinberg, 1982b), but that is strongly positive for ALPactivity, is the anterior lateral mesoderm; however, this region will support CNCC migration (Zackson and Steinberg, 1986). Together, these observations suggest that alkaline phosphatase activity by itself is not sufficient to constitute cell guidance information. These surprisingresults lead to several questions, among which are to determine if ALP is actually involved in directing cell migration, and if so, what sort of role it might have. Before these questions are addressed, however, a brief overview of alkaline phosphatase in development is presented in order to develop a context for the discussion that follows. (Amore extensive presentation of the biochemistryand distribution of this enzyme can be found in McComb et al., 1979.) B. Overview of Alkaline Phosphatase Expression in Development

Alkaline phosphatase is a hydrolytic cell-surface enzyme that exhibits a widespread, but patterned tissue distribution.2Vertebratescontain multiple isozymes of ALP, the number varying with species. In mammals,isozymes have been classified as the “tissue-specific”isozymes (including the intestinal, placental, and germ cell isozymes in humans) and the tissue nonspecific, or boneniverkidney (Bm) isozyme, which is present in other tissues as well. Each of these forms represents a different gene product (Millh, 1990), and posttranscriptional modifications, particularly variationsin glycosylation,generateeven more diversity (Harris, 1982; Moss and Whitaker, 1985).The BLK isozyme is commonly found to be the form expressed in embryonic tissues undergoing morphogenesis (McComb et al., 1979), and is the isozyme predominantly expressed in postimplantation mouse embryos (Hahnel et al., 1990).The expression of the BWK isozyme is highest in differentiated cartilage and bone, being three orders of magnitude greater than in other tissues (Kiledjian and Kadesch, 1991). The tissue-specific forms share greater sequence similarity to one another than to the tissue-nonspecific form (Millh, 1990). Expression patterns of ALP vary between vertebrate species. For example, humans express a placental isozyme, whereas other mammals express the B/WK

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isozyme in the placenta. Erythrocytesfrom some vertebrates,including amphibians and birds, express ALP, whereas mammalian erythrocytes do not. Although the enzymology of ALP has been extensively investigated,its function in developing embryos is not understood. Historically, ALP activity has been correlated variously with growth, differentiation, and cell migration (e.g., Moog, 1944; Hamburger, 1948; Mintz and Russell, 1957). It is known, however, that with few exceptions, ALP is localized to the outer surface of the plasma membrane of cells. One such exception is found in bone tissue, where ALP is both on the cell surface and in extracellular vesicles (Boyan et al., 1989). ALP is further restricted in its membrane distribution in that it localizes to apical surfaces in epithelia in which it is expressed (Lisanti et al., 1988). Some embryonic tissues, such as the developing central nervous system (CNS), retina, lens, olfactory epithelium, and limb bud mesenchyme of the mouse, express gradients of ALP. Within the intermediate zone of the developing CNS, where neuroblasts are actively migrating radially out of the ventricular zone towards the periphery of the neural tube, ALP activity appears arranged in radial fibers with punctate localizations (Kwong and Tam, 1984; and unpublished observations). These fibers probably correspond to the radial glia known to serve as the migration substrata for migrating neuroblasts (Rakic, 1990), as silver staining for neuronal fibers in the same tissue produces an entirely separate pattern (Kwong and Tam, 1984; Tam and Kwong, 1987). The apical ectodermal ridge of limb buds, which is known to have a profound influence on the development of the underlying mesenchyme (Fallon et al., 1983),is strongly positive for ALP activity in the mouse embryo (unpublished observation). A population of migratory cells strongly positive for ALP are the primordial germ cells of mice (Mintz and Russell, 1957). In amphibians, ALP is expressed in a gradient in both developing and regenerating limb blastemas (Karczmar and Berg, 1951). Immunofluorescencestudies have confirmed the cell surface localization of ALP, and have also demonstrated a punctate distribution for ALP on the cell; utilizing indirect immunofluorescence, Berger et al. (1987) reported “definite antigenic clusters” of human placental alkaline phosphatase. Alkaline phosphatase is anchored to the outer surface of cell membranes by a phosphatidylinositol-glycan (PI-G) anchor (Low and Zilversmit, 1980; Low, 1989), and is also sometimes found extracellularly; for example, in serum. The phosphatidylinositol-glycananchor is subject to hydrolysis by the enzyme phosphatidylinositol-specific phospholipase C (PIPLC), and treatment of cells with PPLC in virro will effect release of alkaline phosphatase into the medium (Low, 1987). The phosphatidylinositol-glycananchor affects the targeting of the enzyme; the presence of a PI-G anchor correlates with the apical localizationof cell-surface proteins in polarized epithelial cell monolayers (Lisanti et al., 1988). ALP has two major catalytic activities in vitro: (1) ALP catalyzes the removal of phosphate monoesters from a large number of substrates, and (2) it can also act as a phosphotransferase. Substrates can be defined as R-OP03, where R represents a

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wide variety of moieties, ranging in size from small molecules such as glycerophosphate, to nucleic acids and phosphoproteins. Isozymes of ALP exhibit differ~ ~ various ences from one another primarily with respect to KM and V M for substrates. All three amino acids which are commonly phosphorylated by protein kinases, namely serine, threonine, and tyrosine, can be dephosphorylatedby ALP. Activities for ALP not related to phosphorylation are possible. Millh (1990) has suggested that ALP has collagen-bindingactivity by virtue of sequence homologies between placental ALP, germ cell AW, and the collagen-binding proteins von WillebrandFactor and Cartilage Matrix Protein. From these considerations,as well biochemical measurements of ALP-collagen binding, a structural role for ALP has been suggested. Such a structural role by no means excludes an enzymatic function. To the contrary, binding of ALP to extracellular matrix molecules could be integral to the proper functioning of the molecule, in that substrate specificity could derive from appropriate presentation of substrates, rather than from a preference for particular phosphorylated molecules. This possibility will be discussed further below. ALP may also modulate cell behavior via its phosphatidylinositol-glycanmembrane anchor. Release of ALP from the cell surface by PIPLC would also release diacylglycerol, a known activator of protein kinase C (Cross, 1987; Low, 1987). The inositol-glycan moiety of the P I G anchor of alkaline phosphatase also potentially functions as a “second messenger” for insulin stimulation (Romero et al., 1988). In summary, alkaline phosphatase is expressed on the cell surface, and shows patterned distributions in developing embryos. Its cellular and tissue localization patterns, along with its known enzymatic and binding activities, are compatible with it having a role in modulating or mediating cell-cell interactions in development. C. Is Alkaline Phosphatase Functionally Involved in Guiding Migrating Cells?

