For a cornea to be functional, it must have the correct morphology and the proper physical characteristics including transparency to allow for passage of light and toughness to resist injury. Acquisition of these characteristics during embryonic development requires the synthesis and proper assembly of the extracellular matrices of the corneal stroma and Bowman's and Descemet's membranes. It also requires the precise temporal and spatial control of cellular differentiation within the three component tissue layers: the stroma, epithelium and endothelium. Based on our previous observations, we will directly test the hypothesis that within the avian corneal stroma the collagen fibrils are heterotypic structures composed of mixtures of collagen types I and V. We will also examine how these two collagens are arranged within the individual fibrils. The experiments will involve double-label immunoelectron microscopy with monoclonal antibodies against these collagens, and selective digestions of fibrils with collagen type-specific collagenases. We will also investigate whether, during fibrillogenesis, certain parameter(s) of fibril structure (i.e. diameter) are governed by the interactions of these two different collagen types and whether they are co-synthesized by the same cells. We will determine the tissue interactions and tissue-specific origin(s) of some of the matrix components present in Descemet's membrane, Bowman's membrane, or both, including collagen types I, IV & V and several different basement membrane antigens. We will also test the hypothesis that the developmental information necessary for stromal cell differentiation, and for maintenance of the differentiated state of these cells, is encoded within corneal extracellular matrix. We will culture presumptive corneal stromal cells (and other cell types) in contact with extracellular matrices from corneal stromas from various ages of embryos. As the marker(s) for stromal cell differentiation, we will employ a group of monoclonal antibodies we have produced and characterized as being against a developmentally regulated antigen(s) that is highly selective for corneal matrix. The antigen(s) itself has not yet been identified, but this we also hope to accomplish. If these experiments yield positive results, we will then isolate the differentiation-promoting factor(s) from the corneal matrices, characterize it, and use it in the production of monoclonal antibodies, which will provide probes for further studies.
