Animals with experimentally manipulable genomes have been invaluable for aging research. In model organisms such as worms, flies, and mice, the ability to specifically add or delete a gene enables the characterization of the roles of specific gene(s) in lifespan regulation. While these animal models have contributed, and will continue to contribute, to our understanding of aging biology, there are gaps in the range of experimental strengths provided by each of the existing animal models. This project will focus on a new and promising vertebrate model organism, the annual turquoise killifish (Nothobranchius furzeri), which has a lifespan of just several months. N. furzeri provides many additional advantages, such as the ability to produce many sibling progeny from a single mating, low cost of maintenance, amenability to forward genetic screens, and the ability of their eggs to be stored dry at low temperatures for as long as a year. Despite these advantages, the N. furzeri model has not been widely used in the field of aging research. This is in part because genetic manipulation experiments were not possible in this species. Recently, my lab has succeeded in developing methods for generating stable and inducible transgenesis in this species. Drawing on our research experience with fish biology and the IGF signaling pathway, the goal of this application is to develop new genetic tools for conducting loss-of-function studie and to use them to test the hypothesis that insulin-like growth factor binding protein-3 (IGFBP-3), a major IGFBP, regulates lifespan via IGF-dependent and/or -independent mechanisms. IGFBP-3 regulates IGF availability by binding IGF tightly and releasing it only under certain conditions. IGFBP-3 also has IGF-independent actions. It is possible that this conserved and multifunctional protein may play key roles in modulating aging. In Aim 1, we will develop genetic tools and use them to determine the role of IGFBP-3 in lifespan regulation. Aim 2 will investigate the functional significance of ligand-binding and nuclear localization of IGFBP-3 by generating and studying inducible transgenic lines that express either wild type or a mutant form of IGFBP-3. The completion of the proposed studies will provide novel insights into the roles of IGFBP-3 in the longevity of vertebrates. The anticipated results will also lead to the development of much needed methods and genetic tools to knockout a gene of interest or to induce gene expression in a tissue-specific and temporally restricted manner in this emerging vertebrate model. The methods and transgenic tools developed in this project will be broadly disseminated and distributed to the scientific community. These genetic tools and methods, combined with the unique features and the extremely short-lifespan of N. furzeri, will open many new avenues for investigations and should facilitate new discoveries in vertebrate aging biology. A better understanding of the biology of aging regulation will likely lead to the development of diagnostic and therapeutic tools that will increase the healthy lifespan of the aging population in the United States. PUBLIC HEALTH RELEVANCE: This project will test the hypothesis that insulin-like growth factor (IGF) binding protein-3, a major IGF binding protein, regulates lifespan via IGF-dependent and/or -independent mechanisms using turquoise killifish, a unique vertebrate that has a lifespan of just several months. This project will also develop new genetic tools, which will make it possible to experimentally manipulate the genome in this new aging biology model. The new genetic tools, combined with the extremely short lifespan and other unique features of this new vertebrate model, should help to facilitate new discoveries in vertebrate aging biology. A better understanding of the roles of IGF signaling in lifespan regulation will likely lead to the development of diagnostic and therapeutic tools that will increase the health of the aging population in the United States.