The long-term objectives of this research proposal are to elucidate the mechanisms which underly the formation of prechondrogenic condensations during skeletogenesis of normal mammalian hindlimb-buds and to determine how the brachypod (bpH/bpH) skeletal mutation in mice interferes with these mechanisms to produce congenital limb deformities. It is hypothesized that the glycosylation of cell surface oligosaccharide acceptor molecules by adjacent cell surface galactosyltransferase enzymes is prerequisite to normal condensation formation and that an alteration in cell surface galactosyltransferase activity or the oligosaccharide acceptor molecules themselves forms the basis for regional pattern deformities in brachypod limbs. The specific aims of this proposal are to: 1) assay for plasma membrane-bound galactosyltransferase activity during condensation of normal and brachypod digital limb-bud mesenchyme, 2) determine whether galactosyltransferase activity observed in the normal and mutant plasma membranes is intermediately expressed in heterozygous (+/bpH) cells, and 3) characterize the glycosylated products. To achieve these goals the following experiments will be carried out. 1. An organ culture system will be used which augments normal distal cartilage development in early 12 day autopod (paw primordia) explants. The de novo expression of the bpH allele is enhanced in these cultures, since the digital cartilages do not form due to a failure of prechondrogenic condensation formation. 2. Assay of galactocyltransferase activity spectrophotometrically using plasma membranes prepared from cells in the digital region. In this assay UDP produced from hydrolysis of UDP-galactose is stoichiometrically coupled to the production of NADH as galactose is transferred to the acceptor molecule, N-acetylglucosamine. 3. Analyze qualitatively galactosyltransferase and acceptor molecules by non-denaturing polyacrylamide gel electrophoretic separation. 4. Determine the quantitative levels and size of galactosyltransferase and acceptor molecules by gel permeation using a High Performance Liquid Chromatograph. These experiments will establish whether plasma membrane enzyme or substrate molecules involved in prechondrogenic condensations are defective in the brachypod limb. They will provide new information about the mechanisms which underly formation of prechondrogenic condensations and add to our understanding of the cellular dysfunctions which become manifested as congenital limb deformities.