Investigation of Pax6 Function in Mouse Cornea Using an Inducible Deletion Mouse Model, K12rtTA/tetOCre/Pax6flox

Abstract

Purpose: : Study role of Pax6 in K12⁺ differentiating mouse corneal epithelial cells in vivo.

Methods: : We crossed K12^rtTA/rtTA with tetOCre and Pax6^flox/flox transgenic mice to ablate Pax6 in corneal epithelium of triple transgenic animal, K12^rtTA/rtTA/tetOCre/Pax6^flox/flox upon doxycycline induction. Pax6 was conditionally deleted in K12⁺ differentiating cells in the corneal epithelium of K12^rtTA/rtTA/tetOCre/Pax6^flox/flox and K12^rtTA/rtTA/tetOCre/Pax6^flox/+ mice by feeding them 1g/kg doxycycline chow. Inducible deletion of Pax6 in corneal epithelial cells was performed starting during embryonic development prior to birth, 3 weeks and 6 weeks after birth and ending at 14-15 weeks (three treatment durations total). The effect of homozygous (Pax6^ced/ced) or heterozygous (Pax6^ced/+) corneal epithelial-specific Pax6 deletion was examined by stereomicroscope. Corneal epithelium integrity was examined by immunohistochemistry. All reported research was conducted in compliance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research.

Results: : For mice with embryonic Dox-induced Pax6 deletion, the corneas of Pax6^ced/ced developed corneal haze evident at eight weeks old, earlier than haze seen in corneas of Pax6^ced/+. A change in epithelial cell morphology was already seen at three weeks, as indicated by phalloidin staining. The corneas of some triple transgenic mice induced at 3 weeks-old also developed corneal haze at 14 wks, but most of mice induced at 6 weeks-old did not develop corneal haze.

Conclusions: : The K12^rtTA/rtTA/tetOCre/Pax6^flox/flox transgenic line allows us to study the functional role of Pax6 in the cornea independent of Pax6 effects from other tissues such as the lens, ciliary bodies and iris. Surprisingly, we found that lack of Pax6 expression in K12⁺ cells in adult mice did not affect cornea clarity in most mice, suggesting that Pax6’s contribution to maintenance of corneal epithelial homeostasis may be in progenitor/basal cells.

Keywords: cornea: basic science • cornea: epithelium • genetics