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Current Topics in
Developmental Biology
Volume 51
Series Editor
Gerald P. Schatten
Departments of Obstetrics–Gynecology
and Cell and Developmental Biology
Oregon Regional Primate Research Center
Oregon Health Sciences University
Beaverton, Oregon 97006-3499

Editorial Board
Peter Grüss
Max Planck Institute of Biophysical Chemistry
Göttingen, Germany
Philip Ingham
University of Sheffield, United Kingdom
Mary Lou King
University of Miami, Florida
Story C. Landis
National Institutes of Health/
National Institute of Neurological Disorders and Stroke
Bethesda, Maryland
David R. McClay
Duke University, Durham, North Carolina
Yoshitaka Nagahama
National Institute for Basic Biology, Okazaki, Japan
Susan Strome
Indiana University, Bloomington, Indiana
Virginia Walbot
Stanford University, Palo Alto, California

Founding Editors
A. A. Moscona
Alberto Monroy
Current Topics in
Developmental Biology

Edited by

Gerald P. Schatten
Departments of Obstetrics–Gynecology
and Cell and Developmental Biology
Oregon Regional Primate Research Center
Oregon Health Sciences University
Beaverton, Oregon

San Diego San Francisco New York Boston London Sydney Tokyo
This book is printed on acid-free paper. 
∞

Copyright 
C 2001 by ACADEMIC PRESS

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00 01 02 03 04 05 EB 9 8 7 6 5 4 3 2 1
Contents

Contributors ix

1
Patterning and Lineage Specification in the Amphibian Embryo
Agnes P. Chan and Laurence D. Etkin
I. Introduction 2
II. Xenopus as a Model System for Studying Early Embryogenesis 3
III. Dorsal–Ventral Specification 4
IV. The Spemann Organizer 22
V. The Three Germ Layers 35
VI. Developmental Pathways and Tumorigenesis 47
VII. Perspectives 48
References 49

2
Transcriptional Programs Regulating Vascular Smooth Muscle
Cell Development and Differentiation
Michael S. Parmacek
I. Introduction 69
II. Embryology of the Vascular Smooth Muscle Cell Lineage(s) 70
III. Commitment to the Smooth Muscle Cell Lineage 73
IV. Transcriptional Control of Vascular Smooth Muscle Cell Differentiation 74
V. SRF: A Nuclear Sensor Regulating Growth and Differentiation 76
VI. Modulation of VSMC Phenotype 78
VII. Conclusions and Future Challenges 80
References 82

3
Myofibroblasts: Molecular Crossdressers
Gennyne A. Walker, Ivan A. Guerrero, and Leslie A. Leinwand
I. Myofibroblasts: An Overview 91
II. Myofibroblast Origin and the Role of PDGF 92
III. Cytokines and Myofibroblast Phenotypes 94

v
vi Contents
IV. “Muscle” Structural Protein Expression in Myofibroblasts 95
V. Mechanisms of “Muscle-Specific” Gene Regulation in Myofibroblasts 97
VI. Myofibroblast Contractility 100
VII. Myofibroblasts and the Cell Cycle 101
VIII. Perspectives 104
References 104

4
Checkpoint and DNA-Repair Proteins Are Associated with the Cores
of Mammalian Meiotic Chromosomes
Madalena Tarsounas and Peter B. Moens
I. Introduction 110
II. Structural Characteristics of Meiotic Chromosomes during the Prophase
of Meiosis I 111
III. Meiotic Checkpoint and Recombination Proteins Are Associated
with the Cores of the Meiotic Chromosomes 117
IV. Conclusions and Perspectives 127
References 127

5
Cytoskeletal and Ca2+ Regulation of Hyphal Tip Growth and Initiation
Sara Torralba and I. Brent Heath
I. Introduction: Characteristics of Fungal Growth 136
II. The Cytoskeleton and Apical Growth in Fungi 137
III. Calcium and Apical Growth in Fungi 154
IV. Conclusions 170
References 170

6
Pattern Formation during C. elegans Vulval Induction
Minqin Wang and Paul W. Sternberg
I. Introduction 190
II. Spatial Regulation of VPC Competence 193
III. Temporal Regulation of VPC Competence and Commitment 196
IV. Downstream Events of RAS Signaling 203
V. Negative Regulation of RAS Signaling 206
VI. Lateral Signaling 210
VII. Evolutionary Implications 212
VIII. Conclusions and Future Directions 213
References 214
Contents vii
7
A Molecular Clock Involved in Somite Segmentation
Miguel Maroto and Olivier Pourquié
I. Introduction 222
II. Models for Somite Formation 224
III. A Molecular Clock Linked to Somitogenesis 225
IV. Notch Signaling Pathway 229
V. Other Genes Implicated in Somitogenesis 236
VI. Conservation of the Segmentation Clock in Evolution 242
References 243

Index 249
Contents of Previous Volumes 257
This Page Intentionally Left Blank
Contributors

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

Agnes P. Chan (1), Department of Molecular Genetics, The University of Texas

M. D. Anderson Cancer Center, Houston, Texas 77030
Laurence D. Etkin (1), Department of Molecular Genetics, The University of
Texas M. D. Anderson Cancer Center, Houston, Texas 77030
Ivan A. Guerrero (91), Department of Molecular, Cellular, and Developmental
Biology, University of Colorado, Boulder, Colorado 80309
I. Brent Heath (135), Biology Department, York University, Toronto, Ontario,
M3J 1P3 Canada
Leslie A. Leinwand (91), Department of Molecular, Cellular, and Developmental
Biology, University of Colorado, Boulder, Colorado 80309
Miguel Maroto (221), Laboratoire de Génétique et de Physiologie du Développe-
ment (LGPD), Developmental Biology Institute of Marseille (IBDM), CNRS-
INSERM-Université de la Mediterranée-AP de Marseille, France
Peter B. Moens (109), Department of Biology, York University, Toronto, Ontario,
M3J 1P3 Canada
Michael S. Parmacek (69), Department of Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania 19104
Olivier Pourquié (221), Labatoire de Génétique et de Physiologie du Développe-
ment (LGPD), Developmental Biology Institute of Marseille (IBDM), CNRS-
INSERM-Université de la Méditerranée-AP de Marseille, France
Paul W. Sternberg (189), Howard Hughes Medical Institute and Division of
Biology, California Institute of Technology, Pasadena, California 91125
Madalena Tarsounas (109), Department of Biology, York University, Toronto,
Ontario, M3J 1P3 Canada
Sara Torralba (135), Biology Department, York University, Toronto, Ontario,
M3J 1P3 Canada
Gennyne A. Walker (91), Department of Molecular, Cellular, and Developmental
Biology, University of Colorado, Boulder, Colorado 80309
Minqin Wang (189), Howard Hughes Medical Institute and Division of Biology,
California Institute of Technology, Pasadena, California 91125

ix
This Page Intentionally Left Blank
1
Patterning and Lineage Specification
in the Amphibian Embryo
Agnes P. Chan and Laurence D. Etkin
Department of Molecular Genetics
The University of Texas M. D. Anderson Cancer Center
Houston, Texas 77030

I. Introduction
II. Xenopus as a Model System for Studying Early Embryogenesis
III. Dorsal–Ventral Specification
A. Cytoplasmic Determinants
B. Cortical Rotation
C. Nieuwkoop Center
D. Vegetal Cortical Cytoplasm/␤-Catenin Signaling Pathway
E. Molecular Nature of the Dorsal Determinant in Vegetal
Cortical Cytoplasm
F. Cooperation between TGF-␤ Signaling and Wnt Signaling
G. TGF-␤ Receptors, Smads, and Target Genes
IV. The Spemann Organizer
A. Organizer Genes Expressed in the Dorsal Vegetal Region
B. Organizer Genes Expressed in the Prechordal Mesoderm
C. Organizer Genes Expressed in the Anterior Endomesoderm
D. A Mammalian Structure Analogous to the Anterior Endomesoderm
E. How Is the Organizer Formed after All?
V. The Three Germ Layers
A. Endoderm
B. Mesoderm
C. Ectoderm
D. A Theoretical Model of Germ Layer Formation
VI. Developmental Pathways and Tumorigenesis
VII. Perspectives
References

Xenopus has been widely used to study early embryogenesis because the embryos allow for
efficient functional assays of gene products by the overexpression of RNA. The first asym-
metry of the embryo is initiated during oogenesis and is manifested by the darkly pigmented
animal hemisphere and lightly pigmented vegetal hemisphere. Upon fertilization a second
asymmetry, the dorsal–ventral asymmetry, is established, with the sperm entry site defin-
ing the prospective ventral region. During the cleavage stage, a vegetal cortical cytoplasm

Current Topics in Developmental Biology, Vol. 51

C 2001 by Academic Press. All rights of reproduction in any form reserved.
Copyright 
0070-2153/01 $35.00 1
2 Agnes P. Chan and Laurence D. Etkin
(VCC)/␤-catenin signaling pathway is differentially activated on the prospective dorsal side
of the embryo. The overlapping of the VCC/␤-catenin and transforming growth factor beta
(TGF-␤) pathways in the dorsal vegetal quadrant specifies dorsal–vental axis formation by
regulating formation of the Spemann organizer, including the anterior endomesoderm. The
organizer initiates gastrulation to form a triploblastic embryo in which the mesoderm layer
is located between the ectoderm layer and the endoderm layer.
The interplay between maternal and zygotic TGF-␤s and the T-box transcription factors
in the vegetal hemisphere initiates the specification of germ-layer lineages. TGF-␤ signaling
originating from the vegetal region induces mesoderm in the equatorial region, and initiates
endoderm differentiation directly in the vegetal region. The ectoderm develops from the
animal region, which does not come into contact with the vegetal TGF-␤ signals.
A large number of the downstream components and transcriptional targets of early devel-
opmental pathways have been identified and characterized. This review gives an overview of
recent advances in the understanding of the functional roles and interactions of the molecular
players important for axis determination and germ-layer specification during early Xenopus
embryogenesis.  2001 Academic Press.
C

I. Introduction

The transformation of a single-celled zygote into a highly organized adult organ-

ism requires precise regulation of cell growth and differentiation. The study of
embryogenesis has been an intriguing subject for biologists. Experimental embry-
ology has provided fundamental knowledge of early embryonic interactions by
careful manipulation of embryos. Almost 80 years after the initial discovery of the
amphibian gastrula organizer, our understanding of early development has grown
from a cellular level to a molecular level. A number of developmental processes can
now be explained in terms of activation or repression of gene expression. These
data have provided a molecular basis for understanding the cascade of genetic
regulation during early embryonic development.
This chapter focuses on early embryonic development of Xenopus laevis. The
setting up of the dorsal–ventral axis in embryos has a critical function in deter-
mining the future body plan. In Xenopus, dorsal–ventral polarity is established at
the time of fertilization by the triggering of the translocation of vegetal cortical
cytoplasm (VCC) to the prospective dorsal side of the embryo. Components of
the VCC, via the ␤-catenin-dependent pathway, activate target gene expression
in the dorsal vegetal region in blastula-stage embryos. The VCC/␤-catenin sig-
naling pathway functions cooperatively with the transforming growth factor beta
(TGF-␤) pathway to induce specific gene expression in the Spemann organizer
in gastrula-stage embryos. Induction of the three germ layers takes place con-
comitantly with the establishment of the dorsal–ventral axis. The endodermal and
mesodermal layers of the embryo are induced as a result of active TGF-␤ signaling
initiated by maternally localized transcripts. Bone morphogenetic protein (BMP)
1. Early Xenopus Development 3
and post-MBT Wnt signaling further pattern the dorsal–ventral polarity of the
germ layers by specifying a ventral cell fate.
Examples from other model systems are included throughout the chapter to try
to provide additional data in specific areas of embryonic development. The use
of different developmental model systems can complement the deficiencies of in-
dividual systems. Integration of information obtained from different systems can
allow the elucidation of complex developmental pathways that are evolutionarily
conserved. A better understanding of the mechanisms controlling growth and dif-
ferentiation is a prerequisite to fulfilling the ultimate goal in unraveling the cause
and control of malignancy during tumorigenesis.
This review gives an overview of recent advances in the understanding of the
functional roles and interactions of the molecular players important for patterning
and lineage specification during early embryogenesis in Xenopus laevis.

