Alternative splicing and embryonic expression of the Xenopus mad4 bHLH gene

The cell proliferative activity of the Myc family of basic helix-loop-helix/leucine zipper (bHLHZip) transcription factors is dependent upon binding to the ubiquitous Max protein. In the absence of heterodimerization with Max, Myc protein is unable to efficiently bind to DNA and activate transcription. Members of the Mad family of transcription factors are thought to modulate the cell proliferative effects of the c-myc proto-oncogene by binding to Max, directly competing with the Myc protein for both heterodimerization and DNA binding. Consistent with a role in down-regulating cell division, the murine mad genes are expressed in embryonic tissues undergoing differentiation, often during or shortly after the down-regulation of myc gene expression. Here, we report the isolation and characterization of the first Xenopus mad family member, Xmad4. Maternal Xmad4 transcripts are present at high levels in the oocyte and in the cleavage stage embryo, but almost disappear by the neurula stage. Zygotic expression of the Xmad4 gene is initiated in the epidermis of the late neurula stage, and shortly thereafter, Xmad4 is transiently detectable in the cement and hatching glands. At later stages, expression is also observed in the developing pronephros and liver. Unlike the murine mad4 gene, we find that multiple Xmad4 splice variants exist in Xenopus and that these variants are differentially expressed in both the embryo and the adult. Despite the demonstrated antagonistic role of Mad proteins in the regulation of Myc activity, we show that the over-expression of Xmad4 in the cleavage-stage embryo has no detectable phenotypic effect, suggesting that Myc function is dispensable during early embryonic development.