Dr. Gelbart proposes to continue his studies on the role of intercellular signalling in the establishment and coordination of cell fate during development. His emphasis will be on the dpp gene and the TGF-beta signalling pathway. The TGF-beta family is involved in a variety of signalling events during the development of all metazoan animals. In a number of systems, the TGF-beta protein is cleaved from an inactive proprotein. In mammals TGF-beta functions as a dimer, either homo or hetero. The TGF-beta receptors found thus far function as heterodimers. Little is known about downstream signal transduction in TGF-beta pathways and it is a goal of this proposal to identify downstream components. In Drosophila the dpp gene, which encodes a TGF-beta, is required for a large number of developmental events. This gene, which has been the focus of Dr. Gelbart's laboratory for more than 15 years, is large with cis-acting regulatory sequences spread out over more than 50kb. Previous work by Dr. Gelbart's laboratory identified many of these cis- acting sequences. He also found that, at least in some cases, the response to dpp is graded and dependent on the concentration of gene product. The research plan is divided into four major sections. In the first a series of genetic screens are described to identify mutations that interact with dpp mutations. These new mutations would be in genes that are candidates to encode downstream genes. The second major section of the proposal is a characterization of interacting genes. The newly identified genes would be characterized with respect to phenotypes, epistasis, and genetic location. The investigator then proposes to analyze the most interesting of these in more detail. In addition, Dr. Gelbart will study two already recovered interacting genes in detail. One of these is sax, which encodes a TGF-beta receptor. Further genetic and molecular studies on sax are proposed and the investigator will attempt to construct an inducible gene that encodes a constituitively active receptor. Further studies on a second previously recovered interacting gene (Mad) are also proposed. The third major section of the proposal deals with the cell biology of dpp signalling. A collaborator, Dr. J. Massague, has recovered TGF-beta unresponsive mutations in a mink lung cell line. Some of these are not rescued by transfection with receptor encoding genes and hence are likely to be in downstream genes. Any new Drosophila genes recovered will be tested by transfection to see if they can rescue the mutant phenotype. Mammalian homologs of the Drosophila genes will also be isolated and tested in this way. To address the issue of which receptor binds which ligand (given that as dimers there are many possibilities), the investigator proposes to try to rescue null mutations in a receptor or ligand with transgenes that encode each of the known ligands or receptors. Dr. Gelbart will also attempt to determine if dpp function in the imaginal wing disc involves direct action at a distance or indirect local signalling (dpp expression is required in a strip along the A/P boundary for wing disc development). The investigator will make sax null clones and determine if they are viable in all regions of the wing. If dpp acts as a long range direct signal than these clones should not be viable anywhere in the wing. The fourth major section of the proposal is to examine the role of dpp in egg morphogenesis. For this goal, Dr. Gelbart will first attempt to map the location of the cis-acting regulatory sequences that drive expression of dpp. This should allow the investigator to construct a fly genotype that is specifically deleted for follicle cell expression. This could then serve as a background for introducing engineered dpp genes that express dpp only in specific subsets of follicle cells.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Genetics Study Section (GEN)
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Harvard University
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Twombly, Vern; Bangi, Erdem; Le, Viet et al. (2009) Functional analysis of saxophone, the Drosophila gene encoding the BMP type I receptor ortholog of human ALK1/ACVRL1 and ACVR1/ALK2. Genetics 183:563-79, 1SI-8SI
Myrick, Kyl V; Huet, Francois; Mohr, Stephanie E et al. (2009) Large-scale functional annotation and expanded implementations of the P{wHy} hybrid transposon in the Drosophila melanogaster genome. Genetics 182:653-60
Li, Jinghong; Li, Willis X; Gelbart, William M (2005) A genetic screen for maternal-effect suppressors of decapentaplegic identifies the eukaryotic translation initiation factor 4A in Drosophila. Genetics 171:1629-41
Richards, Stephen; Liu, Yue; Bettencourt, Brian R et al. (2005) Comparative genome sequencing of Drosophila pseudoobscura: chromosomal, gene, and cis-element evolution. Genome Res 15:1-18
Myrick, Kyl V; Gelbart, William M (2002) Universal Fast Walking for direct and versatile determination of flanking sequence. Gene 284:125-31
Huet, Francois; Lu, Jeffrey T; Myrick, Kyl V et al. (2002) A deletion-generator compound element allows deletion saturation analysis for genomewide phenotypic annotation. Proc Natl Acad Sci U S A 99:9948-53
Mohr, Stephanie E; Gelbart, William M (2002) Using the P[wHy] hybrid transposable element to disrupt genes in region 54D-55B in Drosophila melanogaster. Genetics 162:165-76
Conley, C A; Silburn, R; Singer, M A et al. (2000) Crossveinless 2 contains cysteine-rich domains and is required for high levels of BMP-like activity during the formation of the cross veins in Drosophila. Development 127:3947-59
Watson, K L; Chou, M M; Blenis, J et al. (1996) A Drosophila gene structurally and functionally homologous to the mammalian 70-kDa s6 kinase gene. Proc Natl Acad Sci U S A 93:13694-8
Blackman, R K; Grimaila, R; Koehler, M M et al. (1987) Mobilization of hobo elements residing within the decapentaplegic gene complex: suggestion of a new hybrid dysgenesis system in Drosophila melanogaster. Cell 49:497-505

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