Ascl1a regulates Müller glia dedifferentiation and retinal regeneration through a Lin-28-dependent, let-7 microRNA signalling pathway

A Corrigendum to this article was published on 27 March 2015

This article has been updated

Abstract

Unlike mammals, teleost fish mount a robust regenerative response to retinal injury that culminates in restoration of visual function1,2. This regenerative response relies on dedifferentiation of Müller glia into a cycling population of progenitor cells. However, the mechanism underlying this dedifferentiation is unknown. Here, we report that genes encoding pluripotency factors are induced following retinal injury. Interestingly, the proneural transcription factor, Ascl1a, and the pluripotency factor, Lin-28, are induced in Müller glia within 6 h following retinal injury and are necessary for Müller glia dedifferentiation. We demonstrate that Ascl1a is necessary for lin-28 expression and that Lin-28 suppresses let-7 microRNA (miRNA) expression. Furthermore, we demonstrate that let-7 represses expression of regeneration-associated genes such as, ascl1a, hspd1, lin-28, oct4, pax6b and c-myc. hspd1, oct4 and c-myca exhibit basal expression in the uninjured retina and let-7 may inhibit this expression to prevent premature Müller glia dedifferentiation. The opposing actions of Lin-28 and let-7 miRNAs on Müller glia differentiation and dedifferentiation are similar to that of embryonic stem cells3 and suggest novel targets for stimulating Müller glia dedifferentiation and retinal regeneration in mammals.

Figure 1: ascl1a and lin-28 mRNAs are rapidly induced in dedifferentiating Müller glia following retinal injury.
Figure 2: Ascl1a and Lin-28 knockdown inhibit 1016 tuba1a:gfp transgene expression and Müller glia-derived progenitor proliferation.
Figure 3: Ascl1a regulates lin-28 expression.
Figure 4: Lin-28 regulates let-7 miRNA levels in Müller glia-derived progenitors.
Figure 5: let-7 miRNAs suppress expression of regeneration- and pluripotency-associated genes.

Change history

  • 25 February 2015

    In the version of this Letter originally published the first lin-28 targeting morpholino oligonucleotide should have read: 5′-GGGCATCTTTATGATTTAGCCTTCT-3′. This has been corrected in all online versions of the Letter.

References

  1. 1

    Sherpa, T. et al. Ganglion cell regeneration following whole-retina destruction in zebrafish. Dev. Neurobiol. 68, 166–181 (2008).

    Article  Google Scholar 

  2. 2

    Mensinger, A. F. & Powers, M. K. Visual function in regenerating teleost retina following cytotoxic lesioning. Vis. Neurosci. 16, 241–251 (1999).

    CAS  Article  Google Scholar 

  3. 3

    Melton, C., Judson, R. L. & Blelloch, R. Opposing microRNA families regulate self-renewal in mouse embryonic stem cells. Nature 463, 621–626 (2010).

    CAS  Article  Google Scholar 

  4. 4

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

    CAS  Article  Google Scholar 

  5. 5

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

    CAS  Article  Google Scholar 

  6. 6

    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).

    CAS  Article  Google Scholar 

  7. 7

    Thummel, R., Kassen, S. C., Montgomery, J. E., Enright, J. M. & Hyde, D. R. Inhibition of Muller glial cell division blocks regeneration of the light-damaged zebrafish retina. Dev. Neurobiol. 68, 392–408 (2008).

    Article  Google Scholar 

  8. 8

    Ooto, S. [Potential for neural regeneration in the adult mammalian retina]. Nippon Ganka Gakkai Zasshi 110, 864–871 (2006).

    CAS  PubMed  Google Scholar 

  9. 9

    Wan, J. et al. Preferential regeneration of photoreceptor from Muller glia after retinal degeneration in adult rat. Vision Res. 48, 223–234 (2008).

