Self-regulation by Gsc and Vent1/2 V Sander et al Figure 2 The dorsalizing effects of mGsc mRNA injection require Chd. (A–E) Gsc MO reduces Chd expression at gastrula 2.5-fold, whereas overexpression of mGsc mRNA greatly expands Chd expression. (F–H) Injection of 50 pg mGsc mRNA into one ventral blastomere at the four- cell stage leads to a range of dorsalized phenotypes, of which 50% develop secondary axes (38% partial; 12% complete with eyes, notochords, and cement glands). (I, J) Co-injection of Chd MO (34 ng) prevents second axis induction and dorsalization by mGsc mRNA in 97% of the embryos. (K, L) mGsc mRNA microinjection (200 pg total) induces Chd expression in UV-ventralized embryos at gastrula. (M–O) The rescue of head (Otx2) and pan-neural marker (Sox2) in UV embryos by mGsc overexpression (n ¼ 54) has a complete requirement for Chd (co-injection of 136 ng Chd MO; n ¼ 59). treated with 2 ng/ml recombinant human Activin until stage 22, and analyzed by in situ hybridization and quantitative RT–PCR (Figure 3A). Elongation of animal caps by Activin was not blocked in the absence of Gsc, but Gsc-depleted caps failed to undergo neural differentiation, as shown by the lack of Otx2 expression (Figure 3B–E) or that of Sox2 (data not shown). This anti-neural effect in animal caps was stronger than in whole embryos, in which residual neural gene expression was always observed (Figure 3B and D, insets). These results suggest that Gsc is required for neural induc- tion in Activin-treated animal caps, but not for their elongation (which is caused by the differentiation of dorsal mesoderm such as somites). This is in contrast with Chd, which is required for both elongation and neural differentia- tion of animal caps by Activin (Figure 3F and G). Thus, the dorsalizing effects of Activin have a complete requirement for Chd (Oelgeschla¨ger et al, 2003), but only a partial one for Gsc. To gain a better insight into the histotypic differentiation of Gsc-depleted caps, we next performed quantitative RT–PCRs (Figure 3H). The anterior neural markers Otx2 and Rx2a and the organizer gene Admp were downregulated upon Gsc depletion, suggesting that in the wild-type embryo Gsc promotes the expression of these genes. Gsc expression 2958 The EMBO Journal VOL 26 | NO 12 | 2007 itself—measured in samples from whole embryos at gastru- la—was upregulated in Gsc-depleted embryos, in line with the previously described auto-inhibitory regulation of Gsc (Danilov et al, 1998). The somite markers MyoD and a-Actin, as well as the ventral mesoderm markers Vent1, Vent2, and Evx1, a proposed downstream target of Vent (Onichtchouk et al, 1998), were upregulated upon depletion of Gsc (Figure 3H). In addition, the ventrally expressed signaling factor Wnt8 was upregulated in Gsc morphants. Yao and Kessler (2001) described that Wnt8 is directly repressed by Gsc and, underscoring the importance of this interaction, we now found that Wnt8 MO suppresses the phenotype of Gsc morphants (Supplementary Figure 3). Taken together, the results suggest that the wild-type function of Gsc is to repress genes of paraxial and ventral mesoderm, while inducing neural markers and genes expressed in dorsal-most axial mesoderm. Gsc depletion caused a remarkably strong upre- gulation of the homeobox transcription factor Vent1 (up to 25-fold), which prompted us to investigate more deeply the interplay between Gsc and the Vent genes. Loss of Vent leads to dorsalization of the embryo The Vent transcription factors consist of two genes in Xenopus: Vent1 and Vent2 (Gawantka et al, 1995; & 2007 European Molecular Biology Organization