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Identification of gene regulatory enhancers for midbrain dopaminergic progenitors

Project supervisor – Siew-Lan Ang

Division of Developmental Neurobiology

Identification of gene regulatory enhancers for midbrain dopaminergic progenitors.

How the identity of the multitude of cell types in the vertebrate central nervous system is specified remains a central question in Developmental Neurobiology. The mid-hindbrain region, which gives rise dorsally to neurons organized in laminar structures and ventrally to neurons arranged in distinct nuclei, is a good model system to investigate the specification of neuronal identity. Three major neuronal subtypes generated in the ventral midbrain are dopaminergic neurons (DA), neurons of the red nuclei, and oculomotor neurons. DA neurons have been implicated in Parkinson’s disease; hence understanding the intrinsic and extrinsic factors that direct neural stem cells/progenitors towards this neuronal fate will have applications in stem cell therapy of this neurodegenerative disorder.

The project seeks to determine the molecular mechanisms governed by Foxa2 that are involved in the specification and differentiation of midbrain dopaminergic progenitors. We have identified potential direct target genes of Foxa2 in midbrain dopaminergic progenitors at the genome level using chromatin immunoprecipitation experiments and large-scale sequencing (ChIP-Seq). Preliminary characterization of these Foxa2-bound regions shows that many of these regions act as enhancers controlling expression of genes in mDA progenitors. We would like to test more Foxa2-bound regions for enhancer activity by hooking up these regions to reporter constructs and then introducing these constructs into the chick ventral midbrain by electroporation of chick embryos. A few mouse mDA-progenitor enhancers have conserved activity in chick embryos, and this finding has therefore validated this cross-species approach to screen for mouse enhancers. We will use the chick electroporation experiments to identify up to twenty mDA progenitor enhancers. The student will also perform bioinformatics analyses of these enhancers to determine if common DNA binding motifs are enriched in their sequences. Enriched DNA binding motifs suggest that proteins binding to these regions are candidate transcriptional co-facotrs of Foxa2 in mDA progenitors.