Phosphotyrosine signaling pathways controlling tracheal tube geometry

磷酸酪氨酸信号通路控制气管导管几何形状

• 批准号: 8348650
• 负责人: KAI G ZINN
• 金额: $ 28.88万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8348650
• 关键词: Affect; Antibodies; Biochemical; Blood Vessels; Caliber; Cells; Color; Complex; Confocal Microscopy; Cyst; Deletion Mutation; Development; Drosophila Proteins; Drosophila genus; Drosophila melanogaster; Embryo; Enhancers; Enzymes; Epidermal Growth Factor Receptor; Epithelial; Epitopes; Fibroblast Growth Factor; Fibroblast Growth Factor Receptors; Gases; Gene Proteins; Genes; Genetic; Genetic Models; Genetic Screening; Growth; Hand; Human; Hypoxia; Individual; Insertion Mutation; Kidney; Larva; Life; Ligands; Liquid substance; Logic; Lung; Measures; Microscope; Mitogen-Activated Protein Kinases; Morphogenesis; Mutate; Mutation; Organ; Orthologous Gene; Pathway interactions; Pattern; Phenotype; Phosphotyrosine; Preparation; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Proteins; RNA Interference; Receptor Protein-Tyrosine Kinases; Recruitment Activity; Regulation; Research; Screening procedure; Shapes; Signal Pathway; Signal Transduction; Source; Staining method; Stains; Suppressor Genes; System; Targeted Research; Time; Tube; Tubular formation; Tumor Angiogenesis; Tyrosine Phosphorylation; Vascular Endothelial Growth Factor Receptor; Vascular Endothelial Growth Factors; Vascular System; Vesicle; base; mutant; neoplastic cell; new therapeutic target; receptor; respiratory; rho; rho GTP-Binding Proteins; tumor

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. PUBLIC HEALTH RELEVANCE: The developmental logic of the human vascular system is similar to that of the Drosophila respiratory (tracheal system). In both systems, tube growth is driven by interactions between receptor tyrosine kinases (RTKs) and their ligands. In tumor angiogenesis, hypoxic tumor cells recruit new blood vessels through secretion of RTK ligands that are also used during development. An understanding of the RTK signaling pathways that control tracheal tube morphogenesis may help in identification of new targets for research into tumor angiogenesis mechanisms and therapies.

描述(申请人提供)：哺乳动物的血管系统、肺和肾是输送气体或液体的分支管状上皮器官。尽管组装这些器官所需的基因已经被识别出来，但决定它们的管子形状和大小的发育机制还不是很清楚。果蝇幼虫的呼吸(气管)系统为研究复杂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受成纤维细胞生长因子受体酪氨酸激酶(TK)同源基因BTL及其配体BNL之间的模式相互作用的控制。气管系统的发育逻辑与哺乳动物的血管系统相似，在哺乳动物的血管系统中，表达血管内皮生长因子受体TK的血管萌芽向血管内皮生长因子的来源生长。气管分支中的呼吸道是如何塑造成合适的管状形状的？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，这种独特的气管表型是由消除了III型受体酪氨酸磷酸酶(RPTPs)Ptp4E和Ptp10D的双重突变引起的。Ptp4E Ptp10D双突变可将线形单细胞管转化为球形包囊。III型RPTPs是TK受体信号的高度保守的调节因子，我们发现这种表型涉及三种生长因子受体TK同源物的RPTPs的负调控缺失：表皮生长因子受体(EGFR)、BTL和PVR(VEGFR直系同源)。这种表型可能从未在早期的遗传筛查中发现过，因为只有当Ptp4E和Ptp10D都发生突变时才能观察到这种表型。也可能没有RPTP下游的单一成分可以突变来产生这样的表型，因为RTK通过许多途径发出信号。因此，识别调节管状结构的基因可能需要基于Ptp4E Ptp10D表型的敏化遗传筛选。这是第一个特定目标的基础，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查。由于这是一项耗时的筛查，需要使用共聚焦显微镜对单个胚胎进行定量分析，因此我们将使用我们定义的缺失(DF)突变的‘表型筛查试剂盒’来减少需要筛查的品系数量。对于每个增强或抑制表型的缺失，我们将使用插入突变和RNAi系来确定责任基因，这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，并分析它们彼此之间的上位关系，以及与RTK和RPTP的关系，以确定遗传途径。第二个具体目标描述了一种系统的方法，通过它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上(用于活的和抗体染色的制剂中的定位)，并连接到表位标签或酶上(用于生化特性)。这个系统将使我们能够找到定位于细胞中控制管状形状的区域的蛋白质。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。 公共卫生相关性：人类血管系统的发育逻辑与果蝇呼吸道(气管系统)的发育逻辑相似。在这两个系统中，管的生长是由受体酪氨酸激酶(RTK)及其配体之间的相互作用驱动的。在肿瘤血管生成中，缺氧的肿瘤细胞通过分泌RTK配体来招募新的血管，这些配体也在发育过程中使用。了解控制气管形态发生的RTK信号通路可能有助于识别肿瘤血管生成机制和治疗研究的新靶点。