Competition and morphogenesis in tip cell-mediated branching of tubular networks

描述(由申请人提供)：在人类血管系统中产生新分支的关键机制正在萌芽。在萌发血管生成的过程中，来自预先存在的管子的内皮细胞被活跃地迁移的“尖端细胞”引导形成新的血管。同样，在果蝇的呼吸器官(气管系统)中，初级分支通过发芽机制发生，顶端细胞在其中扮演同样重要的角色。在两种分枝萌发模型中，顶端细胞在形态上是不同的，延伸丝状足细胞以感知局部环境，并引导分枝诱导信号(分别为血管内皮生长因子/血管内皮生长因子和成纤维细胞生长因子/成纤维细胞生长因子)的浓度梯度向上迁移。我们已经证明，在果蝇中，气管细胞相互竞争尖端细胞的位置，竞争是基于相对水平的成纤维细胞生长因子受体(FGFR)活性，部分是由侧向抑制信号Notch介导的。如果Notch信号被取消，大量过剩的尖端细胞被选择并迁移到芽的前端，而只有一个或两个跟随细胞组成分枝茎。在萌发的血管生成过程中，也会发生对末端细胞位置的竞争，受体酪氨酸激酶(RTK)和Notch信号在其中起着同样的关键作用。尽管在理解TIP细胞选择方面取得了实质性进展，但Notch信号在茎细胞中对抗TIP细胞行为的直接靶点仍然未知。同样，虽然RTK信号促进了TIP细胞的迁移，但RTK在气管和内皮TIP细胞中的直接靶点尚不清楚。我们已经发现了一种新的突变，我们已经命名了太多的领导者，它赋予突变细胞尖端细胞偏见，以及隔膜/紧密连接基因Polychaetid/ZO-1的突变，赋予Notch样额外尖端细胞缺陷。我们认为，气管末端细胞和内皮末端细胞是通过保守的基于竞争的机制选择的，并将系统地剖析信号通路及其下游靶点，目的是确定信号信息如何转化为细胞形状、细胞在上皮中的位置和细胞迁移的变化，从而使预先存在的管子重组出侧枝。该项目的具体目标是：1.确定介导TIP细胞选择的RTK核心成分，并确定是否需要对RTK信号进行转录反应。2.研究Notch信号在枝条萌发过程中的调控和转导，以及Notch配体表达的选择性缺失对TIP细胞竞争的影响。3.验证在TIP细胞竞争过程中，多毛类生物和过多的先导分子是Notch信号的下游效应者的假说。 公共卫生相关性：血管生成依赖型疾病--包括老年性黄斑变性、冠状动脉疾病、中风和癌症--是当新血管生长过度或不足时产生的。人类血管系统中的新分支是通过一种名为萌芽血管生成的过程产生的，在这种过程中，来自预先存在的管子的内皮细胞被活跃地迁移的尖端细胞引导形成新的血管。这项提议旨在以果蝇气管分支为模型，揭示选择尖端细胞以及它们引导新管生长的遗传和分子机制。

DESCRIPTION (provided by applicant): A critical mechanism for generating new branches in the human vascular system is sprouting. In sprouting angiogenesis, endothelial cells from a pre-existing tube are led by actively migrating "tip cells" to form new vessels. Similarly, in the Drosophila respiratory organ (tracheal system), primary branching occurs through a sprouting mechanism in which tip cells play the same essential role. In both models of branch sprouting, the tip cells are morphologically distinct, extending filopodia to sense the local environment and to lead migration up a concentration gradient of a branch-inducing signal (Vascular endothelial growth factor/VEGF and fibroblast growth factor/FGF, respectively). We have shown that tracheal cells compete with each other for tip cell positions in Drosophila, where competition is based on relative levels of FGF Receptor (FGFR) activity and is mediated in part by the lateral inhibitory signal Notch. If Notch signaling is abrogated, a vast excess of tip cells are selected and migrate to the leading end of the sprout while only one or two follower cells comprise the branch stalk. Competition for tip cell positions also occurs during sprouting angiogenesis, with Receptor Tyrosine Kinase (RTK) and Notch signaling playing the same key roles. Despite substantial progress in understanding tip cell selection, the direct targets of Notch signaling that antagonize tip cell behavior in stalk cells remain unknown. Likewise, while RTK signaling promotes tip cell migration, the direct targets of the RTKs in tracheal and endothelial tip cells are not known. We have identified a novel mutation that we have named too many leaders, that confer a tip cell bias to mutant cells, and mutations in the septate/tight junction gene polychaetoid/ZO-1, that confer a Notch-like extra tip cell defect. We propose that tracheal tip cells and endothelial tip cells are selected by a conserved competition-based mechanism, and will systematically dissect the signaling pathways and their downstream targets, with the goal of determining how signaling information is translated into changes in cell shape, cell position within an epithelium, and cell migration, such that a pre- existing tube reorganizes to sprout a side branch. The specific aims of this project are: 1. To define the RTK core components that mediate tip cell selection, and to determine whether a transcriptional response to RTK signaling is required. 2. To determine how Notch signaling is regulated and transduced during branch sprouting, and to examine how selective loss of Notch ligand expression affects tip cell competition. 3. To test the hypothesis that Polychaetoid and Too Many Leaders are downstream effectors of Notch signaling during tip cell competition. PUBLIC HEALTH RELEVANCE: Angiogenesis-dependent diseases - including age-related macular degeneration, coronary artery disease, stroke, and cancer - result when new blood vessels either grow excessively or insufficiently. New branches in the human vascular system are generated by a process called sprouting angiogenesis, in which endothelial cells from a pre-existing tube are led by actively migrating "tip cells" to form new vessels. This proposal aims to use Drosophila tracheal branching as a model to uncover the genetic and molecular mechanisms by which tip cells are selected and by which they lead outgrowth of new tubes.