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First published online March 9, 2006
doi: 10.1242/10.1242/dev.02304
Review |
Division of Developmental Biology, Cincinnati Children's Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA.
e-mail: heabq9{at}chmcc.org
SUMMARY
Developmental biology teachers use the example of the frog embryo to introduce young scientists to the wonders of vertebrate development, and to pose the crucial question, `How does a ball of cells become an exquisitely patterned embryo?'. Classical embryologists also recognized the power of the amphibian model and used extirpation and explant studies to explore early embryo polarity and to define signaling centers in blastula and gastrula stage embryos. This review revisits these early stages of Xenopus development and summarizes the recent explosion of information on the intrinsic and extrinsic factors that are responsible for the first phases of embryonic patterning.
Introduction
Over the past 5 years, the usefulness of the Xenopus model
organism has grown considerably as a result of the Xenopus Genome
Initiative (see
www.xenbase.org/).
This endeavor has provided a quantum increase in the amount of information
available on Xenopus genes and the resources with which to study
them. The development of loss-of-function technology has also increased our
knowledge of individual gene function
(Heasman et al., 2000
). The
result is that many more molecules have been shown to control early
Xenopus development. The challenge for the modern developmental
biologist is to stay abreast of this information. In this review, I summarize
these new findings and incorporate them with the old. Inevitably, this survey
will be incomplete. [For further information, see De Robertis and Kuroda
(De Robertis and Kuroda,
2004), and for a comparison with zebrafish axis patterning, see
Schier and Talbot (Schier and Talbot,
2005). For research into germ-line establishment, see also Zhou
and King (Zhou and King,
2004).] For example, the nuts and bolts of development, including
the cytoskeletal and adhesion machinery, many components of signaling
pathways, transcriptional and cell cycle regulators are incompletely covered.
The question that drives this review is, `What insight have recent functional
studies given us on the mechanisms that pattern the early Xenopus
embryo?'.
An overview of early Xenopus development
After fertilization, Xenopus embryos undergo cell cycles that have
characteristic features (Fig.
1). During the first, 90-minute cell cycle, cortical cytoplasmic
movements and male and female pronuclear fusion occur. The next eleven
divisions occur at 20- to 30-minute intervals with no gap phases, while the
embryo forms a ball of 4000 cells, which encloses a fluid-filled blastocoel
cavity. This mid-blastula embryo has three regions, the animal cap (which
forms the roof of the blastocoel), the equatorial or marginal zone (the walls
of the blastocoel) and the vegetal mass (the blastocoel floor) (see
Fig. 1B). Although all
mid-blastula cells are pluripotent
(Heasman et al., 1984),
explants of the animal cap form ectodermal derivatives in culture, while
equatorial explants form mesoderm and vegetal explants form endoderm. At the
end of the twelfth cycle, gap phases reappear, the cell cycle lengthens to 50
minutes and zygotic transcription starts (this is called the mid-blastula
transition, MBT). In the 15th cycle, the dorsal lip of the blastopore forms,
the cell movements of gastrulation begin and mitosis stops. Gastrulation
converts the embryonic ball into three layers, and establishes definitive
anteroposterior and dorsoventral axes (Fig.
2, Box 1). In this
review, I retrace this developmental pathway and ask how cells become
committed to specific fates.
Pre-patterning by maternally stored mRNAs and proteins
To what extent does embryonic patterning rely on mRNAs and proteins
inherited from the oocyte, or upon intercellular signaling downstream of
zygotic gene transcription? For Xenopus development, it was predicted
that oocyte stores would be essential for embryonic patterning, because
zygotic transcription does not begin until the 4000-cell stage and because
newly expressed zygotic genes have localized expression patterns. Recent
studies have confirmed this prediction. Included in the essential maternal
pool are: genome-wide transcriptional repressors, such as Xkaiso and the
LEF/TCF family member Xtcf3 (Houston et
al., 2002; Ruzov et al.,
2004); transcriptional activators, including forkhead proteins
(e.g. FoxH1, Foxi1E) (Kofron et al.,
2004a; Suri et al.,
2005); the T box protein VegT
(Zhang et al., 1998); and
cAMP response element-binding protein (CREB)
(Sundaram et al., 2003).
TATA-binding components of basal transcriptional complexes, TBP and TBP2, are
also essential for normal development, and their depletion reduces the
transcription of specific zygotic target genes and disrupts gastrulation
(Jallow et al., 2004).
A simple strategy that provides a blueprint for development is the
localized positioning of maternal mRNAs in the oocyte so that they are
inherited by specific areas of the embryo. Transcripts of the transcription
factors Zic2 and Xenopus grainyhead 1 (Xgrhl1) are
localized to the animal hemisphere of the oocyte and early embryo
(Houston and Wylie, 2005;
Tao et al., 2005a). By
contrast, VegT transcripts are localized in the oocyte vegetal
hemisphere (Zhang and King,
1996), and VegT protein is inherited by only vegetal cells
(Stennard et al., 1999).
The list of vegetally localized mRNAs continues to grow and includes
transcripts of the signaling molecules Vg1
(Weeks and Melton, 1987) and
Wnt11 (Ku and Melton, 1993),
of the transcription factor Otx1 (Pannese
et al., 2000) and of the RNA-binding protein bicaudal C
(Wessely and De Robertis,
2000). The cortical cytokeratin filament network is likely to hold
these transcripts in place, as antibodies specific for cytokeratin disruption
dislodge localized mRNAs (Kloc et al.,
2005). Unexpectedly, the degradation of two of the localized mRNAs
themselves, VegT mRNA and the non-translated mRNA Xlsrts,
also dislodges other mRNAs (Heasman et
al., 2001; Kloc and Etkin,
1994; Kloc et al.,
2005) and disrupts the cytokeratin network. These effects are
rescued by VegT mRNA, suggesting that it has an architectural role,
although the mechanism is unresolved (Kloc
et al., 2005).
