It is our long-term goal to use the fruit fly Drosophila melanogaster as a model system to identify genes that restrict cell proliferation, and to study the functions of these genes in developing tissues. Here we focus on a novel function of the archipelago (ago) gene, which we identified based on its anti-proliferative role in the fly eye, and whose human ortholog Fbw7 was subsequently shown to suppress tumorigenesis in a variety of tissue types. The ago gene encodes an F-box protein (Ago) that targets proteins for proteasomal degradation by binding directly to them, and recruiting them into an SCF E3-ubiquitin ligase for poly- ubiquitination. We have previously identified two Ago targets, the cell cycle regulator Cyclin E, and the proto- oncogenic transcription factor dMyc, both of which are also targeted by Fbw7. However, our lack of knowledge of the full repertoire of ago/Fbw7 targets limits our understanding of the role of the gene in development, and hinders attempts to predict the biological consequences of ago/Fbw7 loss in humans. In the current proposal, we present a substantial body of unpublished work that demonstrates a novel role for the ago ligase in post-mitotic morphogenesis of the fly embryonic tracheal system, a branched network of epithelial tubes similar to the human vasculature which duct oxygen through the developing embryo. We hypothesize that ago acts in this pathway via a novel target: the Trachealess protein (Trh), a member of the Hypoxia Inducible Factor (HIF)-1 alpha family of transcription factors that activates the Fibroblast Growth Factor (FGF) pathway, a known regulator of tracheal development in flies and vascular &lung development in mammals.
In Specific Aim 1, we seek to test a hypothesis suggested by our preliminary data that Ago binds Trh in tracheal cells, and targets it for degradation. We have found that ago may act redundantly in this process with the dVHL gene, which encodes a HIF-1 alpha ubiquitin-ligase known to be involved in tracheal development, and in Specific Aim 2, we propose to test genetic and functional interactions between ago and dVHL. We also find that inactivation of ago in mature tissues is able to non-cell autonomously induce ectopic terminal tracheal branching, a process that is normally controlled by oxygen availability. Thus, we hypothesize that ago plays an important role in the homeostatic mechanisms that restrain tracheal branching. Our goal in Specific Aim 3 is to define this role and elucidate its molecular basis.
