The developing cornea offers an excellent opportunity to study the relation between fibroblast differentiation, nerve axon movement, and extracellular matrix biosynthesis. We propose to study three areas: 1) We have isolated conventional rabbit antisera to the predominant proteoglycan of the mammalian cornea, keratan sulfate-I proteglycan, and shown that they contain antibodies not only to the core protein, but to the keratan sulfate glycosaminoglycan chains as well. We propose to isolate cornea-specific monoclonal antibodies to corneal proteoglycans and corneal fibroblasts to use in determining when and where neural crest cells begin making cornea-specific products as they invade the developing eye, to determine under what circumstances the synthesis of keratan sulfate-I and its core protein can occur in cell and organ culture, and to determine the relation between keratan sulfate biosynthesis, that of its core protein, and the appearance of corneal transparency during development in birds and mammals. 2) We have devised a technique for staining corneal nerves in all phases of their innervation of the embryonic chick cornea. Phase I entails formation of a thick ring of nerves just outside the limbal area and completely encircling the cornea. Phase II entails migration of nerves from the nerve ring radially into the cornea. Phase I can be blocked by in ovo administration of DON, an inhibitor of amino sugar biosynthesis, with concomitant inhibition of sulfated glycosaminoglycan biosynthesis in the cornea, whereas Phase II is not inhibited. We proposed to use inhibitors of matrix biosynthesis and of neural function, as well as microsurgery, to study their effects on normal innervation, possible trophic relations between corneal epithelium and corneal nerves, and the appearance and unelucidated functions of choline acetyltransferase, acetylcholine, and acetylcholinesterase in the cornea. 3) Using a technique for 2-dimensional mapping 125I-labeled peptides, we propose to compare ocular and non-ocular collagens and proteoglycans, to determine if more than one keratan sulfate core protein exists in the cornea and to determine if structurally abnormal collagens and proteoglycans accumulate in the corneas of human patients with various eye diseases.
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