Notch signaling antagonizes a proneural alternative splicing program in the embryonic cerebral cortex

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In embryonic neurogenesis the balance has to be kept between neural stem cell self renewal and neuronal differentiation. Precocious neuronal differentiation causes premature exhaustion of the stem cell pool and can lead to disorders like microcephaly. Notch signaling antagonizes precocious neuronal differentiation. Notch signaling activates the expression of the Hes/Hey transcriptional repressors thereby counteracting the expression of proneural transcription factors, e.g. Neurogenin 2. Although this principle is known already for a long time surprisingly few targets of Hes/Hey repressors have been identified in the embryonic brain and thus the molecular mechanisms downstream of Notch signaling are only poorly understood.With a bioinformatics approach we have predicted Hes/Hey target genes genomwide. A gene ontology analysis of these putative Hes/Hey targets revealed an enrichment of RNA processing factors among the predicted targets. Four of the targets, Ptbp2, Rbfox2, hnRNP F and Srpk1, are all alternative splicing factors. An analysis of published expression data revealed co-expression of all four factors in the embryonic cerebral cortex suggesting a function during neurogenesis. A comparative analysis of the expression of Rbfox2 and Hes5 in the embryonic cerebral cortex via immunostaining revealed a mutually exclusive expression pattern. In addition, luciferase reporter assays confirmed the functionality of the Hes/Hey binding sites in the Rbfox2 promoter. To elucidate the importance of Rbfox2 repression in neural stem cells we overexpressed Rbfox2 in normally Rbfox2 negative, Hes5 positive progenitors in vivo. Rbfox2 expressing Pax positive neural stem cells prematurely migrated out of the ventricular zone. Furthermore Rbfox2 overexpression increased the proportion of Tbr2 positive basal progenitors and decreased the proportion of Tbr1 positive neurons suggesting a shift towards indirect neurogenesis. These preliminary results suggest that Notch signaling plays a so far unknown but important role during the regulation of proneural alternative splicing. Based on these results we want to answer the following questions: Which alternative splicing decisions are regulated by Notch signaling in neural progenitor cells (aim 1)? Are the four splicing factors direct Hes/Hey targets (aim 2)? Is the Notch mediated repression of the four splicing factors essential for neurogenesis (aim 3)?