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

胰岛素样生长因子（IGF）信号系统对脊椎动物的正常生长和发育至关重要。拟议的项目涉及研究IGF信号系统在体内发育的脊椎动物胚胎中控制细胞增殖，分化和细胞死亡（凋亡）的分子机制。脊椎动物IGF信号系统有三个组成部分：IGF配体、IGF受体和IGF结合蛋白（IGFBPs）。 虽然清楚的是IGF配体和受体激活正调节生长的信号，但是每个IGFBP的具体作用及其与IGF配体和受体在体内的相互作用还没有很好地理解。 我们的实验室一直在利用模式硬骨鱼，斑马鱼（Danio rerio）研究IGFs/IGFBP的相互作用及其功能的重要性。 我们使用斑马鱼是因为它们的胚胎容易获得，透明和快速发育。 此外，许多遗传突变体可用于我们的研究。我们已经表征了斑马鱼中的IGF信号系统，并表明IGFBP-2抑制了发育中的斑马鱼胚胎中的IGF作用。我们的进一步研究揭示了斑马鱼中存在其他几种IGFBPs。 这些IGFBPs显示出不同的结构特征和不同的表达模式。 该项目的目标是了解这些IGFBPs如何与IGF配体和受体相互作用，以调节斑马鱼胚胎发育中的细胞增殖，分化和凋亡。要测试的总体假设是，不同的IGFBPs差异调节，他们每个人都发挥了独特的作用，在指定的胚胎组织中的IGF行动。本项目的具体目标是：1）通过在发育中的眼睛中靶向表达IGFBP-2来确定IGFBP-2在体内调节细胞增殖、分化和凋亡中的作用; 2）确定斑马鱼IGFBP-1和IGFBP-3的结构，产生相应的蛋白，并对这些鱼类IGFBP-1和IGFBP-3进行生化和功能分析; 3）确定这些鱼IGFBP的空间和时间表达模式; 4）确定IGFBP-1和IGFBP-3通过在发育中的眼睛中靶向表达在体内控制IGF作用中的特异性作用;和5）使用新的靶向基因“敲低”方法来阐明内源性IGFBPs在体内的生理功能。预期的结果应提供新的见解的各种IGFBPs在控制IGF行动的脊椎动物胚胎中的作用，并将进一步了解如何在体内的IGF信号系统的行为，以控制生长和发育的鱼类，特别是在脊椎动物一般。鱼类IGFBPs结构的确定以及相应蛋白、cDNA探针和抗血清的研制，将为研究IGFBPs在鱼类中的作用提供有价值的工具。比较不同脊椎动物IGFBPs的结构和功能，将为我们理解这一重要基因家族的进化提供新的视角。从这些研究中获得的信息可能对水产养殖业有效生产动物蛋白质以满足不断增长的人口的需求很有价值。

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.