Vegetally localized mRNAs do not all fall into one spatial group. For
example, transcripts of the RNA-binding protein Xdazl
(Houston et al., 1998) and
Xpat mRNAs (Machado et al.,
2005) localize to the germplasm and remain in primordial germ
cells, while VegT mRNA localizes to presumptive endodermal cells
(Stennard et al., 1999).
Vg1 mRNA becomes enriched in the dorsal vegetal quadrant of the early
embryo compared with the ventral vegetal quadrant
(Birsoy et al., 2006;
Tao et al., 2005b). Thus,
several distinct mechanisms of partitioning probably exist.
|
During the first cell cycle, the movement of the cortical cytoplasm
(Fig. 1), has long been known
to be essential for establishing the embryonic dorsoventral (DV) axis
(Vincent and Gerhart, 1987).
Cytoplasmic transfer and ultraviolet (UV) irradiation studies lead to the
hypothesis that a vegetally localized `dorsal determinant' is relocated by
cortical rotation (Scharf and Gerhart,
1980; Holwill et al.,
1987). Several lines of evidence indicate that the dorsal
determinant is a component of a canonical Wnt signaling pathway
(Heasman et al., 1994;
Kofron et al., 2001). The most
likely candidate is Wnt11 mRNA.
Wnt11 mRNA localizes to the vegetal cortex during oogenesis
(Ku and Melton, 1993), and
loss-of-function experiments show that maternal Wnt11 is necessary and
sufficient for specification of the embryonic DV axis
(Tao et al., 2005b). It acts
as a canonical Wnt in this regard, as depletion of the transcriptional
co-activator of Wnt target genes, ß catenin, blocks the dorsalization
caused by Wnt11 mRNA overexpression. Furthermore, ß catenin
overexpression rescues Wnt11-ventralized embryos
(Tao et al., 2005b). In
addition, UV-irradiation of the vegetal pole of the fertilized egg causes a
reduction in the amount of Wnt11 mRNA
(Schroeder et al., 1999).
|
;
Tao et al., 2005b). Once
asymmetrically concentrated, and because there is no new synthesis of
Wnt11 mRNA, the translation and secretion of Wnt11 might be enhanced
in dorsal vegetal cells compared with ventral vegetal cells. Wnt11
mRNA may not be the only `dorsal determinant'. For example, Vg1 mRNA
is also localized and enriched dorsally at the 32-cell stage and is important
in early patterning events (Birsoy et al.,
2006).
Other components of the Wnt signaling pathway, the intracellular
dishevelled protein Xdsh and kinesin-binding protein GBP, also move in
cortical cytoplasm. Xdsh-GFP- and GBP-GFP-containing vesicles move with
cortical rotation towards the dorsal side of the embryo
(Miller et al., 1999;
Weaver et al., 2003). GBP
depletion causes a loss of dorsal structures
(Yost et al., 1998), but Xdsh
has not yet been directly shown to be required for dorsal axis formation.
Unexpectedly, tagged Xdsh protein is localized in Xenopus nuclei,
suggesting Xdsh nuclear localization is required for canonical Wnt signaling
(Itoh et al., 2005).
Until recently, no maternal factors were known to localize along the
embryonic left/right axis. However, the tryptophan derivative, 5
hydroxytryptamine (5-HT or serotonin) was shown to be distributed equally in
the vegetal hemisphere at the two-cell stage, but then to accumulate
specifically in the daughters of the right ventral blastomere from the
four-cell stage onwards, in a gap junction-dependent process
(Fukumoto et al., 2005).
Inhibition of serotonin signaling with receptor blockers shows that it is
required for the later left-sided expression of the nodal-related
Xnr1 mRNA, as well as for correct gut and heart looping. This raises
intriguing questions about how serotonin is localized in this fashion and how
it interacts with canonical signaling pathways.
After the 90-minute marathon of the first cell cycle, the following eleven
division cycles are more rapid (Fig.
1). This is a period of apparent quiescence in terms of cell
signaling and transcriptional events. Signaling through the TGFß and FGF
pathway is low until MBT, as shown by immunostaining for activated forms of
Smad1, Smad2 and MAP kinase (Faure et al.,
2000; Lee et al.,
2001; Schohl and Fagotto,
2002). Heterochronic co-culture assays using pre-MBT and post-MBT
vegetal masses with mid-blastula animal caps also show that no mesoderm
induction can be detected (using the expression of the somite marker MyoD)
from pre-MBT vegetal masses (Wylie et
al., 1996). Furthermore, zygotic expression levels for most genes
are low or undetectable before MBT.
| Box 1. Defining the axes of Xenopus embryos
The first axis of the Xenopus embryo is the animal-vegetal axis,
which passes through the animally localized egg pronucleus, the center of the
egg and the vegetal pole (Fig.
1B, Fig. 2B). The
second axis is defined by the sperm-entry point and by the position of maximal
movement of the cortical cytoplasm away from the sperm-entry point
(Fig. 1B,
Fig. 2B). At gastrulation, the
dorsal lip of the blastopore forms opposite the sperm-entry point. Although
described as `dorsal', this Spemann Organizer region
(Fig. 1A) contains anterior
precursors, including prechordal mesoderm
(Dale and Slack, 1987 As the term `dorsal lip of the blastopore' is established in the literature, it continues to be used here, accepting the fact that the region includes non-dorsal precursors. As shown in Fig. 2, the embryonic dorsoventral axis describes the position of cells relative to the sperm-entry point and to the site of future formation of the dorsal lip blastopore (Fig, 2B). The definitive dorsoventral axis refers to the axis at right angles to the anteroposterior axis, which is established at the end of gastrulation (see Fig. 2B).
|
By contrast, there is accumulating evidence that pre-MBT maternal Wnt
signaling occurs. First, Wnt11-induced dorsalization occurs most effectively
if the mRNA is introduced into the oocyte rather than the fertilized egg
(Tao et al., 2005b),
suggesting that it is required soon after fertilization. Second, nuclear
localization of ß catenin on the dorsal side of the embryo happens before
MBT (Larabell et al., 1997;
Schneider et al., 1996).
Third, depletion of maternal ß-catenin protein
(Heasman et al., 2000) or
activation of a dominant-negative Xtcf3 [the transcription factor activated by
maternal ß catenin (Yang et al.,
2002)] blocks dorsal axis formation at the two- or four-cell
stage, but not later. This indicates that the signaling pathway cannot be
inactivated by the late cleavage stage. Finally, Xtcf3 activity in pre-MBT
embryos is sensitive to the transcription inhibitor actinomycin D, and two of
its target genes, the TGFß nodal-family members Xnr5 and
Xnr6, are expressed from the 256-cell stage onwards
(Yang et al., 2002). These
experiments provide strong circumstantial evidence that the earliest phase of
ß-catenin/Xtcf3 interaction happens during the cleavage to early blastula
stages.
Despite these exceptions, zygotic genes are generally repressed until the
13th cell cycle, and are associated with condensed, hypoacetylated and
H3-methylated chromatin (Meehan et al.,
2005). Several recent studies have shed light on the mechanism of
the transcriptional activation of zygotic genes. Xkaiso, a transcriptional
repressor that binds to specific DNA-binding sequences in a
methylation-dependent manner, maintains pre-MBT repression of an estimated 10%
of zygotic genes (Ruzov et al.,
2004). When Xkaiso is depleted, zygotic transcription starts two
cycles earlier than normal (Ruzov et al.,
2004). The activation of one Xkaiso target, zygotic
Wnt11, depends on the binding of the catenin-family member,
p120-catenin, which causes Xkaiso to dissociate from the Wnt11
promoter (Kim et al.,
2004).
Maternal Xtcf3 acts in a similar way to Xkaiso, by repressing the
expression of Wnt target genes (Brannon et
al., 1997; Houston et al.,
2002; Roose et al.,
1998). New work shows that Xkaiso and Xtcf3 act together to
prevent the transcription of the homeobox transcription factor,
siamois repression (Park et al.,
2005). Whether complexes of Xkaiso and Xtcf3 regulate all Xtcf3
target genes is not known. For many zygotic genes, transcriptional activators
may also be required. For example, the expression of the nodal-related gene
Xnr5 is repressed ventrally by maternal FoxH1 and Sox3, as well as by
ß-catenin/Xtcf3, and is activated dorsally by VegT
(Hilton et al., 2003;
Kofron et al., 2004a;
Zhang et al., 2003). In
addition, depletion experiments suggest that maternal Xtcf4 acts as an
activator of organizer genes, while Xtcf1 has context-dependent activating and
repressing roles (Standley et al.,
2006).
Another aspect of the re-activation process is the availability of
transcriptional co-activators. Until MBT, the transducers of the TGFß and
FGF signaling cascades, Smad1, Smad2 and MAP kinase, are inactive. In
addition, a little explored mechanism of regulation of transcriptional
activation involves the nuclear matrix, which may be required for the
formation of stable transcriptional complexes. Before MBT, transcription
factors may be able to bind to DNA but not able to form stable complexes or to
recruit the basal transcriptional machinery. When chromatin domains are in an
active state, they have a defined, rather than a random, attachment to the
nuclear matrix. Activation of the basic helix-loop-helix (bHLH) transcription
factor Myc has been correlated to specific anchorage sites after MBT,
when compared with its random nuclear matrix attachment before MBT
(Vassetzky et al., 2000).
Whether this mechanism plays a widespread role in transcriptional activation
at MBT remains to be resolved.
From MBT to the beginning of gastrulation
As soon as zygotic transcription starts, the instructive events that set up the framework of the three germ layers rapidly become complex. At least four major signaling pathways are essential that activate the signal transducers Smad2, Smad1, ß catenin and MAP kinase. Earlier reviews have suggested that gradients of the ligands that activate these pathways (Xnr proteins, activin, Vg1, BMP2, BMP4, BMP7, Wnt11, Wnt8, FGF3, FGF4 and FGF8) pattern the blastula in the embryonic animal-vegetal (AV) or DV axis, but such gradients are hard to demonstrate for endogenous ligands. In addition, the number of potential intracellular and extracellular regulators of these pathways continues to grow, including modulators of transcription, translation, processing, cleavage, co-receptors and antagonists, and of the signal transduction intermediaries, many of which are themselves specifically localized in the embryo. Although gradients may be the outcome of these regulations, the challenge at the moment is to understand the signaling context of each location in the embryo, and the results of such signaling in terms of gene expression and embryonic patterning.
