Advances in Developmental Biology

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ADVANCES IN DEVELOPMENTAL BIOLOGY

Volume 2

1993

This Page Intentionally Left Blank

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

Manufactured in the United States of America

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


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


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

ix


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

.

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


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.


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


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


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


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).


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).


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


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


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.

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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.


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


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


20

9

c?

Indifferent Stage

Mesonephric tubules

form genital duct

$:! \
517. Solter, D. (1988). Differential imprinting and expression of maternal and paternal genomes. Ann. Rev. Genet. 22: 127-1 46. Stay, B., and Coop, A. C. (1974). "Milk" secretion for embryogenesis in a viviparous cockroach. Tissue and Cell 6:669-693. Stevens, L. C. (1978). Totipotent cells of parthenogenetic origin in a chimeric mouse. Nature (Lond.) 2 76:266-267. Stevens, 1. C. (1982). Teratocarcinogenesis and parthenogenesis. In: The Mouse in Biomedical Research, pp. 161-167. Academic Press, New York. Stevens, L. C., Varnum, D. S., and Eicher, E. M.(1977). Viable chimeras produced from normal and parthenogenetic mouse embryos. Nature (Lond.) 269:515-5 17. Stewart, C. L., Stuhlman. H., Jahner, D., and Jaenisch, R. (1982). De novo methylation, expression and infectivity of retroviral genomes introduced into embryonal carcinoma cells. Proc. Nat. Acad. Sci. USA 794098-4102. Stewart, C. L., Riither, A., Garber, C., Vanek, M., and Wagner, E. F. (1986). The expression of retrovial vectors in mouse stem cells and transgenic mice. J. Embryol. Exp. Morph. 97 (Supplement): 263-275. Surani, M. A. H., Barton, S. C., and Kaufman, M. H. (1977). Development to term of chimaeras between diploid parthenogenetic and fertilized embryos.Nature (Lond.) 270:601-602. Surani, M. A. H., and Barton, S. C. (1983). Development of gynogenetic eggs in the mouse: Implications for parthenogenetic embryos. Science 222: 1034-1 036. Surani, M. A. H., Barton, S. C., and Norris. M. L. (1984). Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis. Nature 308548-550. Surani, M. A. H., Barton, S. C., and Norris. M. L. (1986a). Nuclear transplantation in the mouse: Heritable differences between parental genomes after motivation of the embryonic genome. Cell 45: 127-136. Surani. M. A. H.. Reik, W., Norris, M. L., and Barton, S. C. (1986b). Influenceofgermline modifications of homologous chromosomes on mouse development. 1. Embryol. Exp. Morph. (Suppl.) 123-136. Surani, M. A. H. (19%~).Evidences and consequences of differences between maternal and paternal genomes during embryogenesis in the mouse. In: Experimental Approaches to Mammalian Embryonic Development (Rossant, J., and Pedersen, R A,, eds.), pp. 401436. Cambridge University Press, UK. Surani, M. A., Barton, S. C., Howlett, S. K., and Norris, M. L. (1988). Influence of chromosomal determinants on development of androgenetic and parthenogenetic cells. Development 103:171178. Surani, M. A. H., Kothary, R., Allen, N. D., Singh, P.B., Fundele. R.,Ferguson-Smith,A. C., andBarton, S. C. (199Oa). Genome imprinting and development in the mouse. Development (Supplement) 89, pp. 89-98. Surani, M. A.. Allen, N. D., Barton, S. C., Fundele, R.,Howlett, S. K., Norris, M. L., and Reik. W. ( 1 99Ob).Developmental consequences of imprinting of parental chromosomes by DNA methylation. Phil. Trans. R. Soc.Lond. B. 326:313-327.

