Unique activities of two overlapping PAX6 retinal enhancers

Enhancers play a critical role in development by precisely modulating spatial, temporal, and cell type-specific gene ex-pression. Sequence variants in enhancers have been implicated in diseases; however, establishing the functional consequences of these variants is challenging because of a lack of understanding of precise cell types and developmental stages where the en-hancers are normally active. PAX6 is the master regulator of eye development, with a regulatory landscape containing multiple enhancers driving the expression in the eye. Whether these enhancers perform additive, redundant or distinct functions is unknown. Here, we describe the precise cell types and regu-latory activity of two PAX6 retinal enhancers, HS5 and NRE. Using a unique combination of live imaging and single-cell RNA se-quencing in dual enhancer-reporter zebrafish embryos, we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping, these enhancers have distinct activities in different cell types and therefore likely nonredundant functions. This work demon -strates that unique cell type-specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo.