Functional analysis of Dlx homeodomain proteins from the zebrafish.

Abstract

The dlx homeobox genes of vertebrates are transcriptional regulators involved in the development of the ventral forebrain, otic vesicle and inner ear, visceral arches, olfactory placodes and fins/limbs. The zebrafish genorne contains eight dlx genes whose expression patterns during development are partially overlapping. Experiments in cultured cells and transgenic zebrafish embryos were used to understand the functional specificity and the biochemical properties of Dlx proteins. Previous work by us and others showed that Dlx proteins can act as transcriptional activators. In order to determine how the various Dlx proteins differ from each other, transfection experiments in cultured cells were done to identify the Dlx transcriptional activation domains. I found that an activation domain was located in the N-terminal region of Dlx1, Dlx2 and Dlx4. In addition, to test the hypothesis that the partially overlapping expression of dlx genes during development is the result, in part, of specific cross-regulatory interactions between the genes, overexpression of mutant versions of the dlx genes into transgenic zebrafish embryos were carried out. Injection into one cell embryo of synthetic mRNA coding for a chimeric protein including the amino-terminal region of Dlx3 and the carboxy-terminal half of Dlx2, resulted in a loss or decrease of endogenous dlx4 expression in the visceral arches, the otic vesicles and the forebrain of 27 hours embryos. Furthermore, I demonstrated that overexpression of that chimera, which disturbed the endogenous dlx4 expression, results in malformations of the craniofacial cartilages. It suggests that Dlx3 is normally a positive regulator of dlx4 expression and that the chimera Dlx3-Dlx2 protein interferes with the function of the endogenous Dlx3. These results suggest that the combinatorial patterns of dlx expression are due at least in part to cross-regulatory interactions between the different dlx genes.