Self-regulation by Gsc and Vent1/2 V Sander et al Figure 4 Double depletion of Vent1 and Vent2 causes severe dor- salization of the embryo. (A–D) Injection of either Vent1 or Vent2 MO expands the neural plate at neurula stage (insets), but only the combination of both MOs strongly dorsalizes tailbud stage embryos, with shortened body axes and large heads and cement glands (n ¼ 122; Supplementary Table I). (E, F) Vent1/2 depletion leads to transcriptional upregulation of Gsc (hemisections at stage 10; n ¼ 15) and Chd (insets in panels E and F; whole embryos, vegetal view; n ¼ 18). (G, H) Loss of Gsc increases Vent1 and Vent2 expression (hemisections at stage 10; n ¼ 21 and 15) (I) Quantitative RT–PCR analyses showing 3- to 4-fold upregulation of the organizer genes Gsc, Chd, and Admp in animal caps at gastrula stage after Vent1/2 depletion. answer this question, we co-injected the MOs for Gsc, Vent1, and Vent2 radially at the four-cell stage. Surprisingly, 80% of triple-depleted embryos were rescued to an almost normal pattern, with well-formed axial structures such as somites (marked by MyoD), spinal cord (HoxB9), notochord (not shown), brain (Six3, Krox20), and heart (Nkx2.5) (Figure 5A–F). At the neurula stage, the expression domains of Sox2 (neural plate), En2 (midbrain/hindbrain border), and XAG1 (cement gland) that were strongly expanded in Vent1/2 morphants, were rescued to normal in triple-depleted embryos (see insets in Figure 5A–C and G–I). To show that the doses of MOs used in the triple knockdown experiments effectively depleted the activities of the three genes, we injected Gsc MO, Vent1/2 MOs and Vent1/2/Gsc MOs in various concentrations; even the lowest doses caused iden- tical phenotypes, indicating that the loss-of-function in the triple morphant embryos was complete (Supplementary Figure 6). This extraordinary rescue in embryos in which Vent1/2 and Gsc were knocked down shows that the dorsalizing effect of the Vent1/2 depletion is mediated by the upregulation of Gsc and vice versa. It is startling that the loss of three transcription factors—shown to have important effects in 2960 The EMBO Journal VOL 26 | NO 12 | 2007 single loss- and gain-of-function situations—is without much consequence on the overall pattern formation of the embryo. The only defect we observed in triple depleted embryos was a marked reduction in blood tissue, marked by Scl (Figure 5G–I). We also observed that the triple MO embryos usually did not survive beyond stage 30. Thus, the loss of Gsc, Vent1 and Vent2 can be compensated to a remarkable extent during early development but not later on. Quantitative RT–PCRs of animal cap explants at gastrula stage supported these ﬁndings. For example, Chd expression was increased four-fold by Vent1/2 knockdown, but was restored to normal levels in triple-depleted cap samples (Figure 5J). Szl, which is expressed in the ventral center as member of the Bmp4 synexpression group, was downregu- lated upon Vent1/2 removal, indicating it is a downstream target of Vent. Vent1/2/Gsc knockdown, however, restored expression levels of Szl to normal (Figure 5K). Similar effects were observed for BAMBI (data not shown). Depletion of Chd had stronger effects than Gsc, for it was epistatic to Vent1/2 (Supplementary Figure 7). The data indicate that the dorsalization effects of Vent1/2 loss-of-function are mediated by the upregulation of Gsc and that, reciprocally, the effects of Gsc depletion are mediated by the upregulation of Vent1/2. Self-regulation in half-embryos Bisection of wild-type embryos at blastula stage along the DV axis (Reversade and De Robertis, 2005) leads to the formation of ventral half belly-pieces (called Bauchstu¨ cke by Hans Spemann) that consist of ventral tissues, whereas dorsal halves self-regulate to form well-proportioned half-sized em- bryos (Figure 6A–D). HoxB9, which is a spinal cord and ventral mesoderm marker (Wright et al, 1990), marks only ventral mesoderm in ventral halves, which are devoid of neural tissue marked by Sox2 (Figure 6D, inset). Knockdown of Gsc did not affect the ventral half and, as expected, reduced head development in the dorsal halves (Figure 6E and F). In contrast, Vent1/2 depletion not only dorsalized the dorsal halves, resulting in large head structures, but also caused dorsalization of the ventral half-embryos. These ven- tral halves formed elongated axial neural structures expres- sing Sox2, HoxB9 and Krox20 (as well as mesodermal MyoD in somites and Xbcan in the notochord, data not shown), which in most cases lacked the forebrain marker Six3 (Figure 6G and H). To investigate whether Gsc was involved in the dorsalization of ventral half-embryos caused by Vent1/2 depletion, we analyzed triple Vent1/2/Gsc morphants. It was found that the depletion of Gsc reversed the phenotypes of Vent1/2 knockdown to the wild-type pattern causing the differentiation of belly-pieces (Figure 6I and J). These results suggest several conclusions. First, because Vent1/2-depleted dorsal halves were only partially dorsalized, we believe additional ventralizing signals must exist on the dorsal side. Second, the development of Vent1/2-depleted ventral halves into embryos with dorsal mesodermal and neural structures is mediated exclusively by Gsc, since in triple knockdowns belly-pieces lacking all dorsal tissues are formed. Finally, it seems that Gsc and the Vent1/2 genes are predominantly required in their normal side of expression, while after removal of all three they seem to be dispensable for embryo- nic pattern formation. & 2007 European Molecular Biology Organization