One possible way to test if ALP provides directional guidance cues in development is to examine the consequences of perturbing its expression or activity. Chemical inhibitors (of which there are many) would seem to provide the obvious choices for reagents. On closer examination, however, chemical inhibitor studies on embryonic function are subject to two major caveats. First, one must be sure that the inhibitor is getting to the relevant sites in the embryo and inhibiting ALP activity under in viva conditions. Second, one must be sure that the inhibitor is specific for ALP. While the first condition may or may not be amenable to fulfillment, the second condition inherently is not. Traditional inhibitor experiments are thus difficult to interpret, due to the possibility of side effects. Another approach involving a compromise between use of small molecule inhibitors, and more specific reagents (such as antibodies or anti-sense synthetic

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transcripts) was to exploit the susceptibility of the phosphatidylinositol-glycan membrane anchor of ALP to hydrolysis by the enzyme PIPLC (Zackson and Steinberg, 1989).This approach permits experimentsin which alkalinephosphatase is enzymatically released from cell surfaces, but has the disadvantage that phosphatidylinositol-glycan anchored membrane proteins other than ALP that are released by P I P E treatment could potentially be the relevant target if migration is disrupted. However, in comparison to the use of small molecule enzyme inhibitors, this approach offers the advantage that the unknown molecules released by P I P E treatment comprise a limited set of cell-surface proteins, which in principle could be identified. In order to test if PIPLC treatment affected pronephric duct cell migration, a slow-release system made from covalently modified polyacrylamide (“hypa beads”), was used to apply PIPLC to stage 24 embryos (Zackson and Steinberg, 1989). In these experiments, single beads l ~ ad e dwith P I P E were placed on the presomitic mesoderm in proximity to the pronephric duct pathway. The embryos were permitted to develop to stage 30-36, then fixed and processed for histochemical staining for ALP activity. Pronephric duct cell migration was entirely inhibited on the operated side of each embryo (26/26 cases examined). Histochemical staining of the embryos for ALP activity indicated virtually no activity in the vicinity of the beads, and greatly diminished activity elsewhere. An important control for nonspecific disruption of cell metabolism was provided in these experiments by the unimpaired formation of somite fissures (which normally occurs with little or no ALP present on the cells) in spite of the P I P E treatment. Thus, the inhibition of pronephric duct cell migration correlated with the release of ALP by PIPLC, while at least one other morphogenetic activity was not affected. This experiment, of course, does not definitively demonstrate an active role for ALP in directing pronephric duct cell migration. The PI-G anchor is used by a growing list of membrane proteins, including developmentallyrelevant molecules such as N-CAM 120 and other members of the immunoglobulin superfamily (Goridis and Wille, 1988; Gennarinni et al., 1989); and several heparan sulfate proteoglycans (Herndon and Lander, 1990),including a low-affinity cell receptor for basic fibroblast growth factor (Brunner et al., 1991). Thus, it is possible that PI-G anchored molecule(s) other than (or in addition to) ALP is/are the relevant target(s) for the PIPLC in these experiments. It is interesting to note that PND cell migration is not the only morphogenetic movement perturbed by PIPLC treatment. In particular, the elongationof the lateral mesoderm is inhibited, so that PIPLC-treated embryos appear shorter than their control sibs. While this elongation process is not well understood, it probably involves an active cell rearrangement of the lateral mesoderm, so that the tissue becomes longer and narrower. The effects of PIPLC on this morphogenetic movement suggest that mesoderm elongation also depends upon PI-G anchored molecules.

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Recent evidence (presented in abstract form) demonstrates that a monoclonal antibody that inhibits axolotl ALP activity,will, when applied to embryos utilizing the hypa bead polyacrylamide bead system, retard pmnephric duct cell migration (Drawbridge et al., 1991).This enmuraging result paves the way for a definitivedemonstration of possible involvement of ALP in directingpronephric duct cell migration. Another approach to determining if ALP is functionally involved in directing embryonic cell migrations is to generate transgenic mice which ectopically express the enzyme. However, efforts to produce such mice have been mostly unsuccessful. The only strains produced to date which express the ALP transgene show elevated levels of germ cell ALP expression in mouse intestine and serum (Millib, 1990). We have attempted to produce transgenics carrying various heat-shock promoteralkaline phosphatase cDNAfusions (Zackson et al., 1990).So far, in well over 100 potential founder mice screened,we have only obtained two transgenic strains, and we have been unable to induce expression in either strain. These two nonexpressing strains were produced from eggs injected with linearized plasmids rather than with a restriction fragment without vector sequences. Plasmid vector sequences are known to inhibit expression of transgenes, and probably account for the lack of expression. We interpret these negative results as suggesting that our constructs, when injected free of vector sequences, are usually expressed at low levels even under noninducing conditions, and that this expression is sufficient to confer embryonic lethality.

VI. HYPOTHESIS: ENZYMES THAT MODIFY PHOSPHORYLATION OF EXTRACELLULAR PROTEINS CONTRIBUTE TO THE REGULATION OF CELL-CELL INTERACTlONS The large number of potential substrates, as well as the widespread but patterned expression of ALP, makes correlationof expression with any particular cell function difficult. Because of the complexity of expression patterns, particularly the tissuespecific waxing and waning of activity observed during development, and the variation of expression levels over orders of magnitude, it is difficult to conclude that ALP has only a “housekeeping” function. A major difficulty, if not frustration, in trying to understand the function of alkaline phosphatase in embryos has been that at a local scale, ALP might seem to correlate with an observable activity (for example, the correlation with neuroblast migration within the CNS), but such correlations are not consistent with all patterns of expression. An hypothesis that takes into account both the distribution and range of activities of ALP is that ALP is involved in intercellular communication, with the substrates consisting chiefly of extracellular and cell-sudacephosphoproteins. An implication of this hypothesis is that in terms of observablecell activities, ALPS cell “function” in any particular instance is dependent upon the history and state of

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the cells, including (but not limited to) the particuky molecules presented as substrates. ALP, according to this hypothesis, resembles the cytokines, which are regarded as messenger molecules whose function can only be understood in terms of individual contexts (Nathan and Sporn, 1991); depending upon the details of particular systems, cytokines can stimulate or inhibit proliferation, and stimulate or inhibit differentiation. In brief, the molecule is the messenger, but not the message. Thus, attempts to correlate the presence of such molecules with particular cell responses can lead to confusing, even apparently contradictory, results. Given the large number of potential substrates for ALP (including the cytokines themselves), it is not surprising that a “function” for ALP has been so elusive. Thus, I am proposing a more generalized function, namely modulation of extracellular phosphorylationof proteins, particularly those proteins mediating cell adhesion and other cell-cell interactions. Phosphorylation has long been recognized as a mechanism for regulating intracellular activities, and recent years have seen an explosion of data regarding intracellular phosphorylation. Recently, intracellular dephosphorylation has been recognized as an important control mechanism (e.g., Tonks and Charbonneau, 1989; Dunphy and Kumagai, 1991; Gautier et al., 1991). Dephosphorylation has as much potential as phosphorylation for controlling cellular activities; in the simplest form, phosphorylationand dephosphorylation can be viewed as alternative states of a binary molecular “switch”; depending upon the particular molecule involved, “on” (or “activated”) status could be represented by either the phosphorylated or nonphosphorylated state. Thus, two potential control mechanisms involve toggling such a switch on, or toggling it off, that is, phosphorylating or dephosphorylatinga molecule. Any amount of complexity potentially can be added to such a switch mechanism; for example,multiple phosphorylationsites, multimer formation, cooperativity, cascades, and positive or negative feedback. One important implication is that kinases, transphosphorylases, and phosphatases together allow for reversible modification of macromolecules. Such reversibility could be important for a migrating cell as it makes and breaks adhesions. Additional implications of enzymatic modification of phosphorylation of extracellular proteins is that such modification potentially can be localized to small domains, and can be effected rapidly. These considerationsof such enzymatic activities potentially make them extremely important in the regulation of cell-cell interactions in migrating cells. Many intracellular regulatory kinases and phosphatases have been identified, along with a large number of substrate proteins. A simple hypothesis that follows from known intracellular phosphorylation and dephosphorylation is that such reactions also occur extracellularly. The most obvious candidate substrates for modulated phosphorylation and dephosphorylation are extracellular and cell surface phosphoproteins, particularly those phosphoproteins known to be mediators of cell adhesion and other cell interactions. Modulation of extracellular phosphorylation presumes a source of phosphoproteins, as well as ectophosphatases,such

