Bix4 is activated directly by VegT and mediates endoderm formation in Xenopus development.

The maternal T-box gene VegT, whose transcripts are restricted to the vegetal hemisphere of the Xenopus embryo, plays an essential role in early development. Depletion of maternal VegT transcripts causes embryos to develop with no endoderm, while vegetal blastomeres lose the ability to induce mesoderm (Zhang, J., Houston, D. W., King, M. L., Payne, C., Wylie, C. and Heasman, J. (1998) Cell 94, 515-524). The targets of VegT, a transcription activator, must therefore include genes involved both in the specification of endoderm and in the production of mesoderm-inducing signals. We recently reported that the upstream regulatory region of the homeobox-containing gene Bix4 contains T-box binding sites. Here we show that expression of Bix4 requires maternal VegT and that two T-box binding sites are necessary and sufficient for mesodermal and endodermal expression of reporter genes driven by the Bix4 promoter in transgenic Xenopus embryos. Remarkably, a single T-box binding site is able to act as a mesoderm-specific enhancer when placed upstream of a minimal promoter. Finally, we show that Bix4 rescues the formation of endodermal markers in embryos in which VegT transcripts have been ablated but does not restore the ability of vegetal pole blastomeres to induce mesoderm. These results demonstrate that Bix4 acts directly downstream of VegT to specify endodermal differentiation in Xenopus embryos.