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

描述（由申请人提供）：在人体血管系统中产生新分支的关键机制正在萌芽。在新生血管生成过程中，内皮细胞由主动迁移的“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，初级分支的发生是通过一种芽化机制，其中尖端细胞起着同样重要的作用。在这两种分支发芽模型中，尖端细胞在形态上是不同的，它们延伸丝状足来感知局部环境，并沿着分支诱导信号（分别是血管内皮生长因子/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，在果蝇中，气管细胞相互竞争尖端细胞位置，竞争基于FGF受体（FGFR）活性的相对水平，并部分由侧抑制信号Notch介导。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，而只有一两个跟随细胞组成枝柄。尖端细胞位置的竞争也发生在发芽血管生成过程中，受体酪氨酸激酶（RTK）和Notch信号也起着同样的关键作用。尽管在了解尖端细胞选择方面取得了实质性进展，但Notch信号在柄细胞中拮抗尖端细胞行为的直接靶标仍然未知。同样，虽然RTK信号促进尖端细胞迁移，但RTK在气管和内皮尖端细胞中的直接作用靶点尚不清楚。我们已经确定了一种新的突变，我们已经命名了太多的先导，它赋予突变细胞尖端细胞偏见，以及分隔/紧密连接基因polychaetoid/ZO-1的突变，赋予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是由保守的竞争机制选择的，并将系统地剖析信号通路及其下游靶标，目的是确定信号信息如何转化为细胞形状、上皮细胞位置和细胞迁移的变化，从而使预先存在的管重组并发芽。本项目的具体目标是：1。定义介导尖端细胞选择的RTK核心成分，并确定是否需要对RTK信号的转录反应。2. 确定Notch信号在分枝发芽过程中是如何被调节和转导的，并研究Notch配体表达的选择性缺失如何影响尖端细胞的竞争。3. 为了验证在尖端细胞竞争过程中，Polychaetoid和Too Many Leaders是Notch信号的下游效应器的假设。

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.