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as alkaline phosphatase. The sources can be the substrates of ectokinases or transphosphorylases,as well as proteins phosphorylated prior to secretion. Several secreted extracellular-matrix phosphoproteins of known importance to cellcell interactions in developments have already identified. In some cases, their activity is modulated by their phosphorylation state, and in other the proteins are known to be in vitro substrates for dephosphorylation by alkaline phosphatase. Some extracellularphosphoproteins which could be relevant in mediating directed cell migration are described below. A. Cytokines

Cytokines (including “growth factors”) are ECM-associated intercellularsignaling proteins, acting nonenzymatically in picomolar to nanomolar concentrations, “whose physiologic role is to coordinate the modeling and remodeling of tissues” (Nathan and Sporn, 1991). The cytokine basicfibroblastgrowthfactor (bFGF) is a secreted, matrix-bound phosphoprotein whose biochemical activity is modulated by phosphorylation. Phosphorylated bFGF has greater potency at displacing I’=-labeled bFGF from its cell receptor, compared to the nonphosphorylated form (Feige and Baird, 1989; Feige et al., 1989). These observationsraise the possibility that phosphorylationl dephosphorylation is involved in the presentation of bFGF or other cytokines to their cell receptors; a change in phosphorylation could cause the release, enhancement, or inhibition of a cytokine’s interaction with its receptor either directly or indirectly through modification of a molecule to which the cytokine is bound (see below). With regard to adhesiondirected cell migration, local dephosphorylation of a cytokine could, in principle, lead to transduction of a signal that causes migrating cells (or cells along the substratum) to establish or release adhesions. As secreted cytokines are usually bound to extracellular matrix, these molecules could provide local guidance information to migrating cells. In addition to bFGF, other cytokines, including transforming growth factors TGFalpha and TGF-beta, insulin-like growth factor-I, and tumor necrosis factor, are in vim substrates for protein kinase C and/or protein kinase A (Feige et al., 1989),raising the possibility that bFGF is just one example of a phosphorylated cytokine.

6. Proteoglycans There has been at least one report of phosphorylation of the proteoglycan dermatan sulfate on a carbohydrate moiety (Gloss1 et al., 1986). The function of this phosphorylation is unknown. Because proteoglycans participate in cell adhesion and play a central role in the presentation of cytokines to their cell receptors, a potentially important role of phosphorylation is to modulate the interactions of proteoglycans, cytokines, and their cell receptors.

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C. Extracellular Glycoproteins

The activity of extracellular glycoproteins could be modified by phosphorylation. For example, the matrix molecule, ostmpontin (OP), is highly phosphorylated (Butler, 1989).OP(or its homologues) is secreted by several mammalian cell types, and has multiple phosphorylation sites. OP has many other names because of independent discoveries, and is a major secreted product not only of osteoblasts, but also of many transformed cell lines (Senger et al., 1979; Senger and Permzzi, 1985; Craig et al., 1988; Butler, 1989). OP can be dephosphorylated by ALP treatment at physiological pH (Nemir et al., 1989).It also contains an RGDS amino acid motif, and its adhesive activity can be competed by an RGDScontaining peptide, suggesting that it binds to cells via an integrin receptor (Oldberg et al., 1986). At least one cell line (normal rat kidney cells) secretes OP in both phosphe rylated (PI3.8) and nonphosphorylated (pI4.5) forms (pp69 and np69, respectively) (Nemir et al., 1989). Phosphorylation modulates the activities of osteopontin; pp69 shows a high affinity for the cell surface, whereas np69 complexes with plasma fibronectin (Nemir et al., 1989). These properties suggest yet another potential mechanism for regulation of cell-cell interactions by alkaline phosphatase; localized modification of phosphorylation of a matrix molecule such as OP could affect its adhesive properties, including its integrin affinity, thus providing a molecular mechanism for haptotaxis. Another phosphorylated molecule central to cell migration and adhesion is fibronectin (HynesJ990, and references therein). This molecule binds to cells via an integrin cell receptor, and also binds to heparin, collagen, osteopontin, and several other proteins. Both cellular and plasma forms of fibronectin are phosphurylated. No functional significance has yet been attributed to the phosphorylation of fibronectin. There are probably many extracellular phosphoproteins yet to be identified. In preliminary experiments in my laboratory, incubation of a Xenopus cell primary fibroblast cell line with 32P-phosphate yielded numerous (>25) radiolabeled phosphopeptides in the culture supernatant, ranging in molecular mass from about 14 kDa to >200 kDa, as visualized by SDS-polyacrylamide gel electrophoresis and autoradiography. The profile of labeled phosphopeptides from culture supernatant did not correspond to that of cells, indicating that the supernatant phosphopeptides are not simply from lysed cells. Many of these bands diminished in intensity or disappeared entirely upon treatment of culture supernatant samples with calf intestinal alkaline phosphatase. Identification of some of these polypeptides is currently underway. The phosphorylation of these molecules could occur either before or after secretion. Although ectokinase activity has been difficult to demonstrateunambiguous demonstration will probably require molecular identification of an ectokinase molecule, because of the presence of potential background activity from

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cytoplasmic kinases-it is interesting to note that the phosphotransferase activity of ALP could, in principle, transfer phosphates onto extracellularproteins. Such an activity would imply that ALP could be used to modulate phosphorylation both positively and negatively, leading to further complexity in terms of requiring a contextual understanding of ALP’S function.

VII. SUMMARY: EXTRACELLULAR PHOSPHORYLATION AND MODULATION OF CELL-CELL INTERACTIONS A fundamental problem of embryonic morphogenesis is to understand the highly regulated intercellularand cell-ECM interactionsproviding the “guidanceinformation” controlling directed cell migrations. As an embryonic cell migrates, signals from the environment specify the cell’s direction and destination. This signaling can be seen as resulting from the integration of information passed between cells via direct cell contact andor contact with the ECM. One example of an embryonic cell migration, that of the elongation of the amphibian pronephric duct, behaves as if the guidance information presented by the environment consists of a gradient of adhesiveness along the migrating pronephric duct cells’ substratum. A cell surface enzyme, alkaline phosphatase, was found to be distributed in the embryo in a manner corresponding to the expected distribution for a molecule contributing to the pronephric duct cell migration guidance information. It is proposed that modulation of phosphorylation of extracellular proteins represents one mechanism by which cell-cell interactions are controlled during development, and that alkaline phosphatase provides a component of this putative regulatory mechanism. Potential extracellularphosphoprotein substrates, including adhesion molecules and cytokines, are present in developing and adult tissues, and alkaline phosphatase is expressed at appropriate times and places to mediate cellcell interactions through modulation of the phosphoryIation state of the extracellular phosphoproteins. Several aspects of the biology of cell migration make enzymatic modifications in general, and enzymatic modification of phosphorylation in particular, attractive mechanisms for regulating morphogenetic cell-cell interactions. Many of a cell’s responses to its environment as it migrates must be rapid, reversible, and localized to small domains within the plasma membrane. Enzymes bound to the surface of a migrating cell, or to cells comprising the migration substratum, could be ideally situated for such purposes; for example, a phosphorylation event that “activates” an adhesion molecule could be an essential step in the establishmentof an adhesion between a migrating cell and its substratum, perhaps through the triggering of a cascade of further modifications. The subsequent release of a localized adhesion could involve the dephosphorylation of the same molecule. If ALP is indeed contributing to the regulation of the complex processes of cell migration and other cell-cell interactions, substrate specificity is likely to be

