The Corneal Epithelial Barrier and Its Developmental Role in Isolating Corneal Epithelial and Conjunctival Cells From One Another.

Purpose: During development, the corneal epithelium (CE) and the conjunctiva are derived from the surface ectoderm. Here we have examined how, during development, the cells of these two issues become isolated from each other. Methods: Epithelia from the anterior eyes of chicken embryos were labeled with the fluorescent, lipophilic dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). DiI was placed on the epithelial surface of the developing anterior eye and its diffusion was monitored by fluorescence microscopy. Concomitant morphologic changes in the surface cells of these epithelial were examined by scanning electron microscopy. Immunofluorescence was used to analyze the expression of cytokeratin K3, ZO-1, N-cadherin and Connexin-43 and the function of gap junctions was analyzed using a cut-loading with the fluorescent dye rhodamine-dextran. Results: Prior to embryonic day 8 (E8), DiI placed on the surface of the CE spreads throughout all the epithelial cells of the anterior eye. When older eyes were similarly labeled, dye diffusion was restricted to the CE. Similarly, diffusion of DiI placed on the conjunctival surface after E8 was restricted to the conjunctiva. Scanning electron microscopy showed that developmentally (1) physical separations progressively form between the cells of the CE and those of the conjunctiva, and (2) by E8 these separations form a ring that completely encompasses the cornea. The functional restriction of gap junctions between these tissues did not occur until E14. Conclusions: During ocular development, a barrier to the diffusion of DiI forms between the contiguous CE and conjunctiva prior to the differential expression of gap junctions within these tissues.