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

 描述（由申请人提供）：在萌芽血管生成中，来自预先存在的管的内皮细胞被主动迁移的“尖端细胞”引导以形成新血管。在果蝇的呼吸器官（气管系统）中，初级分支也由尖端细胞领导。我们和其他人的工作表明，这两个系统中的尖端细胞是通过竞争机制选择的。在这两种模型的分支发芽，尖端细胞形态上是不同的，延长丝状伪足感觉当地的环境，并导致迁移的浓度梯度的分支诱导信号。在引发和引导新的分支生长之后，尖端细胞执行第二个基本功能：它们必须形成管（管腔化）以使新的分支发挥功能。尖端细胞中空形成“无缝”管，缺乏连接“接缝”（粘附连接和紧密/分隔连接）。在果蝇的气管系统中，所有的尖端细胞居住在定型的位置，并形成无缝管，此外，气管终端细胞形成新的无缝管侧支在整个幼虫的生活过程。这些品质，结合果蝇遗传分析的力量，使气管终末细胞成为解剖无缝管形态发生的非常有用的模型。 尖端细胞管的形成是至关重要的，因为它允许血液或气体在细胞中的运输;然而，尽管最近取得了进展（上一个资助周期中的4个主要出版物），重要的问题仍然存在：FGFR信号传导如何在每个阶段触发不同的输出？FGFR是如何诱导无缝钢管的？FGFR如何调节无缝管分支？FGFR下游调节的效应物是什么？ 我们建议利用或公布的初步数据，以解决这些问题，使用创新的方法。为了了解信号传导如何与小管形成机制的变化相结合，以及无缝小管形成机制如何在FGFR下游运作，我们提出了以下具体目标：目标1：确定FGFR激活如何诱导无缝小管形成。我们将把我们的无缝管的研究重点放在新的锌指转录因子，我们确定，在我们的初步数据，作为无缝管形态发生所必需的。 目的2：确定FGFR激活如何调节无缝管的局部分支。我们将专注于使用创新的光遗传学和实时成像工具表征无缝管分支。我们将跟进我们的初步数据，以确定三种蛋白质-PALS 2/MPP 6/2（果蝇静脉曲张），Spectroplakin（果蝇短停）和Moesin -在果蝇和斑马鱼血管系统中的无缝管分支中的作用。 这些特定目标的完成将代表该领域的巨大进步，将信号转导与管形态发生的细胞机制以前所未有的分辨率水平连接起来。

 DESCRIPTION (provided by applicant): In sprouting angiogenesis, endothelial cells from a pre-existing tube are led by actively migrating "tip cells" to form new vessels. In the Drosophila respiratory organ (tracheal system), primary branching is also led by tip cells. Work from us and others showed that tip cells in both systems are selected through a competition-based mechanism. 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. Subsequent to initiating and guiding the outgrowth of a new branch, tip cells execute a second essential function: they must form a tube (lumenize) in order to make the new branch functional. Tip cells hollow out to form "seamless" tubes that lack junctional "seams" (adherens junctions and tight/septate junctions). In the Drosophila tracheal system, all tip cells reside in stereotyped positions and form seamless tubes; furthermore, tracheal terminal cells form new seamless tube side branches throughout the course of larval life. These qualities, combined with the power of genetic analysis in Drosophila, have made tracheal terminal cells an exceptionally useful model for dissecting seamless tube morphogenesis. Tip cell tube formation is critical, as it permits transport of blood or gas throug the cell; however, despite or recent progress (4 major publications in the last grant cycle), important questions remain: how does FGFR- signaling trigger distinct outputs at each stage? how does FGFR induce seamless tubes? how does FGFR regulate seamless tube branching? what are the effectors regulated downstream of FGFR? We propose to exploit or published and preliminary data to address these questions using innovative approaches. With the twin goals of understanding how signaling is coupled to changes in the mechanism of tubulogenesis, and how the seamless tubulogenesis machinery operates downstream of FGFR, we propose the following specific aims: Aim 1: Determine how FGFR activation induces seamless tubulogenesis. We will focus our studies of seamless tubulogenesis on the novel zinc finger transcription factor that we identify, in our preliminary data, as essential for seamless tube morphogenesis. Aim 2: Determine how FGFR activation regulates local branching of the seamless tube. We will focus on characterization of seamless tube branching using innovative optogenetic and live imaging tools. We will follow up on our preliminary data to establish the roles of three proteins - PALS2/MPP6/2 (Drosophila Varicose), Spectroplakin (Drosophila Short stop) and Moesin - in seamless tube branching in Drosophila and in the zebrafish vascular system. Completion of these specific aims will represent a dramatic advance in the field, connecting signal transduction to the cellular machinery of tube morphogenesis at an unprecedented level of resolution.