Restoration of vision after de novo genesis of rod photoreceptors in mammalian retinas

Abstract

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Generation of rod photoreceptors by reprogramming MG in the mouse retina.
Fig. 2: MG-derived rod photoreceptors express essential rod genes and are morphologically similar to native rod photoreceptors.
Fig. 3: MG-derived regeneration of rod photoreceptors in Gnat1rd17Gnat2cpfl3 mice.
Fig. 4: MG-derived rod photoreceptors integrate into retinal circuitry and restore visual function in Gnat1rd17Gnat2cpfl3 mice.

References

  1. 1.

    Goldman, D. Müller glial cell reprogramming and retina regeneration. Nat. Rev. Neurosci. 15, 431–442 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. 2.

    Elsaeidi, F. et al. Notch suppression collaborates with Ascl1 and Lin28 to unleash a regenerative response in fish retina, but not in mice. J. Neurosci. 38, 2246–2261 (2018).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  3. 3.

    Jorstad, N. L. et al. Stimulation of functional neuronal regeneration from Müller glia in adult mice. Nature 548, 103–107 (2017).

    ADS  Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. 4.

    Karl, M. O. et al. Stimulation of neural regeneration in the mouse retina. Proc. Natl Acad. Sci. USA 105, 19508–19513 (2008).

    ADS  Article  PubMed  Google Scholar 

  5. 5.

    Dyer, M. A. & Cepko, C. L. Control of Müller glial cell proliferation and activation following retinal injury. Nat. Neurosci. 3, 873–880 (2000)

    Article  PubMed  CAS  Google Scholar 

  6. 6.

    Ooto, S. et al. Potential for neural regeneration after neurotoxic injury in the adult mammalian retina. Proc. Natl Acad. Sci. USA 101, 13654–13659, (2004).

    ADS  Article  PubMed  CAS  Google Scholar 

  7. 7.

    Ueki, Y. et al. Transgenic expression of the proneural transcription factor Ascl1 in Müller glia stimulates retinal regeneration in young mice. Proc. Natl Acad. Sci. USA 112, 13717–13722 (2015).

    ADS  Article  PubMed  CAS  Google Scholar 

  8. 8.

    Yao, K. et al. Wnt regulates proliferation and neurogenic potential of Müller glial cells via a Lin28/let-7 miRNA-dependent pathway in adult mammalian retinas. Cell Rep. 17, 165–178 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. 9.

    Calvert, P. D. et al. Phototransduction in transgenic mice after targeted deletion of the rod transducin α-subunit. Proc. Natl Acad. Sci. USA 97, 13913–13918, (2000).

    ADS  Article  PubMed  CAS  Google Scholar 

  10. 10.

    Chang, B. et al. Cone photoreceptor function loss-3, a novel mouse model of achromatopsia due to a mutation in Gnat2. Invest. Ophthalmol. Vis. Sci. 47, 5017–5021 (2006).

    Article  PubMed  Google Scholar 

  11. 11.

    Fausett, B. V. & Goldman, D. A role for α1 tubulin-expressing Müller glia in regeneration of the injured zebrafish retina. J. Neurosci. 26, 6303–6313 (2006).

    Article  PubMed  CAS  Google Scholar 

  12. 12.

    Bernardos, R. L., Barthel, L. K., Meyers, J. R. & Raymond, P. A. Late-stage neuronal progenitors in the retina are radial Müller glia that function as retinal stem cells. J. Neurosci. 27, 7028–7040 (2007).

    Article  PubMed  CAS  Google Scholar 

  13. 13.

    Fimbel, S. M., Montgomery, J. E., Burket, C. T. & Hyde, D. R. Regeneration of inner retinal neurons after intravitreal injection of ouabain in zebrafish. J. Neurosci. 27, 1712–1724 (2007).

    Article  PubMed  CAS  Google Scholar 

  14. 14.

    Nishida, A. et al. Otx2 homeobox gene controls retinal photoreceptor cell fate and pineal gland development. Nat. Neurosci. 6, 1255–1263 (2003).

    Article  PubMed  CAS  Google Scholar 

  15. 15.

    Chen, S. et al. Crx, a novel Otx-like paired-homeodomain protein, binds to and transactivates photoreceptor cell-specific genes. Neuron 19, 1017–1030 (1997).

    Article  PubMed  CAS  Google Scholar 

  16. 16.

    Furukawa, T., Morrow, E. M. & Cepko, C. L. Crx, a novel otx-like homeobox gene, shows photoreceptor-specific expression and regulates photoreceptor differentiation. Cell 91, 531–541 (1997).

    Article  PubMed  CAS  Google Scholar 

  17. 17.

    Mears, A. J. et al. Nrl is required for rod photoreceptor development. Nat. Genet. 29, 447–452 (2001).

    Article  PubMed  CAS  Google Scholar 

  18. 18.

    Livesey, F. J. & Cepko, C. L. Vertebrate neural cell-fate determination: lessons from the retina. Nat. Rev. Neurosci. 2, 109–118 (2001).

