From these results, Moretti and co-workers concluded that VENTX—like Vent and Vega—may be involved in mesoderm patterning and maintenance of hematopoietic stem cells in the adult. It appears that the Vent genes have adopted a prominent functional role in Xenopus and zebraﬁsh, while in the mouse and Drosophila the Bar genes might carry out some of the functions of the missing Vents. Gsc homologues, however, are found throughout the animal kingdom, from ﬂatworms to vertebrates, and it is therefore unlikely that this endomesodermal gene is not an important player in embryogenesis, despite the genetic redundant mechanisms that are at play in some animals. In the case of the mouse embryo, the simplest interpretation would be that Gsc lacks a gastrulation phenotype because mice lost the Vent genes. We have analyzed the syntenic region of the mouse genome, and failed to ﬁnd a VENTX murine homologue (V Sander, unpublished observations). Searching for a true murine Vent homologue seems impor- tant, and perhaps such a gene might be found by screening for BMP-inducible genes, since Vent2 is a primary response gene to BMP4 (Rastegar et al, 1999; Trindade et al, 1999; Karaulanov et al, 2004). Self-regulation of the DV mesodermal ﬁeld The interaction between Vent1, Vent2, and Gsc had never been tested in a triple loss-of-function situation, which proved an interesting experiment. The triple knockdown of Gsc and Vent1/2 led to the surprising result of the restoration of normal embryonic patterning, not only in whole embryos but also in bisected dorsal and ventral half-embryos. First, this result argues that the dorsalization caused by Vent1/2 depletion is entirely mediated by Gsc. The Gsc MO, which had only a moderate effect on the dorsal half of the embryo when injected alone, had a very strong effect on Vent1/2- depleted ventral halves, reversing all dorsal cell differentia- tions to ventral mesoderm (Figure 6H and J). Second, it raises the question of how the embryo can compensate for the simultaneous loss of three genes, when single and double knockdowns are strongly affected. Two opposing homeodo- main repressors are transcriptionally upregulated when one signaling center or the other is depleted. Removing both transcription factors negates the phenotype. It should be pointed out that this regulation might not be exclusively transcriptional. Dawid and co-workers have reported that in zebraﬁsh Vega1 and Vega2 can directly bind to Gsc protein in immunoprecipitation experiments (Kawahara et al, 2000b). If binding of Gsc inhibited the activity of Vent/Vega, this could provide a simple mechanism for reinforcing the transcrip- tional regulation at the protein level. This is an aspect of the DV patterning system that deserves more study in the future. One of the properties of the vertebrate embryo that has intrigued researchers since the beginnings of experimental embryology, is its ability to self-regulate pattern after experi- mental perturbation (reviewed by De Robertis, 2006). Recent work has suggested that molecules of similar biochemical activities but under opposite transcriptional control ex- pressed on the dorsal or ventral side of the embryo might explain the formation of a self-regulating morphogenetic ﬁeld. So far, this proposition has been tested for secreted proteins produced in the Spemann organizer, such as Chd and ADMP, and proteins expressed ventrally as part of the Bmp4 synexpression group (also called the ventral center), & 2007 European Molecular Biology Organization Self-regulation by Gsc and Vent1/2 V Sander et al Figure 7 Model of regulatory mechanisms for pattern formation at gastrula. In the dorsal center, Activin/Nodal signals phosphorylate Smad2/3 to activate Gsc expression. The expansion of Chd and ADMP can also be achieved by Gsc-independent pathways. In the ventral center, BMP4/7 signals phosphorylate Smad1/5/8 and lead to the expression of Vent1/2. BMP4 is also able to activate ventral center secreted proteins by Vent-independent mechanisms. The function of Gsc and Vent is to regulate each other, providing an intracellular compensatory mechanism that works in concert with the extracellular networks of growth factors and their antagonists. such as BMP4/7, the Xolloid-related Chordinase (Xlr) and its competitive inhibitor Sizzled (Reversade and De Robertis, 2005; Lee et al, 2006). Both Gsc and Vent are homeodomain proteins that function as transcription repressors. However, they are under the opposite transcriptional control by Smad1/ 5/8 (which are activated by BMP), and Smad2/3 (which are activated by Activin and Nodal) and might be considered part of the intracellular mechanism that maintains the morpho- genetic ﬁeld in the mesoderm. In the triple knockdown situation, a safety net of extra- cellular dorsal and ventral center molecules might still be able to adjust and mediate self-regulation. We have tested this assumption by removing BMP4 or ADMP in addition to Vent1/2/Gsc. As shown in Supplementary Figure 8, both quadruple knockdowns resulted in strongly dorsalized em- bryos. This indicates that removing additional components of the regulatory safety network disrupts the self-regulation that can be still achieved in Vent1/2/Gsc triple morphants. Figure 7 describes a model in which Gsc and Vent are considered central players in the gastrula embryo. The dorsal center is induced by Activin/Nodal signals, and Chordin and ADMP are induced by Gsc-dependent and independent path- ways. On the ventral side, BMP4/Smad1 and Vent positively cross-regulate each other (Onichtchouk et al, 1996; Schuler- Metz et al, 2000; Henningfeld et al, 2002), inducing other components of the Bmp4 synexpression group, such as BAMBI and Sizzled. In the dorsal center, Gsc is a primary response gene to Activin/Nodal signaling (Cho et al, 1991), and in the ventral center Vent2 is a primary response gene to BMP4 (Rastegar et al, 1999; Trindade et al, 1999; Karaulanov et al, 2004). The transcriptional repressors Gsc and Vent strongly oppose each other, in order to establish and maintain a balance between dorsal and ventral pattern formation. DV patterning is a crucial process in early development, and our results suggests that the embryo has enough redundancy to provide a remarkable double assurance mechanism, such that when Gsc and Vent1/2 are removed, they can still be compensated by an extracellular mechanism involving the BMP4 and ADMP morphogens. The EMBO Journal VOL 26 | NO 12 | 2007 2963