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).
Most endodermal and mesodermal gene expression can be rescued in VegT-depleted embryos by the reintroduction of Xnr mRNAs, but not by the reintroduction of FGF or activin mRNAs (Kofron et al., 1999; 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?
An old idea was that no specific signal activates neural fates; that it is the `default state', as disaggregated animal cells express neural markers (Godsave and Durston, 1997). 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).
The classical animal cap assay illustrates how easily mid-blastula animal cells can be diverted to mesodermal or endodermal fates by added growth factors. Moreover, several Xnr proteins have been shown to have long signaling ranges (White et al., 2002; 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.