Abstract
Purpose:
To determine whether Lhx2 is required for maintenance of the corneal epithelial stem cell compartment.
Methods:
Lhx2 expression was evaluated in Lhx2-EGFP, Lhx2 conditional knock out (cKO; k14-cre X lhx2flox/flox) and wild type CD1 mice. Central and limbal corneas were analyzed by qPCR for Lhx2 and GFP, ABCG2 and P63. Corneal sections were immunostained for GFP and Lhx2. In vitro scratch assays using cultured corneal epithelial cells isolated from adult Lhx2cKO and wild type mice were performed. Lhx2 and wild-type mice were subjected to repeated corneal debridements, and epithelial healing was determined by using slit lamp imaging. PAS staining and antibodies against Krt15 were used to detect conjunctival and goblet cells.
Results:
Lhx2 and GFP proteins were located in the basal cells of the central cornea and marked all 3-4 cells layers of peripheral cornea. qPCR revealed that there is a significantly higher expression of GFP in the limbal epithelium versus the central, but no significant difference was seen when testing Lhx2 primers. In the Lhx2cKO mice, stem cell markers ABCG2 and P63 were significantly lower than wild type mice. Scratch assays showed a delay in recovery of injured corneal epithelial cells isolated from Lhx2cKO when compared to wild type cells. A subset of Lhx2 cKO animals demonstrated spontaneous epithelial defects, ocular surface neovascularization and corneal opacities. A comparison of Lhx2 cKO animals without a spontaneous phenotype and wild-type animals subjected to corneal epithelial debridement revealed similar wound healing in both groups after a single injury. However, repeated debridement resulted in overall slower wound healing in cKO animals with an increasing proportion of wound healing delay with each successive debridement. Moreover, a subset of cKO animals developed persistent epithelial defects, neovascularization, or opacification. PAS and Krt15 immunostaining showed that multiple debridements resulted in conjunctivilization in cKO animals, but not wild-type mice.
Conclusions:
Lhx2 can be used as a novel marker of corneal epithelial stem cells given its localization to basal corneal epithelial layers and its co-expression with other putative stem cell markers. Lhx2cKO plays a role in maintaining the stemness capacity of the corneal epithelium and may constitute a novel target for cornea repair.
Keywords: 482 cornea: epithelium •
721 stem cells •
740 transgenics/knock-outs