A study of mesoderm patterning through the analysis of the regulation ofXmyf-5expression

Xenopus laevis has been a particularly useful model organism for identifying factors involved in the induction and patterning of the mesoderm, however, much remains to be learned about how these factors interact. The myogenic transcription factor Xmyf-5 is the earliest known gene to be expressed specifically in the dorsolateral mesoderm of the gastrula, a domain that is established by the interaction of dorsal and ventral signals. For this reason, we have begun to investigate how the expression of Xmyf-5 is regulated. We have identified a 7.28 kb Xenopus tropicalis Xmyf-5 (Xtmyf-5) genomic DNA fragment that accurately recapitulates the expression of the endogenous gene. Deletion and mutational analysis has identified HBX2, an essential element, approximately 1.2 kb upstream from the start of transcription, which is necessary for both activation and repression of Xtmyf-5 expression, implying that positional information is integrated at this site. Electrophoretic mobility shift assays demonstrate that HBX2 specifically interacts with gastrula stage embryonic extracts and that in vitro translated Xvent-1 protein binds to one of its functional motifs. Combined with gain- and loss-of-function experiments, the promoter analysis described here suggests that Xvent-1 functions to repress Xmyf-5 expression in the ventral domain of the marginal zone. Furthermore, the identification of HBX2 provides a tool with which to identify other molecules involved in the regulation of Xmyf-5 expression during gastrulation.