The activin-type TGFß pathway
VegT is inherited equally by all vegetal cells and that activates the
expression of an endodermal-determination network of genes. It also has roles
in mesoderm induction and gastrulation
(Kofron et al., 1999;
Xanthos et al., 2001)
(Fig. 3). VegT regulates the
transcription of pro-endodermal transcription factors, including the HMG-box
gene Xsox17, and GATA factors 4, 5 and 6. It also activates the
transcription of genes encoding mesoderm-inducing molecules (such as
Xnr5 and Wnt8) and of cerberus (the BMP and Wnt
antagonist), raising the issue of how the domains of mesodermal and endodermal
gene expression downstream of VegT are dictated. One likely regulator is the
homeodomain protein Mixer, a target of VegT that induces endodermal
(Xsox17) gene expression while repressing mesodermal genes (such as
those encoding the T-box transcription factor eomesodermin and Fgf8)
(Kofron et al., 2004b).
|
;
Xanthos et al., 2001). This,
as well as many other studies, suggests a pivotal role for Xnr proteins
downstream of VegT in mesoderm and endoderm formation (for a review, see
Agius et al., 2000).
As well as VegT-target TGFß proteins, two other TGFß family
members, Vg1 and activin, play essential roles in patterning the gastrula
(Fig. 3). For many years, Vg1
function was not clear because the original gene product was poorly translated
and processed (Tannahill and Melton,
1989), and did not rescue the Vg1-depleted phenotype
(Birsoy et al., 2006). By
contrast, a second Vg1 allele has recently been characterized, called
Vg1-ser, which is more efficiently processed than the first allele (Vg1-pro)
and does partially rescue the Vg1-depletion phenotype
(Birsoy et al., 2006).
Consistent with the dorsal enrichment of Vg1 mRNA, dorsally localized
BMP antagonist mRNAs (chordin, cerberus, noggin) are severely
depleted in Vg1-depleted embryos, while general endoderm markers are less
affected. Smad2-phosphorylation and gastrulation are delayed in Vg1-depleted
embryos and they develop microcephaly.
The fact that Vg1 activates the same pathway as the nodal proteins raises
the question of why it does not alleviate the phenotype of embryos lacking
VegT function. One likely explanation is that, as discussed above,
VegT mRNA also has a role in the oocyte, maintaining the localization
of other maternal mRNAs. Its depletion reduces Vg1 mRNA and protein,
as well as VegT (Heasman et al.,
2001; Kloc et al.,
2005). Thus the original `VegT phenotype' is likely to be due to
the loss of both Vg1 and VegT. New studies are required to determine the
specific role of VegT alone, using morpholino oligos, which block VegT protein
synthesis but do not degrade VegT mRNA, and do not disrupt
Vg1 mRNA localization (Heasman et
al., 2001).
The function of activin B also took a long time to clarify. Loss of
function studies show it is essential for normal development, and regulates
the dorsal zygotic genes, particularly goosecoid, chordin and the
anterior endodermal marker Xhex. Unlike Vg1, it regulates the
transcription of other TGFß proteins. In particular, Xnr2 mRNA
expression is increased and the Vg1-related derriere mRNA is
decreased by the loss of activin function
(Piepenburg et al., 2004).
Derriere, in turn, regulates the expression of the promesodermal gene
Fgf4 (Sun et al.,
1999).
Although the precise roles of all the TGFß proteins remain to be
resolved, what is clear is that, individually or together, the Xnr proteins,
derriere, Vg1 and activin activate several signal transduction cascades during
the mid-late blastula stages, leading to the transcription of many zygotic
genes. First, they cause the phosphorylation of Smad2 in receiving cells.
Phospho-Smad2 acts as a co-activator of many transcription factors, including
the maternal cell cycle regulator transcription factor, p53
(Cordenonsi et al., 2003), the
transcriptional activator and repressor FoxH1
(Kofron et al., 2004a), and
the VegT target homeodomain transcription factor Mixer
(Kofron et al., 2004b), all of
which are essential for early embryonic patterning. Second, they activate
TGFß-activated kinase 1 (TAK1), which in turn activates [through
nemo-like kinase (NLK)] another essential transcription factor, signal
transducer and activator of transcription (STAT3)
(Ohkawara et al., 2004).
Third, Xnr proteins induce the expression of FGF3, FGF4 and FGF8, which bind
FGF receptors and activate several transcription factors, including activator
protein 1 (AP1).
The FGF signaling pathway
Although some FGF mRNAs are expressed maternally, there are no known
maternal transcripts that localize to the equatorial zone of the oocyte. The
earliest equatorial-specific factors appear at the late blastula stage and
include zygotic T-box genes, brachyury (Xbra),
eomesodermin and antipodean
(Ryan et al., 1996;
Smith et al., 1991;
Stennard et al., 1996). Their
expression is dependent on both Xnr signaling
(Xanthos et al., 2002;
Xanthos et al., 2001), and
the maternal Wnt pathway (Vonica and
Gumbiner, 2002). eomesodermin is expressed first and is
enriched on the dorsal side, and engrailed-repressor experiments suggest it
regulates FGF and Xbra expression, which then act in cross-regulatory
loops (Ryan et al., 1996). The
boundaries of the expression domains of the T-box genes are constrained by
several animally localized regulators (see below), and by Mixer vegetally. In
agreement with this, MAP kinase immunostaining shows that high FGF signaling
is restricted to the equatorial region at this time
(Schohl and Fagotto, 2002),
and FGF loss of function causes reduced somites and notochord and defects in
convergence extension movements (see below)
(Amaya et al., 1991;
Conlon et al., 1996;
Fisher et al., 2002).
