Self-regulation by Gsc and Vent1/2 V Sander et al Figure 6 Knockdown of Gsc and Vent1/2 restores normal develop- ment of dorsal and ventral half-embryos (n ¼ 52 or more per experimental set). (A) Embryos were bisected into dorsal and ventral halves at blastula stage. (B) Control sibling at the same magniﬁcation as the other panels. (C, D) Bisectioned control embryos form smaller but well-proportioned dorsal half-embryos, whereas ventral halves differentiate into belly-pieces that express HoxB9 in the ventral mesoderm (Wright et al, 1990) but are devoid of neural tissue, as indicated by the lack of Sox2 expression (inset). (E, F) Gsc depletion (136 ng MO) causes a reduction of the head in dorsal halves, whereas ventral halves are not affected. (G, H) Dorsal halves of Vent1- and Vent2-depleted embryos (45 ng each) are dorsalized, but retain overall DV patterning. The corresponding ventral halves are strongly dorsalized, including expression of spinal cord (HoxB9), brain (Krox20, Six3), and pan-neural Sox2 marker (inset). (I, J) Remarkably, both halves of triple knock- down embryos (45 ng each) develop as the uninjected control half-embryos. phenotypes included truncations and fusions of the forebrain and eyes, and dose-responsive ventralization of dorsal meso- derm. Quantitative RT–PCR studies in Activin-treated animal cap explants conﬁrmed that Gsc MO causes ventralization, inhibiting the organizer gene Admp and the brain markers Otx2 and Rx2a, and inducing expression of ventral markers, such as Wnt8, Vent2, and Evx1 (Figure 3H). Gsc depletion caused a particularly strong induction of the homeobox transcription factor Vent1 (over 20-fold), providing the initial impetus to investigate the relationship between Gsc and Vent. In addition to the head phenotype, somite formation was affected by the loss of Gsc, and MyoD expression was lost in the tip of the tail (Figure 1I, arrow), a region in which Vent2 is expressed (Onichtchouk et al, 1996). Onichtchouk et al (1998) have described Vent2 as a repressor of muscle formation in this region; perhaps loss of Gsc causes an 2962 The EMBO Journal VOL 26 | NO 12 | 2007 upregulation of Vent2, which in turn may lead to increased repression of MyoD. Experiments using Chd MO showed that the dorsalizing effects of injecting mGsc mRNA into wild-type or UV-treated embryos were mediated by the upregulation of Chd. This was a particularly satisfying result, since Chd was initially isolated as a downstream target gene of Gsc in the Spemann organizer (Sasai et al, 1994). The fact that Gsc is a transcriptional repressor makes a direct induction of Chd transcription unlikely. Chd activation may be mediated in part by a double repression mechanism, whereby Gsc represses Vents which in turn repress Chd expression (Melby et al, 1999). In addi- tion, Chd transcription is also activated by Nodal/Activin signaling. Goosecoid and genetic redundancy Embryos must have highly redundant regulatory systems to ensure that a perfectly proportionate animal is produced time after time. However, our understanding of how genetic redundancy works is very rudimentary. It has been argued that perhaps a second mouse Gsc gene might explain the lack of gastrulation phenotype in the mouse (Belo et al, 1998). However, the present results suggest that an alternative explanation may be possible. In Xenopus, loss of Gsc is devoid of effect in the absence of Vent1 and Vent2. Therefore, it could be that in the mouse the Vent regulatory system might be less prominent than in the more rapidly developing Xenopus embryo. In this respect, it is interesting to note that clear Vent homologues have neither been found in the mouse nor in the Drosophila genome. The genes most closely related to Vent in the mouse are BarX1 and BarH1, members of the Bar family of homeobox genes, that are deﬁned by Drosophila BarH1, which share with Vents a rare amino-acid substitution at position 47 of the third helix of the homeodomain (Thr instead of Val or Ile) (Kappen et al, 1993). Interestingly, in addition to the Niehrs group (Onichtchouk et al, 1996), Vent2 was isolated independently by Papalopulu and Kintner (1996), who named it Xenopus Bar-related (Xbr- 1), as well as by Ladher et al (1996), who named it Xom, for its similarity to Om(1D), the Drosophila annasae homologue of D. melanogaster BarH1. Both groups based the relationship of the newly discovered Xbr-1/Xom and Drosophila BarH1/ Om(1D) on the sequence similarity (approximately 55%) in the homeodomain, as well as on the similarities in the expression pattern of the genes in the eye. As has been proposed for Vents (Onichtchouk et al, 1998), Bar genes also function as antineural agents. They achieve this by inhibiting the transcription of bHLH transcription factors, such as Drosophila Atonal or vertebrate Neurogenin (Saito et al, 1998; Lim and Choi, 2003). Mouse BarX1 and BarH1 are also involved in other processes, such as tooth development and stomach organogenesis (Tissier-Seta et al, 1995; Reig et al, 2006). In humans, a Vent-like homeobox gene, called VENTX, has been described (Moretti et al, 2001). Although VENTX is only distantly related to the Vent genes, it shares the Thr substitu- tion of the Vent- and Bar-subclass of homeobox genes. Two observations also indicate that human VENTX might indeed be a true homologue of the Xenopus and zebraﬁsh Vent/Vega genes: ﬁrst, microinjected VENTX mRNA ventralizes zebra- ﬁsh embryos and, second, VENTX protein was detected in immature bone marrow and erythroleukemia cells. & 2007 European Molecular Biology Organization