    Article  PubMed  CAS  Google Scholar 

  19. 19.

    Ajioka, I. et al. Differentiated horizontal interneurons clonally expand to form metastatic retinoblastoma in mice. Cell 131, 378–390 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. 20.

    Matsuda, T. & Cepko, C. L. Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc. Natl Acad. Sci. USA 101, 16–22 (2004).

    ADS  Article  PubMed  CAS  Google Scholar 

  21. 21.

    Emerson, M. M., Surzenko, N., Goetz, J. J., Trimarchi, J. & Cepko, C. L. Otx2 and Onecut1 promote the fates of cone photoreceptors and horizontal cells and repress rod photoreceptors. Dev. Cell 26, 59–72 (2013).

    Article  PubMed  CAS  Google Scholar 

  22. 22.

    Deng, W. T. et al. Functional interchangeability of rod and cone transducin α-subunits. Proc. Natl Acad. Sci. USA 106, 17681–17686 (2009).

    ADS  Article  PubMed  Google Scholar 

  23. 23.

    Sokolov, M. et al. Massive light-driven translocation of transducin between the two major compartments of rod cells: a novel mechanism of light adaptation. Neuron 34, 95–106 (2002).

    Article  PubMed  CAS  Google Scholar 

  24. 24.

    Majumder, A. et al. Transducin translocation contributes to rod survival and enhances synaptic transmission from rods to rod bipolar cells. Proc. Natl Acad. Sci. USA 110, 12468–12473 (2013).

    ADS  Article  PubMed  Google Scholar 

  25. 25.

    Schmitz, Y. & Witkovsky, P. Dependence of photoreceptor glutamate release on a dihydropyridine-sensitive calcium channel. Neurosci. 78, 1209–1216 (1997).

    Article  CAS  Google Scholar 

  26. 26.

    Thoreson, W. B., Nitzan, R. & Miller, R. F. Reducing extracellular Cl suppresses dihydropyridine-sensitive Ca2+ currents and synaptic transmission in amphibian photoreceptors. J. Neurophysiol. 77, 2175–2190 (1997).

    Article  PubMed  CAS  Google Scholar 

  27. 27.

    Corey, D. P., Dubinsky, J. M. & Schwartz, E. A. The calcium current in inner segments of rods from the salamander (Ambystoma tigrinum) retina. J. Physiol. (Lond.) 354, 557–575 (1984).

    Article  CAS  Google Scholar 

  28. 28.

    Wang, Y. V., Weick, M. & Demb, J. B. Spectral and temporal sensitivity of cone-mediated responses in mouse retinal ganglion cells. J. Neurosci. 31, 7670–7681 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. 29.

    Ke, J. B. et al. Adaptation to background light enables contrast coding at rod bipolar cell synapses. Neuron 81, 388–401 (2014).

    Article  PubMed  CAS  Google Scholar 

  30. 30.

    Nikonov, S. S., Kholodenko, R., Lem, J. & Pugh, E. N. Jr. Physiological features of the S- and M-cone photoreceptors of wild-type mice from single-cell recordings. J Gen. Physiol. 127, 359–374 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

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.

Reviewer information

Nature thanks Z. He, A. Huberman and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Affiliations

Authors

Contributions

K.Y. and S.Q. performed injections, immunohistochemistry and imaging analysis. Y.V.W., S.H.J.P. and J.B.D. performed retinal ganglion cell recordings. E.J.M. and M.C.C. performed cortical recordings. B.M. and D.Z. performed calcium current recordings. X.L. performed transmission electron microscopy analysis. B.Cha. provided Gnat1rd17Gnat2cpfl3 double mutant mice. B.Che. designed the study and wrote the paper with instrumental input from J.B.D.

Corresponding author

Correspondence to Bo Chen.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 MG may undergo only one cell division after β-catenin gene transfer.

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. bg, 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 (af) or presence (gl) 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 df and jl, respectively. Arrowheads, MG-derived rods. Scale bars, 750 µm (ac, gi), 25 µm (df, jl). Experiments were repeated 4 times with similar results.

Extended Data Fig. 3 Additional examples showing the progression of MG-derived rod differentiation.

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 (ac), intermediate (dl) and terminal (mr) stages. Scale bar, 25 µm. Experiments were repeated 6 times with similar results.

Extended Data Fig. 4 MG-derived rods eventually turned off expression of GFAP–GFP over time.

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. ac, 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. ac, Otx2 treatment. df, Crx treatment. gi, Nrl treatment. jl, Otx2 + Crx treatment. mo, Otx2 + Nrl treatment. pr, 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.

Extended Data Fig. 8 MG-derived regeneration of rod photoreceptors decreases in aged mice.

ad, 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.

Extended Data Fig. 9 MG-derived rod photoreceptors express rhodopsin and peripherin-2.

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 (ac) and peripherin-2 (df) 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.

Extended Data Table 1 Viral constructs, packaged viruses and virus titres after purification and concentration

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.