The Wnt signaling pathway
The third major influence in the mid-blastula is the maternal Wnt signaling
pathway. Without its activity, the embryo develops with three layers, but
lacks dorsal, anterior or posterior structures. What information does this
signal provide? Because of the enrichment of Wnt11 and its dorsal secretion,
the expression of siamois, goosecoid, Xhex, Xnr3, and of the
signaling antagonists noggin, chordin and cerberus is
specifically localized. These proteins regulate head, notochord and somite
formation (see below). In addition, chordin, noggin and
siamois are expressed in the embryonic dorsal animal cap, as well as
the marginal zone, and this expression is essential for anterior neural
induction (Kuroda et al.,
2004).
Although the Wnt signal is required for their expression, each zygotic gene
is regulated differently, by multiple factors. For example, cerberus,
is directly regulated by at least four transcription factors: Xlim1, a target
of VegT activation; the orthodenticle-related protein Otx1; and homeodomain
proteins Siamois and Mix1 (Yamamoto et
al., 2003). Such combinatorial regulation may explain why the Wnt
target genes are not expressed in identical locations. For example,
Xnr5 is expressed in dorsal vegetal cells and Xnr3 is
expressed above the dorsal lip of the blastopore. Alternatively, more than one
maternal Wnt signal may regulate their expression.
The BMP signaling pathway
The BMP signaling pathway is initially activated at MBT throughout the
mid-blastula, downstream of maternal BMP2 and BMP7 activity, except in the
embryonic dorsal animal quadrant (Faure et
al., 2000; Schohl and Fagotto,
2002). The restriction of BMP signaling from this quadrant may be
the result of the early expression of the BMP antagonists noggin or
chordin (Kuroda et al.,
2004). Animal cap regions explanted from mid-blastulae follow an
epidermal differentiation pathway that is dictated by BMP signaling
(Fig. 4). When all BMPs and the
dorsally expressed BMP-like molecule anti-dorsalizing morphogenetic protein
(ADMP) are depleted, the entire outer layer of the embryo expresses neural
markers (Reversade and DeRobertis,
2005). What then causes neural specification?
|
).
However, it has recently been shown that cell dissociation actually activates
FGF signaling and inhibits Smad1 by MAP kinase phosphorylation of its linker
region (Kuroda et al., 2005).
This raises the question, `is FGF the activator of neural specification, or
does it act solely as a BMP signaling antagonist?'. Definitive evidence that
FGF has proneural roles would be obtained by identifying specific FGF
transcriptional targets required for neurogenesis. Recent in vivo studies
suggest that this is the case; the proneural genes Sox2 and neural
cell-adhesion molecule (Ncam) expression depend on low levels of FGF
signaling at the blastula stage, independently of BMP antagonism
(Delaune et al., 2005).
The epidermal regulatory network downstream of BMP signaling includes the
transcriptional activators Xvent2
(Onichtchouk et al., 1996) and
Msx1 (Suzuki et al.,
1997), which activate the epidermal pathway and also suppress
pro-neural genes when ectopically overexpressed. These genes in turn activate
more restricted pro-epidermal genes, which can directly regulate epidermal
structural genes, but do not have neural repressive roles
(Tao et al., 2005a),
(Fig. 4). Thus, epidermal fate
is determined by BMP signaling, while neural specification may require FGF
signaling and BMP antagonism (Fig.
5).
|
;
Williams et al., 2004). But
even after suppression of BMP signaling, or after suppressing both BMP signals
and their antagonists, animal cells express neural, rather than mesodermal,
markers (Reversade and De Robertis,
2005). So what prevents animal cells from undergoing mesoderm
induction?
Recently, several intrinsic mesoderm antagonism mechanisms have been
identified. One essential maternal regulator is the RING-type ubiquitin
ligase, ectodermin, which regulates Smad4 degradation
(Dupont et al., 2005). As
Smad4 heterodimerizes with both Smad1 and Smad2, its degradation reduces both
BMP and nodal-type TGFß signaling. The field of influence of ectodermin
is dictated by its localized pattern of expression in the animal half of the
oocyte and blastula, and its depletion causes the ectopic expression of the
mesodermal gene eomesodermin and the expanded expression of the
endodermal gene Mixer into the animal hemisphere. The neural marker
Xsox2 is also downregulated by ectodermin depletion. Thus, the animal
localization of ectodermin dictates the lower margin of the ectoderm precursor
region and favors neural specification. ectodermin expression becomes
asymmetrically enriched in the embryonic dorsal animal quadrant at the
gastrula stage, where the abrogation of Xnr and BMP signaling is required for
neural specification (Dupont et al.,
2005).
Animally localized maternal and zygotic transcription factors also regulate
the boundary between pro-ectodermal and mesodermal areas. The depletion of the
maternal, animally localized, Zic2 transcript results in increased
Xnr expression (Houston and Wylie,
2005), while the Xenopus X-box binding protein 1
(Xbp1) regulates BMP4 expression and suppresses mesodermal and neural
gene expression (Cao et al.,
2006). In addition, the Forkhead-family member Foxi1E (also known
as Xema) activates epidermal differentiation and represses endodermal and
mesodermal gene expression in animal cap cells
(Suri et al., 2005).
BMP signaling occurs in the ventral equatorial zone, as well as in the
animal cap, at the blastula stage. What then are the states of determination
of equatorial cells when BMP function is blocked? A key analysis here has been
to manipulate BMP signaling in a temperature-sensitive manner
(Marom et al., 2005). BMP
suppression at the blastula stage causes upregulation of organizer genes
(goosecoid), causing secondary axis (neural and somite tissue)
formation. This indicates that a major, organizer-suppressing role of the BMP
pathway occurs early at the blastula stage.
