Transcriptional regulation of intestinal stem cells in Drosophila.

Tissue resident Adult Stem Cells (ASCs) are responsible for replenishing cells during homeostasis and in response to stress and tissue damage. Owing to their vital function in tissue maintenance and regeneration, ASC dysfunction is a hallmark of ageing and tumorigenesis. Despite the importance of these cells, our understanding of the pathways and gene regulatory networks (GRNs) that govern them is incomplete. We use Drosophila Intestinal Stem Cells (ISC) as a model to study conserved genes involved in the transcriptional regulation of stem cells.

During this thesis, I have utilised ISCs to expand our understanding of the GRN underlying ISCs in Drosophila. In so doing we have discovered a novel role for the transcription factor Chronophage (Cph) in ISC proliferation and enteroendocrine (EE) cell differentiation. We observed Cph expression is induced during ageing, infection and tumorigenesis in the midgut. cph knockdown in ISCs hampered proliferation and cph overexpression increased EE cells. In addition, cph knockdown in Notch loss-of-function tumours had a tumour suppressive phenotype, increasing lifespan and reducing tumour burden. DamID DNA binding data and qRT-PCR analysis demonstrated Cph directly regulates key EE regulatory genes. Overall, we have identified a hitherto unknown transcriptional regulator of ISCs thereby expanding our understanding of the GRN in ISCs.

Alongside Cph, we investigated the potential role of Dachshund, miR-279, Sema1a and Fer1as putative ISC regulators. We found aberrations in Dac expression led to alterations in the abundance of mitotic ISCs. Furthermore, we demonstrate inhibition and ectopic expression of miR-279 in ISCs impacts ISC proliferation and differentiation, potential by way of repressing regulators of ISC maintenance and proliferation Escargot (Esg) and Nerfin-1. Ultimately, while these results are preliminary, they highlight the variety of remaining factors with yet unknown functions in ISC regulation.

Finally, this thesis also endeavoured to illuminate the interactome of the stemness maintenance factor Escargot (Esg). To this end an esg-BioID line was generated for use in future pulldown experiments to identify protein interactors of Esg.