Self-regulation by Gsc and Vent1/2 V Sander et al Figure 3 Gsc is required for secondary neural induction and mesoderm patterning in Activin-treated animal cap explants. (A) Experimental design (n ¼ 15 or more per experimental set) (B, C) Untreated animal caps develop into atypical epidermis, whereas Activin treatment leads to elongation and brain formation, visualized by Otx2 at the anterior pole (arrows). In addition, Otx2 expression in anterior endoderm can be seen in one of the explants (arrowhead). (D, E) Gsc-depleted caps elongate after treatment with Activin, but lack Otx2 neural staining. (F, G) Chd- depleted caps treated with Activin are unable to elongate, conﬁrming the requirement of Chd for dorsal mesoderm and neural induction by Activin (Oelgeschla¨ger et al, 2003). Insets show whole sibling embryos. (H) Quantitative RT–PCRs showing genes affected by depletion of Gsc include markers of anterior CNS, organizer, somites, and ventral mesoderm. Note that Vent1 expression is increased more than 20-fold by Gsc knockdown. Onichtchouk et al, 1996). To study their roles in the early embryo, MOs against both genes were designed. Radial injection of Vent1 MO into 2- to 4-cell stage embryos lead to a modest increase of Sox2 expression at neurula stage, but seemed to have no effect on tadpole stage embryos (Figure 4B). Vent2 depletion broadened the neural plate and also moderately dorsalized the embryo at later stages (Figure 4C; Supplementary Figure 4). Strikingly, dorsalization was greatly increased when MOs for Vent1 and Vent2 were co-injected, causing the development of enlarged neural plates and head structures. At the tadpole stage, double Vent1/2-depleted embryos consisted of head structures with no tails and very short trunks (Figure 4D). Thus, Vent1 and Vent2 are partially redundant, as had been reported pre- viously in zebraﬁsh (Imai et al, 2001). Co-injection of mRNAs for Vega1 and Vega2, the zebraﬁsh homologues of Vent1 and Vent2 (Kawahara et al, 2000a, b; Melby et al, 2000), rescued the dorsalized phenotype of Vent1/2 knock- downs, indicating that the effect of these MOs was speciﬁc (Supplementary Figure 5). & 2007 European Molecular Biology Organization Gsc and Vent1 and Vent2 have been proposed to repress each other in a cross-regulatory loop (Gawantka et al, 1995; Onichtchouk et al, 1996). Accordingly, Gsc expression would be expected to be upregulated upon Vent1/2 depletion. This was indeed the case, as shown in hemi-sectioned gastrula stage embryos (Figure 4E and F) and quantitative RT–PCRs in animal caps (Figure 4I). In addition, the expression of other dorsal genes, namely Chd (see insets in Figure 4E and F) and Admp, was increased upon Vent1/2 depletion (Figure 4I). The opposite result, upregulation of Vent1 and Vent2 expression in Gsc-depleted embryos, was also observed (Figure 4G and H). We conclude that Vent1 and Vent2 play an important role in repressing dorsal gene expression, since their depletion leads to a severe dorsalization of the embryo and a striking upregulation of Gsc. Triple depletion of Goosecoid and Vent1/2 restores normal DV and AP pattern What would be the result if transcription factors under opposite regulation were knocked down simultaneously? To The EMBO Journal VOL 26 | NO 12 | 2007 2959