Hamilton PW and Henry JJ (2016), The lens regenerative competency of limbal vs. ...

XB-ART-52491

Exp Eye Res 2016 Nov 01;152:94-99. doi: 10.1016/j.exer.2016.08.013.

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The lens regenerative competency of limbal vs. central regions of mature Xenopus cornea epithelium.

Hamilton PW , Henry JJ .

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The frog, Xenopus laevis, is capable of completely regenerating a lens from the cornea epithelium. Because this ability appears to be limited to the larval stages of Xenopus, virtually all the work to understand the mechanisms regulating this process has been limited to pre-metamorphic tadpoles. It has been reported that the post-metamorphic cornea is competent to regenerate under experimental conditions, despite the fact that the in vivo capacity to regenerate is lost; however, that work didn't examine the regenerative potential of different regions of the cornea. A new model suggests that cornea-lens regeneration in Xenopus may be driven by oligopotent stem cells, and not by transdifferentiation of mature cornea cells. We investigated the regenerative potential of the limbal region in post-metamorphic cornea, where the stem cells of the cornea are thought to reside. Using EdU (5-Ethynyl-2'-deoxyuridine), we identified long-term label retaining cells in the basal cells of peripheral post-metamorphic Xenopus cornea, consistent with slow-cycling stem cells of the limbus that have been described in other vertebrates. Using this data to identify putative stem cells of the limbal region in Xenopus, we tested the regenerative competency of limbal regions and central cornea. These regions showed a similarly high ability for the cells of the basal epithelium to express lens proteins when cultured in proximity to larval retina. Thus, the regenerative competency in the post-metamorphic cornea is not restricted to stem cells of the limbal region, but also occurs in the transit amplifying cells throughout the basal layer of the cornea epithelium.

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Fig. 1. EdU label retention in post metamorphic corneas. A) Histological structure of a post-metamorphic cornea. Boxes with dotted lines indicate putative limbal regions, and box with solid line indicates central cornea (above pupillary space). After a 5 Day chase, EdU (red) can be observed throughout the basal layer of the epithelium (B) and the more superficial apical layers (C). Scale bar (100 μm) for both B and C is located in C. DAPI labeled nuclei are blue. D-E) EdU label (red) in 3 week (3W) cornea pelt. Boxes indicate locations of central and peripheral images in F-O. F-J) Corneas after 3 week chase show label retention in the peripheral cornea. F) An orthogonal projection of confocal data taken from cornea shown in D reveals the layers of the cornea epithelium (“CE”) that are retaining signal. The stroma (“S”) lies beneath the cornea. Arrow indicates the direction to the center of the cornea from the periphery. Arrowheads identify label retaining cells of the basal epithelium. G-H) Apical cell layers from the Z-stack used to create the orthogonal projection in (F). I-J) Basal cell layers from the Z-stack used to create the orthogonal projection in (F). K-O) Central cornea after 3 week chase has very little signal in basal layers, but some signal is still present in apical layers. * denotes macrophage-like cell commonly observed in basal layer of cornea epithelium (K, N). After 10 weeks of chase (P-AA), corneas possess label retaining cells in peripheral cornea (R-V), while the central cornea is relatively devoid of signal and the signal that remains is weak (W-AA). B, C, H, J, M, O, T, V, Y, and AA show an overlay of EdU (red) and DAPI (blue). Scale bar for D, E, P, and Q is 1 mm and is located in Q. Scale bar for G-J, L-O, S-V, and X-AA is 50 μm and is located in AA. Dorsal (“D”) Ventral (“V”) axis is shown in D and P.

Fig. 2. Regenerative capacity of the mature cornea. A) Zones of post-metamorphic donor corneas. Black dotted line represents putative limbal region. Scale bar in A is 1 mm. B-G) Representative cases of regeneration as defined by positive staining using an anti-lens antibody. Scale bar in B-G is 200 μm. H) Percent regenerative success as determined by the percentage of examined cases expressing lens proteins. Error bars indicate standard error. I) Mature cornea tucked inside larval retina is expressing lens proteins. J-K) Higher magnification showing expression in the same eye as (I) where lens crystallin protein expression is restricted to the basal layer of the cornea epithelium. Scale bar in I is 200 μm. Scalebar in J and K is 100 μm. J) Structure of mature cornea inside larval eyecup. Abbreviations: DL, dorsal limbal region; VL, ventral limbal region; CC, central cornea; S, stroma; CE, cornea epithelium; LR, larval retina; K, keratocytes; BE, basal cells of cornea epithelium; AE, apical cells of cornea epithelium.

References [+] :

Chung, Localization of corneal epithelial stem cells in the developing rat. 1992, Pubmed