Self-regulation by Gsc and Vent1/2 V Sander et al Figure 1 Gsc knockdown in Xenopus embryos causes loss of head structures and affects patterning of the AP and DV axes. (A) Gsc marks Spemann organizer endomesoderm at early gastrula. (B) Gsc MO targets both pseudoalleles of the X. laevis Gsc gene. (C–I) Gsc MO injection (136 ng total) causes loss of head structures, marked by Otx2 (forebrain), Six3 and Rx2a (forebrain and eyes), and En2 (midbrain/hindbrain border) (n ¼ 106; Supplementary Table I). Expression of the ventral marker Szl is reduced anteriorly and expanded posteriorly in the ventral blood island. (E) Co-injection of mGsc mRNA (200 pg total, radial injection) rescues the Gsc MO phenotype (n ¼ 78). (H, I) Knockdown of Gsc reduces head size and affects patterning of the posterior somites, including loss of MyoD expression at the tip of the tail (arrows). (J, K) Moderately affected embryos survive until tadpole stage and have cyclopic eyes (indicating holoprosencephaly) and no mouth opening. as shown by the expression of the pan-neural marker Sox2 and the forebrain marker Otx2 (Figure 2N). However, UV rescue by mGsc mRNA was completely blocked in Chd- depleted embryos (Figure 2O). Taken together, these results show that the effects of Gsc gain-of-function, which mimic the properties of the Spemann organizer, depend on the expression of its downstream target Chordin. & 2007 European Molecular Biology Organization Goosecoid requirement in Activin-treated animal caps We next addressed the requirement for Gsc in a sensitized ectodermal explant (animal cap) assay. Treatment with Activin leads to dose-dependent induction of mesodermal cell fates, which in animal caps assays result in explant elongation and neural induction. Animal caps from control and Gsc MO-injected embryos were excised at blastula, The EMBO Journal VOL 26 | NO 12 | 2007 2957