    CAS  Article  Google Scholar 

  10. 10

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

    CAS  Article  Google Scholar 

  11. 11

    Takeda, M. et al. α-Aminoadipate induces progenitor cell properties of Muller glia in adult mice. Invest. Ophthalmol. Vis. Sci. 49, 1142–1150 (2008).

    Article  Google Scholar 

  12. 12

    Fausett, B. V., Gumerson, J. D. & Goldman, D. The proneural basic helix-loop-helix gene ascl1a is required for retina regeneration. J. Neurosci. 28, 1109–1117 (2008).

    CAS  Article  Google Scholar 

  13. 13

    Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).

    CAS  Article  Google Scholar 

  14. 14

    Hochedlinger, K. & Plath, K. Epigenetic reprogramming and induced pluripotency. Development 136, 509–523 (2009).

    CAS  Article  Google Scholar 

  15. 15

    Zupanc, G. K. & Horschke, I. Proliferation zones in the brain of adult gymnotiform fish: a quantitative mapping study. J. Comp. Neurol. 353, 213–233 (1995).

    CAS  Article  Google Scholar 

  16. 16

    Nimmo, R. A. & Slack, F. J. An elegant miRror: microRNAs in stem cells, developmental timing and cancer. Chromosoma 118, 405–418 (2009).

    CAS  Article  Google Scholar 

  17. 17

    Eisen, J. S. & Smith, J. C. Controlling morpholino experiments: don't stop making antisense. Development 135, 1735–1743 (2008).

    CAS  Article  Google Scholar 

  18. 18

    Thummel, R. et al. Inhibition of zebrafish fin regeneration using in vivo electroporation of morpholinos against fgfr1 and msxb. Dev. Dyn. 235, 336–346 (2006).

    CAS  Article  Google Scholar 

  19. 19

    Bill, B. R., Petzold, A. M., Clark, K. J., Schimmenti, L. A. & Ekker, S. C. A primer for morpholino use in zebrafish. Zebrafish 6, 69–77 (2009).

    CAS  Article  Google Scholar 

  20. 20

    Li, J. et al. Identification and analysis of the mouse basic/Helix-Loop-Helix transcription factor family. Biochem. Biophys. Res. Commun. 350, 648–656 (2006).

    CAS  Article  Google Scholar 

  21. 21

    Bertrand, N., Castro, D. S. & Guillemot, F. Proneural genes and the specification of neural cell types. Nat. Rev. Neurosci. 3, 517–530 (2002).

    CAS  Article  Google Scholar 

  22. 22

    Rybak, A. et al. A feedback loop comprising lin-28 and let-7 controls pre-let-7 maturation during neural stem-cell commitment. Nat. Cell Biol. 10, 987–993 (2008).

    CAS  Article  Google Scholar 

  23. 23

    Viswanathan, S. R., Daley, G. Q. & Gregory, R. I. Selective blockade of microRNA processing by Lin28. Science 320, 97–100 (2008).

    CAS  Article  Google Scholar 

  24. 24

    Heo, I. et al. TUT4 in concert with Lin28 suppresses microRNA biogenesis through pre-microRNA uridylation. Cell 138, 696–708 (2009).

    CAS  Article  Google Scholar 

  25. 25

    Blackshaw, S. et al. Genomic analysis of mouse retinal development. PLoS Biol. 2, E247 (2004).

    Article  Google Scholar 

  26. 26

    Jadhav, A. P., Roesch, K. & Cepko, C. L. Development and neurogenic potential of Muller glial cells in the vertebrate retina. Prog. Retin. Eye Res. 28, 249–262 (2009).

    CAS  Article  Google Scholar 

  27. 27

    Roesch, K. et al. The transcriptome of retinal Muller glial cells. J. Comp. Neurol. 509, 225–238 (2008).

    CAS  Article  Google Scholar 

  28. 28

    Trimarchi, J. M., Stadler, M. B. & Cepko, C. L. Individual retinal progenitor cells display extensive heterogeneity of gene expression. PLoS One 3, e1588 (2008).

