The most distinctive product of neural crest differentiation in the cornea is keratan sulfate proteoglycan (KSPG). This extracellular, sulfated, N-linked glycoprotein is more abundant in cornea than in any other tissue. Although good chemical characterization of KS polysaccharide has been performed, little is known about the core proteins of KSPG. Some studies suggest multiple forms of corneal KSPG. Our preliminary data suggest as many as 5 proteins associated covalently or non-covalently with KSPG. Synthesis of KS polysaccharide by isolated, differentiated corneal stroma cells (keratocytes) in vitro occurs for only short periods of time. During corneal wound healing, synthesis of antigenically altered KS in the scar is detected. During normal corneal development in avian embryos, neural crest cells do not express antigens of mature KS until after the cells invade the primary corneal stromal. Corneal nerves invade the stroma shortly after extracellular KS is detected. Given this background, we propose to test three theories. Hypothesis 1: There are two classes of KSPG: a """"""""constitutive"""""""" form present in most tissues including cornea, and a """"""""tissue-specific"""""""" form; KSPG-binding proteins are associated with both forms. Chick corneal KSPG core proteins will be isolated, 10- 20 residues of each N-terminal sequence will be determined, peptides duplicating these sequences will be synthesized chemically, polyclonal antibodies will be prepared against each peptide, and expression of the antigens will be localized in developing chick eyes and other tissues. Hypothesis 2: Cornea- specific KSPG synthesis is regulated by the extracellular environment: 1) diffusible factors from other corneal cell-types or aqueous humor, 2) insoluble components of the stromal extracellular matrix, or 3) physical forces and/or dehydration of the stroma. Antibodies will be used to determine whether or when KSPG core protein antigens stop being expressed in vitro. One organ culture system for neural crest differentiation will allow experimental alteration of corneal cell-types and perturbation of extracellular matrix. Another organ culture system will assay stability of keratocyte differentiation within altered matrices and test the effect of constant tensile stress on KSPG synthesis. Hypothesis 3: Nerves invade the corneal stroma during embryogenesis in response to extracellular deposition of KSPG in the stroma. Inhibitors of N-linked glycosylation and processing will be used to perturb synthesis of KSPG, and effects on corneal innervation will be recorded.
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