GERM CELL DIFFERENTIATION IN DROSOPHILA

描述（由申请人提供）： 干细胞补充由于周转或创伤而在组织中丢失的细胞。此外，越来越多的证据表明，几种疾病，包括一些与衰老有关的疾病和许多形式的癌症，都是由干细胞的不当调节引起的。因此，一方面，理解干细胞生物学对于操纵干细胞用于治疗目的具有非常广泛的意义，另一方面，为现代医学提供新的靶点来对抗许多非常高影响的疾病。该奖项支持的研究为果蝇卵子发生过程中生殖干细胞的维持和分化提供了详细的模型。生殖系干细胞生态位已被证明是一个有价值的模型，用于理解选择干细胞并指导其分化为功能配子的信号传导和基因调控。大理石袋（bag-of-marbles，bam）基因是这些研究的主要焦点，因为它在控制干细胞向成囊细胞转化中起主要作用。小生境信号特异性沉默bam转录，成囊细胞表达程序被阻断。当干细胞产生一个子细胞移出信号龛时，bam表达被解除抑制，成囊细胞分化被激活。最近的研究确定了控制bam沉默的其他因素，但更重要的是，已经揭示了生殖系干细胞维持所需的几个基因，而不直接作用于bam转录。因此，这些研究揭示了调节干细胞命运的调节途径的新方面。Pumilio-Nanos翻译抑制复合物在这方面尤其重要，最近的研究结果表明，成囊细胞分化开始，因为Bam通过阻断Nanos的翻译拮抗Pumilio-Nanos复合物的作用。在本申请中提出的研究中，我们试图将这些发现扩展到解释Bam如何抑制Nanos翻译的精细分子机制。拟议的研究将：（1）定义存在于Nanos 3 '-UTR中的Bam响应元件。这些实验将使用经典的缺失和转基因构建体策略来测试实验转基因的生物活性（2）进行遗传修饰剂筛选以鉴定与BAM起作用和/或调节BAM下游成囊细胞转变的因子（3）采用综合策略来鉴定与BAM相互作用以形成拮抗Pumilio-Nanos作用的复合物的多肽。本目标的目的是使用酵母双杂交和免疫共沉淀方法来鉴定BAM多肽复合物的组分。公共卫生相关性：干细胞提供了巨大的治疗和研究机会，但其生物学知之甚少。对果蝇生殖系干细胞和支持它们的基质细胞的研究揭示了调节干细胞的信号传导和基因表达程序的重要特征。本申请中提出的研究将探索有关控制干细胞及其后代分化的因素和过程的新想法。

DESCRIPTION (provided by applicant): Stem cells replenish the cells lost in tissues due to turnover or trauma. In addition there is increasing evidence that several diseases, including some associated with aging and also many forms of cancer, are caused by inappropriate regulation of stem cells. Thus on one hand, understanding stem cell biology has very broad implications for manipulating stem cells for therapeutic purposes and, on the other hand, giving modern medicine new targets to combat a host of very high impact diseases. The studies supported by this award have produced a detailed model for the maintenance and differentiation of the germline stem cell during oogenesis in Drosophila. The germline stem cell niche has proven itself to be a valuable model for understanding the signaling and gene regulation that selects stem cells and guides their differentiation into functional gametes. The bag-of-marbles (bam) gene has been a primary focus of these studies because of the primary role it plays in controlling the stem cell-to-cystoblast transition. Niche signaling specifically silences bam transcription and the cystoblast expression program is blocked. When the stem cell produces a daughter cell that moves out of the signaling niche, bam expression is derepressed and cystoblast differentiation becomes activated. The most recent studies defined additional factors that control bam silencing but, even more importantly, have revealed several genes that are required for germ line stem cell maintenance without acting directly on bam transcription. These have therefore revealed novel aspects of the regulatory pathways regulating stem cell fate. The Pumilio-Nanos translational inhibitory complex is especially important in this regard and the most recent findings show that cystoblast differentiation begins because Bam antagonizes the action of the Pumilio- Nanos complex by blocking translation of Nanos. In the studies proposed in this application we seek to extend these discoveries to a refined molecular mechanism explaining how Bam inhibits Nanos translation. The proposed studies will: (1) define the Bam Response Elements that reside in the Nanos 3'-UTR. These experiments will use classical deletion and transgenic constructs strategies to test the biological activity of experimental transgenes (2) carry out genetic modifier screens to identify the factors that work with Bam and/or that regulate cystoblast transition downstream of bam (3) mount a comprehensive strategy to identify the polypeptides that interact with Bam to form the complex that antagonizes the action of Pumilio-Nanos. The goals of this Aim will use yeast two-hybrid and co- immunoprecipitation methods to identify components of the Bam polypeptide complex. Public Health Relevance: Stem cells offer great therapeutic and research opportunities but their biology is poorly understood. Studies of the Drosophila germ line stem cells and the stromal cells that support them have revealed important features of the signaling and gene expression programs that regulate stem cells. The studies proposed in this application will probe new ideas about the factors and processes that control stem cells and the differentiation of their progeny.