II. Xenopus as a Model System for Studying

Early Embryogenesis

Xenopus laevis is one of several model systems used for studying early em-
bryogenesis (Harland and Gerhart, 1997). Other developmental systems include
Caenorhabditis elegans (Rose and Kemphues, 1998; Labouesse and Mango, 1999),
Drosophila (Baek and Lee, 1999), zebrafish (Kodjabachian et al., 1999; Mullins,
1999), the chick (Bachvarova, 1999), and the mouse (Beddington and Robertson,
1999; Gardner, 2000). The advantages of using Xenopus as an experimental sys-
tem for studying early embryonic development are the ease of maintenance of
the animals, the availability of large quantities of embryos year-round, and the
rapid development of the embryos in simple salt solution at ambient conditions.
The embryos are easy to manipulate for studies involving tissue transplantation,
recombination, and explant cultures.
The relatively large size of the Xenopus embryo allows efficient isolation of
specific regions of embryonic tissues and provides sufficient quantities of starting
materials for the construction of cDNA libraries (Blumberg et al., 1991). Several
genes have been isolated from the screening of a Xenopus dorsal-lip cDNA library
(Cho et al., 1991; Sasai et al., 1994; Bouwmeester et al., 1996). These genes have
subsequently been used to isolate homologs from other developmental systems.
Furthermore, the capacity of Xenopus embryos for large injection volumes has
made possible rapid functional screening using cDNA expression libraries (Smith
and Harland, 1991; Smith et al., 1993; Lemaire et al., 1995).
Overexpression of RNA in Xenopus embryos is an efficient way to assay for
gain-of-function phenotypes. In addition to the wild-type gene products, dominant-
interfering constructs of growth factors and receptors, and transcription factors
fused with activation or repression domains have been overexpressed in embryos
to assay for gene functions (Amaya et al., 1991; Conlon et al., 1996; Ryan et al.,
4 Agnes P. Chan and Laurence D. Etkin
1996; Horb and Thomsen, 1997). The use of hormone-inducible transcription
factor fusion constructs has further aided in the identification of genes that are the
immediate targets of transcription factors (Kolm and Sive, 1995; Tada et al., 1998;
Melby et al., 1999; Saka et al., 2000).
Knockout studies in Xenopus, performed with the use of antisense oligonu-
cleotides or antisense RNA, are another method of assessing gene functions. Mater-
nal RNA transcripts can be depleted using antisense oligonucleotides (Shuttleworth
and Colman, 1988; Kloc et al., 1989; Heasman et al., 1991). Antisense RNA ex-
pression has been shown to repress zygotic gene expression and thus permit demon-
stration of gene functions during normal development (Steinbeisser et al., 1995).
The introduction of transgenic techniques to Xenopus (Etkin and Pearman, 1987;
Kroll and Gerhart, 1994; Chan and Gurdon, 1996; Kroll and Amaya, 1996; Fu et al.,
1998; Amaya and Kroll, 1999; Marsh-Armstrong et al., 1999) has exploited the
potential of this model system for use in studying the zygotic effects of transgene
expression and in characterizing the regulatory sequences of promoters. A gene
trap approach in which transgenic techniques were used to carry out mutagenesis
in Xenopus has been successful (Bronchain et al., 1999). Introduction of mutations
into the Xenopus genome is likely to lead to the identification of novel genes on
the basis of the mutant phenotypes.
Although there are several advantages to using Xenopus laevis as a model of
embryogenesis, the system also suffers from pitfalls. A relatively long generation
time of around 1 year is required for the animal to reach sexual maturity, making
germline transmission of genetic alterations impractical. The pseudotetraploid na-
ture of the animal does not favor genetic analysis. However, the introduction of
Xenopus tropicalis, which is diploid and has a much shorter generation time of
5 months, is likely to circumvent some of the limitations of the Xenopus system
(Amaya et al., 1998).
On the other hand, model systems widely used for genetic studies include
C. elegans, Drosophila, zebrafish, and the mouse. Both C. elegans and the mouse
are reliable systems in which to study the effect of knocking out gene functions.
RNA interference has been demonstrated in C. elegans (Fire et al., 1998), and gene
targeting has been widely applied to manipulate genomic sequences of the mouse
(Koller and Smithies, 1992).

