Computational and experimental approaches for the identification of genes and gene networks in the Drosophila sex-determination hierarchy

UnrestrictedSexual reproduction in most multi-cellular species requires the development of sex-specific adult structures and the potential to perform sex-specific behaviors. These adult sex-specific phenotypes are typically patterned during development. Therefore, a more thorough understanding of how the genome is deployed in a sex-differential manner during development provides insight into how gene networks give rise to sex-specific traits. The fruit fly, Drosophila melanogaster, is an excellent model system for studying how sex-specific differences are established at the molecular-genetic level. The Drosophila sex determination hierarchy is responsible for establishing adult sex-specific morphologies and behaviors through an alternative pre-mRNA splicing cascade that culminates in the production of sex-specific transcription factors encoded by doublesex (dsx) and fruitless (fru). Little is known, however, about the genes regulated by these transcription factors during development.; Metamorphosis is the developmental stage during which the largely asexual larval form is transformed into the sexually dimorphic adult form. We, therefore, focus our analyses on this developmental stage. Using a microarray-based approach, we analyzed gene expression during metamorphosis of all predicted Drosophila genes, for males and females separately, in both wild type animals and animals that lack germline tissues. We then narrowed our focus to the developmental stage 48-hours-after-puparium-formation to identify genes that are regulated downstream of DSX and/or FRU. We identified several genes regulated by DSX and/or FRU during metamorphosis and our results allowed us to propose an extension of the canonical models for how DSX regulates gene expression. In addition, we show that the sex-determination hierarchy and the ecdysoneregulatory hierarchy regulate expression of similar gene sets. We also show that abrogating ecdysone receptor function in the fru neural circuit leads to defects in malecourtship behaviors. Finally, we developed a computational approach to search for genomic regions called cis-regulatory modules that contain clusters of transcription factor binding sites. We used this computational method, in combination with our expression data analyses, to predict direct targets of DSX and FRU. This computational method is publically available as a web-based program (SUPRfly; Significant Upstream Regions of the fly)