NOVEL TRANSCRIPTIONAL PROFILING OF CELLS IN THE DROSOPHILA TESTIS

In the Drosophila melanogaster testis, research has primarily focused on identifying genes expressed within the largest population of cells present within the tissue: the germ cells. However, determining the gene expression profiles of the other cell types that exist within the Drosophila testis, particularly rarer cell types such as the germline and somatic stem cells, would broaden our understanding of how the testis functions. With the advancement of technologies, questions that were previously impossible to tackle due to tool unavailability are now approachable. Improvements in genomic tools like next generation sequencing have allowed scientists to identify differences within seemingly homogenous populations of cells and identify multiple genes expressed in a cell type at a singular time. In this thesis work, two recently developed transcriptional profiling techniques are applied to answer biologically interesting questions about the testis. First, single cell RNA sequencing is used to study the inactivation of sex chromosomes within differentiating male germ cells. This process occurs in mammals, but its existence in Drosophila was debated, due to the inability to discern genome-wide gene expression patterns in discrete populations of cells. Using this method, we determine the expression profiles of populations within the germline lineage that reside at different cellular stages simultaneously. In particular, we were able to identify the specific cell type in which downregulation of X transcripts occurs and determine a potential mechanism of the downregulation of X transcripts in the identified primary spermatocytes, all due to the ability to purify unique germ cell populations from the testis. In addition, we were also able to adapt a new tool called Targeted DNA Adenine Methyltransferase Identification to identify novel candidate sex-biased gene expression within the somatic lineage of the testis. These genes, some of which have newly described functions in the testis, serve as a foundation for future studies determining genes that maintain somatic sex identity in this tissue. Using both of these techniques, we have generated gene expression profiles for discrete subsets of cells in the testis as well as the ovary, which we hope can serve as a new rich gene resource for the fly community