Conserved and novel enhancers in the Aedes aegypti single-minded locus recapitulate embryonic ventral midline gene expression

Transcriptional cis -regulatory modules, e.g., enhancers, control the time and location of metazoan gene expression. While changes in enhancers can provide a powerful force for evolution, there is also significant deep conservation of enhancers for developmentally important genes, with function and sequence characteristics maintained over hundreds of millions of years of divergence. Not well understood, however, is how the overall regulatory composition of a locus evolves, with important outstanding questions such as how many enhancers are conserved vs. novel, and to what extent are the locations of conserved enhancers within a locus maintained? We begin here to address these questions with a comparison of the respective single-minded (sim) loci in the two dipteran species Drosophila melanogaster (fruit fly) and Aedes aegypti (mosquito). sim encodes a highly conserved transcription factor that mediates development of the arthropod embryonic ventral midline. We identify two enhancers in the A. aegypti sim locus and demonstrate that they function equivalently in both transgenic flies and transgenic mosquitoes. One A. aegypti enhancer is highly similar to known Drosophila counterparts in its activity, location, and autoregulatory capability. The other differs from any known Drosophila sim enhancers with a novel location, failure to autoregulate, and regulation of expression in a unique subset of midline cells. Our results suggest that the conserved pattern of sim expression in the two species is the result of both conserved and novel regulatory sequences. Further examination of this locus will help to illuminate how the overall regulatory landscape of a conserved developmental gene evolves. AUTHOR SUMMARY The expression patterns and roles of genes, especially those involved in core developmental processes, are often conserved over vast evolutionary distances. Paradoxically, the DNA sequences surrounding these genes, which contain the cis -regulatory sequences (enhancers) that regulate gene expression, tend to be highly diverged. The manner and extent to which enhancers are functionally conserved, and how the overall organization of regulatory sequences within a locus is preserved or restructured, is not well understood. In this paper, we investigate these questions by identifying enhancers controlling expression of a master nervous system regulatory gene named sim in the mosquito Aedes aegypti , and comparing their functions and locations to those in the well-characterized sim locus of the fruit fly Drosophila melanogaster . Our results suggest that the two species generate identical patterns of sim expression through a mix of conserved and novel regulatory sequences. Continued exploration of the sim locus in these two species will help to build a comprehensive picture of how a regulatory locus for a master developmental regulator has evolved. ### Competing Interest Statement The authors have declared no competing interest.