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generated through mechanisms that restrict the availability or presentation of the enzyme to particular substrates.AlargeECM molecule or cell surfaceprotein might be subject to modification by ALP only under specific conditions that allow or promote enzyme-substrate complex formation. A similar multistep mechanism is found in the binding of cytokines with proteoglycans prior to binding with signaltransducing receptors. One implication of enzymatic regulation of extracellular and cell surface phosphorylation, as presented here, is that the metastatic behavior of cancer cells could include abnormalitiesin the expression of these enzymes. Such abnormalitiescould lead to alterations in a metastatic cell’s adhesive activity, which in turn could affect invasiveness. Understandingextracellularphosphorylation,its control, and its roles in cell-cell interactions, and the potential involvement of ALP in these processes present major challenges for future studies.

NOTES 1. As this review deals with both cell migration and enzymes, the term “substratum” will be used in reference to surfaces involved with cell migration whereas “substrate” will be used in reference to molecules modified by enzymes. 2. The adjective “ubiquitous” has sometimes been applied to ALP; while it might possibly be found in all organisms, it is probably not ubiquitous to all cells or tissues.

ACKNOWLEDGMENTS Much of this work was conducted while a postdoctoral fellow in the laboratory of Malcolm Steinberg, Princeton University and in the laboratory of Ralph Greenspan, Roche Institute ofMolecular Biology. The oppoxtunities to work in these laboratories are gratefully acknowledged. My laboratory is supported by grants from the Alberta Heritage Foundation for Medical Research and the Alberta Cancer Board. I am pleased to acknowledge helpful discussions with many colleagues, particularly Tim Karr, M. John Anderson, and Wendy Dean during the preparation of this manuscript.

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Nuccitelli, R., and Erickson, C. A. (1983). Embryonic cell motility can be guided by physiological eledric fields. Exp. Cell Res. 147 195-201. Oldberg, A., FranzCn, A.. and HeinegArd, D. (1986). Cloning and sequence analysis of rat bone sialoprotein(osteopontin)cDNArevealsan Arg-Gly-Asp cell-bindingsequence. Roc. Natl. Acad. Sci. USA 83:8819-8823. Poole, T. J. (1988). Cell rearrangement and directional migration in pronephric dud development. Scanning Microscopy 2: #I, 411415. Poole, T. J., and Steinberg, M. S. (1981a). Amphibianpronephric duct morphogenesis:segregation, cell rearrangement and directed migration of the Ambystoma duct rudiment J. Embryol. Exp. Morph. 63:l-16. Poole, T. J., and Steinberg, M. S. (1981b). Strategies for specifying form and pattern: adhesion-guided multicellular assembly. Phil. Trans. Roy. Soc. Lond. B 29945 1-460. Poole, T. J., and Steinberg, M. S. (1982a). Cellular adhesive differentials as determinants of morphe genetic movements and organ segregation. In: Developmental Order: Its Origin and Regulation, pp. 351-378. Alan R. Liss, New York. Poole, T. J., and Steinberg, M. S. (1982b). Evidence for the guidance of pronephric duct migration by a craniocaudally traveling adhesion Gradient. Dev. Biol. 92:144-158. Rakic, P. (1990). Principles of neural cell migration. Experientia 46882-891. Romero, G., Luttrell. L., Rogol, A., Zeller, K. Hewlett, E., and Lamer, J. (1988). Phosphatidylinositolglycan anchors of membrane proteins: potential precursors of insulin mediators. Science 242509511. Rovasio, R. A,, Delouvee, A,, Yamada, K. M., Timpl, R., and Thiery, J. P. (1983). Neural crest cell migration: requirements for exogenous fibronectinand high cell density. J. Cell Biol. 96462473. Schreckenberg, G. M. and Jacobson, A. G. (1975). Normal stages of development of the axolotl, Ambystoma mexicanum. Dev. Biol. 42:391-400. Senger, D. R., and Permzzi, C. A. (1985). Secreted phosphoproteinmarkers for neoplastic transfomtion of human epithelial and fibroblastic cells. Cancer Res. 45:5818-5823. Senger. D. R., Wirth, D. F.,and Hynes. R. 0. (1979). Transformed mammalian cells secrete specific proteins and phosphoproteins.Cell 16:885-893. Tam, P. P.L.,andKwong, W. H.(1987). Astudyon thepatternofalkaline phosphataseactivitycorrelated with observations on silver-impregnatedstructures in the developing mouse brain. J. Anat. 1.50: 169-1 80. Tonks, N. K., and Charbonneau,H. (1989). Protein tyrosine dephosphorylationand signal transduction. Trends in Bicchem. Sci. 14497-500. Trinkaus, J. P. (1984). Cells Into Organs, second edition. Prentice-Hall,Englewood Cliffs, NJ. Zackson, S. L., and Steinberg, M. S. (1986). Cranial neural crest cells exhibit directed migration on the pronephric duct pathway: futher evidence for an in viva adhesion gradient. Dev. Biol. 117: 342-353. Zackson, S. L., and Steinberg, M. S. (1987). Chemotaxis or adhesion gradient? Pronephric duct elongation does not depend on distant sources of guidance information. Dev. Biol. 124418422. Zackson, S. L., and Steinberg, M. S. (1988). A molecular marker for cell guidance information in the axolotl embryo. Dev. Biol. 127435-442. Zackson, S. L., and Steinberg, M. S. (1989). Axolotl pronephric duct cell migration is sensitive to phosphatidylinositol-specificphospholipaseC. Development 105:1-7. Zackson, S. L., Greenspan, R. J., Berger J.. and O’Brien, M.C. (1990) Production of transgenic mice containinga cDNAcoding for an alkaline phosphatase under heat shock promoter control. J. Cell. Biochem. 14E130.

