Self-regulation by Gsc and Vent1/2 V Sander et al (a BMP modulator), Smad 6 and 7 (intracellular inhibitors of the BMP signaling pathway), as well as Vent2 (Niehrs and Pollet, 1999; Karaulanov et al, 2004). Since their discovery a decade ago, Vent1 and Vent2 have been proposed to nega- tively cross-regulate Gsc on the dorsal side (Gawantka et al, 1995; Onichtchouk et al, 1996), presumably mediating the negative regulatory loop between BMP4 and Gsc in the mesoderm identiﬁed by Fainsod et al (1994). This inhibitory relationship between ventral and dorsal homeobox genes has been further strengthened by a number of Xenopus and zebraﬁsh studies (Melby et al, 2000; Kawahara et al, 2000a, b; Imai et al, 2001). In the present study, we have generated MOs that deplete Gsc, Vent1 and Vent2 in the Xenopus laevis subtetraploid species. The MOs target both pseudoalleles for each gene, and are expected to be generally useful for investigators working in X. laevis. It was found that Gsc is required for head development and dorsal–ventral (DV) patterning. In gain-of- function experiments, the dorsalizing effects of Gsc mRNA were found to be dependent on the expression of Chordin (Chd). Depletion of either Vent1 or Vent2 had little effect on their own, but in combination a marked expansion of dorsal tissues was observed. This was accompanied by expanded Gsc expression throughout the endomesoderm. In animal cap explants, Vent1/2 MO injection induced the expression of organizer markers such as Gsc and Chd. In ventral half- embryos, which normally develop as belly-pieces, signiﬁcant amounts of neural and axial tissues developed. These dorsa- lizing effects of Vent1/2 MOs were blocked in Vent1/2/Gsc triple knockdowns. Unexpectedly, triple depleted embryos appeared to have normal DV and anterior–posterior (AP) pattern, as if Gsc, Vent1 and Vent2 were dispensable for embryogenesis. One exception was the blood marker Scl (Stem cell leukemia transcription factor), which was not restored in triple MO embryos. Evidently, additional mechan- isms exist in the embryo that are sufﬁcient for embryonic patterning in the absence of these three important homeobox genes. These ﬁndings provide insights into the redundant mechanisms operating in vertebrate development. Taken together, our data suggest that DV patterning is mediated in part by the reciprocal transcriptional repression of Gsc and Vent, whose balanced activities provide a self-adjusting safety net that ensures robust and reproducible embryonic development. Results Depletion of Goosecoid affects head development and DV patterning of the embryo The homeobox gene Gsc marks the Spemann organizer (Figure 1A) and it also executes some of the functions of the organizer when overexpressed, such as induction of secondary axes, recruitment of neighboring cells into axial tissues, and patterning of dorsal mesodermal tissues (Niehrs et al, 1993). We designed a MO targeted against both pseudo- alleles of the X. laevis Gsc gene, which should provide a better tool than the more indirect loss-of-function reagents used by previous workers (Figure 1B). Radial injection of Gsc MO into the vegetal pole of early two-cell stage embryos caused severe truncations of the head, indicated by the forebrain markers Otx2 and Six3, the midbrain/hindbrain border marker Engrailed2 (En2), and the hindbrain rhombo- 2956 The EMBO Journal VOL 26 | NO 12 | 2007 mere 3 and 5 marker Krox20, as well as a complete loss of the eyes marked by Six3 or Rx2a in about 40% of the embryos (Figure 1C–I). Depletion of Gsc also affected DV pattern, ventralizing the embryo, as illustrated by a moderate expan- sion of the ventral marker gene Sizzled (Szl; Figure 1G). Ventralization was cell-autonomous, because dorsal B1 blasto- meres co-injected at the 32-cell stage with lacZ mRNA lineage tracer and Gsc MO adopted somite fates instead of notochord (Supplementary Figure 1) (Niehrs et al, 1993). In dorsal marginal zone (DMZ) explants, increasing concentra- tions of Gsc MO induced notochord, muscle, and ventral mesoderm tissues at the expense of prechordal plate in a dose-responsive manner (Supplementary Figure 2; Niehrs et al, 1994). The loss of Gsc also affected AP axis patterning, as indicated by the reduced head and an altered pattern of MyoD expression, including a signiﬁcant loss of MyoD at the tip of the tail (Figure 1I, arrow), whereas the expression of the spinal cord marker HoxB9 appeared normal (Figure 1D). The phenotypes of Gsc MO injection were completely rescued by co-injection of mouse Gsc (mGsc) mRNA (Figure 1D and E). Less affected embryos had cyclopic eyes and lacked the mouth opening (Figure 1J and K). These results show that Gsc MO works as a speciﬁc tool for Gsc knockdown, and demonstrate that Gsc is required for head formation and patterning of the DV axis in the Xenopus embryo. The effects of Goosecoid overexpression are mediated by Chordin We next investigated downstream effectors of Gsc, in parti- cular Chd, a secreted BMP antagonist expressed in the organizer (Sasai et al, 1994). We observed that expression of Chd at midgastrula was reduced 2.5-fold in Gsc-depleted embryos (Figure 2A–C). The opposite effect was seen in gain- of-function experiments, in which Chd expression was greatly expanded after injection of mGsc mRNA (Figure 2, compare panels D and E). These results indicate that Chd is indeed a downstream target of Gsc. Ectopic Gsc expression leads to axis induction or dorsaliza- tion of the embryo (Cho et al, 1991; Niehrs et al, 1993). To test whether Chd is required for these effects we injected mGsc mRNA into one of the ventral blastomeres at four-cell stage. A range of dorsalized phenotypes was observed (Figure 2J): 35% of the embryos showed dorsalization with enlarged head structures, whereas 50% formed secondary axes, of which 12% had complete secondary eyes (marked by Rx2a) and notochords (marked by Xenopus brevican, Xbcan, an extracellular protein also expressed in rhombomeres 5 and 6 of the hindbrain; Sander et al, 2001) and cement glands (Figure 2F–H). Simultaneous injection of Chd MO (Oelgeschla¨ger et al, 2003) together with mGsc mRNA lead to a rescue of normal development in 97% of co-injected embryos (Figure 2I and J). This indicates that Chd mediates the dorsalizing effects of Gsc. Gsc is also known to rescue DV patterning in embryos ventralized by irradiation with ultraviolet (UV) light (Steinbeisser et al, 1993). Fertilized eggs treated with UV develop into ventral tissue and are devoid of organizer and neural gene expression (Figure 2K and M). As expected, injection of mGsc mRNA induced Chd expression (Figure 2L) and rescued the dorsal axis and anterior tissues, & 2007 European Molecular Biology Organization