The role of post-transcriptional regulation in neural development of Drosophila melanogaster

Brain development is a complex process, during which neural stem cells undergo a highly orchestrated series of divisions to produce a large number and diversity of neurons. It is well established that neural stem cell proliferation and differentiation relies on the precisely regulated expression of transcription factors in time and space. However, how the specific temporal and spatial expression profile of the transcription factors is achieved is currently unclear. Here, I investigate the upstream regulation of a highly conserved transcription factor, Prospero (Pros), in Drosophila melanogaster's neural stem cell (or neuroblast) lineage. In particular, I study how the cell-specific differential expression between neuroblast, where Pros is kept low to permit proliferation, and neuroblast progeny, where Pros is up-regulated to initiate terminal division and differentiation is achieved.
Using single molecule RNA fluorescent in situ hybridisation (smFISH), I show that pros is transcribed at similar levels in all cell types but the pros mRNA level is selectively up- regulated in neurons. I found that pros RNA is intrinsically unstable and has multiple isoforms with near identical exon but significantly different length 3'UTR of approximately 200 nt, 3 kb and 15 kb, respectively. I further show that the 15 kb 3'UTR pros isoform is the most stable isoform and its transcription is specific to neurons. This is suggests that the neuron-specific pros expression up-regulation is achieved through the cell type-specific transcription of the more stable pros isoform.
I identified Syncrip (Syp), a highly conserved RNA binding protein, to be an upstream regulator of pros. I show that in syp mutants, the levels of Pros protein and pros RNA fail to increase in neurons. However the level of pros transcription and the neuron-specific transcription of the 15 kb 3'UTR pros isoform is not affected. This suggests that transcription alone is not sufficient for achieving the cell-specific differential pros expression level. I demonstrate that Syp selectively stabilises the 15 kb 3'UTR pros isoform and that this stabilisation is necessary for the neuron-specific pros expression up-regulation. Collectively, these results show that the differential pros expression level is dependent on both transcriptional and post-transcriptional regulation.
Genome-wide experiments performed in collaboration with Tamsin Samuels and Aino Jarvelin showed that Syp is a potential regulator of transcripts in other key neural developmental pathways. This suggests that an RNA binding protein driven cross- regulatory network exists to co-ordinate gene expression between the different aspects of neural development. To disentangle this cross-regulation, future large-scale RNAi screens examining multiple aspects of neural development are required. However, relying on the current fixed imaging techniques and manual mutant phenotype analysis, a screen of such magnitude and complexity cannot be easily achieved. To address this, I developed a long term ex vivo live imaging protocol of intact developing larval brains and the live imaging data were automatically analysed using a new software tool (Q-Brain) developed in collaboration with Dr. Dominic Waithe, Dr. Richard Parton and Martin Hailstone.
This thesis reveals the importance of the co-ordination between transcriptional and post- transcriptional regulation in determining the precise expression level of key regulatory genes of neural development in time and space. With the development of the novel imaging and automated analysis techniques, the investigation of the cross-regulatory network of gene expression in the developing nervous system can now be expanded to a genome-wide scale.

Advisor: Davis, I