A conditional Pax6 depletion study with no morphological effect on the adult mouse corneal epithelium

The corneas of heterozygous Pax6 mice develop abnormally and deteriorate further after birth but it is not known whether the postnatal deterioration is predetermined by abnormal development. Our objective was to identify whether depletion of Pax6 in adult mice caused any corneal abnormalities, similar to those in Pax6 mice, where Pax6 levels are low throughout development and adulthood. We used two tamoxifen-inducible, Cre-loxP experimental strategies to deplete Pax6 either ubiquitously or in a restricted range of cell types. In a preliminary study, ubiquitous depletion of Pax6 by tamoxifen treatment of E9.5 CAG-CreER ;Pax6 embryos affected eye development. Tamoxifen treatment of 12-week old, adult CAG-CreER ;Pax6 and CAG-CreER ;Pax6 mice resulted in weak and/or patchy Pax6 immunostaining in the corneal epithelium but caused no corneal abnormalities. GFP staining in tamoxifen-treated CAG-CreER ;RCE:loxP reporter mice was also patchy. We attribute patchy Pax6 staining to mosaic deletion of the Pax6 allele, probably caused by mosaic CAG-CreER expression. In a parallel study, we treated adult Krt19-CreER ;Pax6 mice with tamoxifen to try to deplete Pax6 in limbal epithelial stem cells (LESCs) which replenish the corneal epithelium. However, Pax6 staining remained strong after a 12-week chase period so the Krt19-CreER transgene may have failed to target LESCs.