Function of Nephronectin in the corneal ECM during development, homeostasis, and wound healing

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Dysgenesis of the anterior eye together with corneal diseases and injuries are major causes of ocular defects and loss of vision. The periocular neural crest cells (pNC) are multipotent embryonic cell population that provide crucial signals and contribute to the cellular and extracellular components of the cornea, but the molecular mechanisms underlying these processes are still not well understood. We have identified novel expression of a recently discovered extracellular matrix (ECM) protein, nephronectin (Npnt), during corneal development. Although Npnt has been shown to function in various developmental processes, and identified in pathological conditions including cancer metastasis, it has not been studied in the cornea. This project seeks to understand the mechanisms of Npnt function in the cornea by testing the hypothesis that Npnt promotes cell migration and attachment during corneal development, homeostasis and wound healing. Our ongoing studies have identified that Npnt is expressed in the ECM of the presumptive cornea whereas its major receptor Itgα8 is expressed by the pNC prior to and during migration. In addition, we observed spatial differences in the localization of Npnt to the chick epithelial and mouse endothelial basement membranes, and that it is maintained throughout adulthood in mice. Based on these observations, we will take advantage of mouse genetics and the ease of manipulating avian eyes, combined the spatiotemporal differences in Npnt expression in the two models, to provide a comprehensive understanding of the function of Npnt in the cornea. Our preliminary studies of Npnt knockdown and overexpression in chick show corneal thinning and thickening, respectively. In addition, we show that knockdown of Npnt causes corneal epithelial defects and that Npnt/Itgα8 signaling augments pNC migration in vitro. We will further examine the function of Npnt during pNC migration and in the corneal epithelial basement membrane and embryonic corneal wound healing. Analysis of Npnt knockout mice will indicate the function of Npnt during a different pattern of pNC migration, formation and maintenance of the Descemet's membrane, and function in adult corneal wound healing. We will also perform proteomic analysis to identify Npnt interacting partners and determine how the absence of Npnt affects the corneal ECM and basement membrane proteins. All proposed studies are supplemented with micromanipulation of chick embryonic eyes, in vitro culture and molecular biology techniques, and pharmacological inhibition of gene function. The following Specific Aims will test our hypothesis: (1) Determine the role of nephronectin during migration of pNC into the cornea. (2) Investigate the role of Npnt in the corneal epithelial and endothelial basement membranes. (3) Determine the function of Npnt during embryonic and adult corneal wound healing. The proposed studies will reveal novel functions of Npnt in cellular processes that are required for normal development, function, and repair of the cornea.