Genomic structure and functional control of the Dlx3-7 bigene cluster.

The Dlx genes are involved in early vertebrate morphogenesis, notably of the head. The six Dlx genes of mammals are arranged in three convergently transcribed bigene clusters. In this study, we examine the regulation of the Dlx3-7 cluster of the mouse. We obtained and sequenced human and mouse P1 clones covering the entire Dlx3-7 cluster. Comparative analysis of the human and mouse sequences revealed several highly conserved noncoding regions within 30 kb of the Dlx3-7-coding regions. These conserved elements were located both 5' of the coding exons of each gene and in the intergenic region 3' of the exons, suggesting that some enhancers might be shared between genes. We also found that the protein sequence of Dlx7 is evolving more rapidly than that of Dlx3. We conducted a functional study of the 79-kb mouse genomic clone to locate cis-element activity able to reproduce the endogenous expression pattern by using transgenic mice. We inserted a lacZ reporter gene into the first exon of the Dlx3 gene by using homologous recombination in yeast. Strong lacZ expression in embryonic (E) stage E9.5 and E10.5 mouse embryos was found in the limb buds and first and second visceral arches, consistent with the endogenous Dlx3 expression pattern. This result shows that the 79-kb region contains the major cis-elements required to direct the endogenous expression of Dlx3 at stage E10.5. To test for enhancer location, we divided the construct in the mid-intergenic region and injected the Dlx3 gene portion. This shortened fragment lacking Dlx7-flanking sequences is able to drive expression in the limb buds but not in the visceral arches. This observation is consistent with a cis-regulatory enhancer-sharing model within the Dlx bigene cluster.