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

Abstract

PurposeDuring 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.MethodsEpithelia 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.ResultsPrior 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.ConclusionsDuring 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.

Corneal-Committed Cells Restore the Stem Cell Pool and Tissue Boundary following Injury

“…The underlying limbal/conjunctival stroma is probably involved in limbal boundary formation under homeostasis and its re-establishment following injury. Mechanisms engaged to prevent corneal-conjunctival cell mixing (Kubilus et al, 2017) may induce border refinement and should be active throughout life to prevent lineage intermixing. Notably, corneal cells that repaired the marginal conjunctiva (Figures 3G-3I) did not persist long-term, presumably due to extinction in the foreign tissue and/or because they were dragged by ''alike'' cells, in line with differential adhesion or differential interfacial tension models (Batlle and Wilkinson, 2012;Dahmann et al, 2011).…”

“…The corneal epithelium is the outermost layer that covers the eye. Its transparency, high accessibility, and segregated compartments for stem cells (SCs) and differentiated cells makes it an excellent model for studying SCs and boundary maintenance (Kubilus et al, 2017;Secker and Daniels, 2008). The location of corneal epithelial SCs is at the limbus, a narrow ring-shaped boundary between the translucent cornea and the opaque conjunctiva (Amitai-Lange et al, 2015a;Cotsarelis et al, 1989;Di Girolamo et al, 2015;Huang and Tseng, 1991).…”

Understanding cornea epithelial stem cells and stem cell deficiency: Lessons learned using vertebrate model systems

“…LSCD is a debilitating corneal disease that results from functional or anatomical loss of the CESC population. In LSCD, the transparent cornea epithelium cannot be renewed, the corneal epithelial barrier that separates the cornea and the conjunctiva cannot be maintained (Kubilus, Zapater, & Linsenmayer, 2017), and the cornea is replaced by surrounding conjunctival epithelial cells (conjunctivalization; Figure 1a). Pterygium appears to be a related condition, in which fibrovascular tissue grows from the conjunctiva onto the cornea (T. Liu, Liu, Xie, He, & Bai, 2013).…”

“…In one study, DiI labeled cells were cultured on AMs for transplantation onto cat corneas, which was helpful in evaluating efficacy of this treatment method (Fan et al, 2013). Another study utilized DiI labeling of corneal and conjunctival cells during development of chick eyes, and showed the formation of a boundary between corneal and conjunctival cells during development (Kubilus et al, 2017). A more recent method using Quantum dot nanocrystals (Qdots) has been used to track cultured limbal epithelial cells for transplantation (Genicio, Gallo Paramo, & Shortt, 2015).…”

Plasticity of ocular surface epithelia: Using a murine model of limbal stem cell deficiency to delineate metaplasia and transdifferentiation

“…Under steady-state conditions, the limbus provides a physical and biochemical barrier that maintains epithelial identity within the corneal and conjunctival compartments, preventing cells on either side from mixing ( Kubilus et al., 2017 ). Notably, when the limbus is severely damaged to the extent that its LESCs are depleted, as is the case in limbal SC deficiency (LSCD) ( Deng et al., 2019 ; Rama et al., 2010 ), the barrier function imposed by this site is lifted, and a pathological wound-healing response referred to as “conjunctivalization” materializes, which is characterized by the invasion of an inflamed fibrovascular pannus that enshrouds the cornea and obscures vision.…”