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.

Familial exudative vitreoretinopathy (FEVR) and Norrie disease are examples of genetic disorders in which the retinal vasculature fails to fully form (hypovascular), leading to congenital blindness. While studying the role of a factor expressed during retinal development, T-box factor Tbx3, we discovered that optic cup loss of Tbx3 caused the retina to become hypovascular. The purpose of this study was to characterize how loss of Tbx3 affects retinal vasculature formation.

Intermediate filament proteins are structural components of the cellular cytoskeleton with cell-type specific expression and function. Glial fibrillary acidic protein (GFAP) is a type III intermediate filament protein and is up-regulated in glia of the nervous system in response to injury and during neurodegenerative diseases. In the retina, GFAP levels are dramatically increased in Müller glia and are thought to play a role in the extensive structural changes resulting in Müller cell hypertrophy and glial scar formation. In spite of similar changes to the morphology of Xenopus Müller cells following injury, we found that Xenopus lack a gfap gene. Other type III intermediate filament proteins were, however, significantly induced following rod photoreceptor ablation and retinal ganglion cell axotomy. The recently available X. tropicalis and X. laevis genomes indicate a small deletion most likely resulted in the loss of the gfap gene during anuran evolution. Lastly, a survey of representative species from all three extant amphibian orders including the Anura (frogs, toads), Caudata (salamanders, newts), and Gymnophiona (caecilians) suggests that deletion of the gfap locus occurred in the ancestor of all Anura after its divergence from the Caudata ancestor around 290 million years ago. Our results demonstrate that extensive changes in Müller cell morphology following retinal injury do not require GFAP in Xenopus, and other type III intermediate filament proteins may be involved in the gliotic response.

Pluripotent cells such as embryonic stem (ES) and induced pluripotent stem (iPS) cells are the starting point from which to generate organ specific cell types. For example, converting pluripotent cells to retinal cells could provide an opportunity to treat retinal injuries and degenerations. In this study, we used an in vivo strategy to determine if functional retinas could be generated from a defined population of pluripotent Xenopus laevis cells. Animal pole cells isolated from blastula stage embryos are pluripotent. Untreated, these cells formed only epidermis, when transplanted to either the flank or eye field. In contrast, misexpression of seven transcription factors induced the formation of retinal cell types. Induced retinal cells were committed to a retinal lineage as they formed eyes when transplanted to the flanks of developing embryos. When the endogenous eye field was replaced with induced retinal cells, they formed eyes that were molecularly, anatomically, and electrophysiologically similar to normal eyes. Importantly, induced eyes could guide a vision-based behavior. These results suggest the fate of pluripotent cells may be purposely altered to generate multipotent retinal progenitor cells, which differentiate into functional retinal cell classes and form a neural circuitry sufficient for vision.