ROLE OF EXTRACELLULAR MATRIX REMODELING IN MANDIBULAR MORPHOGENESIS

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A major issue in developmental biology is how the temporal and spatial instructions are translated to form during embryogenesis. The first branchial arch undergoes morphogenesis to structures of the lower jaw, including the mandibular cartilage and bone from an amorphous mesenchyme from embryonic day (E)10 to 14 in vivo. In E10 tissue the program of morphogenesis is set, and in subsequent culture for 9 days forms all the tissues of the mandible in serum-less, chemically defined culture medium. Thus, this system is ideal for determining the local factors that regulate the morphogenesis of bone and cartilage. Extracellular matrix remodeling is believed to play a significant role in growth and development of connective tissues. The aim of this proposal is to determine whether extracellular matrix remodeling mediated by metalloproteinases and their inhibitors, which are known to be major components of bone and cartilage, plays a role in the formation and form of the extracellular matrix constituents of bone and cartilage during mandible development. To test this hypothesis it is essential to obtain quantitative and qualitative data on the expression of metalloproteinases (MMPs) and metalloproteinase inhibitors (TIMPs) during mandibular development in vivo and in culture. The approach will sue RT-PCR for initial identification of proteinase and inhibitor mRNA transcripts, followed by analysis of protein products of these genes by enzymatic and immunological means. Once these MMPs and TIMPs have been identified and their temporal patterns of expression defined, the expression of selected MMPs and TIMPs will be mapped to chondrogenic and/or osteogenic foci by in situ hybridization and immunocytochemistry during development in vivo and in culture. Then the function of these MMPs and TIMPs will be studied. One approach will involve addition of purified or recombinant MMPs or TIMPs to mandible cultures to see if these perturb morphogenesis. A second approach will involve production of a hypomorphic phenotype with blocking antibodies or antisense oligonucleotides to MMPs or TIMPs to see if these affect morphogenesis. A third approach will involve modulation of mandibular growth and morphogenesis by addition of exogenous growth and differentiation factors or by ablation of these factors by antibodies or antisense oligonucleotides followed by determination of their effects on MMP and TIMP expression, and on timing, position, and expression of chondrogenesis and osteogenesis. These experiments should lead to an understanding of the molecular factors involved in the extracellular matrix aspects of bone and cartilage morphogenesis, in general, and in the mandible, in particular, and lead to novel therapeutic strategies for mandibular malformations and bone and cartilage repair.