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Sutrare, P., Kelly, A. M., and Hughes, S. H. (1990).Ski can cause selective growth of skeletal muscle in transgenic mice. Genes Dev. 41462-1472. Surti, U. (1987).Genetic concepts and techniques. In: Gestational Trophoblastic Disease (Szulman, A. E., and Buchsbaum H. J., eds.),pp. 111-121. Springer-Verlag, Berlin. Swain, J. L.,Stewart, T. A., andLeder, P. (1987).Parental legacydetermines methylation and expression of an autosomal transgene: A molecular mechanism for parental imprinting. Cell 5iO:719-727. Smlman, A. E. (1987).Partial hydatidiform mole. In:.Gestational Trophoblastic Disease (Szulman, A. E., and Buchsbaum H. J., eds.), pp. 37-44. Springer-Verlag. Berlin. Szulman, A. E. (1987).Complete hydatidiform mole: clinicopathological features. In: Gestational Trophoblastic Disease (Szulman, H. J., and Buchsbaum, H. J., eds.), pp. 27-36. Springer-Verlag. Berlin. Tada, T., and Takagi, N. (1992). Early development and X-chromosome inactivation in mouse parthenogenetic embryos. Mol. Reprod. Dev. 31:20-27. Takagi, N. (1991). Abnormal X-chromosomic dosage Compensation as a possible source of early developmental failure in mice. Dev. Growth and Differ. 33429435. Takagi, N., and Sasaki, M. (1975). Preferential inactivation of the paternally derived X-chromosome in the extraembryonic membranes of the mouse. Nature 256641-642. Thomson, J. A., and Solter, D. (1988).The developmental fate of androgenetic, parthenogenetic, and gynogenetic cells in chimeric gastrulating mouse embryos. Genes Dev. 2:1344-1351. Thomson, J. A., and Solter, D. (1989).Chimeras between parthenogenetic or androgenetic blastomeres and normal embryos: Allocation to the inner cell mass and trophectoderm. Dev. Biol. 1m80-583. Tsai, J.-Y, and Silver, L. M. (1991). Escape from genomic imprinting at the mouse T-associated maternal effect (Tme) locus. Genetics 129:1159-1166. Tsukahara, M., and Kajii, T. (1985).Replication of X chromosomes in complete moles. Hum. Genet. 71:7-10. Turner, C. L. (1947). Viviparity in Teleost fishes. Scientific Monthly 65:50%518. Vetter, U., Zapf, J., Heit, W., Helbing, G., Heinze, E., Froesch, E. R., and Teller, W. M. (1989).Human fetal and adult chandrocytes. Effect of insulin-like growth factors I and 11, insulin and growth hormone on clonal growth. J. Clin. Invest. 77:190>1908. Vrijenhoeck, R. C. (1989).Genetic and ecological constraints on the origins and establishment of unisexual vertebrates. In: Evolution and Ecology of Unisexual Vertebrates (Dawley, A. M., and Bogart, J. P., eds.), Bulletin 466, New York State Museum, Albany. Vrijenhceck, R. C., and Lerman, S. (1982).Heterozygosity and developmental stability under sexual and asexual breeding systems. Evolution 36768-776. Winking, H., and Silver, L. M. (1984). Characterization of a recombinant mouse t-haplotype that expresses a dominant lethal maternal effect. Genetics 108:10I3-lO20. Wright, W. E., Sassoon, D., and Lin, V. K.(1989).Myogenin, a factor regulating myogenesis, has a domain homologous to Myo D. Cell 56:607417. Wrigley, J. M.. and Graves, J. A. M. (1988).Sex chromosome homology and incomplete tissue-specific X-inactivation represent an intermediate stage of mammalian sex chromosome evolution J. Hered. 79:11 5-1 18. WU,X., Hadchouel, M., Farm, H., Ama, R., and Pourcel, C. (1989).Analysis of the sex-dependent imprinting of a chromosome 13 region using a transgene as a molecular probe. In: Chromosomes Today 10 (Fredga, K.. Kihlman, B. A.. and Bennett, M. D., 4 s . ) . pp. 149-156. Allen and Unwin, London.

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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MARIANNE BRONNER-FRASER

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

Neural Crest Cell Migration

121 MESENCEPHALON

METENCEPHALON

DIENCEPWLON

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

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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0

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7

HNK-1

500

50000

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

-

4 9 Somite Embryo

1 lnlect Ab that blocks

cell interactions

I

t

2. Incubate for 2 4 - 4 8 hr

3. Section and stain wrth

HNK-1 Ab

pr lul

Controls: Non-blocking Ab

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.


a

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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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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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

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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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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

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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.

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