Axis Determination and Germ Plasm Assembly in Drosophila

描述(由申请人提供)：在多细胞生物体的发展中，细胞-细胞相互作用在建立细胞多样性方面非常重要。在果蝇中，胚系来源的卵母细胞和覆盖其上的体细胞后毛囊细胞之间的诱导相互作用已经变得明显，建立了卵室和胚胎的主体轴。后卵泡细胞到卵母细胞的信号传递也在卵母细胞的后部建立了一个特殊的细胞质区域，这对于生殖细胞的确定和胚胎(生殖质)的腹部分割是必不可少的。尽管这些诱导相互作用对于确定胚系的轴和确定生殖系至关重要，但到目前为止，在卵母细胞内调节这一重要信号转导途径的成分还很少。研究这些过程将对理解人类发育缺陷和癌症具有重要意义。我们的长期目标是阐明决定果蝇主要体轴和生殖质成分组装的分子机制。Mago nashi(MAGO)基因编码一种位于卵母细胞后极的蛋白质，调节卵母细胞对后卵泡细胞-卵母细胞信号的反应。利用酵母双杂交筛选，Tsunagi蛋白，一种RNA结合蛋白，被鉴定为Mago相互作用因子。Mago：Tsunagi复合体是确定主要身体轴所必需的，对于定位种质的成分是不可或缺的。这项建议的目标如下：(1)将使用生殖系克隆分析、免疫学技术和细胞学方法来研究巨细胞和海啸是否相互作用来确定和/或分化卵母细胞。(2)通过免疫沉淀和生物化学方法确定与Mago蛋白相互作用的因素。(3)两种方法可以鉴定和纯化体内与Mago：Tsunagi复合体结合的RNA。在第一种方法中，从生殖器和/或卵巢cDNA迭代亲和选择文库转录的RNA将与卵巢提取物中的MAGO：Tsunagi复合体进行免疫共沉淀。在第二种方法中，卵巢提取物的免疫沉淀将用于纯化与Mago和Tsunagi形成复合体的内源RNA。

DESCRIPTION (provided by applicant): Cell-cell interactions are important in establishing cellular diversity in the development of multicellular organisms. In Drosophila melanogaster, it has become apparent that inductive interactions between the germline-derived oocyte and the overlying somatic posterior follicle cells establish the major body axes of the egg chamber and embryo. Posterior follicle cell-to-oocyte signaling also establishes a specialized region of cytoplasm within the posterior of the oocyte that is indispensable for germ cell determination and abdominal segmentation of embryos (the germ plasm). Although these inductive interactions are critical for defining the axes and determination of the germline, thus far, few components that function within the oocyte to regulate this important signal transduction pathway have been identified. Investigating these processes will haw important implications for understanding human developmental defects and cancers. Our long-tern objectives are to elucidate the molecular mechanisms that define the major body axes and assembly of germ plasm components in Drosophila. The mago nashi (mago) gene has been demonstrated to encode a protein that localizes within the posterior pole of the oocyte to regulate the response of the oocyte to the posterior follicle cell-to-oocyte signal. Utilizing a yeast two-hybrid screen the Tsunagi protein, an RNA-binding protein, was identified as a Mago interactor. The Mago:Tsunagi complex is required to define the major body axes and is indispensable for localizing components of the germ plasm. The objectives in this proposal are as follows: (1) Germline clonal analysis, immunological techniques and cytological methods will be used to investigate whether mago nashi and tsunagi interact for the determination and/or differentiation the oocyte. (2) To identify factors that interact with Mago protein localized within the posterior pole, immunoprecipitation and biochemical methods will be employed. (3) Two approaches will enable identification and purification of RNAs bound in vivo to Mago:Tsunagi complexes. In the first approach, RNAs transcribed from a germarial and/or ovarian cDNA iterative affinity selection library will be co-immunoprecipitated with Mago:Tsunagi complexes from ovarian extracts. In the second approach, immunoprecipitations of ovarian extracts will be used to purify endogenous RNAs that form a complex with Mago and Tsunagi.