DNA sequences mediating the transcriptional response of the Mix.2 homeobox gene to mesoderm induction

Abstract

Peptide growth factors can initiate changes in cell fate in Xenopus ectodermal explants and induce the formation of mesoderm. Marker genes expressed in mesoderm allow the analysis of whether, or how much, induction has occurred but do not tell us what molecules are involved in carrying out the response. In this report we describe the isolation of genomic and cDNA clones of Mix.2, a gene closely related to the Xenopus homeobox gene Mix.1, and demonstrate that the promoter of the Mix.2 gene is responsive to mesoderm induction signals when linked to a CAT reporter and microinjected into developing Xenopus embryos. Like the chromosomal Mix.1 gene, microinjected Mix.2 gene plasmids respond to activin in the presence of cycloheximide in animal cap assays and also respond to the embryonic inductive signal in Nieuwkoop recombinants. The injected promoter does not respond to TGF-β2 or FGF. Deletion analysis of the Mix.2 promoter demonstrated that sequences required for maximal transcriptional activity in response to mesoderm induction are scattered across a 290-bp region. This is the first report of a microinjected plasmid responding to immediate-early transcriptional activation in developing Xenopus embryos. This assay reduces the complexity of the cellular response to embryonic induction to the simple question of which molecules activate the Mix.2 promoter and provides a sensitive and rapid test with which to pursue the answer.