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.
In tubular networks, such as the human vascular system, fine connections between branches are often made by so-called 'seamless' endothelial cells, which make specialized, subcellular tubes that lack junctional seams. Formation of patent connections is required for blood flow, and misregulation of that process is implicated in human disease (cerebral cavernous malformations). We propose to use a Drosophila model of seamless tube formation (the terminal cells of the tracheal system) to better understand the genetic and molecular mechanisms at play.