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INDEX Aarskog-Scott syndrome, 48 Adhesion molecules, 155, 178-179 fibronectin, 178 neural crest cells, 131-132 osteopontin (OP), 178 Adrenoleukodystrophy, 47 Agammaglobulinemia, 47 Aicardi syndrome, 47 AIS9T, 48-50 Alkaline phosphatase, control of cell migration and, 153-183 adhesiveness, gradient of along PND pathway, 167-170 observations on guidance information, summary of, 167-168 Ambystoma pronephric duct, experimental manipulation Of, 159-166 and chemotaxis, 161 cranial neural crest cells, (CNCCS), 162-166 guidance information, 161-166 lateral mesoderm, 160-161 marker system, genetic, 159 melanin, 159 mesectoderm, 162 other cells, PND guidance information recognized by, 161-166 185

scanning electron microscopy (SEM), 159 hypothesis, 175-179 adhesion molecules, 178-179 cytokines, 177 (see also “Cytokines”) dephosphorylation, 176-178 fibronectin, I78 intercellular communication, involvement in, 175-179 osteopontin (OP), 178 phosphorylation, 176-178 proteoglycans, 177 introduction, 154-156 cadherins, 155 directed cell migration, 154-155 environmental signals for migrating cells, problem of, 156 extracellular matrix, 156 fasciclins, 155 glycosyltransferases, 155 integrins, 155 NCAM, 155 proteoglycans, 155 selectins, 155 as molecular marker, 170-175 B / L / K isozyme, 170-171

186

in bone tissue, 172 in development, 171-173 diacylglycerol, 173 enzymatic function, 173 guidance of migrating cells, 173175 immunofluorescence studies, 172 levamisole, 170 patterned distribution in axolotl embryo, 170-171 phosphatidylinositol-glycan (PIG), 172, 174 phosphatid ylinositol-specific phospholipase C, 172, 174 PIPLC, 172, 174 radial glia, 172 structural role, 173 through transgenic mice, 175 preface, 154 pronephric duct (PND), amphibian, as model for directed cell migration, 157-159 Ambystoma mexicanum, 157158 chemotaxis, 157, 167-168 contact guidance, 157, 167-168 contact inhibition, 157, 166, 167-168 galvanotaxis, 157, 167-168 haptotaxis, 157, 167-168 population pressure, 157, 166, 167-I68 rearrangement of cells, 158 in all vertebrate embryos, 159 in vivo experiments essential, 157 Xenopus laevis, 159 summary, 179-180 Alpha lactalbumin, 141 Alport syndrome, 47 Ambystoma mexicanum, 157-158 (see also “Alkaline phosphatase.. .”)

INDEX

and chemotaxis, 161 cranial neural crest cells, 162-166 lateral mesoderm, 160-161 marker system, genetic, 159 melanin, 159 rnesectoderm, 162 other cells, PND guidance information recognizable by, 161-166 pronephric duct, experimental manipulation of, 159-166 scanning electron microscope (SEM), 159 Arnelogenesis imperfecta, 46-47 AMH, 11,20,25 Androgenates, 74,76, 77-80,81,99101 abnormal development of, 97-10 I ES cells, 86-88 Anemia, hypochromic or sideroblastic, 47, 48 Angelman syndrome, 106 Anhidrotic ectodermal dysplasia, 4748 ANT3 translocase gene, 60 Anti-Mullerian Hormone, 11, 20, 25 Anti-sense oligonucleotides, 136-137 Antibody perturbation experiments, 138-141 AS, 106 Asynchronous DNA replication, 4041 Autosomal imprinting, 89-93

B! L! K isozyme, 170-I7 I bFGF of cytokines, 177 Ballistics, 167-168 Banded krait minor sequences (Bkm), 5 Barr body, 38, 40-41, 53-54 Basal lamina, 19, 21 Basic fibroblast growth factor (bFGF) of cytokines, 177

lndex

Beckwith-Wiedemann syndrome (BWS), 106-107 Bkm sequences, 5 Bone/liver/ kidney isozyme, 170-171 BWS, 106-107 Cadherins, 155 Cartilage Matrix Protein, 173 Cell-marking techniques, using to analyze neural crest migratory pathways, 126-128 DiI, injection of, 127 advantages of, 127-128 HNK-1 results, results of similar to, 127 orderly pattern, 127 Chemotaxis, 157, 167-168 Chimeric embryos, analysis of, 80-85 advantage in using chimeras, 80 between androgenetic and wildtype embryos, 83-85 between parthenogenetic and wildtype embryos, 80-83 parthenogenetic derivatives, selection against, 82 Chondrodysplasia punctata, 46 Choroideremia, 48 Chou-Fasman algorithm, 28 CNCCs, 162 Collagen type IV, 129, 132-134, 139, 173 Collagenase, 156 Competence of male gonadal tissue, 22 Contact guidance, 157, 167-168 Contact inhibition, 157, 167-168 CpG islands, 40-41 Cranial neural crest cells, 162 Cytokines, 177 basic fibroblast growth factor (bFGF), 177 TGF-alpha and -beta, 177 Cytotactin, 128-129

187

Deletion mapping, Z F Y and, 5-7 Dephosphorylation, intracellular activities and, 176 Diacylglycerol, 173 Diandric embryos, 79, 86 Digynic embryos, 79, 80 DiI, injection of to analyze neural crest cell migration, 127 advantages of, 127-128 HNK-1 antibody labeling, results similar to, 127 orderly pattern, 127 Diploid chromosomal combinations: altered, developmental consequences of eggs containing, 78-80 derivation of, 76-78 DMD, 47 DNA methyl transferase, 103 DNA methylation, 39, 40-41 DNA replication, asynchronous, 4041 DNA sequencing of SR Y/ Sry genes, 25-30 Dosage compensation, 49 EC cells, 52, 85 ECM molecules, 120, 126 (see also “Neural crest cell.. .”) along pathways, 128-130 collagen type IV, 129 fibronectin, 128-129 heparan sulfate proteoglycans, 129 laminin, 128-129 T-cadherin, 129 tenascinl cytotactin, 128-129 EK cells, 52 Embryonal carcinoma (EC) cells, 52, 85 Embryonic morphogenesis, 153-183 (see also “Alkaline phosphatase.. .”) Embryonic stem (ES) cells, 81, 85-88

188

Endogenous genes, imprinting and, 103-105 Enzymes and morphogenesis, 153183 (see also “Alkaline phosphatase.. .’3 Epithelial-mesenchyme interaction, 22 ES cells, 81, 85-88 Extracellular matrix, neural crest cells and, 120-152 (see also “Neural crest cell. . .”) and migratory pathways, 156 along pathways, 128-130 collagen type IV, 129 fibronectin, 128-129 heparan sulfate proteoglycans, 129 laminin, 128-129 T-cadherin, 129 tenascin/ cytotactin, 128-129 phosphoproteins, 177 adhesion molecules, 178-179 cytokines, 177 proteoglycans, 177 F-actin filaments, adhesion molecules and, 155 Fabry’s disease, 47 Fasciclins, 155 Fibronectin, 22, 128-129, 132-137, 139-141, 178 G6PD, 43, 104-105 Galvanotaxis, 157, 167-168 Genetic switch, 2 Genital ridges, 3 Genomic imprinting and regulation of mammalian development, 73-1 18 abnormal development of androgenetic and parthenogenetic embryos and expression of androgenous imprinted genes, 97-101

INDEX

androgenetic development, 98100 IGFBP, 100 muscle formation in androgenetic teratocarcinomas, 100101 MyoD, 100-101 parthenogenetic development, 97-98 altered diploid chromosomal combinations, developmental consequences of eggs containing, 78-80 hydatidiform males, 80 triploid embryos, 79, 80 chimeric embryos, analysis of, 80-

85 advantage in using chimeras, 80 between androgenetic and wildtype embryos, 83-85 parthenogenetic derivatives, selection against, 82 between parthenogenetic and wild-type embryos, 80-83 conclusions, 107-108 diploid chromosomal combinations, derivation of, 76-78 ES cells in imprinting analysis, 8588 androgenetic, 86-88 advantage, 85 parthenogenetic, 86, 88 genetic basis of imprinting, 88-93 autosomal, 89-93 H19,92,93,96-97 IGF II,92,93-95, 99-100, 103104, 107 IGF2r/ M6Pr, 92,93,95-96 uniparental disomias, 89-93 X chromosome, 89 H19 gene, 96-97,99, 103-104 and BWS in humans, 106-107

Index

Insulin-Like Growth Factor I1 (IGF II), 92, 93-95, 99-100, 103-104, 107 and BWS in humans, 106-107 expression of, 94 function of during embryogenesis, 94 what it is, 93-94 introduction, 75 “imprinting,” 75 mechanisms of imprinting, 101105 criteria, four, 101 DNA methyl transferase, 103 and endogenous genes, 103-105 “imprinting box,” 101 methylation, 101-103 and transgenes, 101-103 Xce, 104 Xist, 104 other species, imprinting in, 105107 Beckwith-Wiedemann syndrome, 106-107 in birds, 105 in fish, 105 in humans, 106 Huntington’s chorea, 107 in marsupials, 105 in plants, flowering, 105 Prader-Willi/ Angelman syndromes, 106 in vertebrates, 105 preface, 74-75 androgenates, 74, 76, 77-80, 81, 86-88 definition, 74 gynogenates, 74, 76, 77-80 imprinted genes, three, 74-75, 92 parthenotes, 74, 76, 77-82, 86, 88

189

Type 2 IGF receptor, 95-96 binding with higher affinity, 95 function, 95-96 structurally different, 95 Germ cells of adult testes, 20, 21 Glucose-6-phosphate dehydrogenase gene, 104-105 Glycosyltransferases, 155 Goeminne syndrome, 48 Gonadal blastema, 16, 17, 18, 21, 24 Gonads, indifferent, differentiation of, 3 (see also “Sry gene.. .”) Sry, expression of, 14-25 germ cells, 20, 21 gonadal blastema, 16, 17, 18, 21,24 inducing signal, 22 kidney systems, three, 14-15 laminin, 22 Leydig cells, 19-20, 21 mesonephros, 14-20, 24 metanephros, 14-15 Miillerian duct, 16, 20 organogenesis, 22 peritubular myoid cells, 19, 20, 21 pronephros, 14 seminiferous tubules, 20 Sertoli cells (sc), 19-23 spermatogenesis, 25 testosterone, 20, 21 uniqueness of, 14 Wolffian duct, 14-20 Gynogenates, 74, 76, 77-80 H19,92, 93,96-97, 99, 103-104 and BWS in humans, 106-107 H-Y antigen, 4-5 Haptotaxis, 157, 167-168 Heparan sulfate proteoglycans (HSPG), 129, 132-134, 139 Hepatoblastoma, 106-107 HMG box, 8, 14, 25-29

190

INDEX

HNK-1 antibody, 130-131, 134, 136137, 143 labeling, results of similar to DiI labeling results, 127 HPRT, 43-46 HSPG, 129, 132-134, 139 hUBF, 25 Hunter syndrome, 47 Huntington’s chorea, 107 Hyaluronic acid, 128 Hydatidiform moles, 80, 106, 107 Hypo- and hypermethylation and transgenes in imprinting, 102-103 Hypochromic anemia, 47,48 Hypoglycemia, elevated IGF TI levels and, 100 Hypomanesemia, 47 HYa, 5 ICMs, 80 IGF II,92,93-95, 99-100, 103-104 and BWS in humans, 106-107 IGF2r/ M6Pr, 92,93,95-96 IGFBP, 100 Immunodeficiency disease, 47 Immunofluorescence studies, ALP and, 172 Imprinted genes, 74-75 “Imprinting,” 58, 75 genetic basis of, 88-93 genomic, and regulation of mammalian development, 73-1 18 (see also “Genomic imprinting.. .”) Incontinentia pigmenti, 47 Indifferent gonads, differentiation of, 3 Insulin-Like Growth Factor II,92,93 type 2 receptor, 92, 93 Integrin receptor, neural crest cells and, 130 Inteprins. 155 Y

I

KALIG, 48-50 Laminin, 22, 120, 128-129, 132-137, 139 Lesch-Nyhan syndrome, 47 Levamisole, 170 Leydig cells, 19, 20, 21 “Localized expression” of X-linked genes, 46 Lowe syndrome, 48 Lyon hypothesis, 38,46, 60 (see also “X chromosome inactivation.. .”) Mammalian development, genomic imprinting and regulation of, 73-1 18 (see also “Genomic imprinting.. .”) Marsupials, inactivation of paternal X chromosome in, 89 matl, 28 Menkes disease, 48 Mesonephros, 14-20, 24 Metanephros, 14-15 Methylation, imprinting and, 101103 MIC2,4 1,49 Monosomy, 78 Morphogenetic cell migrations, 153183 (see also “Alkaline phosphatase.. .”) Mosaic expression of X-linked genes, 42,4345,46-47 Mosaicism, sex reversal and, 11 mtTFI, 25 Miillerian duct, 16, 20 Muscle formation in androgenetic teratocarcinomas, 100-101 Mutation studies, sex-reversal, 9 XY females, 29 MyoD, muscle formation and, 100101 Myotonic dystrophy, 107

Index

191

N-cadherins, 131 preface, 120 N-CAM, 131-132, 155 extracellular matrix, 120 Neural crest cell migration, cell intersummary and future directions, actions in, 119-152, 157 147-148 cell adhesion molecules, 131-132 surface of cell, 130-131 N-cadherin, 131 HNK-I antibody, 130-131, 134, N-CAM, 131-132 136-137, 143 cell-marking techniques, using to integrin receptors, 130 analyze, 126-128 tissues, surrounding, role of in DiI, injection of, 127 (see also determining pattern of, 141“DiI.. .”) 147 orderly pattern, 127 dorsoventral patterning, 146cell-matrix interactions in, 132-141 147 adhesion assay, 132-133 notochord, inhibitory effects of, alpha lactalbumin, 141 143-146 anti-sense oligonucleotides, 136within somites, 141-143 137 Norrie disease, 47 migration assay, 132-134 Notochord, neural crest cells and, quantitative cell adhesion assay, 120-126 132-133 inhibitory effects of, 143-146 surface receptors, 134-137 tissue culture analysis, 132-137 Ocular albinism, 46 in vivo perturbation analysis, OP, 178 138-141 Organogenesis, 22 CNCCS, 162-166 Osteopontin (OP), 178 cranial, 162-166 Osteosarcomas, imprinting and, 107 extracellular matrix molecules (see also “Sry gene.. .”) Ovotestes, 13 along pathways, 128-130 collagen type IV, 129, 132-134, Parent-of-origin effects, 102, 107 139 Parthenotes, 74, 76, 77-82 fibronectin, 128-129, 132-137, abnormal development of, 97-101 139-141 ES cells, 86, 88 heparan sulfate proteoglycans, of, 76-77 production 129, 132-134, 139 Passive migration, 167-168 laminin. 128-129. 132-137, 139 “Patchy expression” of X-linked T-cadherin, 129, 143 genes, 42 tenascini cytotactin, 128-129, Peritubular myoid cells, 19, 20, 21 132-134, 141 Pgc, 19, 102 introduction, 120-126 Phosphatidylinositol-glycan (PI-G), migrations, extensive, 120 172, 174 notochord, 120-126 Phosphatidylinositol-specific phossclerotome, 125-126 pholipase C, 172, 174 somites, 121-126

