EMBRYONIC DORSOVENTRAL POLARITY IN DROSOPHILA

果蝇胚胎的背腹极是由一种 涉及母体分子的跨膜信号通路。二 关于这一途径的悬而未决的问题涉及到细胞外如何 诱导极性的信号是非对称产生的，以及它是如何产生的 传到胚胎内部。细胞外信号是 跨膜Toll蛋白的配体，与其相似的 哺乳动物白介素L受体的胞质结构域。激活 无论是蛋白质，还是通过一种不清楚的机制，都会导致核靶向 Rel原癌基因转录因子。早期的研究表明， 蛋白酶的局部激活限制了Toll‘s的生产 配基到胚胎的腹侧，这是定位的 激活由不对称铺设的部件决定 胚胎未来胞外室的卵子发生。我们 最近发现了一条可能的线索，说明托尔的配体是如何不对称地 制作。我们克隆了Nudel基因，并对其进行了测序，该基因在 在卵子发生过程中，需要激活产生 托尔的配基。Nudel基因产物被预测为一个大的(更大的 大于或等于300kD)胞外糖蛋白，模块化样蛋白 细胞外基质分子，首次发现富含半胱氨酸的基序 哺乳动物的低密度脂蛋白受体。努德尔最耐人寻味的结构主题是 丝氨酸蛋白酶结构域，因此它可能启动了蛋白酶的级联产生 托尔的配基。由于核糖核糖核酸似乎在腹侧富含 在卵子发生过程中，Nudel蛋白也可能起到 胚胎背腹方向的空间决定因素。 我们的长期目标是详细了解Nudel是如何 蛋白质激活并定位产生托尔氏配体的蛋白酶， 以及Toll如何激活细胞内的信号反应。具体目标 L)将Nudel蛋白免疫定位于卵室和胚胎中 确定它是否腹壁浓缩；并进行分馏 确定Nudel是否与胚胎血浆结合的实验 膜或周围的卵黄膜，因为它必须是非 可扩散的，以定位蛋白酶的激活。2)测试Nudel 丝氨酸蛋白酶和其他功能在胚胎中的应用 体外诱变、生殖系转化和RNA注射方法。3) 鉴定Nudel的蛋白水解酶底物和其他与Nudel结合的分子 使用体外酶分析、免疫沉淀法和亲和力 层析法。4)鉴定与Toll细胞质结合的蛋白质 通过免疫分离、亲和层析和酵母 交互陷阱屏幕。关于Nudel和Toll的拟议研究及其 相互作用的蛋白质，将增加我们对相似分子的理解 与癌症和心血管疾病有关。

Dorsoventral polarity of the Drosophila embryo is established by a transmembrane signaling pathway involving maternal molecules. Two outstanding questions about this pathway concern how the extracellular signal that induces polarity is asymmetrically generated and how it is transmitted to the interior of the embryo. The extracellular signal is the ligand for the transmembrane Toll protein, which is similar in its cytoplasmic domain to the mammalian interleukin l receptor. Activation of either protein causes, by an unclear mechanism, the nuclear targeting of a rel proto-oncogene transcription factor. Earlier studies suggested that localized activation of a protease cascade restricts production of Toll's ligand to the ventral side of the embryo, and that this localized activation is determined by a component asymmetrically laid down during oogenesis in the future extracellular compartment of the embryo. We recently uncovered a possible clue to how Toll's ligand is asymmetrically produced. We cloned and sequenced the nudel gene, which is expressed during oogenesis and is required to activate the proteases that produce Toll's ligand. The nudel gene product is predicted to be a large (greater than or equal to 300 kD) extracellular glycoprotein, modular like extracellular matrix molecules, with cysteine-rich motifs first seen in the mammalian LDL receptor. Nudel's most intriguing structural motif is a serine protease domain, so it may initiate the protease cascade producing Toll's ligand. Since the nuclei RNA appears to be ventrally enriched in the egg chamber during oogenesis, the nudel protein may also function as a spatial determinant of embryonic dorsoventral polarity. Our long-term goal is to understand in biochemical detail how the nudel protein activates and localizes the proteases that produce Toll's ligand, and how Toll activates intracellular signaling reactions. Specific aims are: l) Immunolocalize the nudel protein in egg chambers and embryos to determine if it is ventrally enriched; and perform fractionation experiments to determine if nudel is bound to the embryonic plasma membrane or the surrounding vitelline envelope, since it must be non- diffusible in order to localize protease activation. 2) Test nudel's serine protease and other functions in embryos using a combination of in vitro mutagenesis, germline transformation, and RNA injection methods. 3) Identify nudel's protease substrate and other molecules that bind to nudel using in vitro protease assays, immunoprecipitation methods, and affinity chromatography. 4) Identify proteins that bind to Toll's cytoplasmic domain through immunoisolation, affinity chromatography, and the yeast interaction trap screen. The proposed studies on nudel and Toll, and their interacting proteins, will increase our understanding of similar molecules involved in cancer and cardiovascular disorders.