Thus, at the late blastula stage, the animal cap is already divided into areas of BMP signaling and BMP antagonism, the equatorial zone is a site of dynamic interactions of all four pathways, and the vegetal mass has high Smad1 and Smad2 activity and low Wnt and FGF signaling. During gastrulation, the results of these interactions begin to be realized.
Gastrulation
The timing of gastrulation
During gastrulation, the cell cycle expands from 55 minutes to 4 hours, a
lengthening that is essential for further development
(Fig. 1). The control of
cytostasis is not understood, although TGFß signaling is likely to be
involved as it is known to limit re-entry into the cell cycle
(Siegel and Massague, 2003)
and is necessary downstream of VegT for gastrulation to occur
(Zhang et al., 1998). WEE1,
an antagonist of M-phase re-entry, is clearly required because its depletion
causes an increased mitotic index from 10% to 25% during gastrulation and
results in abnormalities in gastrulation movements. Zygotic gene expression
continues, although the positioning of Xbra and chordin
expression is disrupted, which may be crucial for correct cell movements (see
below) (Murakami et al.,
2004). WEE1 may regulate the mitotic activity of bottle cells, the
shape changes of which in response to Xnr/Vg1/activin are responsible for the
first invagination movements of gastrulation. These cells are the earliest
non-mitotic population at gastrulation, and promoting mitosis arrests bottle
cell formation (Kurth, 2005).
WEE1 is a maternal protein, which may explain a long-standing observation that
the timing of MBT and gastrulation onset are not linked
(Smith and Howard, 1992). The
timing of gastrulation is also dependent on several other maternal inputs.
Abrogating either the maternal Wnt or Vg1 pathway delays formation of the
dorsal lip (Birsoy et al.,
2006; Heasman et al.,
1994), and maternal CREB depletion slows ventral lip formation
(Sundaram et al., 2003).
|
), and driving firstly the involution of the prechordal (head)
mesoderm, followed by chordamesoderm (presumptive notochord)
(Fig. 6). The fact that these
two domains can be physically separated suggests that AP patterning in the
dorsal mesoderm is established by this time. After involution, prechordal
mesoderm cells become actively migratory and move animally
(Shook et al., 2004). This
behavior is regulated by multiple factors but the definitive endogenous
combination is not yet known.
One secreted protein known to be involved in chordamesoderm cell behavior
is platelet-derived growth factor, PDGFA, which is secreted by blastocoel roof
cells. PDGFA depletion causes random protrusive activity of prechordal
mesoderm and loss of head structures
(Nagel et al., 2004). A second
regulator of prechordal mesoderm is the Wnt antagonist dickkopf
(Kazanskaya et al., 2000).
Inhibition of its activity results in microcephaly, while overexpression of
dickkopf expands the size of the prechordal plate at the neurula stage,
without affecting chordamesoderm formation. An important issue here is which
Wnt signal is being antagonized by dickkopf. Third, the migrating prechordal
mesoderm zone is an area of active repression of Xbra expression.
Repression is achieved by several inputs, including the binding of the
transcriptional repressor goosecoid to the Xbra promoter
(Yao and Kessler, 2001).
Xbra overexpression in the prechordal mesoderm prevents cell adhesion
to the extracellular matrix protein fibronectin
(Kwan and Kirschner, 2003). As
with PDGFA depletion, abrogation of goosecoid activity or of its
transcriptional activator siamois blocks prechordal migration,
presumably by increasing Xbra expression. Last, the size, shape and
correct placement of the entire involuted region is regulated by nodal, BMP
and Wnt antagonists, particularly by the TGFß family member antivin/lefty
(Branford and Yost, 2002).
Antivin-depleted embryos have increased and expanded
Gsc/Xnr3 and Xbra expression, exogastrulate at the
mid-gastrula stage and fail to form heads
(Branford and Yost, 2002).
The period of prechordal mesoderm migration is brief and is limited by the
direct adhesion of the prechordal mesoderm to the head neuroectoderm
(Koide et al., 2002)
(Fig. 2), so the major force in
extending the AP axis in gastrulation is the involution and convergence
extension of the chordamesoderm (Figs
2 and
6). Many factors regulate this
convergence extension, as described in Box
2. In summary, many essential early zygotic patterning genes are
not tissue differentiation genes, but are involved in regulating cell
behavior. For example, loss of dickkopf, siamois or
goosecoid activity abrogates prechordal mesoderm migration and
prevents the formation of head structures, while Xbra is required for
convergence extension of the chordamesoderm.
| Box 2. Factors regulating chordamesoderm formation Convergence extension (CE) movements of the chordamesoderm close the blastopore during gastrulation (see Fig. 2A and Fig. 6). Several factors have essential roles in this process:
|
Events in the non-organizer marginal zone
Meanwhile, away from the organizer region, convergence extension movements
are delayed. Several pathways are involved in establishing muscle precursor
fates. First, FGF signaling and Xbra expression are required and
maintain each others expression, as described above
(Fig. 7). The expression
pattern of Xbra mRNA, which forms a ring around the blastopore of the
gastrula, is evidence that its role is not confined to dorsal convergence
extension activity. Promoter studies show that dorsal and ventrolateral
Xbra expression are differently regulated
(Latinkic and Smith, 1999;
Lerchner et al., 2000;
Papin et al., 2002). By
mid-gastrulation the myogenic markers Mespo, Myf5 and MyoD
mRNA are all expressed in an equatorial ring similar to Xbra and FGF,
and depletion of FGF4, FGFR1 or Xbra causes severe reduction in their
expression (Conlon et al.,
1996; Fisher et al.,
2002; Yokota et al.,
2003).
