The microRNA pathway is an important regulator of stem cell proliferation, maintenance and self-renewal in eukaryotes. Drosophila germline stem cells (GSCs) are a premier model for stem cell behavior. In this system, two to three GSCs are situated within a niche that is formed by somatic cells. A BMP signal is released from this niche, which acts as the primary factor in suppressing stem cell differentiation. More recently, it was shown that the miRNA pathway is cell-autonomously required in GSCs, where it acts either downstream or in parallel to BMP signalling. However, only one miRNA, bantam, has been identified to directly participate in the regulation of GSC maintenance, though the downstream targets remain unknown. This thesis aims to examine how the miRNA pathway influences stem cell maintenance by examining the profiles of miRNAs and their targets in Drosophila GSCs.Using a comparison of microRNA profiles from GSCs with high and low levels of BMP signalling, the extend to which BMP signalling is able to influence miRNA profiles was studied. The identification of miRNA targets was determined through a combination of bioinformatic and experimental approaches. It was revealed that the impact of BMPs on microRNA expression in the GSC system is only of minor importance. The interaction sites between Ago1 and targets of miRNAs were determined on a whole genome level using Ago1 iCLIP, which allows single nucleotide resolution. Integration of this dataset with whole genome Ago1 RNA immunoprecipitation from ovary tissue revealed possible genes and processes that might regulate the fate of GSCs. These results indicate that in the GSC niche, the miRNA pathway may regulate cell death and cell cycle genes and may interact with EGFR signalling and receptor protein tyrosine kinase pathways. Furthermore, potential targets of five miRNAs, including bantam, that are highly expressed in GSCs were identified. Subsequent analysis of miRNA mutant germaria revealed a previously unknown function of miR-14 and miR-989 in normal development of GSCs and germarial morphology. This work gives new insights into miRNA regulation of stem cell biology. As miRNAs are known to be important stem cell regulators of other eukaryotes, including humans, these results can be extrapolated to serve as a platform for understanding the role of miRNAs in other stem cell systems.
|Date of Award
|1 Aug 2015
- The University of Manchester
|Hilary Ashe (Supervisor) & Sam Griffiths-Jones (Supervisor)