The hypothesis on which this proposal is based is that the presumed proto-oncogene int-2 plays an important role in the control of early mammalian embryogenesis. Data from our previous study of int-2 expression in mouse embryos and teratocarcinoma cells demonstrated that there are four embryonic transcripts and that their expression is differentially regulated during normal embryogenesis and during the course of differentiation of teratocarcinoma stem cells in vitro. We propose to clone and characterize these four embryonic int-2 RNAs. Their protein products will be obtained by the use of a bacterial expression vector system, and anti-int-2 antibodies will be raised. The nucleic acid and immunological probes obtained in these studies will then be used to determine the stage- and cell type- specificity of int-2 expression in the embryo. In particular, we will determine which cells in the embryo express the gene and the subcellular location of its product(s). If it is a secreted protein, we will determine where in the embryo it becomes localized. Finally, we propose a series of experiments aimed at determining the effect of altering or abolishing the expression of int-2 in teratocarcinoma cells and/or embryos. The results of this study should provide us with information that will enable us to determine the function of int-2 in the embryo, which in turn should lead to a better understanding of the processes that control normal embryonic development. Furthermore, we anticipate that knowledge of the normal function of the gene will provide some insight into why abnormal expression of this proto-oncogene in the mammary gland results in carcinogenesis.
