Investigating the underlying mechanisms of axis formation in Drosophila melanogaster
The establishment of the body axes has been well-studied in Drosophila melanogaster, and is known to occur prior to fertilisation in the developing egg chamber as a result of crosstalk between the germline and the surrounding somatic epithelium. Whilst it is known that the formation of the anterior-posterior and dorsal-ventral axes occurs due to the establishment of oocyte cell polarity and repositioning of the oocyte nucleus, respectively, the nature of the signals sent from the soma to trigger these two events, and the method through which these signals are transduced, remain unclear. In an attempt to elucidate these underlying mechanisms, studies of both the follicle epithelium and germline were undertaken. Posterior follicle cells are known to be responsible for the formation of both body axes through signalling, but very few factors have been identified which perturb this process. The transcriptomic method Targeted DamID was used to quantify gene expression in these cells, compared to their lateral follicle cells counterparts. This resulted in the identification of 140 genes which were either uniquely expressed or upregulated in posterior follicle cells. Specific subsets of these genes were knocked down via RNAi, and the impact of this knockdown on axis formation determined. However, an attempted validation of these observed phenotypes through CRISPR-based mutagenesis or analysis of extant alleles failed to recapitulate many of the expected defects. Subsequent experiments have attempted to further narrow this list of differentially-expressed genes to provide a more specific profile of the posterior follicle cell transcriptome. In parallel, a forward genetic screen was undertaken to identify novel factors required for axis formation in the germline. Whole genome sequencing of a mutant which fails to establish cell polarity identified the causative mutation as a SNP in the enzyme CDP-diacylglycerol synthase (CdsA), required for the production of a number of phospholipid species. An initial characterisation of this mutant phenotype suggested the activity of CdsA is required to recruit the polarity kinase Par-1 to the posterior of the oocyte. A further analysis established that the production of phosphatidylglycerol-based species is dispensable for oocyte polarity establishment, suggesting that the phenotype of cdsa alleles is produced due to its impact on phosphatidylinositol synthesis. Additionally, an analysis of the localisation of the PIP2 derivative diacylglycerol using a fluorescent biosensor revealed its localisation was not uniform within the oocyte. To better understand potential downstream functions of oocyte cell polarity in the context of axis formation, several previously-identified Par-1 phosphorylation target sites were examined. The six sites of interest were located in the eIF4E-binding protein Cup, which is required to repress translation of osk mRNA until it is posteriorly-localised. To analyse the function of these phosphorylation sites, several Cup-GFP transgenes expressed under the endogenous Cup promoter were created, in which these phosphorylation sites were perturbed. Expression of these constructs in a cup mutant background rescued their premature egg chamber degradation phenotype. However, due to technical difficulties in the creation of these phosphorylation mutant transgenes, it remains unclear to what extent Par- 1 phosphorylation affects the function of Cup in the context of osk mRNA translation.
Networked Digital Library of Theses and Dissertations