Self-regulation by Gsc and Vent1/2 V Sander et al Figure 5 Gsc is required for the dorsalization caused by Vent1/2 knockdown. (A–I) Co-injection of Gsc MO restores normal pattern in Vent1/ 2-depleted whole embryos (n ¼ 53; Supplementary Table I). At the neurula stage, knockdown of Vent1/2/Gsc reduces the neural plate (Sox2) back to normal size (insets in panels A–C). In addition, the expansion of the cement gland and midbrain in Vent1/2 morphants is rescued in triple knockdown embyros (insets in panels G–I). Note that blood formation (Scl) is not rescued in the triple depletions (I). All MOs were injected at the same dose (45 ng each). (J) The upregulation of Chd expression by Vent1/2 MO is restored to control levels in Vent1/2/Gsc- depleted animal caps at gastrula stage. (K) Expression of Szl is downregulated by Vent1/2 MO, but restored to normal levels when Gsc is also depleted. Discussion The results presented in this loss-of-function study by MO knockdown strengthen the view that Gsc and Vent homeobox genes have mutually opposing roles in patterning the meso- derm of the Xenopus embryo. First, single Gsc knockdown produced head truncations and increased ventral tissue in whole embryos. Second, all the effects of Gsc mRNA over- expression could be blocked by Chd MO. Third, Vent1 and Vent2 MOs strongly synergized with each other, causing severe dorsalizations accompanied by the massive upregula- tion of Gsc expression over the entire endomesodermal region. Fourth, triple Vent1/2/Gsc knockdown embryos developed with an almost completely normal pattern, with- out either the ventralized phenotype of the Gsc MO, or the dorsalizing inﬂuence of Vent1/2 MOs. This lead to the surprising conclusion that the basic DV and AP patterning can be achieved in the absence of these three important transcription factors. Thus, it appears that these homeobox genes are engaged principally in cross-regulatory interactions with each other to ensure robust development. & 2007 European Molecular Biology Organization Goosecoid is required for early patterning in Xenopus embryos The discovery of Gsc was a very exiting moment, because it provided a marker for the Spemann organizer that, when overexpressed, could induce secondary axes and other as- pects of the inducing activities of organizer tissue (reviewed in De Robertis, 2006). Gsc homologues have been found in all animals that have been studied, ranging from ﬂatworms to humans (Blum et al, 1992; De Robertis, 2004). Therefore, the lack of a gastrulation phenotype of Gsc knockouts in the mouse (Rivera-Pe´rez et al, 1995; Yamada et al, 1995; Belo et al, 1998) and in Drosophila (Goriely et al, 1996; Hahn and Jaeckle, 1996) was very puzzling. In zebraﬁsh, however, Seiliez et al (2005) recently reported cyclopia and anterior truncations in Gsc morphants. In Xenopus, work using anti- morphic Goosecoids, VP16 fusions, or antisense RNA, all had indicated a role for Gsc in patterning the early mesoderm (Steinbeisser et al, 1995; Ferreiro et al, 1998; Latinkic and Smith, 1999; Yao and Kessler, 2001). Using a Gsc MO, we have now conﬁrmed that Gsc is required for early patterning in Xenopus. The Gsc knockdown The EMBO Journal VOL 26 | NO 12 | 2007 2961