Novel PAX6 variants and their roles in corneal epithelial regulation and homeostasis

Abstract

Purpose : Patients with aniridia associated PAX6 mutations develop corneal and anterior segment defects. This study aims to understand the mechanisms of PAX6 regulation at the limbus and its effects on cornea-specific gene targets.

Methods : Multiple ocular cell lines, primary limbal cultures, human corneo-scleral tissues and hiPSC-derived ocular tissues were evaluated for differential expression of PAX6 variants and their spatial distribution by qRT-PCR, Sanger sequencing, luciferase reporter assays, ChIP-PCR, Immunocytochemistry (ICC), Immunohistochemistry (IHC) and RNA-FISH assays. Statistical analysis was performed using Student’s t test.

Results : We report the identification of four novel splice variants of PAX6 in human ocular tissues and are generated by alternative splicing at two major splicing hotspots near Exon 5-6-7 and Exon 12-13 splice junctions. These variants carry in-frame deletions, possibly affecting either the N-terminal paired domain-mediated DNA binding or C-terminal PST domain-mediated transactivation functions. qRT-PCR evaluations revealed that these novel splice variants are driven by the PAX6 pA promoter in all ocular tissues and was highly expressed in the limbal and corneal epithelium. Activation of canonical Wnt signals in limbal cultures induced the expression of novel splice variants. Luciferase reporter assays using recombinant PAX6 isoforms has confirmed negative auto-feedback regulation on PAX6 pA promoter. While PAX6A could activate K3 promoter, it had no effect on K12, TAp63 and dNp63 promoters. However, a paired domain truncated isoform significantly activated the dNp63, K12 and K3 promoters. In situ localization studies using RNA-FISH combined with ICC/IHC has confirmed that the novel variants are co-expressed along with PAX6A transcripts in a sub-set of cells that are predominantly located at the basal and suprabasal layers of limbal and corneal epithelium. These cells are identified as PAX6^Low, p63α^High, BrdU⁺ and K3/K12^- proliferating and migrating TACs.

Conclusions : The novel PAX6 splice variants alter the relative stoichiometry of wild type transcripts and ensure low levels of PAX6 expression in basal cells. This enables the self-renewal of activated LSC and expansion of TACs, while preventing pre-mature differentiation, thus promoting optimal epithelial stratification during normal corneal development and wound healing.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.