How does Xbra function in both myogenic and convergence extension
regulation? The expression of one target gene, the cytoplasmic regulator
sprouty, may be particularly important in this regard. Sprouty is a
cytoplasmic antagonist of FGF signaling that blocks convergence extension
movements by interfering with protein kinase C (PKC) function, without
blocking activation of myogenic gene expression. It can thus separate the
convergence extension and muscle specification functions of Xbra
(Sivak et al., 2005). A
second receptor tyrosine kinase regulator, Spred, has the opposite effect; it
is required for somite specification but not for convergence extension
movements (Sivak et al.,
2005). Muscle specification during gastrulation also depends on
the repression of BMP signaling, as triple depletions of noggin, chordin and
follistatin eliminate muscle precursor gene expression
(Khokha et al., 2005).
|
). The
early Hox expression determines the length of the trunk region, and depletion
of HoxA1, HoxB1 and HoxD1 results in reduced MyoD expression
(Wacker et al., 2004a;
Wacker et al., 2004b). Hox
expression depends on the FGF/Xbra pathway
(Fig. 7), and is limited by BMP
signaling (Wacker et al.,
2004b). Another important mesodermal regulator is retinoic acid
(RA). Depletion of retinoic acid receptor Xrar
2 causes both
microcephaly and tailless embryos, and reduces expression of both FGF
receptors FGFR1 and FGFR4, and posterior Hox gene expression (HoxB9)
(Shiotsugu et al., 2004). The
Xenopus caudal family member Xcad3 is essential for tail
somite formation, and its transcriptional regulation is complex and involves
maternal CREB, FGF signaling and RA activity
(Isaacs et al., 1998;
Shiotsugu et al., 2004;
Sundaram et al., 2003).
Unexpectedly, a key BMP antagonist, the frizzled-related protein sizzled,
is expressed not dorsally, but in the ventral lip of the blastopore. Sizzled
acts as a competitive inhibitor of the chordin metalloproteinases, Xlr1 and
BMP1 (Lee et al., 2006). Its
expression is activated by BMP signaling and is repressed by VegT/Vg1 via the
BMP antagonists (Birsoy et al.,
2006; Lee et al.,
2006; Salic et al.,
1997). Depletion of sizzled causes an expansion of ventral blood
islands and does not affect the expression of MyoD or of organizer
genes. It has an essential role in regulating epidermal versus neural cell
fates (Lee et al., 2006).
Events in the animal cap
While these complex events are occurring in mesodermal precursors, cells in
the animal cap remain set to become epidermis, as dictated by continuing BMP
signaling, providing that they are not underlain by involuted prechordal
mesoderm or chordamesoderm. Switching from epidermal to proneural fate
absolutely requires BMP suppression, as described previously
(Khokha et al., 2005); the
entire ectoderm becomes neural when all BMP signaling is depleted
(Reversade et al., 2005;
Reversade and De Robertis,
2005). The epidermis is a bilayer at the gastrula stage, and both
layers overlying the chordameosderm express proneural genes, although only the
inner layer undergoes primary neuronal differentiation at the neurula stage
(Chalmers et al., 2002)
(Fig. 5).
It is clear that neural specification, like that of muscle, is an active
process, involving a complex network of transcription factors
(Fig. 5). Transcription factors
of the Sox class are expressed in ectoderm from the late blastula stage, and
dominant-negative Sox2 inhibits neural induction when it is expressed
specifically during the gastrula stage
(Kishi et al., 2000). SoxD and
Smad interacting protein 1, SIP1, a member of the EF1/ZFH family, maintain
expression of each other after the gastrula stage. Loss-of-function
experiments show that they are necessary for neural induction, with SIP1
preventing the activation of pro-epidermal genes
(Nitta et al., 2004). The
three paralogous Hox group 1 genes, HoxA1, HoxB1 and HoxD1,
are essential for neural patterning and begin to be expressed at the gastrula
stage; their simultaneous depletion has severe effects on hindbrain and neural
crest patterning (McNulty et al.,
2005). FGF8 and RA are the most likely candidate activators of Hox
gene expression (Christen and Slack,
1997; Shiotsugu et al.,
2004). Cell cycle withdrawal is also important for further neural
differentiation. Depletion of the maternally expressed SWI/SNF remodeling
protein BRG1 causes the proliferation and expansion of proneural gene
expression (Sox2), but reduces neural differentiation. BRG1 also
directly binds to and co-activates several bHLH transcription factors in the
differentiation pathway (Seo et al.,
2005).
As gastrulation proceeds, anterior- and posterior-specific neural genes
begin to be expressed in the neural anlagen, a process that depends on the
anterior repression of Wnt signaling via both intrinsic and extrinsic
pathways. An important regulator of neural AP patterning is the zinc-finger
protein Xsalf, which is activated in the anterior neural ectoderm in the late
gastrula. Xsalf both activates the expression of anterior neural genes
(Otx1) and represses posteriorizing Wnt signals by activating the
expression of Wnt repressors Xtcf3 and GSK3ß, so that
Xsalf-depleted embryos have reduced heads and lack forebrain gene expression
(Onai et al., 2004). The
picture that emerges is different from the original activation/transformation
model of neural induction, which suggested that the activation step turns the
entire neural anlagen into a pro-anterior neural state, which is later
transformed by a posteriorizing gradient. The activation/transformation model
predicts that Xsalf would be expressed throughout the neural ectoderm, which
it is not. This lends support to an alternative, regional activation model, as
described further elsewhere (Onai et al.,
2004).
