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体发育中建立细胞多样性非常重要。在黑腹果蝇中，很明显，生殖系来源的卵母细胞和覆盖的体细胞后囊细胞之间的诱导相互作用建立了卵室和胚胎的主要体轴。后卵泡细胞到卵母细胞的信号传导也在卵母细胞的后部内建立了一个专门的细胞质区域，这对于生殖细胞决定和胚胎的腹部分割（生殖质）是不可或缺的。虽然这些诱导的相互作用是至关重要的定义轴和决定的种系，到目前为止，很少有组件，在卵母细胞内的功能，以调节这一重要的信号转导途径已被确定。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果蝇主要体轴和种质组分组装的分子机制。mago nashi（mago）基因已被证明编码一种定位于卵母细胞后极的蛋白质，以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的Tsunagi蛋白，RNA结合蛋白，被确定为Mago相互作用。Mago：Tsunagi复合体是确定主要体轴所必需的，并且是定位种质成分所不可或缺的。本研究的主要目的如下：（1）利用生殖系克隆分析、免疫学技术和细胞学方法，研究卵母细胞的决定和/或分化过程中是否存在相互作用。(2)为了鉴定与位于后极内的Mago蛋白相互作用的因子，将采用免疫沉淀和生物化学方法。(3)两种方法将能够识别和纯化RNA结合在体内的Mago：Tsunagi复合物。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形成复合物的内源性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.