The avian embryo is a powerful experimental system for exploring the tissue interactions and molecular mechanisms underlying vertebrate development. The combination of tissue transplantation methodologies and retrovirus-mediated ectopic gene expression or direct recombinant protein application have allowed enormous advances in the past few years. These experimental approaches are limited, however, by an inability to regulate the activity of the introduced gene or protein. In the experiments described in this proposal, the Laufer lab will adapt two conditional signaling systems for use in the avian system. The LBD (hormone receptor ligand binding domain) system uses mutant ligand binding domains of hormone receptors to reversibly inactivate constitutively activate protein: LBD fusions. Both the estrogen and progesterone receptor LBDs will be used. These are regulated by 4- hydroxy-tamoxifen and RU486, respectively. The FK506 binding protein (FKBP) system uses modular FKBP binding domains fused to partner proteins, and a bivalent synthetic ligand (AP20187) to regulate protein activity. Reagents will be developed using each system, and their properties investigated in vitro. Avian retroviruses expressing nuclear GFP:LBD fusions will be constructed, and ligand-dependent cytoplasmic to nuclear translocation used as a monitor of activation. Retroviruses that express a truncated FGF receptor: FKBP fusion protein will also be constructed; activation is caused by ligand-dependent dimerization. Activation in cell culture will be assessed through receptor phosphorylation and induction of DNA synthesis. For each of these reagents, activation kinetics and dose responses will be determined. The in vivo regulatory parameters of each fusion protein-ligand pair will then be established. Chick embryos will be infected with the recombinant retroviruses, and after allowing for expression of the target protein, embryos will be treated with the appropriate drug. nGFP:LBD activation will be determined via subcellular localization. FGFR activity will be assessed by FGF-dependent target gene expression using non-radioactive in situ hybridization histochemistry and induction of tissue growth. Various drug doses and application regimens (via superficial vasculature, intravenous or intralumenal injection) will determine the most efficacious means of target activation, as well as kinetics and dose responses. Together these systems will provide the means to regulate protein function in a spatial, temporal and quantitative fashion during avian development.
