Long-term objectives: Identify factors causing differentiation of tissue-specific populations of fibroblasts in the eye, with emphasis on the cornea. The transparent cornea is remarkable because it contains the highest concentration of keratan sulfate proteoglycan (KSPG) of any tissue and the highest density of nerve endings of any body surface region.
Specific Aims and Methods: 1) Determine how cornea-specific (and shared) isoforms of KSPG core proteins are synthesized. They may arise from alternative RNA splicing or from separate but related genes. Analyses of corneal polyA+RNA will eliminate one of these hypotheses. 2) Experimentally determine roles of each KSPG isoform during corneal development. Freshly isolated quail cranial neural crest first will be transfected with a plasmid carrying sequence for one KSPG isoform in reverse (antisense) orientation and then will be used for orthotopic orthochronic unilateral transplants into chick embryos. Chimeric corneas with altered transparency or innervation will provide data on roles of KSPGs. 3) Characterize factors regulating synthesis of each corneal KSPG isoform core protein. Growth factors and periocular cells will be added to neural crest and keratocytes in vitro to test for synthesis of KSPG isoform-specific mRNAs and proteins. 4) Determine relationships between molecular features of KSPG and development of corneal nerves. Specific KSPG isoforms of high or low degree of KS glycosaminoglycan chain sulfation will be used as substrates to test for effects on neural crest differentiation into fibroblasts or nerves, and to test neurite outgrowth from crest- and/or ectodermal placode-derived cells of the trigeminal ganglion (corneal nerve source). 5) Characterize putative receptors for KSPG on corneal cell types. Corneal cell surface proteins will be allowed to bind (reversibly) to KSPG, solubilized, characterized (native and peptide N-terminal sequencing), and used to make antibody against the whole protein and/or peptides. Using oligonucleotide probes based on receptor amino acid sequence, the cDNAs for these receptor proteins will be isolated, and expression in chimeric corneas perturbed with antisense probes. Health relevance: Factors that would stimulate keratocyte synthesis of specific KSPG isoforms or would make the stromal matrix more permissive/penetrable by neurite growth cones might hasten reinnervation of transplanted human corneas, a process currently requiring 12-18 mos.
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