Dr. Mark Krasnow proposes to continue his genetic analysis of trachea development in Drosophila. The tracheal system is composed of a highly branched network of epithelial tubes. During the last 4 years Dr. Krasnow worked out procedures and markers for describing the development of the tracheal system in the Drosophila embryo. His results showed that the successive branching that occurs is not due to a single mechanism used repeatedly during development. Rather each stage in the branching appears to have a different cellular basis. Dr. Krasnow carried out an enhancer trap screen to identify genes expressed at various stages in tracheal development. In addition, he identified mutations in about a dozen genes that give a strong and consistent abnormal tracheal phenotype. Among these are mutations that affect the primary branching, the secondary branching, or the terminal branching. One of the genes essential for normal terminal branching, the pruned, locus was cloned and found to encode the Drosophila homolog of the mammalian serum response factor. This is a well studied transcription factor in mammals that is activated via a signal transduction pathway. The research plan consists of 4 specific aims. The first is to test the role of pruned/DRSF in the regulated outgrowth of terminal branches. In mammals SRF is activated by a phosphorylation stimulated by a signal transduction cascade. The PI proposes to generate constitutively activated DSRF and to test the consequences of expression of this protein on tracheal branching. The PI's model suggests that this should cause excessive terminal branching. The second specific aim is to determine if RAS and other components of the mammalian SRF signalling pathway are involved in terminal branching. The PI proposes to first obtain stocks that carry loss of function or gain of function mutations in the RAS/MAPK pathway and to determine if they alter terminal branching in the embryo. The PI will express dominant negative and constitutively activated RAS in the terminal cells, determine if they have a phenotype, and then determine if pruned mutations are epistatic to the RAS mutations as would be expected if pruned is downstream of RAS. A similar set of experiments will be carried out with both activated and dominant negative versions of the breathless tyrosine kinase receptor. The third specific aim is the molecular cloning of two additional genes trimmed and misguided. Trimmed has a pruned like mutant phenotype. The trimmed gene will be cloned by virtue of the fact that it is defined by a P insertion mutation. The function of trimmed and its interaction with pruned will be studied using standard Drosophila genetics approaches and will be guided by the sequence of the trimmed protein. The misguided enhancer trap is expressed in the same cells as pruned and trimmed. Mutations in this gene result in branches that grow in variably inappropriate target territories. The misguided gene will be cloned and its function analyzed in a variety of ways. The final specific aim is the identification of additional genes involved in terminal branching. Both enhancer trap and EMS mutagenesis strategies will be used.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Genetics Study Section (GEN)
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Stanford University
Schools of Medicine
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Peterson, Soren J; Krasnow, Mark A (2015) Subcellular trafficking of FGF controls tracheal invasion of Drosophila flight muscle. Cell 160:313-23
Ghabrial, Amin S; Levi, Boaz P; Krasnow, Mark A (2011) A systematic screen for tube morphogenesis and branching genes in the Drosophila tracheal system. PLoS Genet 7:e1002087