192

Phosphorylation, intracellular activities and, 176-178 PI-G, 172, 174 PIPLC, 172, 174 Plasminogen activator, 156 Pluripotent cells, 52 PND, 157 (see also “Alkaline phosphatase.. .” and “Pronephric duct.. .”) Population pressure, 157, 167-168 Prader-Willi/ Angelman syndromes, 106 Pre-Sertoli cells (psc), 19, 20, 21 Primordial germ cells (pgc), 19, 102 Proliferin, 96 Pronephric duct, elongation of, 153183 (see also “Alkaline phosphatase.. .”) and chemotaxis, 161 experimental manipulation of, 159-166 lateral mesoderm, 160-161 marker system, genetic, 159 melanin, 159 mesectoderm, 162 other cells, PND guidance information recognizable by, 161-166 cranial neural crest cells, 162-166 scanning electron microscopy (SEM), 159 Proteoglycans, 155, 177 Psc, 19 Pseudoautosomal pairing region, 42 PWS, 106 Relaxin, 93 Retinitis pigmentosa, 46 Reverse transcriptase-polymerase chain reaction technique, 23 Rhabdomyosarcomas, 106-107 Robertsonian translocations, 90 RPS4X, 49-50

INDEX

RPS4- Y, 8 RT-PCR technique, 23 analysis of cDNA from human cell line. 55

Sc, 19 Scanning electron microscopy (SEM), 15 Sclerotome, 125-126 Selectins, 155 SEM, 159 Semini ferous tubules, 20 Sertoli cells (sc), 19-23 Sex chromatin body, 38,40,41 Sex determination in mammals, Sry gene and, 1-35 (see also “Sry gene. ..”) Sex-reversal studies, Sry/ SR Y and, 9 XY females, 28-29 Sideroblastic anemia, 48 snRNP-associated polypeptide, 108 Somites, 121-126 and neural crest cells migration pattern, 141-143 SOX-I,-2, -3,28 Spermatogenesis, 25, 56 Spino cerebellar ataxia, 107 SPY, 4 SR Y gene, 2 (see also “Sry gene. . .”) Sry gene and sex determination in mammals, 1-35 candidates for, 4-7 Bkm sequences, 5 deletion mapping and ZFY, 5-8 H-Y antigen and Sxr, 4-5 Ubely-I, 4 XyTdYml 57 evidence, direct, of Sry/ S R Y as sex-determining gene, 9-13 Anti-Mullerian Hormone (AMH), 11,20,25 mutation studies, 9 transgenic studies, 10-13

Index

gonadal differentiation and expression of Sry, 14-25 germ cells, 20, 21 gonadal blastema, 16, 17, 18, 21,24 inducing signal, 22 kidney systems, three, 14-15 laminin, 22 Leydig cells, 19-20, 21 mesonephros, 14-20, 24 metanephros, 14-15 Miillerian duct, 15-16, 20 organogenesis, 22 peritubular myoid cells, 19, 20, 21 pronephros, 14 seminiferous tubules, 20 Sertoli cells (sc), 19-23 spermatogenesis, 25 testosterone, 20, 21 uniqueness of, 14 Wolffian duct, 14-20 introduction, 2-3 developmental decisions, role in, 3 genetic switch, 2 genital ridges, 3 TDF, 3-35 Tdy, 3-35 testes, 3 isolation and properties of, 7-8 HMG box, 8, 14 R PS4- Y, 8 S R Y and Sry genes, 2, 8 Tdyml,8 , 9 Y chromosome, 8 molecular structure and biochemistry of, 25-30 Chou-Fasman algorithm, 28 DNA sequencing, 25-30 HMG box,25-29 hUBF, 25 matl, 28

193

SOX-I,-2, -3, 28 S, pombe gene, 28 Stel I, 28 TCFI, 28 preface, 2 SRY and Sry genes, 2, 8 Y chromosome, 2 , 4 (see also "Y chromosome") summary and conclusions, 30 X and autosomal testisdetermining genes, 13-14 Tas, 13-14 Tda-1 and Tda-2, 13-14 in wood lemmin, 14 X-linked genes, 14 Yak', 14 Ypos,14 Steroid sulfatase gene, 43-46, 104 Stel I, 28 STS gene, 43-46,48-50, 104 Sxr, 4-5

T(X:16) 16H, 23 T-cadherin, 129, 143 Talin, 155 Tas, 13-14 TCFI, 28,29 Tda-1 and Tda-2, 13-14 TDF, 3, 5-6, 7 Tdy, 3-35 (see also "Sry gene.. .") Tdy"", 8 , 9 Tenascin/cytotactin, 128-129, 132134, 141 Testes, 3 cell types, four, 20 Testosterone, 20, 21 Thyroglobulin, 96 Tissue-specific ligands, 167-168, 171173 Transgenes, imprinting and, 101-103 Transgenic experiments of Sry/ SR Y as sex-determining gene, 1013

194

Triploid embryos, 79, 80 Trisomy, 78 Turner syndrome, RPS4- Y and, 8 Ube ly-l,4, 8 “Unfortunate lyonization,” 48 Uniparental disomics, 89-93 Vinculin, 155 Vitronectin, 130 Von Willebrand Factor, 173 Wilm’s tumor, 106-107 Wiskott-Aldrich syndrome, 47 Wolffian duct, 14-20 Wood lemming, X* chromosome in, 14 X:autosome ratio, 2, 3 (see also “Sry gene.. .”) X/ autosome translocations, 47 X chromosome inactivation, human, molecular and genetic studies of, 37-71 activity of X-linked genes, 42-48 direct analysis of gene expression, 43-46 dosage of gene product, 45 evidence for genes being subject to, 42 HPRT, 43-46 indirect analysis of gene expression, 46-48 mosaic expression, 42,4345, 46-47 number of, 42 “patchy expression,” 42 pseudoautosomal pairing region, 42 in somatic cell hybrids, 45-46 “unfortunate lyonization,” 48 X/ autosome translocations, 47 conclusions, 59-60

1N DEX

features of, 4042 asynchronous DNA replication, 4041 Barr body, 38, 4041 CpG islands, 40-41 DNA methylation, 40-41 MIC2,41 sex chromatin body, 38, 40,41 genes that escape, 48-50 definition, 48 dosage compensation, 49 X chromosome, human, diagram of, 49 inactivation process, 50-53 cycles, 50, 51 EC cells, 52 EK cells, 52 embryonal carcinoma cells, 52 extraembryonic tissues, 5 1-52 in marsupials, 52 primordial germ cells, 50, 5 1 pluripotent cells, 52 stable, 53 introduction, 38-39 components of, 38-39 DNA methylase, 39, 40 Lyon hypothesis, 38,46, 60 models for, 56-59 “imprinting,” 58 initiation, 57, 58 maintenance, 57, 59 promulgation, 57, 58-59 preface, 38 X-linked genes, 38 XIC, 38, 53-56 XIST gene, 38,53-60 X I S T gene, X inactivation center and, 53-56, 60, 104 mapping, 54 X chromosomes, 2 (see also “Sry gene.. .”) imprinting, 89

