Distinct cis-acting regions control six6 expression during eye field and optic cup stages of eye formation

The eye field transcription factor, Six6, is essential for both the early (specification and proliferative growth) phase of eye formation, as well as for normal retinal progenitor cell differentiation. While genomic regions driving six6 optic cup expression have been described, the sequences controlling eye field and optic vesicle expression are unknown. Two evolutionary conserved regions 5' and a third 3' to the six6 coding region were identified, and together they faithfully replicate the endogenous X. laevis six6 expression pattern. Transgenic lines were generated and used to determine the onset and expression patterns controlled by the regulatory regions. The conserved 3' region was necessary and sufficient for eye field and optic vesicle expression. In contrast, the two conserved enhancer regions located 5' of the coding sequence were required together for normal optic cup and mature retinal expression. Gain-of-function experiments indicate endogenous six6 and GFP expression in F1 transgenic embryos are similarly regulated in response to candidate trans-acting factors. Importantly, CRISPR/CAS9-mediated deletion of the 3' eye field/optic vesicle enhancer in X. laevis, resulted in a reduction in optic vesicle size. These results identify the cis-acting regions, demonstrate the modular nature of the elements controlling early versus late retinal expression, and identify potential regulators of six6 expression during the early stages of eye formation.