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Retinitis pigmentosa (RP) is the most common inherited retinal disease (IRD) and is characterized by photoreceptor degeneration and progressive vision loss. We report 4 patients presenting with RP from 3 unrelated families with variants in TBC1D32, which to date has never been associated with an IRD. To validate TBC1D32 as a putative RP causative gene, we combined Xenopus in vivo approaches and human induced pluripotent stem cell-derived (iPSC-derived) retinal models. Our data showed that TBC1D32 was expressed during retinal development and that it played an important role in retinal pigment epithelium (RPE) differentiation. Furthermore, we identified a role for TBC1D32 in ciliogenesis of the RPE. We demonstrated elongated ciliary defects that resulted in disrupted apical tight junctions, loss of functionality (delayed retinoid cycling and altered secretion balance), and the onset of an epithelial-mesenchymal transition-like phenotype. Last, our results suggested photoreceptor differentiation defects, including connecting cilium anomalies, that resulted in impaired trafficking to the outer segment in cones and rods in TBC1D32 iPSC-derived retinal organoids. Overall, our data highlight a critical role for TBC1D32 in the retina and demonstrate that TBC1D32 mutations lead to RP. We thus identify TBC1D32 as an IRD-causative gene.

Retinal neurodegenerative diseases are the leading causes of blindness. Among numerous therapeutic strategies being explored, stimulating self-repair recently emerged as particularly appealing. A cellular source of interest for retinal repair is the Müller glial cell, which harbors stem cell potential and an extraordinary regenerative capacity in anamniotes. This potential is, however, very limited in mammals. Studying the molecular mechanisms underlying retinal regeneration in animal models with regenerative capabilities should provide insights into how to unlock the latent ability of mammalian Müller cells to regenerate the retina. This is a key step for the development of therapeutic strategies in regenerative medicine. To this aim, we developed several retinal injury paradigms in Xenopus: a mechanical retinal injury, a transgenic line allowing for nitroreductase-mediated photoreceptor conditional ablation, a retinitis pigmentosa model based on CRISPR/Cas9-mediated rhodopsin knockout, and a cytotoxic model driven by intraocular CoCl2 injections. Highlighting their advantages and disadvantages, we describe here this series of protocols that generate various degenerative conditions and allow the study of retinal regeneration in Xenopus.

Retinitis pigmentosa is an inherited retinal dystrophy that ultimately leads to blindness due to the progressive degeneration of rod photoreceptors and the subsequent non-cell autonomous death of cones. Rhodopsin is the most frequently mutated gene in this disease. We here developed rhodopsin gene editing-based models of retinitis pigmentosa in two Xenopus species, Xenopus laevis and Xenopus tropicalis , by using CRISPR/Cas9 technology. In both of them, loss of rhodopsin function results in massive rod cell degeneration characterized by progressive shortening of outer segments and occasional cell death. This is followed by cone morphology deterioration. Despite these apparently similar degenerative environments, we found that Müller glial cells behave differently in Xenopus laevis and Xenopus tropicalis . While a significant proportion of Müller cells re-enter into the cell cycle in Xenopus laevis , their proliferation remains extremely limited in Xenopus tropicalis . This work thus reveals divergent responses to retinal injury in closely related species. These models should help in the future to deepen our understanding of the mechanisms that have shaped regeneration during evolution, with tremendous differences across vertebrates.