Novel approaches to studying alternative splicing in Drosophila Melanogaster: Insights into sex-specific gene expression and the evolution of sex determination

Males and females exhibit numerous differences, from the initial stages of sex determination to the development of secondary sexual characteristics. In Drosophila, these differences have been thoroughly studied. Extensive research has been performed to understand the role and molecular mode of action of central sex in determining switch genes, such as transformer (tra) and Sex-lethal (Sxl). Furthermore, studies have highlighted differential gene expression as an essential mechanism to create sexual dimorphism. An alternative path to sexual dimorphism that has been less explored is alternative splicing, the mechanism through which genes can produce multiple transcripts with distinct properties and functions. The primary switch sex-determining gene Sxl is a good example of the role of alternative splicing for sex-specific functions: the inclusion of a specific exon determines the male or female form of the protein, which in turn switches on either the male or female developmental pathway. The genes that act upstream of Sxl and determine which form is expressed - the counter genes - have received less attention. This thesis addresses two critical questions about the molecular encoding of sexes in the Drosophila melanogaster genome: First, the use of splice forms in male and female tissues in D. melanogaster is examined, inferring the molecular and evolutionary parameters shaping the diversity of the splicing landscape. Second, the behaviour of counter genes in Drosophila-related species is investigated, shedding light on potential changes leading to their incorporation into the sex-determination pathway. For the alternative splicing analyses, long-read RNA sequencing of testes, ovaries, female and male midguts, heads, and whole bodies was performed. A novel pipeline was developed to assign unique transcript identifiers for each sequence of exons and introns in the read, enabling detailed comparisons of splicing variants in each tissue/sex. Alternative splicing was found to be more pervasive in females than males (22,201 exclusive splice forms in females versus 12,631 in males), especially when comparing ovaries to other tissues. The ovaries alone displayed 15,299 exclusive splice forms, suggesting most female exclusive splice forms originate there. Genome location and gene age were also correlated with the number of splice forms per gene. In particular, the X and 4th chromosomes (Muller elements A and F) showed more splice forms per gene than other chromosomes. Additionally, genes older than 63 million years exhibited more splice forms per gene than younger genes. Our results suggest that alternative splicing is more prevalent than previously believed, with numerous female-exclusive forms, age, and location playing significant roles in shaping its prevalence. For the counter genes analyses, we combined published gene expression, genomic, and gene interaction data from various clades (Bactrocera jarvisi, B. oleae, Ceratitis capitata, Mus musculus, Caenorhabditis elegans, Homo sapiens, and D. melanogaster). The counter genes scute (sc), extra macrochaetae (emc), groucho (gro), deadpan (dpn), daughterless (da), runt (run), Sxl, hermaphrodite (her), and tra maintain conserved Muller element locations between C. capitata and D. melanogaster, which are most of the counter genes identified in the C. capitata genome. Their expression patterns during early embryogenesis in B. jarvisi and D. melanogaster are also similar for counter genes dpn, gro, da, and emc. However, Sxl and sc are also found to have more extreme expression ratios between the species. Lastly, gene interactions within the counter genes are conserved, with da-sc and gro-dpn interactions occurring in Drosophila, worms, humans, and mice. Interactions such as dpn-sc, dpn-da, da-emc, and gro-run are present in Drosophila, mice, and humans, suggesting these genes were recruited by ancestral characteristics, primarily during embryogenesis. The conserved expression, location, and interactions of counter genes suggest serendipitous recruitment of such genes instead of a change in those characteristics as they were recruited for this function.