Functional Roles of Long Noncoding RNAs During Neuronal Development

描述(申请人提供)：在这里，我们建议使用我们最近建立的20个小鼠lincRNA敲除模型的独特资源来阐明候选lincRNAs在哺乳动物大脑发育中的作用。我们特别关注了我们通过之前的研究假设的在神经元发育中发挥作用的候选人。事实上，20名候选人中有15人表现出细胞类型和大脑特有的表达模式。更重要的是，我们已经在选定的突变体中观察到了大脑发育过程中的致命性表型和大体解剖异常。我们的目标是探索观察到的表型以及我们可能在这项工作中遇到的其他表型的分子和生理基础。我们建议采取全面和系统的方法来解决以下问题：在神经元发育过程中，在体内lincRNA表达的时空动力学是什么？哪些基因途径受到调控？(目标1)。我们将通过利用敲打LacZ记者来监测lincRNA在整个大脑发育过程中的表达模式和动态来解决这些问题。我们将通过下一代野生型和敲除菌株之间的RNA测序研究来解决第二个问题，以确定体内由lincRNAs调控的特定基因和途径。在选定的lincRNA突变体中观察到的致命性和脑发育表型的生理和分子基础是什么？(目标2)。在这里，我们将通过一系列体内实验来解决这个问题，以确定在选定的突变体中对正常神经细胞命运的扰动。我们将首先关注三个已经显示了大脑特定表型的lincRNA，它们导致了神经发生的致命性和异常，其中一个在神经元前体细胞中表达。如果时间和资源允许，我们将把这些表型研究扩展到更多的菌株。LincRNAs如何在分子和机制水平上工作，以确保适当的神经细胞命运规范和活性？(目标3)。在这里，我们使用多方面的和新颖的实验方案来定义恢复表型所需的lincRNAs的特定区域。此外，我们将确定lincRNA蛋白质伙伴以及它们如何相互作用来调节细胞命运的决定。这项拟议的研究是由我们的愿景推动的，该愿景旨在统一实验基因组学和计算基因组学，以推动理解lincRNAs在神经元发育过程中的生理和分子调控作用的新前沿。总之，这些研究将首次提出lincRNA在神经元发育过程中的功能作用的表型，并将在体内探索其生化机制。

DESCRIPTION (provided by applicant): Here, we propose to elucidate the role of candidate lincRNAs during mammalian brain development using a unique resource of 20 mouse lincRNA knockout models that we have recently generated. We specifically focused on candidates we hypothesized, through previous studies, to play a role in neuronal development. Indeed 15 of the 20 candidates exhibit cell-type and brain specific expression patterns. More importantly, we have already observed lethality phenotypes and gross anatomical abnormalities during brain development in selected mutants. We aim to explore the molecular and physiological underpinnings of the observed phenotypes as well as of others that we may encounter through this work. We propose to take a comprehensive and systematic approach to address the following questions: What are the spatial temporal dynamics of lincRNA expression in vivo during neuronal development and what gene pathways are regulated? (Aim 1). We will address these questions by leveraging a knock-in lacZ reporter to monitor lincRNA expression patterns and dynamics throughout brain development. We will address the second question through next generation RNA-sequencing studies between wild-type and knockout strains to identify specific genes and pathways regulated by lincRNAs in vivo. What are the physiological and molecular underpinnings of lethality and brain development phenotypes observed in selected lincRNA mutants? (Aim 2). Here, we will address this question through a battery of in vivo experiments to define perturbations to normal neuronal cell fate specification in selected mutants. We will focus first on three lincRNAs that have already shown brain specific phenotypes resulting in lethality and abnormalities of neurogenesis and one expressed in neuronal precursor cells. If time and resources allow we will expand these phenotypic studies to additional strains. How are lincRNAs working on a molecular and mechanistic level to ensure proper neuronal cell fate specification and viability? (Aim 3). Here, we use multifaceted and novel experimental protocols to define the specific regions of lincRNAs required to recover phenotypes. Moreover, we will identify lincRNA protein partners and how they interact to modulate cell fate decisions. The proposed research is driven by our vision, which seeks to unify experimental and computational genomics to push new frontiers in understanding the physiological and molecular regulatory roles of lincRNAs during neuronal development. Collectively, these studies will present the first phenotyping of lincRNA functional roles during neuronal development and will explore their biochemical mechanisms in vivo.