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

Notch信号传导拮抗胚胎大脑皮层中的原神经选择性剪接程序

• 批准号: 369055984
• 负责人: Privatdozentin Dr. Jennifer Winter, Ph.D.
• 金额: --
• 依托单位: Institut für Humangenetik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/369055984?language=en
• 关键词: Notch; signaling; antagonizes; proneural; alternative

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)?

在胚胎神经发生过程中，必须在神经干细胞自我更新和神经元分化之间保持平衡。神经细胞的早熟分化会导致干细胞库的过早耗尽，并可能导致小头畸形等疾病。Notch信号拮抗性早熟神经元分化。Notch信号可激活Hes/Hey转录抑制因子的表达，从而抑制神经转录因子，如神经原蛋白2的表达。虽然这一原理早已为人所知，但令人惊讶的是，在胚胎脑中发现的Hes/Hey抑制因子的靶标很少，因此Notch信号下游的分子机制还不清楚。对这些假定的HES/HY靶标进行的基因本体论分析显示，在预测的靶标中，RNA加工因子丰富。其中四个靶点Ptbp2、RBFox2、hnRNP F和SRPK1都是替代剪接因子。对已发表的表达数据的分析显示，所有四种因子在胚胎大脑皮层中共同表达，表明在神经发生过程中发挥了作用。通过免疫染色对比分析RBFox2和Hes5在胚胎大脑皮层中的表达，揭示了一种相互排斥的表达模式。此外，荧光素酶报告分析证实了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，我们在体内正常的RBFox2阴性和Hes5阳性的祖细胞中过表达了RBFox2。表达Pax阳性神经干细胞的RBFox2过早地迁移出脑室带。此外，RBFox2的过表达增加了Tbr2阳性基础祖细胞的比例，降低了Tbr1阳性神经元的比例，提示向间接神经发生转变。这些初步结果表明，Notch信号在神经选择性剪接的调控中发挥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：神经前体细胞中的Notch信号调节哪些选择性剪接决定(目标1)？这四个剪接因子是否直接针对HES/嘿(目标2)？Notch介导的对四种剪接因子的抑制对神经发生是必要的吗(目标3)？