The aim of this proposal is to elucidate the role in mammalian development of the gene product known as Bone Morphogenetic Protein-4 (BMP-4). This secreted protein is a member of the TGFbeta superfamily and is closely related to Drosophila Decapentaplegic (DPP). BMP-4 RNA is first expressed in the posterior ventral mesoderm of the early neurula stage mouse embryo, and then later in many regions where mesenchymalepithelial interactions are required for morphogenesis. There is also strong genetic and immunocytochemical evidence that, in Drosophila, DPP acts as an intercellular signaling molecule, transferring positional information from the mesoderm of the larval midgut to the underlying endodermal cells. A variety of genetic and biochemical techniques will be used to test the hypothesis that BMP-4 plays a similar role in mediating inductive tissue interactions in the mammalian embryo. Homologous recombination in embryonic stem (ES) cells will be used to introduce null mutations into the BMP-4a gene on Chromosome 14 and the BMP-4b gene on the X chromosome. Chimeric mice will be produced by blastocyst injection and used to generate hemizygous, heterozygous and homozygous mutant mice. These will be analyzed for defects in morphogenesis and pattern formation. A similar analysis will be made of transgenic mice in which BMP-4 is misexpressed in ectopic sites in the embryo and adult under the control of heterologous gene regulatory sequences. For studying the synthesis, secretion and localization of BMP-4 in vivo and in transfected cell lines in vivo, polyclonal rabbit antibodies will be prepared against both the variable and conserved regions of the protein. Finally, the effect of BMP-4 protein will be investigated on Xenopus and chick embryos in vivo. Together, these different approaches will throw light on the role of BMP-4 in morphogenesis and pattern formation in the embryo, and provide basic information relevant to congenital defects and teratogenesis in humans.
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