Axis Determination and Germ Plasm Assembly in Drosophila

描述（由申请人提供）：细胞-细胞相互作用对于在多细胞生物发育中建立细胞多样性非常重要。在果蝇中，很明显，种系来源的卵母细胞和覆盖的体细胞后卵泡细胞之间的诱导相互作用建立了卵室和胚胎的主轴。后卵泡细胞到卵母细胞信号传导还在卵母细胞后部建立了一个特殊的细胞质区域，这对于生殖细胞测定和胚胎腹部分割（种质）是必不可少的。尽管这些诱导性相互作用对于定义轴和种系的确定至关重要，但迄今为止，在卵母细胞内发挥作用以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产生重要影响。我们的长期目标是阐明定义果蝇主要身体轴和种质成分组装的分子机制。 mago nashi (mago) 基因已被证明编码一种位于卵母细胞后极内的蛋白质，以调节卵母细胞对后卵泡细胞间信号的反应。利用酵母双杂交筛选，Tsunagi 蛋白（一种 RNA 结合蛋白）被鉴定为 Mago 相互作用蛋白。 Mago:Tsunagi 复合体需要定义主要的身体轴，并且对于定位种质成分是必不可少的。本提案的目标如下：（1）将使用种系克隆分析、免疫学技术和细胞学方法来研究mago nashi和tsunagi是否相互作用以确定和/或分化卵母细胞。 (2) 为了确定与位于后极内的Mago蛋白相互作用的因素，将采用免疫沉淀和生化方法。 (3) 两种方法将能够识别和纯化体内与 Mago:Tsunagi 复合物结合的 RNA。在第一种方法中，从生殖细胞和/或卵巢 cDNA 迭代亲和选择文库转录的 RNA 将与来自卵巢提取物的 Mago:Tsunagi 复合物进行共免疫沉淀。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 形成复合物的内源 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.