III. Dorsal–Ventral Specification

The basic body plan of an embryo is elaborated upon the dorsal–ventral axis
established during early embryogenesis. In Xenopus, dorsal–ventral polarity is
set up at the time of fertilization. In the fertilized egg, cytoplasmic determinants
are translocated to the future dorsal side by a rotation of the egg cortex. The
VCC/␤-catenin signaling pathway is activated on the prospective dorsal region
as a consequence of the cortical rotation. This pathway functions cooperatively
1. Early Xenopus Development 5
with TGF-␤ signaling to activate expression of organizer genes at the mid-blastula
transition (MBT), when zygotic gene expression first commences. In the past few
years, the knowledge of vegetally localized maternal determinants and molecular
components of this signaling pathways has grown dramatically.

A. Cytoplasmic Determinants

In some embryos, cell fate is determined by the response of a cell to preexisting

cytoplasmic factors. Embryonic cells containing such cytoplasmic factors can un-
dergo autonomous differentiation according to their normal fate in the absence of
cell–cell interactions.
In Xenopus, the existence of cytoplasmic determinants with dorsalizing activity
has been demonstrated in the vegetal cortex of unfertilized eggs (Fujisue et al.,
1993; Holowacz and Elinson, 1993). Vegetal deposition of determinants in Xenopus
probably results from an asymmetric distribution of maternal components such as
RNAs and proteins along the animal–vegetal axis during oogenesis.
Wild-type Xenopus embryos exhibit prominent external polarity beginning at
mid oogenesis (stage III), when pigment granules become more highly concen-
trated in the animal cortex than in the vegetal cortex. However, animal–vegetal
polarity can be traced back to the stage when the secondary oogonium undergoes
mitotic divisions to give rise to nests of 16 oocytes. The secondary oogonium
contains a large aggregate of mitochondria on only one side of the nucleus. It has
been suggested that this aggregate is the precursor of the mitochondrial cloud, also
known as the Balbiani body (Al-Mukhtar and Webb, 1971; Coggins, 1973).
Differential localization of maternal RNAs in Xenopus follows one of two path-
ways, the message transport organizer (METRO or early) pathway and the late
pathway (Forristall et al., 1995; Kloc and Etkin, 1995; Kloc et al., 2000). RNAs
that follow the METRO pathway first localize to the mitochondrial cloud in stage
I oocytes. Between late stage I and early stage II, the localized RNAs translocate
together with the mitochondrial cloud to the vegetal region and become localized
to the cortex, where they remain throughout oogenesis. RNAs that follow the late
pathway are excluded from the mitochondrial cloud and are found throughout the
cytoplasm in stage I oocytes. Between late stage II and early stage III, late-pathway
RNAs localize to specific domains of the vegetal hemisphere including a crescent-
shaped region in proximity to the nucleus (Chan et al., 1999), a wedge-shaped
structure in the vegetal cytoplasm (Kloc and Etkin, 1995), and at the vegetal corti-
cal region (Melton, 1987). The RNAs eventually localize to the vegetal cortex and
occupy a broader region than do METRO-pathway RNAs.
A subtle difference during the process of vegetal localization has been observed
between two late-pathway RNAs, Vg1 and fatvg. Whereas Vg1 mRNA shows no
association with the mitochondrial cloud during oogenesis (Kloc and Etkin, 1995;
Chan et al., 1999), fatvg mRNA has been found to localize to the mitochondrial
6 Agnes P. Chan and Laurence D. Etkin
cloud transiently in stage II oocytes (Chan et al., 1999). fatvg mRNA also associates
with the germ plasm during early embryogenesis, similarly to METRO-pathway
RNAs (A. P. Chan and L. D. Etkin, unpublished observation). Although the func-
tional role of fatvg mRNA in the germ plasm is yet to be determined, the association
of a late-pathway RNA to the germ plasm suggests that the association of localized
