The molecular mechanisms of mesenchymal morphogenesis in the embryonic vertebrate limb will be analyzed using a multi-leveled approach. Interaction between cell surface heparan sulfate and the amino-terminal domain (NTD) of extracellular fibronectin has been shown previously to play an essential role in promoting precartilage cell condensation and subsequent cartilage differentiation. Therefore we will design site-specific reagents that interfere with this interaction in living cell cultures in order to determine the precise residues on fibronectin required for skeletal morphogenesis. The strategy consists of using protein engineering to produce mutant forms of the fibronectin-NTD. Sites chosen for mutagenesis will include those predicted to interact with heparin/heparan sulfate. The mutated proteins will be tested in several positive assays for changed ability to interact with heparin: (i) matrix-driven translocation, an in vitro assay for fibronectin-dependent morphogenesis in model extracellular matrices. Domains of fibronectin required for activity in this assay have previously correlated well with domains required for mesenchymal morphogenesis; (ii) circular dichroism, and (iii) polarization of fluorescence, spectroscopic techniques that can be used to probe fibronectin-NTD conformation in the presence and absence of heparin. Sites in the fibronectin-NTD the mutagenesis of which alters interactions of the protein with heparin in these assays will be the basis for designing oligopeptide and antibody reagents for use in tissue cultures of developing limb mesenchyme. Inhibition of mesenchymal condensation and cartilage-specific gene expression by these reagents will identify sites in fibronectin required for normal development, and help define the molecular mechanism of tissue morphogenesis in the embryonic limb.
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