Expression of a stabilized β-catenin mutant in keratocytes retards corneal epithelial stratification

Abstract

Purpose : Mouse corneal epithelium stratification is highly dependent on the intense communication between epithelium and stromal mesenchyme. We previously reported that deletion of β-catenin in mouse corneal stromal cells triggered precocious corneal epithelial stratification. In this study, the consequence of expression of a stabilized β-catenin mutant (Ctnnb1^ΔE3) in keratocytes was examined to further explore the role of Wnt/β-catenin signaling from corneal stromal keratocytes during the corneal development.

Methods : An inducible triple transgenic mouse line Kera^RT; TetO-Cre (TC); Ctnnb1^fE3 was generated to specifically express Ctnnb1^ΔE3 in keratocytes after Dox-induction. A quadruple transgenic mouse line Kera^RT; TC; Ctnnb1^fE3; Axin2^LacZ was further generated to detect Wnt/β-catenin signaling activity after Ctnnb1^ΔE3 expression in keratocytes. Mice were administrated Doxycycline (Dox) from embryonic day 0 (E0) to postnatal day 21 (P21). Collected eyeballs were subjected to examination by X-gal, H&E, immunofluorescence staining and RT-qPCR.

Results : X-gal staining showed that the Wnt/β-catenin signaling activity in the Dox-induced Kera^RT; TC; Ctnnb1^fE3; Axin2^LacZ mice was significantly augmented in the corneal stroma compared to Axin2^LacZ littermate controls. H&E staining indicated the central corneal epithelium in littermate controls stratified to 5-6 cell layers, while the Ctnnb1^ΔE3 corneal epithelium consisted of 1-3 cell layers. Interestingly, K12 immunostaining revealed the Ctnnb1^ΔE3 mice had small, flattened corneal basal cells at P21, which implied they maintained at a very early developmental stage. Reduced cell proliferation in the corneal basal layer of Ctnnb1^ΔE3 was revealed by Immunofluorescence staining against Ki-67 and PCNA compared to control. Notably, Bmp4 and ΔNp63 expression in the Ctnnb1^ΔE3 corneal epithelium was decreased at both protein and mRNA level. The expression of Pax6 and K14 in Ctnnb1^ΔE3 corneas was not altered.

Conclusions : Corneal epithelial stratification was retarded by the ectopic expression of Ctnnb1^ΔE3 in keratocytes, which was associated with downregulation of Bmp4 and ΔNp63 in corneal epithelium. These data combined with our previous loss of β-catenin function study confirmed that Wnt/β-catenin signal transduction in the corneal stroma dictates corneal epithelial stratification.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.