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

Ghabrial -项目摘要 为了建立一个功能性的血管系统，必须通过组合产生内皮管网络， 血管生成和血管生成的过程。在萌芽血管生成过程中，内皮尖端细胞导致 新的分支尖端细胞与其他尖端细胞或与预先存在的管重叠。与此同时， 还必须内腔化，使得管变得开放。对斑马鱼的研究表明， 在横截面上没有接合缝的细胞内管（无缝管）的过程。甚至尖端细胞 随后经过无缝管的中间阶段，重新形成多细胞管。 同样，在果蝇呼吸系统的初级分支过程中，气管尖端细胞导致了 新的分支为了形成网络，一些气管尖端细胞相互融合（融合细胞），而另一些细胞（终末细胞 细胞）广泛地分支，以产生数十个盲端管，这些盲端管在靶组织上分叉，并充当 气体交换场所。像内皮细胞一样，气管尖端细胞形成无缝管。细胞生物学研究 已经导致了目前通过反向膜起泡形成无缝管的模型;然而， 了解所需的遗传和分子途径。我们一直在利用 在果蝇中可能的遗传方法，以满足我们的长期目标， 了解制造、成型和维护无缝钢管所需的遗传和分子框架。 我们的方法的基本原理是，果蝇的基本规则和遗传途径是 很可能在整个动物王国都是保守的。当我们通过识别新奇的事物来建立我们的理解时， 无缝管形态发生所需的基因（目的1：确定 囊腔基因），我们还深入研究--使用分子遗传学、细胞学和蛋白质组学方法-- 为了确定我们以前已经确定的遗传途径的其他成分和作用机制， 确定，并将我们的研究结果扩展到内皮无缝管（目的2：阐明细胞和 TBC 1D 10和Rab 35在无缝管生长中作用的分子机制）。此外，一名 无缝管发生途径（脑海绵状血管畸形3-Germination中心激酶III 通路），已知在内皮细胞中是关键的，因为在人类基因的正向突变中， 导致家族性血管疾病。利用果蝇遗传学和蛋白质组学方法，我们已经鉴定出 CCM 3-GCKIII通路上游和下游都需要的因子，现在提出更多的 明确定义干扰途径的生物学后果，并确定下游靶点， 信号通路。鉴于CCM 3在肾小管发生中的保守作用，我们还试图扩展我们的结果， 脊椎动物的内皮系统，利用斑马鱼，其独特的性质将允许遗传 操作和血管病变形成的活体成像（目的3：表征 NDR激酶，Tricornered，在调节无缝管形状中。

Ghabrial – Project Summary To build a functional vascular system, a network of endothelial tubes must be generated through a combination of vasculogenesis and angiogenesis. During sprouting angiogenesis, endothelial tip cells lead the outgrowth of new branches. Tip cells anastomose with other tip cells or with pre-existing tubes. At the same time, tip cells must also lumenize so that the tubes become patent. Studies in zebrafish indicate tip cells lumenize by a process that an intracellular tube that, in cross section, lacks junctional seams (seamless tube). Even tip cells that later remodel to contribute to multicellular tubes, pass through a seamless tube intermediate stage. Likewise, during primary branching of the Drosophila respiratory system, tracheal tip cells lead the outgrowth of new branches. To form a network, some tracheal tip cells anastomose (fusion cells) while others (terminal cells) branch extensively to produce dozens of blind ended tubes that ramify on target tissues and act as the sites of gas exchange. Like endothelial tip cells, tracheal tip cells form seamless tubes. Cell biological studies have led to the current model of seamless tube formation by inverse membrane blebbing; however, very little is known about the genetic and molecular pathways that are required. We have been exploiting the powerful forward genetic approaches possible in Drosophila to meet our long-term objective of pioneering an understanding of the genetic and molecular framework required to make, shape and maintain seamless tubes. The rationale of our approach is that the fundamental rules and genetic pathways operative in Drosophila are likely to be conserved throughout the animal kingdom. As we build our understanding by identifying novel genes required for seamless tube morphogenesis (AIM 1: Determine the molecular identity and function of the cystic lumens gene.), we also go deeper – using molecular genetic, cellular, and proteomic approaches – to identify additional components and mechanisms of action for genetic pathways we have previously identified, and extending our findings to endothelial seamless tubes (AIM 2: Elucidate the cellular and molecular mechanisms of TBC1D10 and Rab35 action in seamless tube growth). Additionally, one seamless tubulogenesis pathway (the Cerebral Cavernous Malformations 3-Germinal Center Kinase III pathway) we identified, is known to be critical in endothelial cells, as mutations in orthologous human genes lead to familial vascular disease. Using Drosophila genetic and proteomic approaches, we have identified factors required both upstream and downstream in the CCM3-GCKIII pathway, and now propose to more clearly define the biological consequences of perturbing the pathway and to identify the downstream targets of the signaling pathway. Given the conserved role of CCM3 in tubulogenesis, we also seek to extend our results to the vertebrate endothelial system, making use of zebrafish, whose unique properties will allow both genetic manipulation and live imaging in vivo of vascular lesion formation (AIM 3: To characterize the role of the NDR kinase, Tricornered, in regulating seamless tube shape.).