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).
However, the movement of cortical Wnt11 mRNA during the first cell cycle has not been directly visualized. Indirect evidence comes from the greater abundance of both Wnt11 mRNA and protein on the dorsal side compared with the ventral side of the embryo at the 32-cell stage (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.
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.