Genetic insights into the role of PAX6 in ocular development

The fovea is a small retinal indentation packed with specialized cone photoreceptors. Despite its key-role in central vision, little is known about foveal pathologies and development at the cellular and molecular levels. Therefore, no treatment is yet available for vision loss resulting from underdeveloped-fovea (foveal hypoplasia (FH)). First, I used aniridia as a disease model to better understand FH at the cellular and molecular levels. Thirty-three aniridia subjects from British Columbia underwent a thorough ophthalmic examination with in-vivo imaging of foveal structure. Molecular investigations include sequencing of PAX6, candidate genes, in addition to 11p chromosomal analysis. In those in whom imaging was possible, FH was seen in the majority (80%) of cases. Best corrected visual acuities in the cohort ranged from normal vision to no light-perception. Molecular genetic defects involving PAX6 were identified in 30 participants, including 4 novel PAX6 mutations and 4 novel chromosome 11p deletions inclusive of PAX6 or its regulatory region. Then as a proof-of-principle, we employed the SMaRT (spliceosome-mediated RNA trans-splicing) method to rescue Pax6 defects in homozygous-mutant mouse embryonic-fibroblasts and then in-vivo in a naturally occurring Pax6 mouse model. We showed that by using SMaRT technology we were able to rescue Pax6 expression in-vitro and in-vivo, paving the way for potential future therapies for FH. Finally, we tested the feasibility of using Anolis carolinensis (green anole lizard) as a novel foveated model. With its complete published genome, bioinformatic analysis revealed that 85% of human candidate FH genes had an orthologous gene or DNA sequence in the anole. Eyes were collected at various stages of prehatching development for histological analysis, immunofluorescence, and apoptosis analysis. We demonstrated that embryonic foveal development in green anoles resembles human foveal development during infancy. Additionally, at embryonic stage (ES) 14 Pax6 was localized across the entire retina. However, at ES17 Pax6 expression in the ganglion cells of the central retina was markedly reduced. These findings provide the first insight into foveal morphogenesis in the green anole and suggest that it could be an ideal model for perturbing the molecular signals driving foveal development, thus informing on human foveal development and disease.Medicine, Faculty ofExperimental Medicine, Division ofMedicine, Department ofGraduat