Exploring a Nuclear Insulin Receptor Signalling Pathway in Drosophila Neural Stem Cells
The insulin receptor (InR) is a central regulator of metabolism, growth, and proliferation in both humans and *Drosophila*. Integrating a range of signals, InR coordinates pleiotropic, tissue‐specific outputs. Neural stem cells (NSCs) give rise to the vast range of cell types that comprise the central nervous system (CNS) in a manner highly regulated in both space and time. With a comparatively simple CNS that nonetheless exhibits remarkable biological conservation, combined with superior genetic tractability, *Drosophila melanogaster* is an ideal model organism for studying the principles of neurogenesis and NSC biology. In *Drosophila*, insulin signalling is necessary and sufficient for the reactivation of NSCs from quiescence, a conserved state of mitotic dormancy. InR has been observed in the nucleus of a range of cell types *in vitro* for decades, but it is only more recently that the significance of this is becoming clear. Data reveal a novel nuclear arm of the insulin signalling pathway, in which InR associates with chromatin genome‐wide, strongly localizing to promoters and activating the transcription of relevant genes. This finding represents a growing paradigm shift, encompassing other receptor tyrosine kinases (RTKs), whereby direct nuclear signalling complements canonical membrane‐localised signal transduction. Given the complexity of insulin signalling and its dysregulation across an array of chronic human diseases, this nuclear pathway constitutes an exciting avenue for basic and translational science. Characterising the nuclear actions of InR might offer a more complete understanding of the longer‐term effects of insulin signalling. In the context of the CNS, it may offer a more complete understanding of NSC dynamics, which in turn may guide future therapeutic strategies for brain repair and difficult‐to‐treat malignancies. In this thesis, nuclear insulin signalling in *Drosophila* NSCs is studied via a variety of *in vivo* methods. Cyan fluorescent protein (CFP)‐tagged InR was expressed with the GAL4 system to examine its subcellular localization. InR‐CFP is present in the nucleus of *Drosophila* NSCs throughout CNS development. This localization pattern is not upheld in NSC progeny, where InR‐CFP exhibits strong membrane localisation. Next, the endogenous InR locus was tagged with green fluorescent protein (GFP) knock‐in using the CRISPR/Cas9 system. At endogenous InR expression levels, no clear nuclear localization was visible on confocal microscopy. However, the presence of nuclear InR was later confirmed with NanoDam. NanoDam, a recently published technology based on Targeted DamID, was used to profile the chromatin association of InR in NSCs *in vivo* genome‐wide. NanoDam was performed at endogenous levels of InR expression in the InR‐GFP background, and under misexpression conditions in the UAS‐*InR‐CFP* background. InR associates with chromatin genome‐wide in both genetic backgrounds. These data represent the first known finding that InR associates with chromatin in any cell type in *Drosophila*, and that InR associates with chromatin in NSCs in any organism. More genes are reproducibly and significantly bound in the endogenously tagged InR‐GFP background than the misexpression UAS‐*InR‐CFP* background, which may be due to higher expression levels in the latter background saturating physiological binding patterns and leading to more random interactions. Consistently, significantly more genes are bound in reactivating than quiescent NSCs; and consistently, InR binding is enriched in promoters. Amongst the most significantly bound genes in reactivating NSCs are genes with known roles in NSCs, signalling pathways, cell growth, and proliferation. Given the known role of canonical insulin signalling in the reactivation of quiescent NSCs, this work hypothesizes that nuclear insulin signalling contributes to this process. Exploring the transcriptomes of quiescent and reactivating NSCs with scRNA‐seq and intersecting this data with the NanoDam data revealed genes differentially expressed and bound by InR in reactivating NSCs. These constitute candidate genes that may act downstream of nuclear InR to mediate reactivation. They include genes with known roles in NSC development; genes that encode important RNA‐modifying proteins; and genes that modulate the activity of central signalling pathways known to affect NSC dynamics. Amongst these candidate genes, several stand out as being of particular biological interest. *Mettl3* encodes a conserved RNA methyltransferase. The m(6)A modification METTL3 catalyses is the most prevalent mRNA modification in vertebrates and influences mRNA stability, translational dynamics, and RNA pol II pause release. The latter is an important mechanism of enabling rapid, synchronous cell state changes, of which reactivation is a prime example. Preliminary data show that *Mettl3* knockdown is associated with a reactivation delay (Cian Doherty and Andrea Brand, unpublished), making METTL3 acting downstream of nuclear InR to facilitate reactivation an exciting model to investigate. *dachs*, a negative regulator of Hippo signalling constitutes another intriguing candidate gene; a switch between Hippo signalling and insulin signalling is vital for reactivation to occur. Dpp is an established signal for stem cell self‐renewal and prevents premature differentiation, and genes pertinent to its signalling, including the *tkv* receptor and *shn* transcription factor are also candidate genes. Overall, the findings support a model in which nuclear InR signalling serves as a point of integration of important NSC signalling pathways and orchestrates their cross‐regulation to fine‐tune the balance between pro‐quiescence and pro‐proliferation signals. The binding of a broad range of relevant genes and a resultant modest and combinatorial transcriptional activation was a picture also seen in mammalian cells. Future work will be necessary to validate the candidate genes and models proposed by this work and to elucidate the mechanisms of InR nuclear import and transcriptional activation. Some pertinent experimental avenues are suggested in the conclusions.
Drosophila, Insulin signalling, Neural stem cells
Networked Digital Library of Theses and Dissertations