Virtually all aspects of vertebrate embryonic development are dependent on signaling between cells in neighboring tissues. Much of this signaling is mediated by Receptor Tyrosine Kinases (RTKs). In the adult, disruptions in RTK signaling can have profound effects, resulting in various forms of disease including tumor formation. Because of its central importance in both the embryo and adult, considerable attention has been devoted to understanding the mechanisms that regulate RTK signaling. Studies in Drosophila and in mammalian cell culture have demonstrated that members of the Sprouty gene family are inhibitors of RTK signaling, and studies in vertebrate embryos have indicated that three members of the Sprouty family are expressed at various stages of development. The studies proposed here employ a genetic approach to determine the functions of the Sprouty (Spry) genes in the mouse. In the course of the current grant period, mice carrying null and conditional loss of function alleles of both Spry2 and Spry4, as well as a line of mice carrying a conditional Spry2 gain of function allele that can be activated in specific tissues by Cre-mediated recombination have been produced. Analyses of these mice have revealed that loss of Spry2 function causes a severe hearing deficit and abnormal mammary gland development, and that Spry4 null mice have abnormal forelimbs. The experiments proposed here are aimed at elucidating the functions of Spry2 in the inner ear and mammary gland, and of Spry4 in the developing limb. Additional studies are aimed at assessing the functional equivalence of the mouse Sprouty genes by generating mice that lack different combinations of Sprouty null alleles, and by producing a set of mouse lines, each carrying a different Sprouty gene conditional gain of function allele inserted into precisely the same site in the mouse genome. When activated, the Sprouty genes in these mouse lines will be identically expressed, making possible a meaningful comparison of the activities of the different Sprouty genes. Finally, experiments are described aimed at determining whether Sprouty genes function as suppressors of neoplastic cell transformation in vitro and tumorigenesis in the mammary gland. The results of the proposed studies will lead to a deep understanding of the function of the Sprouty genes, which should have important implications for preventing human congenital defects and treating disease. ? ? ?
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