Effect of Versican Mutation on Limb development In Vitro

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DESCRIPTION (provided by applicant): The long-term objective of the proposed research is to define the role of cell-extracellular matrix interactions in differentiation and patterning of the cartilage model of the limb skeleton during embryonic development. The chondroitin sulfate proteoglycan, versican, has been implicated in limb chondrogenesis, but its precise role in this process has yet to be defined. Previous work has shown that versican is highly expressed in early chondrogenic and joint-forming regions in vivo and that cartilage differentiation is inhibited in vitro in versican-deficient limb mesenchyme derived from the hdf (heart defect) mutant mouse. To better understand versican's function during limb development in vivo, this proposal will test the hypothesis that specific domains of versican facilitate differentiation of the cartilaginous skeletal template and are critical for establishment of interzone regions required for synovial joint formation. The specific aims are to assess effects of: 1) over-expression of specific versican domains on chick limb development in ovo; and, 2) knock down of mature versican expression on chick limb development in ovo. These studies will provide new information to help ensure that in the future children are free of skeletal/articular disability due to disruption of normal development of the appendicular skeleton. Recombinant adenoviral constructs for over-expression of versican hyaluronan-binding (G1) and selectin-like (G3) domains and a full length versican isoform (V3) containing both G1 and G3 domains will be delivered directly to limb mesenchyme at various stages by microinjection into the limb in ovo. In complementary experiments, knock down of versican expression in ovo will be performed by microinjection of adenovirally encoded small interfering RNA constructs into limb mesenchyme. Effects on morphogenesis of the skeletal/joint template will be evaluated by in situ hybridization, Western blotting, and immunohistochemistry for endogenous or ectopic versican, augmented by correlation with peanut agglutinin-binding, Alcian blue histochemistry, and localization of collagen Type II and hyaluronan. The proposed studies bear important relevance to public health by providing new insights into factors controlling normal limb skeletal development. It is through understanding these molecular mechanisms that prevention or correction of birth defects affecting the developing appendicular skeleton can be achieved.