Conserved and novel enhancers in theAedes aegypti single-mindedlocus recapitulate embryonic ventral midline gene expression

Abstract

Transcriptionalcis-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 respectivesingle-minded (sim)loci in the two dipteran speciesDrosophila melanogaster(fruit fly) andAedes aegypti(mosquito).simencodes a highly conserved transcription factor that mediates development of the arthropod embryonic ventral midline. We identify twosimenhancers inA. aegyptiand demonstrate that they function equivalently in both transgenic flies and mosquitoes. OneA. aegyptienhancer is highly similar to a knownDrosophilacounterpart in its activity, location, and autoregulatory capability. The other differs from any knownDrosophila simenhancers 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 ofsimexpression 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 SUMMARYThe 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 thecis-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 namedsimin the mosquitoAedes aegypti, and comparing their functions and locations to those in the well-characterizedsimlocus of the fruit flyDrosophila melanogaster. Our results show that the two species generate identical patterns ofsimexpression through a mix of conserved and novel regulatory sequences. Continued exploration of thesimlocus in these two species will help to build a comprehensive picture of how a regulatory locus for a master developmental regulator has evolved.

Improved methodology for fixation and preparation of Aedes aegypti embryos

“…Embryos prepared using our method are suitable for immunohistochemistry (Fig 1 ), and for in situ hybridization using the hybridization chain reaction [12] (Fig 2). We recently demonstrated that digoxigenin-labelled in situ hybridization staining in A. aegypti is problematic and often results in unexpected false positive signals [13]; although embryos processed using our protocol can be stained in this manner, we advise caution and extensive controls. In addition, we found that immunocytochemistry in older embryos frequently leads to false-positive staining in the abdominal sensory setae and their associated trichome cells (Fig 3).…”