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）发育过程中神经干细胞和祖细胞（NSC和NPC）行为的基本特征，调节不当可导致脑异常或肿瘤形成。在不对称细胞分裂过程中，分子决定因子被优先分离到一个子细胞中，以确定其命运。一个重要的目标是确定NSCs和npc中的不对称决定因素，这些决定因素可能是肿瘤抑制因子或特定神经命运的诱导剂。在果蝇中，Staufen是一种携带Prospero等决定因子mRNA的双链RNA结合蛋白，从茎样神经母细胞中不对称分离到神经节母细胞中诱导分化。在这里，我们建议研究密切相关的小鼠Stau2蛋白，验证它在哺乳动物中枢神经系统祖细胞分裂中不对称分离的假设，并且它携带的货物rna是中枢神经系统谱系的重要决定因素。我们的初步证据表明，在大脑皮质祖细胞有丝分裂过程中，Stau2是不对称分离的，我们从之前的研究中知道，大脑皮质祖细胞经过反复的不对称细胞分裂来产生各种神经元，然后产生神经胶质后代。此外，接受Stau2的细胞通常是Tbr2+中间祖细胞（IPC），相当于神经节母细胞。我们通过免疫沉淀E13.5皮层的Stau2发现，其中一个相关mrna是Trim32，这是一种已知的不对称决定因素，另一个是Insm1，它已被证明可以促进IPC的命运。在这里，我们建议更充分地表征Stau2在皮质祖细胞分裂中的作用，并全面鉴定其载货rna。该实验将与Stau2专家Michael Kiebler和rna结合蛋白及其结合基元的生物信息学分析专家Scott Tenenbaum合作进行。我们将使用经过验证的抗体和细胞类型特异性标记物来检测不同发育阶段的皮质祖细胞是否分离Stau2。shRNA慢病毒构建体和过表达构建体将用于操纵体内和体外的Stau2水平，确定对祖细胞行为的影响。此外，我们将研究一种新的Stau2基因诱捕器突变小鼠的皮质发育。通过长期延时显微镜观察皮层祖细胞的行为，将对Stau2的功能进行详细分析。最后，我们将进行Stau2免疫沉淀和测序，以鉴定相关rna，包括信使rna和非编码rna，并寻找结合基序。候选的新型不对称决定因素将通过单分子FISH和Stau2免疫定位进行验证，然后对其对皮质发育的影响进行功能测试。了解大脑皮层中Stau2的功能将增加我们对NSCs和npc中RNA结合蛋白和不对称细胞分裂机制的理解，并为其他干细胞系统提供有价值的信息。

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.