A conserved PAX6 enhancer links cis-regulatory control to aniridia and vertebrate eye diversity
PAX6 is a master regulator of eye development, and its haploinsufficiency causes aniridia, a congenital disorder characterized by iris hypoplasia. Yet, the cis-regulatory mechanisms that control PAX6 expression in anterior eye structures remain poorly defined. Here, we identify an evolutionarily conserved enhancer, IrisE, by analyzing chromatin accessibility profiling of the ciliary margin and retinal pigment epithelium —tissues that give rise to the iris and ciliary body. Deletion of IrisE in mice selectively reduces Pax6 expression in the ciliary margin and RPE, leading to iris hypoplasia while sparing other ocular tissues. IrisE integrates transcriptional inputs from Meis1/2, Otx1, Vsx2, and Wnt/ß-catenin signaling, as demonstrated by ChIP-seq and conditional knockout analyses. This regulatory logic extends to additional ciliary margin–specific genes, suggesting a shared gene regulatory network for anterior eye morphogenesis. Cross-species reporter assays in chick and zebrafish reveal that vertebrate IrisE enhancers robustly drive expression in retina, though with subtle species-specific activity patterns. Together, these findings identify IrisE as a conserved cis-regulatory element essential for Pax6 dosage control in the retina and suggest that evolutionary tuning of IrisE contributed to morphological diversity of the iris across vertebrates.