Altered fate of corneal epithelial cells in Dstn^corn1 mice

Abstract

Purpose : Dstn^corn1 mice, deficient mutants of the actin depolymerizing factor destrin, display epithelial hyperproliferation, inflammation and neovascularization in the cornea. In the Dstn^corn1 corneal epithelium, we observed keratin 12 (Krt12)-positive cells that are also positive for keratin 15 (Krt15), a marker of corneal epithelial progenitor cells that normally reside in the limbus. The purpose of this study is to test the hypothesis that abnormal actin dynamics lead to transformation of Krt12-positive corneal epithelial cells into the less differentiated state in Dstn^corn1 mice.

Methods : The mTmG double fluorescent cre reporter system was used to genetically label specific cell populations for cell lineage tracing. Specifically, we generated mice with the genotypes of Dstn^corn1/corn1; Krt12^rtTA/rtTA; tetO-cre; mTmG/+, and Dstn^corn1/+; Krt12^rtTA/rtTA; tetO-cre; mTmG/+. Doxycycline (dox) was administered through diet for 10 days to label the differentiated Krt12-positive cells with green fluorescence (mG), during which mT reporter gene cassette was deleted by cre expressed in Krt12-positive cells. The mG was chased upon the withdraw of dox from the diet for 0, 2 and 4 days, and excised corneas were subjected to immunohistochemistry using the Krt15 antibody. The corneal area positive for mG and the total corneal area were quantified using ImageJ. Analysis of variance was used with Tukey’s honest significant difference test for statistical analysis.

Results : The mG-positive area normalized to the total corneal area was the largest immediately after the completion of dox administration (day 0), and decreased in time both in control and Dstn^corn1 mice. The mG-positive corneal area was less in Dstn^corn1 mice compared to control mice at each time point. In the Dstn^corn1cornea, we observed mG labeled cells that were also stained by Krt15 antibody.

Conclusions : Our findings indicate that the Krt12-positive terminally differentiated corneal epithelial cells transform to the less differentiated state in the corneal epithelium of Dstn^corn1 mice. The mG-positive area was larger in control mice compared to Dstn^corn1 mice at each time point, suggesting that cells in the Dstn^corn1 corneal epithelium may have a higher renewal rate compared to control mice.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.