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Using single cell transcriptomics to uncover mechanisms of neural development, neural stem cell quiescence and tumourigenesis



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Tang, Lok Yee Jocelyn 


Neural stem cells produce all the cells of the nervous system and regulation of proliferative behaviour is crucial for proper brain development. Over-proliferation of NSCs can also lead to disease, resulting in the formation of tumours. There is a delicate balance between NSC proliferation, differentiation and quiescence. Studying the mechanisms that regulate this balance is important for understanding development and disease, providing a basis for new therapeutic strategies for brain injury or cancer. In Drosophila melanogaster, a small pool of NSCs produce all of the 200 000 neurons present in the adult brain. The precise mapping of NSCs in the brain, short developmental time, genetic tractability and wide variety of experimental tools make Drosophila an ideal model for investigating NSC behaviour. My project utilises single-cell transcriptomics to study two aspects of brain development: 1) the regulation of developmental NSC quiescence and its relevance to glioblastoma, and 2) identifying novel players regulating the temporal identity of neural progenitors and how this generates neuronal diversity.

NSCs in the larval Drosophila brain undergo quiescence, a reversible, cell-cycle arrested state, after a period of neurogenesis and reactivate in response to extrinsic signals, namely feeding. In order to investigate how quiescence is regulated, I used scRNA-seq to profile quiescent NSCs. One of the genes found to be enriched during quiescence was tis11, involved in regulating RNA stability. Functional studies of tis11 suggest that it is involved in the maintenance of quiescence. Interestingly, ZFP36, the orthologue of tis11 is also expressed in human glioblastoma cells that are slow-cycling and express genes characteristic of mammalian NSC quiescence. Glioblastoma is a cancer with poor prognosis, as chances of tumour recurrence are high. This may be due to the presence of a subpopulation of cancer stem cells which divide slowly and are therefore resistant to conventional chemotherapy. Expression of ZFP36 (involved in developmental quiescence) in glioblastoma cells suggests that they are quiescent and may be a source of tumour recurrence. Understanding the mechanisms of tis11 during developmental quiescence may therefore lead to a better understanding of action in the context of glioblastoma and inform better treatment strategies.

Brain development requires different subtypes of neurons to be produced at the right time and in the right numbers, in order to achieve functional connectivity. The Drosophila brain has a class of NSCs that produce intermediate neural progenitors (INPs) that transition through a ‘temporal cascade’. The temporal cascade is a series of sequentially expressed transcription factors that cross-regulate one another and determine the subtypes of neuronal progeny that are produced. The INP temporal cascade was originally identified by antibody screens but the cross-regulatory interactions were not comprehensive, suggesting that there are unknown players yet to be identified. Using a newly developed technique from our lab, NanoDam, the genome-wide binding targets of the previously identified temporal factors were profiled in the INPs, in their endogenous windows. NanoDam enables profiling of chromatin-binding proteins with just one genetic cross. Combined with scRNA-seq of the INPs, two new candidate temporal factors were identified: Homeobrain and Scarecrow. These novel temporal factors, in addition, have roles in regulating NSC longevity. These NSCs divide in a similar manner to the outer radial glia of the mammalian brain and both factors have orthologues, namely ARX and NKX2.1. Remarkably, we showed that these factors have conserved roles in the NSCs of the visual system, which have a different developmental origin.





Brand, Andrea


stem cell, neural stem cell, tumourigenesis, scRNA-seq, transcriptomics, neural development


Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge