MOLECULAR GENETIC ANALYSIS OF ASYMMETRIC CELL DIVISIONS

不对称的细胞分裂，产生不同的子代细胞 命运对于胚胎期间细胞多样性的产生是重要的 发育，并可能调节干细胞在许多组织中的功能(如 皮肤、肠道、血液、肝脏、生殖细胞和神经系统)。 尽管临床上很重要的一点是要了解 不对称的细胞分裂，令人惊讶的是，人们对细胞分裂的方式和方式知之甚少 在哺乳动物中发生不对称分裂的地方。我们的长期目标是 识别控制果蝇细胞不对称分裂的基因，以及 确定同源小鼠基因是否控制不对称细胞 胚胎发育期间或成体干细胞群体中的分裂。这个 拟议中的研究可能在哺乳动物干细胞方面有临床应用 干细胞肿瘤的永生化、基因治疗和治疗。 我们将重点研究果蝇中枢神经系统干细胞(神经母细胞)，它可以分裂 不对称地产生新的神经母细胞和更分化的 子细胞(GMC)。至少有4种蛋白质被分割成 女儿GMC，包括Propero转录因子，它是 从神经母细胞特异性向GMC特异性转变所必需的 基因表达。这项建议的具体目的是：(1) 识别调控神经母细胞不对称细胞分裂的新基因。我们 将筛查Propero蛋白的错误定位，并进行特征分析 用标准的分子遗传学方法进行突变。(2)刻画 Miranda，一种编码一种新的螺旋卷曲蛋白的基因， 普洛斯彼罗在神经母细胞中的不对称定位。我们将按顺序排列 影响米兰达定位的蛋白阳性米兰达EMS等位基因 (3个等位基因)或Propero结合(2个等位基因)，以映射每个功能 结构域；使用酵母双杂交筛选来特异性地鉴定蛋白质 结合米兰达本地化结构域；筛选新的米兰达- 结合“货物”蛋白质；并确定米兰达的结构 功能领域(与M.丘吉尔博士合著)。(3)进一步刻画 普洛斯彼罗功能。我们将确定Propero RNA是否具有翻译能力 神经母细胞(但不是转基因细胞)中的抑制是建立 不同的兄弟姐妹的命运；分析磷酸化在繁荣中的作用 在成人中的定位；并检测Propero的表达和功能 感觉神经系统。(4)分离米兰达的小鼠同源基因(和 在特定目的1)中识别的基因，并表征它们在 调控胚胎发育和成体茎的不对称分裂 细胞。我们将进行RNA和蛋白质本地化研究；我们的 合作者G.奥利弗博士将产生并分析基因敲除 老鼠。

Asymmetric cell divisions, which produce daughter cells with different fates, are important for generating cell diversity during embryonic development, and may regulate stem cell function in many tissues (e.g. epidermis, intestine, blood, liver, germ cells, and the nervous system). Despite the clinical importance of understanding the regulation of asymmetric cell divisions, remarkably little is known about how and where asymmetric divisions occur in mammals. Our long-term goal is to identify genes controlling asymmetric cell divisions in Drosophila, and to determine if homologous murine genes control asymmetric cell divisions during embryogenesis or in adult stem cell populations. The proposed research may have clinical applications in mammalian stem cell immortalization, gene therapy, and treatment of stem cell neoplasms. We will focus on Drosophila CNS stem cells (neuroblasts), which divide asymmetrically to produce a new neuroblast and a more differentiated daughter cell (GMC). At least 4 proteins are partitioned into the daughter GMC, including the Prospero transcription factor, which is necessary for the transition from neuroblast-specific to GMC-specific gene expression. The specific aims of this proposal are: (1) To identify new genes regulating neuroblast asymmetric cell divisions. We will screen for mis-localization of Prospero protein, and characterize mutants by standard molecular genetic methods. (2) To characterize miranda, a gene encoding a novel coiled-coil protein required for the asymmetric localization of Prospero in neuroblasts. We will sequence protein-positive miranda EMS alleles that affect Miranda localization (3 alleles) or Prospero binding (2 alleles) to map each functional domain; use a yeast two hybrid screen to identify proteins specifically binding the Miranda localization domain; screen for novel Miranda- binding "cargo" proteins; and determine the structure of Miranda functional domains (with Dr. M. Churchill). (3) To further characterize prospero function. We will determine if prospero RNA translational repression in neuroblasts (but not GMCs) is necessary to establish distinct sibling fates; assay the role of phosphorylation in Prospero localization; and assay prospero expression and function in the adult sensory nervous system. (4) To isolate murine homologues of miranda (and genes identified in Specific Aim 1), and to characterize their role in regulating asymmetric divisions during embryogenesis and in adult stem cells. We will do RNA and protein localization studies; our collaborator, Dr. G. Oliver will generate and assay the gene knock-out mice.