RNA with the germ plasm in embryos is not limited to METRO-pathway RNAs.
Both Vg1 and VegT mRNAs are localized by the late pathway and have been
identified as possible cytoplasmic determinants. Xenopus Bicaudal-C (xBic-C)
mRNA has also been found to localize to the vegetal cortex and shares a similar
time course of localization as Vg1 and Veg T (Wesseley and De Robertis, 2000) Vg1
belongs to the TGF-␤ superfamily. Processed Vg1 can induce dorsal mesoderm
and secondary axis (Dale et al., 1993; Thomsen and Melton, 1993). It has been sug-
gested that the processing of vegetally localized Vg1 mRNA is spatially regulated
so that Vg1 is active only in the dorsal vegetal region of the embryo (Thomsen and
Melton, 1993). VegT is a T-box transcription factor (Zhang and King, 1996). This
factor is also known as Antipodean (Apod) (Stennard et al., 1996), Xombi (Lustig
et al., 1996b), and Brat (Horb and Thomsen, 1997). Antisense oligonucleotide
knockout experiments have indicated that VegT acts as a maternal determinant in
the specification of both the endoderm and mesoderm lineages (Zhang and King,
1996; Kofron, et al., 1999).
Several METRO-pathway RNAs have been identified as possible candidates of
cytoplasmic determinants. These include the Xwnt-11, Xcat2, DEADSouth (for-
merly Xcat3), Xpat and Xdazl mRNAs, all of which follow the METRO pathway
(Kloc and Etkin, 1995) and have been found to associate with the germ plasm in
cleavage stage embryos. Xcat2 is a zinc-finger protein (Mosquera et al., 1993).
High-resolution electron microscopic studies have shown that Xcat2 mRNA is as-
sociated with the germinal granules of the germ plasm (Kloc et al., 1998, 1999).
The Xcat2 protein is related to the Drosophila morphogen Nanos, which is in-
volved in germ cell development, including formation and migration (Kobayashi
et al., 1996; Forbes and Lehmann, 1998). The DEADSouth protein is a DEAD-box
RNA-dependent helicase (MacArthur et al., 2000) Xpat mRNA is expressed in the
primordial germ cells until the cells enter the dorsal mesentery. The Xpat pro-
tein does not contain any identifiable functional domains (Hudson and Woodland,
1998) Xdazl is an RNA-binding protein required for spermatogenesis (Houston
et al., 1998). Depletion of maternal Xdazl mRNA has resulted in defective germ
cell migration within the endoderm during early differentiation (Houston and King,
2000). Xwnt-11 mRNA encodes a maternal Wnt molecule (Ku and Melton, 1993).
Although the RNA is localized to the entire vegetal cortex, the protein differen-
tially accumulates on the dorsal side of the embryo because of regulated translation
of the localized RNA along the dorsal–ventral axis (Schroeder et al., 1999). The
control of Xwnt-11 mRNA translation is mediated by differential polyadenylation.
Xwnt-11 or other maternal Wnt molecules may be the cytoplasmic determinants
required for the VCC/␤-catenin pathway that specifies the dorsal identity of the
embryo, provided that the pathway is dependent on a maternal ligand.
1. Early Xenopus Development 7
The presence of a dorsalizing activity in Xenopus embryos has been demon-
strated by blastomere transplantation studies (Gimlich and Gerhart, 1984; Takasaki
and Konishi, 1989; Kageura, 1990). The dorsal vegetal blastomeres of a 64-cell
embryo can rescue axis formation in UV-irradiated embryos, which would other-
wise develop with a ventralized phenotype lacking any axis (Gimlich and Gerhart,
1984). When the same blastomeres are transplanted to the ventral side of a recipi-
ent embryo, an ectopic secondary axis forms in addition to a normal primary axis.
The transplanted dorsal vegetal cells therefore have a dorsalizing or axis-inducing
activity.
The spatial and temporal origins of the dorsalizing activity have been further
determined by cytoplasmic-transfer and deletion experiments. Vegetal cytoplasm
taken from activated eggs is capable of inducing an ectopic axis in recipient em-
