Pax6 as a key regulator of lens development

ABSTRACT Transcription factor (TF) dysfunction is a direct cause of multiple inherited ocular diseases. Understanding the precise cellular and molecular functions of each individual ocular TFs is key towards understanding the mechanistic foundation for mammalian ocular development and the development of therapies to manage human congenital eye diseases. PAX6 encodes a sequence-specific DNA-binding TF that plays pivotal roles in the earliest stages of lens and retinal development. Genetic data of the microphthalmia- anophthalmia-coloboma (MAC) syndrome identified prominent roles of TFs PAX6 and SOX2 and retinoic acid (RA) signaling. Heterozygous mutations of PAX6 such as G18W, A33P, N50K and R128C, alter DNA-binding of mutated PAX6 proteins and cause variable aniridia. Nonsense R317X mutation causes haploinsufficiency. Heterozygous R74P and H101R SOX2 mutations cause microphthalmia and anophthalmia, respectively. Homozygous mutations in PAX6 cause anophthalmia. This application seeks to elucidate abnormal functions of PAX6 in aniridia and MAC syndrome to test the overarching hypothesis that specific mutations of PAX6 and SOX2 disrupt critical functions of these TFs during early stages of eye development, including RA-signaling. These early stages of human eye development, including formation of lens and retinal progenitor cells, will be modelled in iPS-derived organoids pioneered by our laboratory. Three mouse models of Pax6 will be used to directly compare human and mouse transcriptomic and chromatin data. Preliminary studies include functional analyses of four missense and a nonsense PAX6 mutations in cell cultures to study haploinsufficiency, analysis of lens formation in Pax6+/lacZ embryos, genome-wide Pax6-binding in the retina, and disrupted brain development in Leca4 (N50K) and Leca2 (R128C) Pax6 null mouse mutants. Aim 1 will uncover the molecular mechanisms of how PAX6 missense and nonsense (R317X) mutations disrupt the early steps of human eye development. Aim 2 will elucidate Pax6-dependent gene control in mouse optic cup stage embryos. Aim 3 will determine function of SOX2, PAX6/SOX2 functional overlaps, and vitamin A’s potential role in “compensating” PAX6 R317X haplo- insufficiency. These results will reveal how individual PAX6 mutations alter gene control in multiple ocular tissues, i.e. in the lens and retinal progenitor cells, identify in vivo PAX6- and SOX2-binding requirements in human cells, compare function of N50K and R128C mutated proteins between human and mouse, identify candidate genes driving cataract, glaucoma, foveal hypoplasia and other diseases within the variable aniridia phenotypes, and mechanistically link PAX6 and SOX2 with RA signaling. The successful completion of these studies will provide novel insights into the mechanisms and gene control by PAX6 and SOX2 in ocular development and provide a basis for the development of therapies to manage aniridia and potentially other human inherited eye diseases.