The EMBO Journal (2007) 26, 2955–2965 | & 2007 European Molecular Biology Organization | Some Rights Reserved 0261-4189/07 www.embojournal.org The opposing homeobox genes Goosecoid and Vent1/2 self-regulate Xenopus patterning THE EMBO JOURNAL EMBO open This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation or the creation of derivative works without speciﬁc permission. Veronika Sander, Bruno Reversade and EM De Robertis* Howard Hughes Medical Institute and Department of Biological Chemistry, University of California, Los Angeles, CA, USA We present a loss-of-function study using antisense mor- pholino (MO) reagents for the organizer-speciﬁc gene Goosecoid (Gsc) and the ventral genes Vent1 and Vent2. Unlike in the mouse Gsc is required in Xenopus for mesodermal patterning during gastrulation, causing phenotypes ranging from reduction of head structures— including cyclopia and holoprosencephaly—to expansion of ventral tissues in MO-injected embryos. The overexpres- sion effects of Gsc mRNA require the expression of the BMP antagonist Chordin, a downstream target of Gsc. Combined Vent1 and Vent2 MOs strongly dorsalized the embryo. Unexpectedly, simultaneous depletion of all three genes led to a rescue of almost normal development in a variety of embryological assays. Thus, the phenotypic effects of depleting Gsc or Vent1/2 are caused by the transcriptional upregulation of their opposing counter- parts. A principal function of Gsc and Vent1/2 homeobox genes might be to mediate a self-adjusting mechanism that restores the basic body plan when deviations from the norm occur, rather than generating individual cell types. The results may shed light on the molecular mechanisms of genetic redundancy. The EMBO Journal (2007) 26, 2955–2965. doi:10.1038/ sj.emboj.7601705; Published online 24 May 2007 Subject Categories: development Keywords: BMP; Chordin; embryonic induction; Goosecoid; Vent Introduction The isolation of the homeobox gene Goosecoid (Gsc) initiated the molecular exploration of the inductive activities of the Spemann organizer or dorsal lip of the blastopore (Cho et al, 1991). This venerable gene has been the subject of extensive studies in many organisms (reviewed in De Robertis, 2004). In Xenopus, overexpression studies showed that Gsc mRNA has axis inducing activities, promotes dorso-anterior migra- *Corresponding author. Howard Hughes Medical Institute and Department of Biological Chemistry, University of California, 675 Charles Young Drive South, Los Angeles, CA 90095-1662, USA. Tel.: þ 1 310 206 1401; Fax: þ 1 310 206 2008; E-mail: ederobertis@mednet.ucla.edu Received: 24 January 2007; accepted: 5 April 2007; published online: 24 May 2007 & 2007 European Molecular Biology Organization tion of cells, and causes dose-dependent dorsalization of mesodermal tissues (Niehrs et al, 1993, 1994). Loss-of-func- tion analyses indicated a requirement of Gsc for head forma- tion in Xenopus by a variety of indirect methods such as antisense Gsc mRNA (Steinbeisser et al, 1995), antimorphic Goosecoids generated by the addition of epitope tags (Ferreiro et al, 1998), and fusion of the transcriptional activation domain of VP16 to the Gsc transcriptional repres- sor (Latinkic and Smith, 1999; Yao and Kessler, 2001). With the advent of antisense morpholinos (MOs) as powerful new tools for loss-of-function studies in Xenopus (Heasman, 2002), we decided to revisit the functional role of Gsc, and also of Vent1 and Vent2, two ventral homeobox genes that mediate part of the BMP activity during gastrulation (Onichtchouk et al, 1998). This seemed worthwhile because knockout studies of Gsc in the mouse had shown no gastrulation phenotype, with death shortly after birth accompanied by a modest reduction in the midline of the base of the cranium (Rivera-Pe´rez et al, 1995; Yamada et al, 1995; Belo et al, 1998). Compound Gsc / ; HNF3b/FoxA2 þ / or Gsc / ;Dkk1 þ / mice showed severe disruptions of early embryonic patterning (Filosa et al, 1997; Lewis et al, 2006). Although Gsc knockout embryos gastrulate normally, Gsc / mouse nodes have a decreased neural inducing activity when transplanted into chick primitive streak embryos, indicating that the lack of gastrulation phenotype seen in Gsc mutant mice results from regulatory mechanisms that can compensate for the loss of this gene (Zhu et al, 1999). In Drosophila, mutation of D-gsc is embryonic lethal, but, as in the mouse, also fails to show early phenotypes, with the main abnormalities being restricted to the invaginating fore- gut (Goriely et al, 1996; Hahn and Jaeckle, 1996). In zebra- ﬁsh, a recent study from the Thisses’ lab found that a Gsc MO caused head defects in 14% of the knockdown embryos, but, interestingly, together with a FoxA3 MO, which on its own resulted in no head abnormalities, led to defects ranging from cyclopia to anterior head deletions in 54% of the embryos (Seiliez et al, 2005). Gsc is thought to promote dorsal endomesodermal devel- opment in Xenopus, while Vent genes expressed at the ventral side of the embryo mediate mesodermal patterning on the opposite side (reviewed by Niehrs, 2001). Vent1 (also known as PV.1) and Vent2 (also known as Vox, Xom or Xbr-1) are homeobox genes strongly induced by BMP4 on the ventral side of the embryo (Gawantka et al, 1995; Ault et al, 1996; Ladher et al, 1996; Onichtchouk et al, 1996; Papalopulu and Kintner, 1996; Schmidt et al, 1996). Vent1 was the founding member of the Bmp4 synexpression group, which includes other ventral genes such as BAMBI (BMP and Activin mem- brane-bound inhibitor), Sizzled (a ventrally expressed metal- loproteinase inhibitor), Bmp receptor 2, Twisted Gastrulation The EMBO Journal VOL 26 | NO 12 | 2007 2955