Index

X-linked genes, 38 activity of, 4248 (see also "X chromosome.. .") sex determination and, 14 XCE, 104 XE7 gene, 60 XE59 gene, 60 XE113 gene, 60 Xenopus laevis, 159 XIC, 38, 53-56 mapping, 54 X inactivation center, 38, 53-56, 104 mapping, 54

195

X I S T gene, 38,53-60, 104 xyTdy"' 37

XY females, 29

Yak', 14 YPos,14 Y chromosome, 2, 4 , 8 (see also "Sry gene.. .") in marsupials, 6

ZFX,49-50 ZFY,5-8 Zfy-1and Zfy-2,4

J

A I

P R E

Advances in Developmental Biology Edited by Paul Wassarman, Department of Cell and Developmental Biology, Roche lnstitute of Molecular Biology Volume 1,1991,192 pp. ISBN 1-55938-348-8

CONTENTS: introduction. Y Chromosome Function in MammalianDevelopment,Paul S. Burgoyne, MRC Mammalian Development Unit, London, England. A Super Family of PutativeDevelopmentalSignalling Molecules Related to the Proto-Oncogene Wnt-llint-1, Andrew P. McMahon, Roche lnstitute of Molecular Biology Segmentation in Drosophila, Kenneth R. Howard, Roche lnstitute of Molecular Biology. Pattern Formation in Caenorhabditis Elegans, Min Han and Paul W. Sternberg, California lnstitute of Technology, Pasadena. Gap Junctional Communication During Mouse Development, Norton 6. Gilula, Miyuki Nishi, and Nalin Kumar, Research lnstitute of Scripps Clinic, La Jolla, California. Lens Differentiation and Oncogenesisin Transgenic Mice, Heiner Westphal, National lnstitute of Child Health and Human Development, Maryland. Subject Index.

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Advances in Structural Biology Edited by Sudarshan Malhotra, Depaflment of Zoology, University of Alberta Volume 1,1991,357 pp. $90.25 ISBN 1-55938-292-9 CONTENTS Preface, Sudarshan K. Malhotra. Brain Extracellular Matrix, Amico Bignami, Spinal Cord lnjury Research Laboratory, Boston and George Perides. Neuroglia Cells in Neocortical Transplants: Their Genesis and Morphology, Gopal D. Das, Purdue University. Cholinergic Receptors in the An lmmunocytochemical Central Nervous System Approach, Hannsjorg Schroder, Universitat Koln, Germany. Rapid Organelle Transport in Axons, Richard S. Smith, University of Alberta. MolecularMechanismsof Cell Adhesion: Recent Advances, R. Rajaraman, Dalhousie University. Striated Muscle Endosarcomeric and Exosarcomeric Lattice, Maureen G. Price, Rice University. Gap Junctions: A Multigene Family, Nalin M. Kumar, Research lnstitute of Scripps Clinic, La Jolla, California. Morphogenesis of Endoplasmic Reticulum and Golgi Apparatus as Demonstrated by Membrane Transplants, Jacques Paiement, Universite de Montreal. Activation-Induced Cell Death in Developing T Cells and T Cell Hybridomas, Douglas R. Green and Yufang Shi, University of Alberta. The Molecular Interaction of Leishmania with its Host Cell, the Macrophage, Rebecca A. Guy and Miodrag Belosevic, University of Alberta. Structural and Biochemical Bases of the Blackspot Disease of Crucifers, Jalpa P. Tewari, University of Alberta. Subject Index.

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Volume 2, In preparation, Spring 1993 Approx. $90.25 ISBN 1-55938-584-7 CONTENTS. Preface, Sudarshan K. Malhotra. The Role nf Calcium-Dependent Cell-Cell Adhesion Molecules in the Normal Function of Epithelial Cells, WarrenJ. Gallin, University ofAlberta. The Biology of the HyaluronateReceptor (CD44):A Member of the Link Protein Family, Shakti P. Kapur, Martine Culty, and Charles B. Underhill, Georgetown University Medical Center. Molecular Structure of the Eye Lens Gap Junctions, Joerg Kistler, University of Auckland and Stanley Bullivanf, Georgetown University Medical Center. Cytoskeleton Phosphorylation and Cell Morphology, Philip L. Mobley, University of Texas Health Science Center and Beth C. Harrison, Pennsylvania State University. Adaptation of Sarcoplasmic Reticulum to Environmental and Dietary Changes, Anthony N. Martonosi, State University of New York. Structure of Membrane Proteins by Electron Microscopy, Manoj Misra, Duke University Medical Center. Cholesterol Metabolism in Brain, Shantilal N. Shah, University of California. Neuronal and Glial Cell Responses, Hakan Aldskogius and Mikael Svensson, Karolinska lnstitutet, Sweden. Structure and Function of the Nicotinic Acetycholine Receptor, Susan M.J. Dunn, The University of Alberta. The Autonomic Ganglia and the Modulation of Ganglionic Transmission, Peter A. Smith, University of Alberta. Subject Index.

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Advances in Developmental Biochemistry Edited by Paul Wassarman, Department of Cell and Developmental Biology, Roche Institute of Molecular Biology Volume 1,1991,256 pp. ISBN 1-55938-347-X

$90.25

CONTENTS: Introduction.Organelle Assembly and Function in the Amphibian Germinal Vesicle, Joseph G. Gall, Carnegie Institution. DNA Replication and the Role of Transcriptional Elements During Animal Development,Melvin L. Defamphilis, Roche lnstifufe of Molecular Biology, New Jersey. Transcriptional Regulation During Early Drosophi/a Development, K. Prakash, Joanne Topol. C.R. Dearolf, and Carl S. Parker, California lnstitute of Technology, Pasadena. Translational Regulation of Maternal Messenger RNA, L. Dennis Smith, University of Ca/ifornia, lrvine. Gut Esterase Expression in the Nematode Caenorhabditis Elegans, James D. McGhee, University of Calgary. Transcriptional Regulationof Crystallin Genes: Cis Elements,Trans-factors and Signal Transduction Systems in the Lens, Joram Piatigorksy and Peggy S. Zelenka, National €ye Institute, National lnstitutes of Health, Maryland. Subject Index. Volume 2, In preparation, Summer 1993 ISBN 1-55938-609-6

Approx. $90.25

CONTENTS Preface, Paul M. Wassarman, Roche lnstitute of Molecular Siology. Drosophi/a Homeobox Genes, William McGinnis, Yale University. Structural and Functional Aspects of Mammalian HOX Genes, Denis Duboule, European Molecular Biology Laboratory. Developmental Control Genes in Myogenesisof Vertebrates, Hans Henning-Arnold, University of Hamburg. Mammalian Fertilization:Sperm ReceptorGenes and Glycoproteins, Paul M. Wassarman, Roche lnstitute of Molecular Biology. The Fertilization Calcium Signal and How It Is Triggered, Michael Whitaker, University College London. Subject Index.

JAI PRESS INC. 55 Old Post Road - No. 2 P.O. Box 1678 Greenwich, Connecticut06836-1678 Tel: (203)661-7602

Fax: (203)661-0792