Regulation and Function of the dlx Genes in the Zebrafish Forebrain

The Dlx genes encode a family of transcription factors important to the development of the vertebrate forebrain. The expression of the Dlx genes is controlled by a number of highly conserved regulatory elements, which have been extensively studied in the mouse. The Dlx genes have been shown to have highly overlapping expression during the development of the telencephalon/diencephalon and playa role in GABAergic interneuron differentiation and in the regulation of the Gad genes, responsible for GABA synthesis. Using fluorescent in situ hybridizations, we show the dlx genes are expressed in highly overlapping domains within the zebrafish forebrain and appear to follow a conserved pathway involved in the differentiation of GABAergic interneurons. We have identified one of the dlx regulatory elements, URE2, in the three major branches of jawed vertebrates and tested the orthologous sequences for regulatory function. These regulatory sequences have similar activity in the forebrain of mice and zebrafish. Transgenic and dlx expression data indicate there may be similar genetic cascades controlling Dlx expression in the mammalian and teleost forebrain. To elucidate the genetic cascade regulating the expression of the dlx and gad1 genes in zebrafish, we knocked down the function of ascl1a, a Mash1 ortholog known to regulate Dlx expression in the mouse. Knockdown of ascl1a function resulted in a loss of dlx and gad1 expression in the prethalamus. We also impaired the function of the dlx genes in the zebrafish forebrain. Double knockdown of the dlx1a/2a genes resulted in a loss of gad1 expression in the prethalamus, but not in the telencephalon, in contrast to observations made in the mouse. We propose the ascl1a and dlx genes are involved in an evolutionarily conserved genetic cascade controlling GABAergic interneuron differentiation in the prethalamus