Regulation of Insulin-Like Growth Factor (IGF) Actions by IGF Binding Proteins in Fish

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The insulin-like growth factor (IGF) signaling system is essential for normal growth and development in vertebrates. The proposed project involves studying the molecular mechanisms by which the IGF signaling system acts to control cell proliferation, differentiation and cell death (apoptosis) in developing vertebrate embryos in vivo. There are three components to the vertebrate IGF signaling system: the IGF ligands, IGF receptors, and IGF binding proteins (IGFBPs). While it is clear that the IGF ligands and receptors transduce signals that positively regulate growth, the specific role of each of the IGFBPs and their interactions with the IGF ligands and receptors in vivo is not well understood. Our laboratory has been utilizing a model teleost fish, the zebrafish (Danio rerio) to investigate the IGFs/IGFBP interactions and their functional importance. We use zebrafish because of their accessible, transparent and fast-developing embryos. In addition, numerous genetic mutants are available for our studies. We have characterized the IGF signaling system in zebrafish and shown that IGFBP-2 inhibits IGF actions in developing zebrafish embryos. Our further studies have revealed the presence of several other IGFBPs in zebrafish. These IGFBPs show different structural characteristics and distinct expression patterns. The goal of this project is to understand how each of these IGFBPs interact with IGF ligands and receptors to regulate cell proliferation, differentiation, and apoptosis in developing zebrafish embryos. The overall hypothesis to be tested is that different IGFBPs are differentially regulated, and they each play a distinct role in specifying the IGF actions in defined embryonic tissues. The specific aims of the project are: 1) to determine the actions of IGFBP-2 in regulating cell proliferation, differentiation, and apoptosis in vivo by targeted expression of IGFBP-2 in the developing eyes; 2) to determine the structure of zebrafish IGFBP-1 and IGFBP-3, produce the corresponding proteins, and analyze these fish IGFBPs biochemically and functionally; 3) to determine the spatial and temporal expression patterns of these fish IGFBPs; 4) to determine the specific effects of IGFBP-1 and IGFBP-3 in controlling IGF actions in vivo by targeted expression in the developing eyes; and 5) to elucidate the physiological functions of the endogenous IGFBPs in vivo using a novel targeted gene "knockdown" approach. The expected results should provide novel insights for the roles of various IGFBPs in controlling IGF actions in a vertebrate embryo and will further our understanding of how the IGF signaling system acts in vivo to control growth and development in fish specifically and in vertebrates generally. Determination of the structures of fish IGFBPs and development of the corresponding protein, cDNA probe and antisera will make available valuable tools for investigation of IGFBP actions in fish. A comparison of the structure and function of IGFBPs from different vertebrate species will provide novel insight to our understanding of the evolution of this important gene family. Information gained from these studies may prove to be valuable to aquaculture for efficient production of animal protein to meet the needs of a continually growing human population.