The long-term goal of this research is to understand the cellular and molecular factors that determine normal secondary palate morphogenesis. The extracellular matrix(ECM) molecules that compose the palatal interstitial matrix and basement membranes are important to the process. The palatal interstitial ECM is arranged as a scaffolding, composed principally of fibronectin(Fn), collagen III(Col ]III) and hyaluronan(Ha), that is functional by one day before expected in vivo palatal shelf reorientation. If shelves are explanted into organ culture at this time, they reorient and show accompanying changes in ECM distribution. Changes in distribution of collagen IV(Col IV) and laminin(Ln) in local regions of the epithelial basement membrane and changes in syndecan(Sn) distribution in the mesenchyme immediately subjacent to these regions also accompany shelf remodelling. Apparently, once these ECM structures are established, the palatal shelves are capable of remodelling to achieve reorientation. Pilot data suggest the necessary alterations may involve the metalloproteinases(MMPs) and plasminogen activators(PAs). Existing work also suggests, particularly in development, the interplay between ECM, MMPs, PAs and their activators and inhibitors determine the character of an ECM. However, to date no work has been done on the role of these enzymes in secondary palate morphogenesis. In light of current knowledge, the following hypothesis is proposed: The Ha-mediated shelf expansion that leads to reorientation is guided by alterations in tissue architecture resulting from temporo- spatially localized changes in the distribution of Fn and Col III in the interstitial matrix and of Col III and IV, Ln and Sn in specific local areas of the basement membrane of the covering epithelium and the mesenchyme immediately subjacent to it. These changes involve proteinase-mediated degradation of the ECM molecules.
Three specific aims are proposed to test this hypothesis: (1) Describe the temporo-spatial development of the palatal shelf ECM scaffolding from the time of emergence of the palatal shelves from the maxillary process until the structure is functional; (2) Identify the MMPs, PAs and their inhibitors that are present in the palatal shelves during palate morphogenesis in vivo and in vitro and determine their temporal sequence of appearance and spatial distribution; (3) Determine the effects of inhibiting or activating the matrix-degrading proteinases on palate morphogenesis and the composition and organization of the interstitial ECM structure and the basement membrane. Organ culture, SDS-PAGE, zymography, RNA phenotyping, in situ hybridization, and immunohistochemistry analyzed by image processing and analysis combined with morphometry will be used to achieve these aims. The results of these studies will increase our knowledge of the interactions of ECM and matrix-degrading enzymes in the process of palate morphogenesis. Such an understanding may lead to more precise identification of agents that can disrupt the process as well as those that might reverse or compensate for the effects of disruptive agents or defective genes.
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