Events in the vegetal mass
The vegetal mass, the cells of which are determined towards endodermal
fates by the early gastrula stage (Heasman
et al., 1984), is the least studied region of the gastrula.
Immunostaining of the vegetal mass identifies the major signaling activities
as those continuing from the blastula stages: TGFß proteins that activate
Smad2 and Wnt signaling (Faure et al.,
2000; Schohl and Fagotto,
2002). It remains to be shown whether the nuclear ß-catenin
present at this time is the result of maternal Wnt11 signaling or of new
zygotic Wnt pathways. FGF signaling is very low in this region, consistent
with the fact that overexpressed FGF causes reduced endoderm formation, and
blocking FGF signaling expands the expression of endodermal genes into the
equatorial zone (Cha et al.,
2004). BMP signaling activity is almost completely excluded from
the dorsal vegetal area, suggesting its antagonism is necessary for endoderm
specification. In agreement with this, chordin and noggin treatment of animal
caps causes ectopic endodermal gene expression
(Sasai et al., 1996).
Gene expression in the gastrula stage is dictated by the four pathways that
are activated in the blastula vegetal mass. These pathways can be placed into
three groups; those involved in boundary formation between mesoderm and
endoderm [such as the Bix and Mix homebox transcription factors
(Casey et al., 1999;
Kofron et al., 2004b), which
are expressed during gastrulation only]; a dorsally localized group whose
expression domain includes the dorsal prechordal mesoderm (often described as
anterior mesendodermal genes), including cerberus, Xlim1, dickkopf
and Xhex, the main function of which may be in establishing correct
gastrulation movements; and those that are distributed throughout the vegetal
mass (Xsox17, Gata4, Gata5 and Gata6). This last group of
transcription factors continues to establish regulatory networks required for
endoderm specification and maintenance after gastrulation
(Afouda et al., 2005;
Xanthos et al., 2002;
Xanthos et al., 2001). A new
player on the signaling scene in the presumptive endoderm is the short-range
signal receptor Notch. Notch suppression leads to the expansion of mesodermal
molecular markers and to the loss of endodermal markers, endodermin and the
HMG box transcription factor Xsox17
(Contakos et al., 2005).
The timing and order of patterning events in the endoderm, from the
gastrula stage onwards, is not clear. Although early explant studies showed
that AP patterning is autonomous to the endoderm
(Gamer and Wright, 1995), this
has not been supported by more recent work, which suggests that mesodermal
signals as late as the tailbud stage are necessary to specify foregut versus
hindgut fates (Horb and Slack,
2001). It is nevertheless the case that blocking the establishment
of the embryonic dorsal axis by UV treatment of the fertilized egg causes a
loss of expression of the late anterior gut marker Pdx1, as well as
of early gastrula dorsal endoderm markers, indicating that the early
regionalization of the endoderm foreshadows later events
(Henry et al., 1996). Future
studies will determine the extent to which endoderm patterning and shape
changes are regulated by the same signaling networks and their antagonists
that operate in the mesoderm and ectoderm.
|
Important advances have been made in our understanding of the events that pattern the early Xenopus embryo. We appreciate the central importance of maternal regulators in this process and recognize the crucial roles of antagonists, such as transcriptional and cell cycle repressors, and a surprising number of signaling inhibitors. We are perceptibly nearer to understanding the essential roles of the four signaling pathways in ectoderm, mesoderm and endoderm specification (Fig. 8), and the mechanism of gastrulation. Nevertheless, early development continues to surprise. Outstanding mysteries include: the mechanism by which VegT mRNA carries out an architectural role in mRNA localization during oogenesis; the control of vegetal rotation movements in the endoderm; and how Wnt11 can have both canonical and non-canonical roles. And what would be the state of determination of gastrula cells in which BMP, Xnr, Wnt and FGF signaling was prevented?
The next phase of investigation involves an embarrassment of riches. The
Xenopus Genome Initiative and array technology mean that we will all
have long lists of target genes to study. Mutagenesis studies in Xenopus
tropicalis (Grammer et al.,
2005) and easy transgenesis methods
(Pan et al., 2006) will
provide us with a new wave of phenotypes to analyze. Some reassurance that it
will be possible to place the new knowledge of gene function into correct
regulatory networks comes from the fact that we can validate our findings by
rescue experiments. Furthermore, basic principles are emerging that show a
similarity of process among all the germ layers. Examples include the
regulation of AP patterning by Hox genes, repression of cell division
preceding cell movement and differentiation, and the recurring theme of global
transcriptional repression and localized activation. And, if we finally master
gastrulation, the next challenge will be organogenesis.
ACKNOWLEDGMENTS
I am extremely grateful to Stephanie Lang for help with references, and to Chris Wylie, Bilge Birsoy, Jane Alfred and anonymous reviewers for critically reading the work. Work in my laboratory is supported by the NIH RO1 HD33002 and HD38272.
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14 cell cycles
later the dorsal lip of the blastopore forms) marks the beginning of
gastrulation (red arrow in Bc). Immediately above the dorsal lip is the region
of the Spemann Organizer (shown as a blue trapezoid in Ad). (Bb) At the
mid-blastula stage, the embryo is described as having three regions, the
animal cap, equatorial or marginal zone and vegetal mass, explants of which
are dissected along the broken black lines shown.
