Nephronectin Promotes Migration of the Periocular Mesenchyme via EGFR Signaling

Purpose: Nephronectin (Npnt) is an extracellular protein with various functions during development and homeostasis of various tissues, but its role during early mouse cornea development remains unexplored. We characterized the expression of Npnt and investigated its function during cornea development. Methods: We performed in situ hybridization and immunohistochemistry on ocular sections obtained from embryonic day (E)11.5-E16.5 mouse embryos. Wild-type and Npnt mutant embryos were analyzed for corneal thickness, cell number, and cell proliferation. We assessed the expression of Npnt receptors, Integrin alpha 8 (Itgα8) and Epidermal growth factor receptor (EGFR). We performed in vitro migration assays using periocular mesenchyme (POM) explants in the presence of EGFR and STAT3 inhibitors. Results: Npnt mRNA was expressed in the ectoderm, lens, optic cup, and subsequently in the cornea endothelium. Npnt protein correlated with mRNA staining with additional localization in the epithelial basement membrane and POM. Npnt mutants exhibited reduced corneal thickness and cell numbers compared to wild-type littermates, but there were no differences in cell proliferation. Itgα8 mRNA was diffusely expressed in the POM, but not in the migratory regions. EGFR mRNA was expressed in the region of migratory POM. Robust cell migration occurred from POM explants cultured on either Npnt or Fibronectin (Fn) substrates, but the EGFR pathway inhibitors only attenuated migration on the Npnt substrate. Conclusions: Npnt is expressed in the periocular region during ocular development and contributes to POM migration into the nascent cornea via a mechanism that involves activation of the EGFR signaling pathway.