| Abstract |
During my thesis, I studied the role of bHLH-Orange (bHLH-O) proteins and Notch pathway in the self-renewal of post embryonic neuroblasts (NBs) in D. melanogaster. bHLH-O proteins are a subfamily of the basic-helix-loop-helix transcription factors characterized by an ‘Orange’ protein-protein interaction domain. These proteins act as repressors of transcription and typical members are the Hairy/E(spl), or Hes, proteins. They usually serve as targets of Notch signalling and they are well studied in their ability, among others, to suppress neuronal differentiation in both invertebrates and vertebrates. Drosophila larval neurogenesis is an excellent system for studying the balance between self-renewal and differentiation of a somatic stem cell (neuroblast). Post-embryonic neuroblasts (NBs) are neural stem cells that remain in quiescence after embryogenesis and, upon nutrition signals early in larval life, they resume asymmetric divisions. NBs generate differentiated neurons and glia from intermediate precursors cells named GMCs or INPs. We showed that in the larval Central Nervous System (CNS), E(spl)mγ, E(spl)mβ, E(spl)m8 and Deadpan (Dpn) are all expressed both in Type I and Type II NBs, in INPs but not in differentiated cells. A double mutation for the E(spl) complex and dpn severely affected the ability of NBs to self-renew, causing premature termination of proliferation. We also discovered that expression of E(spl)mγ and m8, but not of dpn, depends on Notch signalling coming from the GMC/INP daughter to the NB. When Notch is overactivated in NB progeny cells, overproliferation defects become apparent. We found that this depends on the abnormal induction of E(spl) genes and E(spl) overexpression can partly mimic the Notch-induced overproliferation phenotype. Therefore, we discovered that E(spl) and Dpn act together to maintain the NB in a self-renewing state, a process in which they are assisted by Notch, which sustains expression of the E(spl) subset.
We then used a genome-wide approach to seek the full complement of Notch target genes that affect NB self renewal. We performed transcriptomic analysis on larval CNS where Notch was overactivated for 24 hours leading to hyperplasia of NBs. We identified 1289 gene probesets whose transcript levels were upregulated. To further discern direct Notch targets from this list, we performed Chromatin immunoprecipitation to search for sequences bound by the Nicd/Su(H) complex. These sequences were isolated and hybridized to Drosophila tiling genomic arrays. As a result 169 genes were found to be direct targets of Notch. Among these genes were many transcription factors and factors involved in asymmetric cell division. We selected 10 genes from the first category and 2 from the second for further studies. We found that 5 out of 12 genes [E(spl)mγ, dpn, grh, wor, Mira, numb] were downregulated upon loss of Notch signalling whereas 9 out of 12 [E(spl)mγ, grh, wor, Mira, numb, svp, hth and antp] were upregulated when Notch was over-activated. Finally, besides the E(spl) genes, we found that two more genes ,lola and svp, were necessary for causing the pathological Notch induced hyperplasia. Svp, a gene that codes for an orphan nuclear receptor (homologous to the COUP-TF in mammals) was also sufficient for causing hyperplasia. Four more genes (cas, dpn, grh and hth) were found to play a more minor role in the Notch induced hyperplasia.
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