Regulation of the tinman homologues in Xenopus embryos.

Vertebrate homologues of the Drosophila tinman transcription factor have been implicated in the processes of specification and differentiation of cardiac mesoderm. In Xenopus three members of this family have been isolated to date. Here we show that the XNkx2-3, Xnkx2-5, and XNkx2-10 genes are expressed in increasingly distinctive patterns in endodermal and mesodermal germ layers through early development, suggesting that their protein products (either individually or in different combinations) perform distinct functions. Using amphibian transgenesis, we find that the expression pattern of one of these genes, XNkx2-5, can be reproduced using transgenes containing only 4.3 kb of promoter sequence. Sequence analysis reveals remarkable conservation between the distalmost 300 bp of the Xenopus promoter and a portion of the AR2 element upstream of the mouse and human Nkx2-5 genes. Interestingly, only the 3' half of this evolutionarily conserved sequence element is required for correct transgene expression in frog embryos. Mutation of conserved GATA sites or a motif resembling the dpp-response element in the Drosophila tinman tinD enhancer dramatically reduces the levels of transgene expression. Finally we show that, despite its activity in Xenopus embryos, in transgenic mice the Xenopus Nkx2-5 promoter is able to drive reporter gene expression only in a limited subset of cells expressing the endogenous gene. This intriguing result suggests that despite evolutionary conservation of some cis-regulatory sequences, the regulatory controls on Nkx2-5 expression have diverged between mammals and amphibians.