Effects Of Age-specific And Tissue-specific Knockdown Of Pax6 On Adult Corneal Homeostasis In Mice

Abstract

Purpose: : Reduced PAX6 expression causes aniridia, abnormal eye development and corneal deterioration in adults. Heterozygous Pax6^+/- mice provide a good model of aniridia and display a comparable range of eye abnormalities and corneal deterioration but also have small eyes. Some eye abnormalities occur during development while others arise or worsen in adulthood. Through the use of Pax6 conditional knockouts we aim to identify the roles of Pax6 in the adult cornea. Specifically, we aim to dissect when, and in what tissues, Pax6-deficiency causes corneal abnormalities.

Methods: : Pax6 expression was manipulated through the use of mice with a floxed Pax6 allele (Pax6^fl) in which loxP sites have been inserted in introns 4 and 6. Following exposure to Cre recombinase Pax6^fl is converted to Pax6^Δfl null allele. We employed Le-Cre; Pax6^fl/+ transgenic mice to investigate the effects of tissue-specific knockdown of Pax6 in the surface ectoderm derived tissues of the eye. We also treated CAGG-CreER^TM; Pax6^fl/+ mice with tamoxifen to investigate the effects of an age-specific (but ubiquitous) knockdown of Pax6 after the eyes have developed normally. Tamoxifen was injected (100 µg/g body weight; i.p.) on 5 consecutive days beginning at 8-9 weeks of age. A panel of phenotypic endpoints was investigated in experimental and control eyes 6 weeks later to determine the consequences of age/tissue specific manipulation of Pax6 expression.

Results: : Le-Cre; Pax6^fl/+ mice had small eyes, fewer corneal epithelial cell layers and goblet cells accumulated in the corneal epithelium. Pax6 and K12 immunostaining levels were reduced in the corneal epithelium. Unexpectedly, these mice also showed abnormal lens and retina morphology. Treatment of adult CAGG-CreERTM; Pax6^fl/+ mice with tamoxifen also resulted in reduced corneal Pax6 and K12 immunostaining but eye size and corneal epithelial stratification were normal and goblet cells were absent from the cornea.

Conclusions: : These preliminary results imply that deletion of one Pax6 allele solely in the surface ectoderm derivatives is sufficient to cause some of the corneal abnormalities reported for Pax6^+/- heterozygotes. Small eyes, reduced corneal epithelial stratification and accumulation of goblet cells in the cornea are more likely to occur when Pax6 is depleted before birth.

Keywords: cornea: epithelium • cornea: basic science • development