bryos (Fujisue et al., 1993; Holowacz and Elinson, 1993). Only the vegetal cortical
cytoplasm—not the deep vegetal cytoplasm or the cortical cytoplasm from other
regions—contributes to the dorsalizing activity. The cortical localization of a dor-
salizing activity has also been demonstrated by the transplantation of cortical peels
isolated from the vegetal region of activated eggs (Kageura, 1997). The segrega-
tion of dorsal determinants along the animal–vegetal axis has been investigated
by removing cytoplasm from different regions by egg ligation and deletion ex-
periments (Kikkawa et al., 1996; Sakai, 1996). When the vegetal region of an
embryo is deleted just after fertilization, the embryo does not develop any dorsal
identity. Vegetally deleted embryos can be rescued by injecting vegetal cytoplasm
but not animal cytoplasm; again, this finding indicates the presence of a dorsal-
izing activity in the vegetal cytoplasm. The dorsalizing activity is present before
oocyte maturation in prophase I oocytes (Elinson and Pasceri, 1989; Holowacz and
Elinson, 1993, 1995). Exposure of prophase I oocytes to UV irradiation produces
ventralized phenotypes even though cortical rotation has taken place. In these em-
bryos, no dorsalizing activity can be detected after cytoplasmic transplantation
(Elinson and Pasceri, 1989). This shows that dorsal determinants in the oocytes
are destroyed by UV irradiation. UV irradiation of one-cell embryos also results
in ventralized phenotypes, but the UV target is different from that in prophase I
oocytes and is believed to be the microtubule array required for cortical rotation
(Elinson and Pasceri, 1989).
Studies in ascidian embryos have demonstrated the existence of prelocalized
ooplasmic factors in different regions of the fertilized eggs. The animal, vegetal,
and posterior regions of the embryo contain tissue-specific determinants for the
development of epidermis, endoderm, and muscle, respectively (Nishida, 1997).
Blastomeres isolated from the posterior region of ascidian embryos develop au-
tonomously to form muscle (Deno et al., 1984; Nishida, 1992). A search for
localized maternal RNAs in ascidian embryos led to the identification of such
RNAs specifically localized to the myoplasm (Swalla and Jeffery, 1995) and ec-
toplasm (Swalla and Jeffery, 1996). A maternal transcript, pem-3, has also been
shown to localize to the posterior-vegetal cytoplasm of the egg after fertiliza-
tion (Satou, 1999). The protein product of pem-3 contains putative RNA-binding
8 Agnes P. Chan and Laurence D. Etkin
domains known as the KH-domain. These findings provide a molecular basis for
studying cytoplasmic determinants in ascidians.
The possible existence of cytoplasmic determinants in mouse embryos is still be-
ing vigorously investigated (Gardner, 1998, 1999). It has been shown that embryos
subject to centrifugation or mechanical mixing of cytoplasm develop normally
(Mulnard and Puissant, 1984; Evsikov et al., 1994). This finding argues against
specific localization of components in the cytoplasm. Deletions of different regions
of a one-cell mouse embryo have no effect on normal development (Zernicka-
Goetz, 1998). Again, this finding provides no evidence for there being early re-
gional asymmetry in the mouse egg cytoplasm. In addition, extensive cell mixing
that occurs in the mouse epiblast prior to gastrulation is inconsistent with an
early segregation of cell lineages by the inheritance of cytoplasmic determinants
(Beddington and Robertson, 1989). However, STAT3 and leptin have been shown
to localize to the cortex of mouse and human oocytes, and potentially function to
specify asymmetry during early cleavage (Antczak and Van Blerkom, 1997). The
alignment of the animal–vegetal axis of the mouse zygote with the axis of bilateral
symmetry in blastocysts and the proximal–distal axis in egg cylinders suggests
that some degree of regional specification or polarity might already exist in the
egg cytoplasm (Gardner, 1997; Weber et al., 1999).

