CHARACTERIZATION OF GENES IMPORTANT IN LIMB DEVELOPMENT

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The long term objectives of this project are to understand the mechanisms involved in the differentiation of different cell types within the developing limb. Previous studies of the molecular biology of limb development have emphasized the study of the structure and regulation of phenotypic genes expressed during limb development particularly those of cartilage (proteoglycan core protein and type ii collagen) and muscle (actin and myosin), whereas the genes responsible for regulating or promoting differentiation of individual cells are unknown. An integrated approach combining monoclonal antibody technology and recombinant DNA technology will be used to identify and characterize genes and gene products important in the early cell diversification during limb development. Complementary DNA libraries corresponding to mRNAs unique to specific cell populations in the early limb will be constructed. Single stranded cDNA (first strand) for construction of these libraries will be selected by lack of hybridization to excess mRNA from a closely related cell population. The resulting cloned cDNAs will be analyzed by hybridization to RNA from various cell populations and by in situ hybridization to tissue. Clones corresponding to genes of particular interest will be expressed in E. coli and the resulting polypeptides will be used to generate antibodies for further protein and tissue characterization. A complementary approach will be to use selected antibodies obtained from other laboratories which show interesting patterns of localization during limb development to isolate the corresponding genes either by screening expression libraries or by immunoprecipitation of polysomes and subsequent cDNA construction. Both antibodies and DNA probes will then be used to identify and characterize previously unidentified genes angd gene products which are important in regulating early limb development.