Targeted Deletion of AP–2 in the Corneal Epithelium Results in Defects in Bowman's Layer and Subsequent Activation of Stromal Fibroblasts

Abstract

Abstract: : Purpose: Our earlier studies have shown a requirement for AP–2α transcription factor in early eye and lens development and in the regenerating corneal epithelium. However, we have been unable to address the specific role for AP–2α in corneal epithelial differentiation due to the lethality of AP–2α null mice at birth. Our recent creation of a conditional knockout of AP–2α in the lens placode has provided us with a model for examining the requirement of the AP–2α gene in corneal epithelial differentiation. Methods: A line of mice expressing Cre–recombinase specifically in the early lens placode (Le–Cre) was crossed with mice in which the AP–2α allele has been flanked by two loxP sites. Conditional knockout of AP–2α in the developing lens placode resulted in a targeted deletion of AP–2α in the lens placode and its derivatives including the differentiating corneal epithelium. Results: Embryonic Le– AP–2α mutant mice exhibited isolated ocular phenotypes including microphthalmia and a lens stalk in which the lens had failed to separate from the overlying ectoderm. In postnatal Le– AP–2α mutants, adhesion of the lens and cornea persisted in a small region of the peripheral cornea. The remaining corneal epithelium was stratified similar to wild–type littermates. However, PAS staining revealed that the basement membrane, or Bowman’s layer, in the mutants was substantially thinner than wild–type mice and in many regions was discontinuous. Immediately beneath the basement membrane of the mutant corneas stromal fibroblasts accumulated and showed immunoreactivity for PCNA and αSMA indicative of fibroblast activation and a wound healing response. Conclusion: These findings suggest that AP–2α may be required for regulating expression of a basement membrane component(s). The activation of the stromal fibroblasts was likely a secondary event related to the disrupted Bowman’s layer. Further studies will determine the specific alteration(s) in basement membrane component expression in the mutants and whether one or more of the components are directly regulated by AP–2α.

Keywords: cornea: epithelium • transgenics/knock–outs • gene/expression