Phosphotyrosine signaling pathways controlling tracheal tube geometry

描述（由申请人提供）：哺乳动物血管系统、肺和肾是运输气体或液体的分支管状上皮器官。虽然已经确定了组装这些器官所需的基因，但决定其管的形状和大小的发育机制还不清楚。果蝇幼虫的呼吸（气管）系统为研究复杂的分支管状网络的发育提供了一个有用的遗传模型。胚胎气管系统中的分支形态发生由成纤维细胞生长因子（FGF）受体酪氨酸激酶（TK）直系同源物Breathless（Btl）及其FGF配体Branchless（Bnl）之间的模式化相互作用控制。气管系统的发育逻辑与哺乳动物血管系统的发育逻辑相似，其中表达血管内皮生长因子（VEGF）受体TK的血管芽朝向VEGF来源生长。气管分支中的气道是如何塑造成适当的管状形状的？当我们发现一种独特的气管表型时，我们获得了这个问题的切入点，该表型是由消除III型受体酪氨酸磷酸酶（RPTPs）Ptp4E和Ptp10D的双重突变引起的。Ptp4E Ptp10D双突变将线性单细胞管转化为球形囊肿。III型RPTP是受体TK信号传导的高度保守的调节剂，并且我们发现该表型涉及三种生长因子受体TK直系同源物的RPTP的负调节的丧失：表皮生长因子受体（Egfr）、Btl和Pvr（VEGFR直系同源物）。这种表型可能从未在早期的遗传筛选中发现过，因为只有当Ptp4E和Ptp10D都突变时才能观察到。RPTP下游也可能没有单一组分可以突变以产生这种表型，因为RTK通过许多途径进行信号传导。因此，鉴定调控管几何形状的基因可能需要基于Ptp4E Ptp10D表型的致敏遗传筛选。这是第一个具体目标的基础，该目标描述了对赋予表型增强或抑制的隐性突变的系统筛选。由于这是一项耗时的筛选，需要使用共聚焦显微镜对单个胚胎进行定量分析，因此我们将通过使用我们定义的缺失（Df）突变的“表型筛选试剂盒”来减少需要筛选的细胞系数量。对于每一个增强或抑制表型的缺失，我们将使用插入突变和RNAi线来识别负责的基因，这些基因存在于大多数果蝇基因中。当我们手头有单个基因的突变时，我们将详细检查它们的表型，并分析它们之间的上位关系，以及与RTK和RPTP的上位关系，以确定遗传途径。第二个具体目标描述了一种系统的方法，通过这种方法，我们可以定位和标记在屏幕上识别的基因的蛋白质产物。我们可以将蛋白质连接到各种颜色的荧光标记物（用于活的和抗体染色的制备物中的定位）和表位标签或酶（用于生物化学表征）。这个系统将使我们能够找到蛋白质，这些蛋白质定位于细胞中控制管形状的区域。我们还可以分析蛋白质的酪氨酸磷酸化，并确定它们在胚胎中是否相互作用。

DESCRIPTION (provided by applicant): The mammalian vascular system, lung, and kidney are branched tubular epithelial organs that transport gases or fluids. Although genes required for assembly of these organs have been identified, the developmental mechanisms that determine the shapes and sizes of their tubes are not well understood. The respiratory (tracheal) system of the Drosophila larva has provided a useful genetic model for the study of the development of complex branched tubular networks. Branching morphogenesis in the embryonic tracheal system is controlled by patterned interactions between a fibroblast growth factor (FGF) receptor tyrosine kinase (TK) ortholog, Breathless (Btl), and its FGF ligand, Branchless (Bnl). The developmental logic of the tracheal system is similar to that of the mammalian vascular system, where vascular sprouts expressing the vascular-endothelial growth factor (VEGF) receptor TK grow toward sources of VEGF. How are the airways in tracheal branches sculpted into the appropriate tubular shapes? We obtained an entry point into this problem when we discovered a unique tracheal phenotype caused by a double mutation eliminating both of the Type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. The Ptp4E Ptp10D double mutation converts linear unicellular tubes into spherical cysts. Type III RPTPs are highly conserved regulators of receptor TK signaling, and we found that the phenotype involves the loss of negative regulation by the RPTPs of three growth factor receptor TK orthologs: epidermal growth factor receptor (Egfr), Btl, and Pvr (VEGFR ortholog). This phenotype may have never been found in earlier genetic screens because it is only observed when both Ptp4E and Ptp10D are mutated. There may also be no single component downstream of the RPTPs that could be mutated to generate such phenotypes, since the RTKs signal through many pathways. Thus, the identification of genes that regulate tube geometry may require a sensitized genetic screen based on the Ptp4E Ptp10D phenotype. This is the basis of the first specific aim, which describes a systematic screen for recessive mutations that confer enhancement or suppression of the phenotype. Because this is a time-consuming screen, requiring quantitative analysis of individual embryos using confocal microscopy, we will reduce the numbers of lines that need to be screened by using a 'phenotypic screening kit' of deletion (Df) mutations that we have defined. For each deletion that enhances or suppresses the phenotype, we will identify the responsible gene using insertion mutations and RNAi lines, which exist for most Drosophila genes. When we have mutations in individual genes in hand, we will examine their phenotypes in detail and analyze their epistatic relationships with each other, as well as with the RTKs and RPTPs, in order to define genetic pathways. The second specific aim describes a systematic approach by which we can localize and tag the protein products of genes identified in the screen. We can attach the proteins to fluorescent markers of various colors (for localization in live and antibody-stained preparations) and to epitope tags or enzymes (for biochemical characterization). This system will allow us to find proteins that are localized t the regions of cells where tube shape is controlled. We can also analyze tyrosine phosphorylation of the proteins and determine if they physically interact with each other in the embryo.