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Lingling Zhang, Yujin Zhang, Yen-Chiao Wang, Yuka Okada, Chia-Yang Liu; Expression of a stabilized β-catenin mutant in keratocytes retards corneal epithelial stratification. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2246. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
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 (Ctnnb1ΔE3) in keratocytes was examined to further explore the role of Wnt/β-catenin signaling from corneal stromal keratocytes during the corneal development.
An inducible triple transgenic mouse line KeraRT; TetO-Cre (TC); Ctnnb1fE3 was generated to specifically express Ctnnb1ΔE3 in keratocytes after Dox-induction. A quadruple transgenic mouse line KeraRT; TC; Ctnnb1fE3; Axin2LacZ was further generated to detect Wnt/β-catenin signaling activity after Ctnnb1ΔE3 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.
X-gal staining showed that the Wnt/β-catenin signaling activity in the Dox-induced KeraRT; TC; Ctnnb1fE3; Axin2LacZ mice was significantly augmented in the corneal stroma compared to Axin2LacZ littermate controls. H&E staining indicated the central corneal epithelium in littermate controls stratified to 5-6 cell layers, while the Ctnnb1ΔE3 corneal epithelium consisted of 1-3 cell layers. Interestingly, K12 immunostaining revealed the Ctnnb1ΔE3 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 Ctnnb1ΔE3 was revealed by Immunofluorescence staining against Ki-67 and PCNA compared to control. Notably, Bmp4 and ΔNp63 expression in the Ctnnb1ΔE3 corneal epithelium was decreased at both protein and mRNA level. The expression of Pax6 and K14 in Ctnnb1ΔE3 corneas was not altered.
Corneal epithelial stratification was retarded by the ectopic expression of Ctnnb1ΔE3 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.
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