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Title: Investigating novel direct Notch targets in Drosophila neural stem cells
Author: Feng, Shiyun
ISNI:       0000 0004 7230 5241
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2018
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Notch signalling is an evolutionary highly conserved signalling pathway. It plays various important roles in the regulation of many fundamental cellular processes such as proliferation, stem cell maintenance and differentiation during embryonic and adult development. Notch signalling has a simple transduction pathway. Upon Notch ligand binding to the receptor, the Notch intracellular domain (NICD) is released into the nucleus. The nuclear NICD interacts with the DNA-binding protein Suppressor of Hairless (Su(H)) to activate the expression of target genes, which are silenced by the Su(H)-corepressor complex in the absence of Notch activity. The functions of Notch are very context-dependent, making it important to identify the Notch regulated genes in different processes. Neural stem cells (NSCs) are cells that can divide and differentiate into all kinds of cells within the brain while they self-renew. Notch signalling is one of the key regulators in maintaining NSCs and performs a similar function in both Drosophila and vertebrate NSCs. Drosophila NSCs serve as an ideal model for studying the relationship between Notch function and stem cell behaviours. Although many target genes, such as the Hes genes, have been identified, they cannot fully account for the diversity of Notch responses. Therefore, further functional study of more potential target genes is needed to gain understanding about Notch-regulated NSC maintenance. In this thesis, a group of potential direct Notch target genes are examined for their responsiveness to Notch regulation and their functions in Drosophila NSCs. Previous genome-wide study in the Bray lab has found a number of potential Notch target genes in the Drosophila larval brain, with the characteristics of Notch transcription factor Su(H) binding and mRNA upregulation by Notch over-activation (Zacharioudaki et al. 2016). I first examined the Notch responsive element (NRE) activity of these potential Notch targets and their regulation by Notch both in vivo and in cell lines. The presented findings validated path, cables and Asph as direct Notch target genes in Drosophila NSCs, while syp, lola and Fer2 do not exhibit characteristics of Notch responsive targets in NSCs. The functional roles of two of the responsive genes, path and cables, were subsequently explored in Drosophila larval brains. Firstly, I found that Path, a potential amino acid transporter, is not only important for protecting NSC proliferation under normal and abnormal conditions through integrating growth pathways, but is also required for protecting brain growth under nutrition deprivation. Secondly, the cables gene was connected to a distal NRE through knocking out the suspected NRE region and the gene itself using the CRISPR/Cas9 technique. Subsequent experiments revealed that cables is also required for NSC proliferation. In summary, a group of direct Notch target genes were validated and as a consequence two genes that are important for protecting NSC proliferation were identified.
Supervisor: Bray, Sarah Sponsor: China Scholarship Council ; Cambridge Oversea Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Notch target ; neural stem cells ; proliferation ; nutrition restriction