Distal mRNA localization and translation in neural stem cells of the developing brain

摘要 信使核糖核酸的本地化和局部翻译是一种保守的机制，能够在时间和空间上控制 基因表达。这些过程在神经系统中尤为重要，在神经系统中，局部调节 基因表达影响轴突引导和突触活动。我们对信使核糖核酸的大部分理解 本地化和本地化翻译一直依赖于体外研究。我们最近建立了一种新的可驯服的 研究神经系统发育过程中体内局部基因调控的活体范例。具体来说，我们 发现mRNA亚细胞定位和局部翻译存在于胚胎放射状胶质细胞的基底结构中 细胞(RGC)，称为末端足。这些基础结构对于组织完整性和神经元迁移是必不可少的， 从而影响视网膜节细胞在神经元生成中的中枢功能。我们开发了实时成像方法来 发现信使核糖核酸可以在视网膜节细胞中主动转运，并在基底端进行局部翻译。我们也 开发了分离RGC末端脚的新方法，并用这些方法发现了末端脚的局部转录组， 由RNA结合蛋白FMRP结合。我们的研究使我们假设RNA的局部化和局部化 翻译在RGC末端受到内在和外在的调节，以影响信号传递和 细胞骨架。在这项提议中，我们的目标是实施这些新的分子成像和基因组学工具，以实现以下目标： (1)发现RGC末端在发育过程中的局部转录，(2)确定内在和外在 RGC末端翻译的调节，以及(3)RGC末端RNA定位的定义功能。 成功完成，我们将大大推进我们对信使核糖核酸本地化和 通过在活体模型中研究这些过程，在发育中的神经系统进行翻译。

Abstract mRNA localization and local translation are conserved mechanisms enabling spatial and temporal control of gene expression. These processes are particularly important in the nervous system, where local regulation of gene expression influences axon guidance and synaptic activity. Much of our understanding of mRNA localization and local translation has relied upon in vitro studies. We recently established a novel tractable in vivo paradigm for investigating local gene regulation in vivo in the developing nervous system. Specifically we found that mRNA is sub-cellularly localized and locally translated in basal structures of embryonic radial glial cells (RGCs), called endfeet. These basal structures are essential for tissue integrity and neuronal migration, thus impacting the central function of RGCs in neuron generation. We developed live imaging approaches to discover that mRNA can be actively transported in RGCs and locally translated in basal endfeet. We also developed novel methods to isolate RGC endfeet and used these to discover a local transcriptome in endfeet, bound by the RNA binding protein, FMRP. Our studies lead us to hypothesize that RNA localization and local translation are intrinsically and extrinsically regulated in RGC endfeet to influence signaling and the cytoskeleton. In this proposal we aim to implement these new molecular imaging and genomic tools towards: (1) discovering local transcriptomes of RGC endfeet across development, (2) determining intrinsic and extrinsic regulation of translation in RGC endfeet, and (3) defining functions for RNA localization in RGC endfeet. Successfully completed, we will have significantly advanced our understanding of mRNA localization and translation in the developing nervous system, by studying these processes in an in vivo model.