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

在胚胎神经发生中，必须保持神经干细胞自我更新和神经元分化之间的平衡。早熟的神经元分化会导致干细胞池过早衰竭，并可能导致小头畸形等疾病。Notch信号可拮抗早熟神经元分化。Notch信号激活Hes/Hey转录抑制因子的表达，从而抵消前膜转录因子（如Neurogenin 2）的表达。尽管这一原理早已为人所知，但令人惊讶的是，在胚胎大脑中发现的he /Hey阻遏物靶点很少，因此Notch信号下游的分子机制尚不清楚。利用生物信息学方法，我们预测了he /Hey靶基因的全基因组。对这些推测的Hes/Hey靶点的基因本体论分析显示，在预测的靶点中，RNA加工因子富集。其中四个靶标Ptbp2、Rbfox2、hnRNP F和Srpk1都是可选的剪接因子。对已发表的表达数据的分析显示，胚胎大脑皮层中所有四种因子的共同表达表明在神经发生过程中具有功能。通过免疫染色对比分析Rbfox2和Hes5在胚胎大脑皮层的表达，揭示了一种互斥的表达模式。此外，荧光素酶报告基因检测证实了Rbfox2启动子中Hes/Hey结合位点的功能。为了阐明Rbfox2抑制在神经干细胞中的重要性，我们在体内正常Rbfox2阴性和Hes5阳性祖细胞中过表达Rbfox2。表达Pax阳性Rbfox2的神经干细胞过早迁移出心室区。此外，Rbfox2过表达增加了Tbr2阳性基底祖细胞的比例，并降低了Tbr1阳性神经元的比例，表明间接神经发生的转变。这些初步结果表明，Notch信号在原细胞选择性剪接的调控中起着迄今未知但重要的作用。基于这些结果，我们想要回答以下问题：在神经祖细胞中，哪些可选择的剪接决定是由Notch信号调节的（目的1）？这四种剪接因子是直接的Hes/Hey靶点吗（目标2）？Notch介导的四种剪接因子的抑制对神经发生至关重要吗（目的3）？

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)?