Role of transcription factor activating protein-2 beta (AP-2β) in corneal epithelial cell fate determination and stratification - Corneal surface pathologies such as corneal conjunctivalization arise mainly from the loss of limbal epithelial stem cells (LESCs) and can lead to corneal neovascularization, opacity and ultimately, blindness. Although the role of neural crest cells (NCC)-derived periocular mesenchyme (POM) has been established in development of the corneal stroma, its role in development of the corneal limbus and corneal epithelium has yet to be determined. It has been shown that transcription factors including activating protein-2 beta (AP-2β) play key roles in the development and differentiation of the POM. However, the role of AP-2β in POM-mediated corneal epithelial development remains largely unknown, as AP-2β null mice die soon after birth. To address this, we have specifically deleted AP-2β in the NCC of mice, using the Wnt1Cre-recombinase system (AP-2β NCC KO). Two major defects observed in these mice were corneal thinning and vascularization and contributing to this phenotype were an absence of the corneal endothelium and impairment in corneal epithelial stratification. Our scRNA-seq analyses along with RNAscope and immunohistochemistry revealed an absence of keratin-12 (K12), a corneal epithelial marker, and expansion of keratin-15 (K15) and K13, conjunctival epithelial specific markers, into the corneal epithelium of the mutant when compared to controls. Further investigations revealed an absence of ABCB5, a LESC marker, from the limbal region of the mutants indicating a conjunctival-like phenotype due to the absence of AP-2β in the NCC. In addition, bone morphogenetic protein (BMP) 4, a key player in corneal mesenchymal-to-epithelial signaling known to be modulated by Wnt/β-catenin during corneal epithelial stratification, was absent in the epithelium of the mutants further suggesting a crucial role of AP-2β in regulating corneal epithelial cell fate and stratification. Thus, our overarching hypothesis is that expression of AP-2β in the POM is critical for corneal epithelial cell fate determination and stratification through modulation of the Wnt/β-catenin signaling pathway. In the current proposal we aim to determine: 1) the developmental timing and fate of LESC in the AP-2β NCC KO mutants and 2) whether Wnt/β-catenin/BMP4 –signaling axis-is disrupted in the mutant and contributes to the ocular surface defects. Overall, these studies will contribute to our understanding of the gene regulatory network (GRN) controlling corneal epithelial cell fate determination and stratification, and the pathogenesis of diseases marked by corneal thinning, neovascularization and opacification.
