Evolution of auto- and cross-regulatory elements of members of the distal-less-related family of homeobox-containing genes

The Vertebrate Dlx gene family consists of at least three linked pairs of convergently transcribed genes. The linked Dlx genes demonstrate a striking degree of overlap in expression patterns during development that is coincident with their genomic organization and is relatively well conserved between teleost fish, such as the zebrafish, and mammals. Two explanations for this overlap of expression are that there are cross-regulatory interactions between the Dlx genes or, in the case of the paired Dlx genes, that they share cis-acting regulatory elements. To test this second hypothesis, we compared the homologous intergenic regions of the paired Dlx genes between mouse and zebrafish. We have identified elements in the intergenic regions of all the paired Dlx genes that are well conserved between zebrafish and mice and that may, at least partially, account for the overlap in expression patterns of their neighboring Dlx genes by acting as shared enhancer elements. At least two of these elements are able to direct specific expression in transgenic mice and, remarkably, also in analogous anatomical regions in transgenic flies. Two of the elements identified are also the sites of regulation by the Dlx proteins themselves. An additional site of regulatory interactions between members of the Dlx family has been identified in the region upstream of the zebrafish dlx4 gene which seems to be dependant on expression of dlx3 . These results indicate that interactions between members of the Dlx family are part of their normal function during development and that there has been some degree of conservation of the elements directing expression of the Dlx genes during metazoan evolution. These results also suggest a mechanism by which duplicated genes may be stabily maintained during evolution as well as a possible explanation for the maintenance of the Dlx genes as linked pairs in the genome.