MOLECULAR GENETIC ANALYSIS OF ASYMMETRIC CELL DIVISIONS

不对称的细胞分裂，产生不同的子细胞 在胚胎发育过程中， 干细胞在胚胎发育过程中起重要作用，并且可以调节许多组织中的干细胞功能（例如， 表皮、肠、血液、肝脏、生殖细胞和神经系统）。 尽管了解调节的临床重要性 不对称的细胞分裂，非常少的是知道如何和 在哺乳动物中发生的不对称分裂。 我们的长期目标是 鉴定果蝇中控制不对称细胞分裂的基因， 为了确定同源鼠基因是否控制不对称细胞， 在胚胎发生期间或在成体干细胞群体中分裂。的 拟议的研究可能在哺乳动物干细胞方面具有临床应用价值 永生化、基因治疗和干细胞肿瘤的治疗。 我们将重点研究果蝇中枢神经系统干细胞（神经母细胞）， 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种蛋白质被分配到 子GMC，包括Prospero转录因子， 从成神经细胞特异性转化为GMC特异性所必需的 基因表达。 这项建议的具体目标是：（1） 鉴定调节神经母细胞不对称细胞分裂的新基因。 我们 将筛选Prospero蛋白的错误定位，并表征 通过标准的分子遗传学方法。(2)表征 米兰达，一个基因编码一种新的卷曲螺旋蛋白所需的 Prospero在成神经细胞中的不对称定位。 我们将测序 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Prospero结合（2个等位基因），以绘制每个功能 结构域;使用酵母双杂交筛选， 结合米兰达定位结构域;筛选新的米兰达- 结合“货物”蛋白;并确定米兰达的结构 功能域（与M.丘吉尔）。(3)为了进一步表征 函数。 我们将确定， 神经母细胞（但不是GMC）的抑制是建立 不同的同胞命运;分析磷酸化在Prospero中的作用 定位;并测定在成人中的表达和功能 感觉神经系统(4)分离米兰达的小鼠同源物（和 在特定目标1）中鉴定的基因，并表征它们在 在胚胎发生和成体茎中调节不对称分裂 细胞 我们将进行RNA和蛋白质定位研究;我们的 合作者G博士奥利弗将产生并检测基因敲除 小鼠

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.