Variant-specific disruption to notch signalling in PAX6 microphthalmia and aniridia patient-derived hiPSC optic cup-like organoids

Biochim Biophys Acta Mol Basis Dis. 2025 Aug;1871(6):167869. doi: 10.1016/j.bbadis.2025.167869. Epub 2025 Apr 23.

Abstract

The homeobox-containing transcription factor PAX6 is a key regulator of eye development. Pathogenic heterozygous PAX6 variants lead to variable ocular phenotypes, most commonly haploinsufficiency-induced aniridia. Missense variants are typically associated with milder ocular conditions, although variants in the DNA-binding paired domain which alter target binding lead to severe ocular phenotypes including bilateral microphthalmia, similar to SOX2-anophthalmia syndrome. However, the variant-specific pathway disruption resulting in phenotypic heterogeneity is not well understood. To investigate pathogenic mechanisms of PAX6 variants, transcriptomic and chromatin accessibility analysis was performed on hiPSC derived 3D optic cup-like organoids generated from patients with variants (i) PAX6N124K displaying combined microphthalmia, aniridia and optic nerve coloboma, and (ii) PAX6R261X exhibiting typical aniridia. Total RNA sequencing analysis revealed downregulation of SOX2 in missense PAX6N124K cups compared to both wildtype and PAX6R261X haploinsufficient aniridia controls, along with Notch signalling components and markers of proliferation and differentiation. Transcription factor binding motifs of Notch-related genes were also found to be differentially bound in PAX6N124K cups through ATACseq footprinting analysis. Our analysis of PAX6-related oculopathies using in vitro models reveals disruption to DNA binding perturbs SOX2 and Notch signalling, contributing to severe ocular phenotypes in patients with missense changes in the paired domain. This work reveals a previously unestablished role for PAX6 in SOX2 and Notch signalling regulation during early oculogenesis, as well as illuminating disease mechanisms underlying variant-specific ocular phenotypes and genotype-phenotype correlations. These novel insights can influence clinical care, and provide valuable data on potential therapeutic targets, which can guide future translational research.

Keywords: Eye development; Microphthalmia; Notch signalling; Optic cups; Organoids; PAX6; RNAseq.

MeSH terms

  • Aniridia* / genetics
  • Aniridia* / metabolism
  • Aniridia* / pathology
  • Humans
  • Induced Pluripotent Stem Cells* / metabolism
  • Induced Pluripotent Stem Cells* / pathology
  • Microphthalmos* / genetics
  • Microphthalmos* / metabolism
  • Microphthalmos* / pathology
  • Mutation, Missense
  • Organoids* / metabolism
  • Organoids* / pathology
  • PAX6 Transcription Factor* / genetics
  • PAX6 Transcription Factor* / metabolism
  • Receptors, Notch* / genetics
  • Receptors, Notch* / metabolism
  • SOXB1 Transcription Factors / genetics
  • SOXB1 Transcription Factors / metabolism
  • Signal Transduction

Substances

  • PAX6 Transcription Factor
  • PAX6 protein, human
  • Receptors, Notch
  • SOXB1 Transcription Factors
  • SOX2 protein, human