In zebrafish, Müller glia (MG) are a source of retinal stem cells that can replenish damaged retinal neurons and restore vision1. In mammals, however, MG do not spontaneously re-enter the cell cycle to generate a population of stem or progenitor cells that differentiate into retinal neurons. Nevertheless, the regenerative machinery may exist in the mammalian retina, as retinal injury can stimulate MG proliferation followed by limited neurogenesis2,3,4,5,6,7. Therefore, there is still a fundamental question regarding whether MG-derived regeneration can be exploited to restore vision in mammalian retinas. Gene transfer of β-catenin stimulates MG proliferation in the absence of injury in mouse retinas8. Here we report that following gene transfer of β-catenin, cell-cycle-reactivated MG can be reprogrammed to generate rod photoreceptors by subsequent gene transfer of transcription factors essential for rod cell fate specification and determination. MG-derived rods restored visual responses in Gnat1rd17Gnat2cpfl3 double mutant mice, a model of congenital blindness9,10, throughout the visual pathway from the retina to the primary visual cortex. Together, our results provide evidence of vision restoration after de novo MG-derived genesis of rod photoreceptors in mammalian retinas.
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This research was supported by National Institutes of Health grants R01 EY024986, R01 EY021502, R01 EY014454, R01 EY021372, R01 EY015788, R01 EY023105, R01 EY021195, R01 EY014990, P30 EY026878, Pew Scholars Program in the Biomedical Sciences, Research to Prevent Blindness and the McGraw Family Foundation for Vision Research.
Nature thanks Z. He, A. Huberman and the other anonymous reviewer(s) for their contribution to the peer review of this work.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, A schematic of the EdU/BrdU double-labelling experiment. Wild-type retinas were injected with ShH10-GFAP-β-catenin (0 day), followed by an injection of EdU (10 days). BrdU was either co-injected with EdU (0 h) or injected 24 h after EdU injection (24 h). Retinas were collected 14 days after β-catenin gene transfer. b–g, Detection of EdU and BrdU labelled MG. Scale bar, 20 µm. h, Percentage of MG labelled by EdU (EdU+BrdU−, green), BrdU (red), or both (EdU+BrdU+, yellow). Experiments were repeated 3 times with similar results. Data are presented as mean ± s.e.m., n = 4 retinas.
Extended Data Fig. 2 A MG-derived rod differentiation was observed across the whole retinal section.
Wild-type retinas at 4 weeks of age were first injected with ShH10-GFAP-GFP (label transduced MGs), and ShH10-rhodopsin-tdTomato (label MG-derived rods) in the absence (a–f) or presence (g–l) of ShH10-GFAP-β-catenin (to stimulate MG proliferation), followed 2 weeks later by the second injection of ShH10-GFAP-mediated gene transfer of Otx2, Crx and Nrl for rod induction. Retinal samples were analysed by confocal microscopy 10 days after the second injection. The boxed areas in c and i are enlarged in d–f and j–l, respectively. Arrowheads, MG-derived rods. Scale bars, 750 µm (a–c, g–i), 25 µm (d–f, j–l). Experiments were repeated 4 times with similar results.
Wild-type retinas at 4 weeks of age were first injected with ShH10-GFAP-β-catenin, ShH10-GFAP-GFP and ShH10-rhodopsin-tdTomato, followed 2 weeks later by a second injection of ShH10-GFAP-mediated gene transfer of Otx2, Crx and Nrl for rod induction. MG-derived rod differentiation progressed through the initial (a–c), intermediate (d–l) and terminal (m–r) stages. Scale bar, 25 µm. Experiments were repeated 6 times with similar results.
Wild-type retinas at 4 weeks of age were first injected with ShH10-GFAP-β-catenin, ShH10-GFAP-GFP and ShH10-rhodopsin-tdTomato, followed 2 weeks later by the second injection of ShH10-GFAP-mediated gene transfer of Otx2, Crx and Nrl for rod induction. a–c, Retinas were collected 12 weeks after the second injection and analysed for the expression of GFAP–GFP in tdTomato+ MG-derived rods. Scale bar, 25 µm. Experiments were repeated 3 times independently with similar results. d, Percentage of tdTomato+ cells also expressing GFP. Data are presented as mean ± s.e.m., n = 7 retinas.
Extended Data Fig. 5 Treatment with Otx2, Crx and Nrl individually or in pairs is not sufficient for rod induction.
Wild-type retinas were injected with ShH10-GFAP-β-catenin (for MG proliferation), ShH10-GFAP-GFP (to label transduced MG) and ShH10-rhodopsin-tdTomato (to label MG-derived rods) at 4 weeks of age, followed 2 weeks later by a second injection of ShH10-GFAP-mediated gene transfer of transcription factors for rod induction. Samples were analysed by confocal microscopy in retinal sections at 4 weeks after the second injection. a–c, Otx2 treatment. d–f, Crx treatment. g–i, Nrl treatment. j–l, Otx2 + Crx treatment. m–o, Otx2 + Nrl treatment. p–r, Crx + Nrl treatment. Scale bar, 20 µm. Experiments were repeated 4 times with similar results.
Extended Data Fig. 6 Time-course analysis of MG-derived rod differentiation in wild-type retinas treated with Crx and Nrl.
Wild-type retinas at 4 weeks of age were first injected with ShH10-GFAP-β-catenin, ShH10-GFAP-GFP and ShH10-rhodopsin-tdTomato, followed 2 weeks later by a second injection of ShH10-GFAP-mediated gene transfer of Crx and Nrl. tdTomato+ cells were only detected in the initial stage of rod differentiation at 1, 2 and 4 weeks after the second injection. Data are presented as mean ± s.e.m., n = 3 retinas at each time point.
Extended Data Fig. 7 Fate-mapping experiments indicate that the two-step reprogramming method may occasionally produce cells with a horizontal cell morphology.
The boxed area is enlarged to show an MG-derived tdTomato+ cell with a horizontal cell morphology located in the outer region of the inner nuclear layer. Arrowhead, cell soma; arrows, cell processes. Scale bar, 5 µm. Experiments were repeated 4 times with similar results.
a–d, Generation of tdTomato+ MG-derived rod photoreceptors in 7-month-old-mouse retinas in the dorsal (a), nasal (b), temporal (c) and ventral (d) quadrants in retinal flat-mount preparations. Scale bar, 20 µm. Experiments were repeated 4 times with similar results. e, Quantification of tdTomato+ MG-derived rods per mm2 in the four retinal quadrants. Data are presented as mean ± s.e.m., n = 4 retinas.
Wild-type retinas were injected with ShH10-GFAP-β-catenin (for MG proliferation) and ShH10-rhodopsin-tdTomato (to label MG-derived rods) at 4 weeks of age, followed 2 weeks later by the second injection of ShH10-GFAP-mediated gene transfer of Otx2, Crx and Nrl for rod induction. Treated retinas were dissociated 4 weeks after the second injection and analysed for the expression of rhodopsin (a–c) and peripherin-2 (d–f) using immunohistochemistry and confocal microscopy. MG-derived rods were immunoreactive for rhodopsin (arrowheads, c) and peripherin-2 (arrowheads, f). Scale bar, 20 µm. Experiments were repeated 3 times with similar results.
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Yao, K., Qiu, S., Wang, Y.V. et al. Restoration of vision after de novo genesis of rod photoreceptors in mammalian retinas. Nature 560, 484–488 (2018). https://doi.org/10.1038/s41586-018-0425-3
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