Whole-genome analysis of the transcriptional network underlying male sexual behaviour in Drosophila melanogaster

The robust behavioural courtship ritual displayed by Drosophila melanogaster males is governed by their underlying nervous system (NS). Two key genes of the Sex Determination Hierarchy, fruitless (fru) and doublesex (dsx), determine most neuronal substrates for sexual behaviour. In this study we aim to better understand the role fru plays in determining these neural substrates, as a means of better understanding the relationships between brain, behaviour and genes, and thus how the development of neuronal networks shape innate and species-specific behaviours.
Fru has two major functions: control of male sexual behaviour, and viability in both sexes. Alternative splicing of fru produces transcription factors driven by four promoters: P1 transcripts are sex-specifically spliced (only viable in the male), and P2-4 transcripts are crucial to both sexes survival. The resulting proteins contain a BTB protein-protein interaction domain at the N-terminus, and one of four C-terminal zinc-finger (ZnF) DNA binding domains. Male-specific proteins (FruM) contain an additional 101 amino acid N-terminal domain, and one of three alternative C2H2 ZnF domains (FruMA, FruMB, FruMC). These male-specific isoforms are expressed in the central nervous system (CNS) beginning in the late stage larvae (L3), peaking during pupation and on into adulthood. Little is known, however, about the roles of the individual isoforms, and no clear transcriptional targets have been identified. The central aims of this thesis are to document the wild-type expression patterns of Fru isoforms throughout development in the CNS, create and characterise isoform-specific mutants, and to identify and evaluate putative transcriptional targets of Fru. Combining these findings will lead to a better understanding of the underlying molecular functions of individual FruM isoforms, as a means to understanding their roles in sexual behaviour.
Expression analysis of FruM isoforms throughout development in the NS is described. To further characterise the role of individual FruM isoforms, isoform-specific mutants in fruA and fruB exons were generated using site-specific homologous recombination (HR). These novel mutants were validated by PCR and Fru isoform-specific antibody stainings. Mutants were analysed in fruM- and fru-null genetic backgrounds, to distinguish the roles of sex-specific vs. common isoforms. These analyses included: fertility, viability and morphology. FruA was found to have a role in wing extension with possible repercussions for song production. FruB was found to be developmentally lethal, in addition to having defects in male courtship behaviour.
To understand the role of fru in the NS, downstream transcriptional targets of FruM isoforms were identified. DNA adenine methyltransferase identification (DamID) was used to identify putative transcription targets of FruM isoforms. The Dam protein methylates DNA in Drosophila in a sequence-specific manner allowing targets of Fru to be isolated. Candidate genes were identified using computational analysis (including gene ontology, peak analysis and motif analysis) along with a biologically significant connection with fru. The relationship between fru and six candidate genes were characterised using RNAi. The results of these studies advance our knowledge of how FruM isoforms influence the development and physiology of the NS underlying male sexual behaviour in Drosophila.

Keywords: Drosophila melanogaster
courtship
Fruitless
DamID
Advisor: Goodwin, S