A conserved enhancer cluster regulates efnb2 expression in the vertebrate dorsal retina

The vertebrate retina is patterned along its dorsal-ventral (DV) axis by conserved signaling centers, yet the cis-regulatory architecture maintaining these territories remains unclear. Using the four-eyed fish Anableps anableps, which enables physical separation of dorsal and ventral retina, we integrated RNA-seq, ATAC-seq and Hi-C to map DV regulatory landscapes. We identify a 29-kb dorsal-enriched enhancer cluster within a gene poor region between efnb2a and gtpbp8, embedded in a conserved topologically associating domain spanning efnb2a-gtpbp8-slc10a2. Dorsal-biased chromatin contacts link this cluster to the efnb2a promoter. The orthologous human region contains a genome-wide significant association with retinal thickness (rs2575134; P = 2e-14). Leveraging the compact genome of Tetraodon nigroviridis, we cloned the orthologous interval and show that it drives dorsal retina-specific reporter expression in zebrafish and mouse. Together, these results identify an evolutionarily conserved regulatory domain controlling retinal axial identity and linking enhancer architecture to human ocular variation. Funding, National Science Foundation.