Asymmetric cell division of CNS progenitor cells

描述（由申请人提供）：不对称细胞分裂是中枢神经系统（CNS）发育期间神经干细胞和祖细胞（NSC和NPC）行为的基本特征，调控不当可导致脑异常或肿瘤形成。在不对称的细胞分裂过程中，分子决定簇被优先分离到一个子细胞中以指定其命运。一个重要的目标是确定在神经干细胞和NPC的不对称决定因素，这可能是肿瘤抑制剂或特定的神经命运的诱导剂。在果蝇中，Staufen是一种双链RNA结合蛋白，携带Prospero等决定簇的mRNA，并从干细胞样神经母细胞不对称分离到神经节母细胞中以诱导分化。在这里，我们建议调查密切相关的小鼠Stau2蛋白，测试的假设，它是不对称分离的哺乳动物中枢神经系统祖细胞分裂，它携带货物RNA是重要的决定因素的中枢神经系统谱系。我们的初步证据表明，Stau2在大脑皮质祖细胞的有丝分裂过程中是不对称分离的，我们从以前的研究中知道，这些祖细胞经历反复的不对称细胞分裂，产生各种神经元，然后产生神经胶质后代。此外，接受Stau2的细胞通常是Tbr 2+中间祖细胞（IPC），相当于神经节母细胞。我们通过免疫沉淀来自E13.5皮质的Stau2发现，其相关mRNA之一是Trim32，一种已知的不对称决定簇，另一种是Insm1，已显示其促进IPC命运。在这里，我们建议更充分地描述Stau2在皮质祖细胞分裂中的作用，并全面识别其货物RNA。这些实验将与Stau2专家Michael Kiebler和RNA结合蛋白及其结合基序的生物信息学分析专家Scott Tenenbaum合作进行。我们将使用经验证的抗体和细胞类型特异性标记物来检查皮质祖细胞在不同发育阶段是否分离Stau2。shRNA慢病毒构建体和过表达构建体将用于在体内和体外操纵Stau 2水平，确定对祖细胞行为的影响。此外，我们将研究皮质发育的一种新的Stau2基因陷阱突变小鼠。Stau2功能的详细分析将通过在长期延时显微镜下观察皮质祖细胞行为来进行。最后，我们将进行Stau2免疫沉淀和测序，以确定相关的RNA，信使和非编码，并寻求结合基序。候选的新的不对称决定簇将使用单分子FISH和Stau2免疫定位进行验证，然后功能测试对皮质发育的影响。了解Stau2在大脑皮层中的功能将增加我们对NSC和NPC中RNA结合蛋白和不对称细胞分裂机制的理解，并提供可翻译到其他干细胞系统的有价值的信息。

DESCRIPTION (provided by applicant): Asymmetric cell divisions are a fundamental feature of neural stem and progenitor cell (NSC and NPC) behavior during central nervous system (CNS) development, and misregulation can lead to brain abnormalities or tumor formation. During an asymmetric cell division, molecular determinants are segregated preferentially into one daughter cell to specify its fate. An important goal is to identify the asymmetric determinants in NSCs and NPCs, which could be tumor suppressors or inducers of specific neural fates. In Drosophila, Staufen is a double-stranded RNA binding protein that carries the mRNA of determinants such as Prospero, and is asymmetrically segregated from the stem-like neuroblast into the ganglion mother daughter cell to induce differentiation. Here we propose to investigate the closely-related mouse Stau2 protein, testing the hypothesis that it is asymmetrically segregated in mammalian CNS progenitor divisions, and that it carries cargo RNAs that are important determinants of CNS lineages. Our preliminary evidence shows that Stau2 is asymmetrically segregated during mitosis in cerebral cortical progenitor cells, which we know from prior studies undergo repeated asymmetric cell divisions to generate a variety of neurons then later glial progeny. Moreover, the cell that receives Stau2 is typically a Tbr2+ intermediate progenitor (IPC) cell, the equivalent to the ganglion mother cell. We found by immunoprecipitating Stau2 from E13.5 cortex, that one of its associated mRNAs is Trim32, a known asymmetric determinant, and another is Insm1, which has been shown to promote the IPC fate. Here we propose to more fully characterize the role of Stau2 in cortical progenitor cell divisions, and to comprehensively identify its cargo RNAs. The experiments will be carried out in collaboration with Michael Kiebler, a Stau2 expert and Scott Tenenbaum an expert in RNA-binding proteins and bioinformatic analysis of their binding motifs. We will examine whether cortical progenitor cells at different stages of development segregate Stau2, using verified antibodies and cell-type specific markers. shRNA lentiviral constructs and overexpression constructs will be used to manipulate Stau2 levels in vivo and in vitro, determining the effect on progenitor cell behavior. In addition, we will examine cortical development in a novel Stau2 genetrap mutant mouse. Detailed analysis of Stau2 function will be carried out by following cortical progenitor cell behavior under long-term time-lapse microscopy. Finally, we will perform Stau2 immunoprecipitation and sequencing to identify the associated RNAs, both messenger and non-coding, and seek binding motifs. Candidate novel asymmetric determinants will be validated using single molecule FISH and Stau2 immunolocalization, then functionally tested for impact on cortical development. Knowledge of Stau2 function in the cerebral cortex will increase our understanding of RNA binding proteins and asymmetric cell division mechanisms in NSCs and NPCs, and provide valuable information translatable to other stem cell systems.