Conditional Deletion of AP-2β in the Periocular Mesenchyme of Mice Promotes Corneal Conjunctivalization

Abstract

Purpose : The corneal endothelium and stroma derive from the periocular mesenchyme (POM), which originates from neural crest cells (NCCs), while the corneal epithelium stems from the anterior surface ectoderm. Signalling between the POM and surface ectoderm regulates epithelial proliferation and cell fate. Activating Protein-2 beta (AP-2β), encoded by Tfap2b, is a transcription factor expressed in the POM. We have previously shown that conditional knock out AP-2β generates a unique anterior segment phenotype that includes a closed iridocorneal angle, absent corneal endothelium and reduced corneal epithelial stratification. The purpose of this investigation was to further determine the impact of AP-2β deletion in the POM on the corneal epithelial phenotype.

Methods : To generate the AP-2β NCC KO model, male mice heterozygous for Tfap2b, Tfap2b^+/-, and the wnt1-cre transgene, Wnt1Cre^+/-, were bred with female mice in which the Tfap2b gene was floxed, Tfap2b^lox/lox. AP-2β expressing offspring from this breeding scheme were used as age-matched controls. Eyes were enucleated from euthanized 2-3-month-old mice and processed before being embedded in paraffin. Immunohistochemistry was conducted using primary antibodies specific to Keratin-12 (K12), a corneal epithelial marker, and Keratin-15 (K15), a marker of conjunctival and limbal epithelia.

Results : Mature AP-2β NCC KO mice have a distinct corneal phenotype, with significantly reduced stratification, an absent endothelium and disorganized, vascular stroma. 2-3-month-old eye sections from wild-type mice demonstrated consistent immunostaining of K12 across the corneal epithelium, while K15 was confined to the limbal region and continued throughout the conjunctival epithelium. In contrast, AP-2β NCC KO mice demonstrated significantly reduced expression of K12 across the corneal epithelium, while K15 was observed consistently across the entire corneal epithelium, continuous with the conjunctiva.

Conclusions : The observed phenotypic changes in keratin expression indicate a shift towards conjunctival cell fate during development as a result of AP-2β NCC KO. This change to the epithelial phenotype is consistent with other signs of corneal conjunctivalization seen in the AP-2β NCC KO model, including stromal neovascularization.

This is a 2021 ARVO Annual Meeting abstract.