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

基本信息

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
项目类别：
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

项目摘要

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（由申请人提供）：哺乳动物血管系统，肺和肾脏是传输气体或液体的分支管状上皮器官。尽管已经确定了组装这些器官所需的基因，但确定管子的形状和大小的发育机制尚不清楚。果蝇幼虫的呼吸道（气管）系统为研究复杂的分支管网的发展提供了有用的遗传模型。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，喘不过气（BTL）与其FGF配体，无分支（BNL）之间的图案相互作用。气管系统的发育逻辑类似于哺乳动物血管系统的发育逻辑，在该系统中，表达血管 - 内皮生长因子（VEGF）受体TK的血管芽源性朝向VEGF的来源生长。气管分支中的气道如何雕刻成适当的管状形状？当我们发现由双重突变引起的独特气管表型时，我们获得了该问题的切入点，从而消除了III型受体酪氨酸磷酸酶（RPTPS），PTP4E和PTP10D。 PTP4E PTP10D双突变将线性单细胞管转换为球形囊肿。 III型RPTP是受体TK信号传导的高度保守调节剂，我们发现表型涉及RPTPs由三种生长因子受体TK直播的负调控的丢失：表皮生长因子受体（EGFR），BTL），BTL和PVR（VEGGR Ortholog offer）。这种表型可能从未在早期的遗传筛选中找到，因为仅当PTP4E和PTP10D都突变时才能观察到。由于RTKS通过许多途径信号，因此RPTPS下游的单个成分也可能被突变为产生这种表型。因此，调节管几何形状的基因的鉴定可能需要基于PTP4E PTP10D表型的敏感遗传筛选。这是第一个特定目标的基础，它描述了赋予表型增强或抑制的隐性突变的系统屏幕。因为这是一个耗时的屏幕，需要使用共聚焦显微镜对单个胚胎进行定量分析，因此我们将减少需要使用我们定义的“缺失表型筛选试剂盒”进行筛选的线数量。对于增强表型或抑制表型的每种缺失，我们将使用插入突变和RNAi系确定负责的基因，这是大多数果蝇基因存在的。当我们手头上有各个基因的突变时，我们将详细检查它们的表型，并与彼此以及RTK和RPTPS进行分析，以定义遗传途径。第二个特定目的描述了一种系统的方法，我们可以通过该方法定位并标记屏幕上鉴定的基因的蛋白质产品。我们可以将蛋白质连接到各种颜色的荧光标志物（用于现场和抗体染色的制剂中）以及表位标签或酶（用于生化表征）。该系统将使我们能够找到位于控制管形的细胞区域的蛋白质。我们还可以分析蛋白质的酪氨酸磷酸化，并确定它们是否在胚胎中相互相互作用。公共卫生相关性：人血管系统的发育逻辑与果蝇呼吸道（气管系统）相似。在这两个系统中，管的生长都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，低氧肿瘤细胞通过在发育过程中也使用的RTK配体的分泌募集了新的血管。对控制气管形态发生的RTK信号通路的理解可能有助于识别用于研究肿瘤血管生成机制和疗法的新靶标。