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.

National Institute of Health (NIH)
National Eye Institute (NEI)
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Visual Sciences A Study Section (VISA)
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Kansas State University
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Mao, Xiuli; Zhang, Yuntao; Schwend, Tyler et al. (2015) Effects of polysialic acid on sensory innervation of the cornea. Dev Biol 398:193-205
Zhang, Yuntao; Mao, Xiuli; Schwend, Tyler et al. (2013) Resistance of corneal RFUVA–cross-linked collagens and small leucine-rich proteoglycans to degradation by matrix metalloproteinases. Invest Ophthalmol Vis Sci 54:1014-25
Littlechild, Stacy L; Zhang, Yuntao; Tomich, John M et al. (2012) Fibrinogen, riboflavin, and UVA to immobilize a corneal flap--molecular mechanisms. Invest Ophthalmol Vis Sci 53:5991-6003
Zhang, Guodong; Boyle, Daniel L; Zhang, Yuntao et al. (2012) Development and mineralization of embryonic avian scleral ossicles. Mol Vis 18:348-61
Littlechild, Stacy L; Brummer, Gage; Zhang, Yuntao et al. (2012) Fibrinogen, riboflavin, and UVA to immobilize a corneal flap--conditions for tissue adhesion. Invest Ophthalmol Vis Sci 53:4011-20
Zhang, Yuntao; Sukthankar, Pinakin; Tomich, John M et al. (2012) Effect of the synthetic NC-1059 peptide on diffusion of riboflavin across an intact corneal epithelium. Invest Ophthalmol Vis Sci 53:2620-9
Schwend, Tyler; Lwigale, Peter Y; Conrad, Gary W (2012) Nerve repulsion by the lens and cornea during cornea innervation is dependent on Robo-Slit signaling and diminishes with neuron age. Dev Biol 363:115-27
Schwend, Tyler; Deaton, Ryan J; Zhang, Yuntao et al. (2012) Corneal sulfated glycosaminoglycans and their effects on trigeminal nerve growth cone behavior in vitro: roles for ECM in cornea innervation. Invest Ophthalmol Vis Sci 53:8118-37
Brummer, Gage; Littlechild, Stacy; McCall, Scott et al. (2011) The role of nonenzymatic glycation and carbonyls in collagen cross-linking for the treatment of keratoconus. Invest Ophthalmol Vis Sci 52:6363-9
Zhang, Yuntao; Conrad, Abigail H; Conrad, Gary W (2011) Effects of ultraviolet-A and riboflavin on the interaction of collagen and proteoglycans during corneal cross-linking. J Biol Chem 286:13011-22

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