Changes in connexin43 in early ocular surface development

Purpose. In the limbo-corneal epithelium the stem and early precursor epithelial cell pool is confined to the limbal rim. Among the features associated with this spatial segregation is the general paucity of connexin43 (Cx43) within the limbal basal cell population and its complete absence in resident stem cells. The limbo-corneal epithelial lineage derives from a Cx43-positive (Cx43⁺) embryonic outer ectoderm. Accordingly, as a means of identifying the process through which limbal cell phenotypes emerge, we investigated the expression of Cx43 in the ocular surface of embryonic rats. Methods. Ocular surface expression of Cx43 or K12 was determined in cryostat sections of rat embryos and eyes using immunohistological methods. Results. Changes in Cx43 expression revealed the early phenotypic divergence of three main epithelial cell phenotypes of the ocular surface. An analysis of the level and distribution pattern of Cx43 puncta lead to the identification of two distinct domains by embryonic day 10 (E10), a stage that occurs soon after formation of the lens vesicle. Additionally, at E12, ectodermal cells directly adjacent to the edges of the developing retina no longer express connexin. A comparison of anatomical and expression changes throughout embryonic development demonstrated that the two early zones represent the rudiments for the epithelia of the central cornea and conjunctiva, respectively, and that the isolated Cx43-negative (Cx43^-) cells represent the precursors of the basal and, putatively, stem cells of the limbal epithelium. Conclusions. Changes in Cx43 expression revealed that the phenotypic divergence of ocular surface epithelial cells and the generation of limbo-corneal stem cell precursors takes place at a very early stage in ocular development, ahead of the establishment of any identifiable anatomical or differentiation features for these domains.