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.2017 Mar 1;58(3):1665-1672.
doi: 10.1167/iovs.16-20737.

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

Affiliations

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

James K Kubilus et al. Invest Ophthalmol Vis Sci..

Abstract

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.

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Figures

Figure 1
Figure 1
DiI labeling in embryonic chicken cornea. Brightfield (A) and immunofluorescent (B) images of the same DiI-labeled E10 cornea.Arrow inA shows crystals on CE surface.Arrowhead inB indicates sharp border of DiI diffusion (red);arrows inB show DiI-labeled nerves.Inset inB shows a section of DiI (red)-labeled cornea with nuclei stained with DAPI (blue);arrow ininset indicates Bowman's layer separating CE (above) from stroma (below).
Figure 2
Figure 2
Analysis of DiI diffusion in chick cornea. (A) Section of DiI (red)-labeled E9 cornea in which one small crystal placed on the CE surface shows gradual diffusion of red fluorescence within CE. (B) DiI diffusion from a large crystal of DiI in section of E13 cornea at the CE–conjunctival border shows an abrupt loss of red fluorescence (arrow) just peripheral to the end of Bowman's layer (arrowhead).Dashed line indicates Bowman's layer separating the epithelium from the stroma below. Section inC of E13 CE–conjunctival border immunolabeled for type V collagen (green) shows termination of Bowman's layer as thegreen label is lost. (D) Low-magnification image corneal–conjunctival border marked in the underlying stroma by *-labeled cytokeratin K3 (red) shows absence of label in the ocular surface at E8.E shows E10 cornea labeled for K3 (red) with heterogeneous staining in the apical layers of the corneal epithelium. At E17 (F), K3 (red) marks the corneal epithelium (arrow); at the corneal–conjunctival junction, label is lost in the basal cell layer (arrowheads) and completely lost more peripherally (*). Nuclei stained with DAPI (blue) in all images.Scale bar inC denotes 50 μm and is the same forA–C andE.Scale bar inD denotes 200 μm;scale bar inF denotes 100 μm.
Figure 3
Figure 3
DiI diffusion in the conjunctiva. DiI crystals placed on the conjunctival surface of an E10 anterior eye and imaged after 1 (A), 2 (B), and 4 weeks (C). Images are brightfield with overlayed red fluorescence to show DiI.Arrows inB andC show DiI diffusing along the CE–conjunctival border but into CE.
Figure 4
Figure 4
Temporal analysis of DiI diffusion. DiI (red) placed on the corneal surface in whole mounts of the head of the embryo was imaged by brightfield with red fluorescent overlay (A) and fluorescent channel alone (B) to show DiI diffusion throughout anterior eye at E6. InC, 20× images and a section E6 DiI-labeled cornea were stitched together to show DiI diffusion from the cornea into the conjunctiva (arrows), past the developing ciliary body (arrowheads). Similarly, merged images at E7 (D) show DiI still able to diffuse into conjunctiva (arrows).E andF are DiI-labeled E8 whole mounts imaged similarly toA andB but show DiI-restricted to the CE.Scale bar inB (also forA,E, andF) denotes 500 μm;scale bars inC andD denote 200 μm.
Figure 5
Figure 5
Scanning electron microscopy of anterior eye surface from E6 through E8 and ZO-1 expression. Anterior eyes were imaged en face by SEM at E6 (A), E7 (B), and E8 (C).Inset inB is higher power of region enclosed bydashed box. Arrows inB show fenestrations at E7 that elongate into connections at E8 (arrows inC) at the corneal–conjunctival border.D andE are the same section labeled for nuclei with DAPI (blue inD) and ZO-1 (red inE), with an * marking the position of the corneal–conjunctival transition. Note no difference inred punctate labeling between apical cells in the conjunctival (arrowhead) versus corneal (arrow) epithelium.Scale bars inA denote 213 μm; inB denote 111 μm; ininset inB denote 48 μm; and inC denote 23 μm.
Figure 6
Figure 6
Gap junctions in the embryonic chicken anterior eye. Sections of the corneal–conjunctival border at E8 (A) and E14 (B) were immunolabeled for CX43 (red) with nuclei stained with DAPI.Arrowhead andarrow inA show labeling throughout the corneal epithelium and conjunctiva (respectively) at E8, but at E14 (B), label is lost in the conjunctiva (arrows inB) peripheral to the corneal–conjunctival border (*). Note strong punctatered label central to border at E14 but absent peripherally.Green label inB shows cells labeled for the limbal stem cell marker N-cadherin, peripheral to the junction. Cut loading with rhodamine-dextran 3000 at E8 (C) and E14 (D) show diffusion of the dye (red) through gap junctions from the CE (arrowhead) to the conjunctiva (arrowhead) through the corneal–conjunctival junction (*) at E8 inC. At E14 (D), rhodamine-dextran 3000 does not diffuse into the basal layers of limbal epithelium (arrows) from the CE (arrowhead), peripheral to the corneal–conjunctival border (*).Dashed line in all image shows the location of the basement membrane separating the epithelium from the underlying stroma.
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