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

描述（由申请人提供）：人体血管系统中产生新分支的关键机制是发芽。在发芽血管生成过程中，来自预先存在的管的内皮细胞在主动迁移的“尖端细胞”的引导下形成新血管。同样，在果蝇呼吸器官（气管系统）中，初级分支是通过发芽机制发生的，其中尖端细胞发挥着相同的重要作用。在两种分支发芽模型中，尖端细胞形态不同，延伸丝状伪足以感知局部环境并引导分支诱导信号（分别为血管内皮生长因子/VEGF和成纤维细胞生长因子/FGF）的浓度梯度向上迁移。我们已经证明，果蝇中气管细胞相互竞争尖端细胞位置，这种竞争是基于 FGF 受体 (FGFR) 活性的相对水平，并且部分由横向抑制信号 Notch 介导。如果Notch信号被废除，大量的尖端细胞会被选择并迁移到芽的前端，而只有一两个跟随细胞构成分枝茎。尖端细胞位置的竞争也发生在血管生成过程中，受体酪氨酸激酶 (RTK) 和 Notch 信号传导发挥着相同的关键作用。尽管在了解尖端细胞选择方面取得了实质性进展，但拮抗茎细胞中尖端细胞行为的 Notch 信号传导的直接目标仍然未知。同样，虽然 RTK 信号传导促进尖端细胞迁移，但 RTK 在气管和内皮尖端细胞中的直接靶标尚不清楚。我们已经发现了一种新的突变，我们已经命名了太多的前导序列，它赋予突变细胞尖端细胞偏好，以及隔膜/紧密连接基因多毛类/ZO-1的突变，它赋予了Notch样的额外尖端细胞缺陷。我们提出，气管尖端细胞和内皮尖端细胞通过保守的竞争机制进行选择，并将系统地剖析信号通路及其下游靶点，目的是确定信号传导信息如何转化为细胞形状、上皮内细胞位置和细胞迁移的变化，从而使现有的管道重组以萌发侧枝。该项目的具体目标是： 1. 定义介导尖端细胞选择的 RTK 核心组件，并确定是否需要对 RTK 信号传导的转录反应。 2. 确定Notch信号在枝条发芽过程中如何调节和转导，并检查Notch配体表达的选择性丧失如何影响尖端细胞竞争。 3. 检验多毛类和太多领导者是尖端细胞竞争期间Notch 信号下游效应器的假设。 公众健康相关性：血管生成依赖性疾病——包括年龄相关性黄斑变性、冠状动脉疾病、中风和癌症——是当新血管生长过度或不足时导致的。人体血管系统中的新分支是通过一种称为发芽血管生成的过程产生的，其中来自预先存在的管的内皮细胞在积极迁移的“尖端细胞”的引导下形成新血管。该提案旨在利用果蝇气管分支作为模型，揭示选择尖端细胞并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.