The roles of three signaling pathways in the formation and function of the Spemann Organizer

Since the three main pathways (the Wnt, VegT and BMP pathways) involved in organizer and axis formation in the Xenopus embryo are now characterized, the challenge is to understand their interactions. Here three comparisons were made. Firstly, we made a systematic comparison of the expression of zygotic genes in sibling wild-type, VegT-depleted (VegT(-)), beta-catenin-depleted (beta-catenin(-)) and double depleted (VegT(-)/beta-catenin(-)) embryos and placed early zygotic genes into specific groups. In the first group some organizer genes, including chordin, noggin and cerberus, required the activity of both the Wnt pathway and the VegT pathway to be expressed. A second group including Xnr1, 2, 4 and Xlim1 were initiated by the VegT pathway but their dorsoventral pattern and amount of their expression was regulated by the Wnt pathway. Secondly, we compared the roles of the Wnt and VegT pathways in producing dorsal signals. Explant co-culture experiments showed that the Wnt pathway did not cause the release of a dorsal signal from the vegetal mass independent from the VegT pathway. Finally we compared the extent to which inhibiting Smad 1 phosphorylation in one area of VegT(-), or beta-catenin(-) embryos would rescue organizer and axis formation. We found that BMP inhibition with cm-BMP7 mRNA had no rescuing effects on VegT(-) embryos, while cm-BMP7 and noggin mRNA caused a complete rescue of the trunk, but not of the anterior pattern in beta-catenin(-) embryos.     

The competence of Xenopus blastomeres to produce neural and retinal progeny is repressed by two endo-mesoderm promoting pathways

Only a subset of cleavage stage blastomeres in the Xenopus embryo is competent to contribute cells to the retina; ventral vegetal blastomeres do not form retina even when provided with neuralizing factors or transplanted to the most retinogenic position of the embryo. These results suggest that endogenous maternal factors in the vegetal region repress the ability of blastomeres to form retina. Herein we provide three lines of evidence that two vegetal-enriched maternal factors (VegT, Vg1), which are known to promote endo-mesodermal fates, negatively regulate which cells are competent to express anterior neural and retinal fates. First, both molecules can repress the ability of dorsal-animal retinogenic blastomeres to form retina, converting the lineage from neural/retinal to non-neural ectodermal and endo-mesodermal fates. Second, reducing the endogenous levels of either factor in dorsal-animal retinogenic blastomeres expands expression of neural/retinal genes and enlarges the retina. The dorsal-animal repression of neural/retinal fates by VegT and Vg1 is likely mediated by Sox17alpha and Derriere but not by XNr1. VegT and Vg1 likely exert their effects on neural/retinal fates through at least partially independent pathways because Notch1 can reverse the effects of VegT and Derriere but not those of Vg1 or XNr1. Third, reduction of endogenous VegT and/or Vg1 in ventral vegetal blastomeres can induce a neural fate, but only allows expression of a retinal fate when both BMP and Wnt signaling pathways are concomitantly repressed.     

Relationship of vegetal cortical dorsal factors in the Xenopus egg with the Wnt/beta-catenin signaling pathway

In Xenopus, the dorsal factor in the vegetal cortical cytoplasm (VCC) of the egg is responsible for axis formation of the embryo. Previous studies have shown that VCC dorsal factor has properties similar to activators of the Wnt/beta-catenin-signaling pathway. In this study, we examined the relationship of the VCC dorsal factor with components of the pathway. First, we tested whether beta-catenin protein, which is known to be localized on the dorsal side of early embryos, accounts for the dorsal axis activity of VCC. Reduction of beta-catenin mRNA and protein in oocytes did not diminish the activity of VCC to induce a secondary axis in recipient embryos. The amount of beta-catenin protein was not enriched in VCC compared to animal cortical cytoplasm, which has no dorsal axis activity. These results indicate that beta-catenin is unlikely to be the VCC dorsal axis factor. Secondly, we examined the effects of four Wnt-pathway-interfering constructs (dominant-negative Xdsh, XGSK3, Axin, and dominant-negative XTcf3) on the ability of VCC to induce expression of the early Wnt target genes, Siamois and Xnr3. The activity of VCC was inhibited by Axin and dominant negative XTcf3 but not by dominant negative Xdsh or XGSK3. We also showed that VCC decreased neither the amount nor the activity of exogenous XGSK3, suggesting that the VCC dorsal factor does not act by affecting XGSK3 directly. Finally, we tested six Wnt-pathway activating constructs (Xwnt8, Xdsh, dominant negative XGSK3, dominant negative Axin, XAPC and beta-catenin) for their responses to the four Wnt-pathway-interfering constructs. We found that only XAPC exhibited the same responses as VCC; it was inhibited by Axin and dominant negative XTcf3 but not by dominant negative Xdsh or XGSK3. Although the connection between XAPC and the VCC dorsal factor is not yet clear, the fact that APC binds Axin suggests that the VCC dorsal factor could act on Axin rather than XGSK3.     

Multiple interactions between maternally-activated signalling pathways control Xenopus nodal-related genes

We have investigated the induction of the six Xenopus nodal-related genes, Xnr1-Xnr6, by maternal determinants. The beta-catenin pathway was modelled by stimulation using Xwnt8, activin-like signalling was modelled by activin, and VegT action was studied by overexpression in animal cap explants. Combinations of factors were examined, and previously unrecognised interactions were revealed in animal caps and whole embryos. For the induction of Xnr5 and Xnr6 in whole embryos, using a beta-catenin antisense morpholino oligonucleotide or a dominant negative XTcf3, we have demonstrated an absolute permissive requirement for the beta-catenin/Tcf pathway, in addition to the requirement for VegT action. In animal caps Xnr5 and Xnr6 are induced in response to VegT overexpression, and this induction is dependent upon the concomitant activation of the beta-catenin pathway that VegT initiates in animal caps. For the induction of Xnr3, VegT interacts negatively so as to inhibit the induction otherwise observed with wnt-signalling alone. The negative effect of VegT is not the result of a general inhibition of wnt-signalling, and does not result from an inhibition of wnt-induced siamois expression. A 294 bp proximal promoter fragment of the Xnr3 gene is sufficient to mediate the negative effect of VegT. Further experiments, employing cycloheximide to examine the dependence of Xnr gene expression upon proteins translated after the mid-blastula stage, demonstrated that Xnrs 4, 5 and 6 are 'primary' Xnr genes whose expression in the late blastula is solely dependent upon factors present before the mid-blastula stage.