B. Cortical Rotation

A critical step in determining the prospective dorsal region of the embryo is trig-
gered by cortical rotation. The mechanism and consequences of such movement
has attracted considerable attention. An unfertilized Xenopus egg is radially sym-
metrical along the animal–vegetal axis. Upon fertilization, the dorsal–ventral axis
is defined by the site of sperm entry in the animal region. The sperm entry site
marks the future ventral side of the embryo and overlaps with the first cleavage
plane, which divides the egg bilaterally into right and left halves. After fertiliza-
tion, cortical rotation takes place one-third of the way through the first cell cycle
(100 min), when the outer cortical layer of the fertilized egg rotates 30◦ with respect
to a stationary core (Vincent et al., 1986; Vincent and Gerhart, 1987). However,
this degree of rotation seems to be inconsistent with results from cytoplasmic
transfer studies, which have demonstrated the presence of a dorsalizing activity
around the equatorial region 90◦ away from the vegetal cortex (Yuge et al., 1990;
Fujisue et al., 1993). In fact, axis-inducing activity has also been detected above
the equatorial region in the animal dorsal sector (Gallagher et al., 1991; Hainski
and Moody, 1992; Kageura, 1997).
A possible explanation for the apparent discrepancy between the degree of
cortical rotation and the localization of dorsal activity comes from studies of
microtubule-dependent movement in the vegetal cortical region (Elinson and
Rowning, 1988; Rowning et al., 1997). A set of parallel microtubules is found
in a transport zone 4–8 ␮m below the cortex associated with the inner cytoplasmic
1. Early Xenopus Development 9
core (Larabell et al., 1996). These multiple layers of microtubules align with the
direction of rotation (Elinson and Rowning, 1988). Since the rotation movement
precedes the formation of the microtubule arrays, it has been suggested that the
microtubules are not responsible for the rotation of the cortex (Larabell et al., 1996).
However, small organelles can be propelled along the parallel array of microtubules
that function independently of the cortex. Small organelles may therefore move
along the microtubules by motor molecules toward the plus-end to the dorsal side
of the embryo. Evidence suggests that endogeneous organelles can be translocated
60–90◦ away from the vegetal pole (Rowning et al., 1997). Transport by micro-
tubules thus accounts for the apparent differences in the localization of dorsalizing
activity as predicted from the degree of rotation of the egg cortex. In keeping with
the microtubule transport model, a downstream component of the Wnt pathway,
Dishevelled (Dsh), has been shown to associate with small vesicle-like organelles
that are translocated to the prospective dorsal side by microtubules during cortical
rotation (Miller et al., 1999).
Microtubule transport is not only specific for dorsal–ventral specification in
Xenopus embryos. A dynamic distribution of microtubules has also been observed
in the yolk cells of zebrafish embryos (Jesuthasan and Stahle, 1996). In zebrafish
embryos, a set of parallel microtubules at the vegetal pole region is required for set-
ting up initial asymmetry at the one-cell stage. At the eight-cell stage, microtubule
tracks originating from the dorsal equatorial blastomeres extend toward the vegetal
pole. These microtubule tracks may function to mediate directional transport of
organelles or determinants required for dorsal development.

C. Nieuwkoop Center

After cortical rotation takes place, it is thought that a signaling center—the

Nieuwkoop center—is activated in the dorsal vegetal region that subsequently in-
duces the formation of the organizer in the overlying cells in a non-cell-autonomous
manner. In a series of tissue recombination experiments, different regions of the
yolky vegetal mass of Urodele embryos were tested for their inductive capacity
on animal caps (Boterenbrood and Nieuwkoop, 1973). The dorsal vegetal region
induced dorsal axial structures, whereas the lateral and ventral vegetal regions in-
duced only ventral structures. This tissue recombination assay has been referred to
as the Nieuwkoop recombinant assay. The dorsal vegetal region carrying a dorsal
endomesoderm-inducing property is commonly referred to as the Nieuwkoop cen-
ter (Gerhart et al., 1989). This region is a signaling center required for specifying
the dorsal–ventral axis. The inductive effect of the dorsal vegetal cells is active
between the early cleavage stage and the late blastula stage (Boterenbrood and
Nieuwkoop, 1973). Cell progenies from the Nieuwkoop center do not contribute
to the dorsal lip or axial structures formed during gastrulation. The progenies are
located vegetal to the dorsal lip, in the endoderm, and are fated to become part
of the anterior gut endoderm, as shown by lineage labeling (Bauer et al., 1994;
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