Abstract
Purpose :
Corneal developmental abnormalities that occur due to absence of limbal region often leads to corneal thinning and opacity due to an invasion of conjunctival cells into the cornea epithelium resulting in corneal conjunctivalization. Recently, by deleting transcription factor activating protein 2 beta (AP-2β) in neural crest cell (NCC) using the Wnt1Cre recombinase system that conditionally deletes alleles in the migrating NCC in mice (AP-2β NCC KO), we observed a similar pathological condition as seen in human corneal conjunctivalization. The purpose of this investigation was to determine the impact of AP-2β deletion in the NCC on the corneal epithelial phenotype.
Methods :
To generate the AP-2β NCC KO mouse model, male heterozygous for Tfap2b, tfap2b+/-, and the wnt1-cre transgene, Wnt1Cre+/-, were bred with female mice having floxed Tfap2b gene, tfap2blox/lox. Wild-type littermates were used as controls. Mice were euthanized, eyes enucleated. processed and embedded in paraffin. Immunohistochemistry (IHC) was conducted using primary antibodies specific to Keratin 13 (K13), a marker of conjunctival epithelium, ABCB5 or ABCG2, limbal epithelial stem cell (LESC) markers and active β-catenin (p-489). Pulse-chase experiments were performed by injecting BrdU (5-Bromo-2-Deoxyuridine) (50μg/g body weight) in the control and the mutant at various time-points to determine the status of label retaining cells (LRCs)/stem cells.
Results :
K13 expression was confined to the conjunctival epithelium in the control mice , while AP-2β NCC KO mice showed an expansion of K13 across corneal epithelium, continuous with the conjunctiva. Furthermore, ABCB5 expression was reduced in the limbal region of the mutants. The pulse-chase experiments show a change in the pattern of distribution of LRCs in the corneal epithelium of the mutant when compared to the control. Analysis of signaling cascade reveals an increase in expression and nuclear localization of active β-catenin in the central corneal epithelium of AP-2β NCC KO when compared to control.
Conclusions :
The observed changes in K13 expression indicate a shift towards conjunctival cell fate whereas the decrease in ABCB5 expression and a change in LRC distribution in AP-2β NCC KO suggests modulation of stem cells due to the absence of AP-2β. The increased expression of active β-catenin indicates changes in signaling cascade required for corneal epithelial development by AP-2β.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.