The Xvent homeobox multigene family is essential for the patterning of the ventral mesoderm in Xenopus embryos. We have identified two novel members of this family, Xvent-1B and Xvent-2B, and have characterized their genomic structures. These two genes show a clustered organization and have probably arisen by gene duplication with subsequent inversion. Cis-regulatory elements within the promoters of both genes have been identified which contribute to their spatial activation. Xvent-2B is activated by BMP-2/4 in the absence of de novo protein synthesis, suggesting that this gene is a direct target of BMP-signalling. In contrast, Xvent-1B does not directly respond to BMP-2/4, but is activated by Xvent-2B. This activation is documented by Xvent-1B promoter/reporter studies, Xvent-2B overexpression and loss-of-function analysis using a dominant-negative Xvent-2 mutant. However, cycloheximide experiments reveal that Xvent-2B by itself is not sufficient to activate transcription of the Xvent-1B gene, but that there is a requirement for additional factor(s) being synthesized after midblastula transition.

The Xvent family of homeobox transcription factors is essential for the establishment of the dorsal-ventral body axis during Xenopus embryogenesis. In contrast to Xvent-2B and other members of the Xvent-2 subfamily, Xvent-1B is not a direct response gene of bone morphogenetic protein-4 signaling. Xvent-1B is activated by Xvent-2, but CHX experiments revealed the requirement of additional factors. In this study, we report on the cooperative effect of Xvent-2 and the zinc finger transcription factor GATA-2 on the promoter of the Xvent-1B gene. We show that GATA-2 is a direct target gene of bone morphogenetic protein-4 and that GATA-2 interacts with Xvent-2 to activate transcription of Xvent-1B. Both transcription factors bind to distinct elements within the Xvent-1B promoter, and GATA-2 physically interacts with the C-terminal domain of Xvent-2. Promoter/reporter studies in Xenopus embryos revealed that full activation of Xvent-1B requires both Xvent-2 and GATA-2. Moreover, the two factors are sufficient to direct transcription of Xvent-1B in the presence of CHX at the ventral side of the embryo. The failure of both factors to activate Xvent-1B on the dorsal side suggests the existence of a dorsal inhibitor. This inhibitor is likely a component of the dorsal Wnt signaling pathway because nuclear translocation of ␤-catenin before midblastula transition results in a suppression of Xvent-1B transcription.

Members of the Xvent-2 homeodomain transcription factor family are immediate response genes of BMP-4 signaling. The bone morphogenetic protein response element (BRE) of Xvent-2B was previously identified and characterized with respect to Smad1 and Smad4 binding sites. In this study, we further report on the transcriptional regulation of Xvent-2B. We provide evidence that Xvent-2B (Xvent-2) maintains its own expression through autoregulation. This activity was demonstrated for the endogenous gene by reverse transcriptase-PCR analysis and was found to be insensitive to cycloheximide. Localized by DNase I footprinting were several Xvent-2 binding sites within the proximal upstream region including the BRE. In the early Xenopus embryo, the BRE was shown to be sufficient to drive expression of a green fluorescent protein reporter in a similar pattern compared with the endogenous gene. Furthermore, Xvent-2B was able to activate the BRE in luciferase reporter assays, and in co-injection experiments Xvent-2B and Smad1 were found to synergistically activate the BRE. Moreover, glutathione S-transferase pull-down experiments demonstrated that Xvent-2B directly and specifically interacts with Smad1. This association was mediated by the MH1 domain of Smad1 and required the C-terminal domain of Xvent-2. The failure of an Xvent-2 mutant lacking the C terminus to stimulate the BRE underlines the significance of the C-terminal domain in the described autoregulatory loop.Bone morphogenetic proteins (BMPs), 1 as well as other members of the TGF-␤ superfamily of extracellular signaling molecules, are essential regulators of critical cellular events including cellular differentiation and apoptosis in early embryonic development (1, 2). BMP-4 has been shown to act as a morphogen in the patterning of the dorsal-ventral axis of the amphibian embryo, with high concentrations specifying the most ventral tissues (such as blood) and lower concentrations specifying muscle (3). Within the ectoderm, BMP-4 suppresses neural differentiation and promotes formation of epidermis (4). The Xvent-2 (Xvent-2B, Vox, Xbr-1, and Xom) and Xvent-1 (Xvent-1B and PV.1) homeodomain transcription factors have been identified as both targets and pivotal mediators of BMP function in Xenopus and Zebrafish (5-11). Consistent with the Xvents as downstream effectors of BMP, these genes are expressed in the ventral and lateral marginal zones and are excluded from the dorsal-most regions of early Xenopus embryos. Ectopic expression of the Xvents in the dorsal blastomeres mimics the BMP-induced ventralization of Xenopus embryos and results in the activation of BMP-induced genes. Moreover, these proteins can rescue the dorsalizing effects of the dominant negative BMP receptor.The intracellular transducers of TGF-␤ signaling are the Smad family of proteins (reviewed in Refs. 12 and 13). Ligand binding to TGF-␤ receptors stimulates the transient association and subsequent activation of receptor-specific Smads by phosphorylation of C-terminal serine residues. Smad1, Smad5,...

Transcription of the early response gene XFD‐1′ (XFKH1) in the dorsal lip (Spemann organizer) of Xenopus embryos is activated by dorsal mesoderm inducing factors. Promoter studies revealed the presence of an activin A response element (ARE) which is both necessary and sufficient for transcriptional activation of reporter genes in animal cap explants incubated with activin A. Surprisingly, this ARE is also active within vegetal explants in the absence of exogenously added inducers, but an additional inhibitory response element prevents transcription of the XFD‐1′ gene in the ventral/vegetal region of the embryo in vivo. This element is located upstream of the ARE, it responds to bone morphogenic proteins 2 and 4 (BMP‐2/4) triggered signals and it overrides the activating properties of the ARE. Expression patterns of BMP‐2 and BMP‐4 in the late blastula stage embryo and, especially, their absence from the dorsal blastopore lip may thus control the spatial transcription of the XFD‐1′ gene. Accordingly, the temporal activation and the spatial restriction of XFD‐1′ gene activity to the Spemann organizer is regulated by antagonistic actions of two distinct members of the TGF‐beta family (activin and BMP) which act on different promoter elements.

Ectopic expression of the ventralizing morphogen BMP-4 (bone morphogenetic protein-4) in the dorsal lip (Spemann organizer) of Xenopus embryos blocks transcription of dorsal-lip-specific early response genes. We investigated the molecular mechanism underlying the BMP-4-induced inhibition of the fork head gene XFD-1'. The promoter of this gene contains a BMP-triggered inhibitory element (BIE) which prevents activation of this gene at the ventral/vegetal side of the embryo in vivo. In the present study, we show that BMP-4-induced inhibition is not direct but indirect, and is mediated by Xvent homeobox proteins. Micro-injections of Xvent-1 RNA and XFD-1' promoter deletion mutants demonstrate that Xvent-1 mimics the effect of BMP-4 signalling not only by suppression of the XFD-1' gene, but also by utilizing the BIE. Suppression could be reverted using a dominant-negative Xvent-1 mutant. The repressor domain was localized to the N-terminal region of the protein. Gel-shift and footprint analyses prove that Xvent-1 binds to the BIE. Moreover, PCR-based target-site selection for the Xvent-1 homeodomain confirms distinct motifs within the BIE as preferential binding sites. Thus, biological and molecular data suggest that Xvent-1 acts as direct repressor for XFD-1' transcription and mediates BMP-4-induced inhibition.