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.).
This project is relevant to human health on two levels. First, we pioneer a basic science understanding of the formation and maintenance of tubular networks that is relevant for all organs. Second, we focus on a poorly understood pathway that is directly affected in human vascular disease (cavernous angiomas).
