Overlapping enhancer activity robustly regulates rax expression during ocular development in zebrafish

Vertebrate eye development relies on the tightly regulated yet highly dynamic spatiotemporal expression of eye field transcription factors that coordinate developmental programs required for the formation of a functional eye. The gene rax is among the earliest expressed eye field transcription factors and is specifically associated with ocular-fated tissue. It encodes a transcription factor that is essential for optic vesicle evagination, the precursor to the optic cup and retina. Although the developmental functions of rax are well characterized, the cis -regulatory elements that control its expression remain poorly understood. Using zebrafish ( Danio rerio ), I identified five novel ocular enhancers and demonstrate that they drive similar spatiotemporal expression patterns as rax at three key stages of eye development. Furthermore, I performed an in-depth characterization of two enhancers and demonstrate that they exhibit substantial transcript-level overlap with endogenous rax expression during development. Additionally, through the generation of enhancer deletion lines, I further demonstrate that the gene regulatory network (GRN) controlling rax expression is largely robust to individual and combinatorial enhancer perturbations. However, perturbation of this regulatory network can result in rax -associated ocular abnormalities, including microphthalmia and anophthalmia. Together, these findings highlight the importance of studying developmental ocular enhancers to better understand the functions of noncoding genomic regions and provide insight into potential causes of unexplained congenital eye disease.