Genetic analysis of tracheal terminal cell development. M.M. Metzstein, A.S. Kirby, M.A. Krasnow. HHMI/Dept Biochem, Stanford Univ Medical School, Stanford, CA.

   The Drosophila tracheal system is an interconnected network of epithelial tubes through which oxygen is delivered to internal tissues. A number of genes involved in the outgrowth and patterning of the tracheal system have been previously characterized, but it is likely that many more genes involved in tracheal development are yet to be found. In particular, few genes have been identified that are required in the post-embryonic development of the fine tracheal terminal branches. Terminal branches are formed by specialized cells called terminal cells, each of which forms dozens of tubes by a process that involves cytoplasmic extension and branching, intracellular lumen formation within each extension, and clearance of liquid from these lumens.
    To identify genes involved in each of these processes we have carried out a large scale genetic mosaic screen. We generated a collection of 750 X-linked lethals and examined the effects of these mutations in terminal cell clones. From this screen we have identified 38 mutations with effects on cytoplasmic outgrowth, branch morphology and lumen formation. Genetic and molecular characterization of the genes identified by these mutations is underway.
    One class of mutations, including 11L, give essentially normal branch outgrowth but form no air-filled lumens. We constructed a SNP of the X chromosome and used it to map 11L to a 25 kb region. Subsequent sequence analysis identified a mutation in DmX, the Drosophila homolog of the mammalian gene rabconnectin-3. Rabconnectin-3 interacts with Rab-3 GEF and GAP, which regulate the activity of Rab-3, a small GTPase involved in vesicle trafficking. We are now testing the model that the subcellular localization of the DmX protein guides a specialized set of Rab-3-associated vesicles to form the intracellular lumen.