    Article  Google Scholar 

  29. 29

    Kumar, M. S., Lu, J., Mercer, K. L., Golub, T. R. & Jacks, T. Impaired microRNA processing enhances cellular transformation and tumorigenesis. Nat. Genet. 39, 673–677 (2007).

    CAS  Article  Google Scholar 

  30. 30

    Qin, Z., Barthel, L. K. & Raymond, P. A. Genetic evidence for shared mechanisms of epimorphic regeneration in zebrafish. Proc. Natl Acad. Sci. USA 106, 9310–9315 (2009).

    CAS  Article  Google Scholar 

  31. 31

    Thummel, R. et al. Pax6a and Pax6b are required at different points in neuronal progenitor cell proliferation during zebrafish photoreceptor regeneration. Exp. Eye Res. 90, 572–582 (2010).

    CAS  Article  Google Scholar 

  32. 32

    Miranda, K. C. et al. A pattern-based method for the identification of microRNA-binding sites and their corresponding heteroduplexes. Cell 126, 1203–1217 (2006).

    CAS  Article  Google Scholar 

  33. 33

    Thummel, R. et al. Characterization of Muller glia and neuronal progenitors during adult zebrafish retinal regeneration. Exp. Eye Res. 87, 433–444 (2008).

    CAS  Article  Google Scholar 

  34. 34

    Xu, B., Zhang, K. & Huang, Y. Lin28 modulates cell growth and associates with a subset of cell cycle regulator mRNAs in mouse embryonic stem cells. RNA 15, 357–361 (2009).

    CAS  Article  Google Scholar 

  35. 35

    Kassen, S. C. et al. Time course analysis of gene expression during light-induced photoreceptor cell death and regeneration in albino zebrafish. Dev. Neurobiol. 67, 1009–1031 (2007).

    CAS  Article  Google Scholar 

  36. 36

    Chung, K. H. et al. Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155. Nucleic Acids Res. 34, e53 (2006).

    Article  Google Scholar 

  37. 37

    Lindeman, L. C., Vogt-Kielland, L. T., Alestrom, P. & Collas, P. Fish'n ChIPs: chromatin immunoprecipitation in the zebrafish embryo. Methods Mol. Biol. 567, 75–86 (2009).

    CAS  Article  Google Scholar 

  38. 38

    Senut, M. C., Gulati-Leekha, A. & Goldman, D. An element in the β1-tubulin promoter is necessary for retinal expression during optic nerve regeneration but not after eye injury in the adult zebrafish. J. Neurosci. 24, 7663–7673 (2004).

    CAS  Article  Google Scholar 

  39. 39

    Jowett, T. Double in situ hybridization techniques in zebrafish. Methods 23, 345–358 (2001).

    CAS  Article  Google Scholar 

  40. 40

    Wang, W. X. et al. The expression of microRNA miR-107 decreases early in Alzheimer's disease and may accelerate disease progression through regulation of β-site amyloid precursor protein-cleaving enzyme 1. J. Neurosci. 28, 1213–1223 (2008).

    Article  Google Scholar 

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Acknowledgements

We thank D. Hyde for sharing gfap:gfp transgenic fish, R. Thompson, D. Turner and M. Uhler for sharing expression vectors and reagents, J. Beals for help with confocal microscopy, the UM Flow Cytometry Core for help purifying GFP-labelled Müller glia, V. Kapuria for help with western blots; B. R. Wilfred and D. Turner for advice on miRNAs, P. Macpherson for help with statistics, T. Melendez for expert care of fish and D. Turner, J. Parent and members of the Goldman lab for their support and comments on this work. This work was supported by funds from the NIH NEI RO1 EY018132 (to D.G.) and NIH NICHD T32HD007507 (to R.R.).

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D.G. and R.R. designed and analyzed the research and wrote the paper. D.G. generated the 1016 tuba1a:gfp transgenic fish. R.R. performed all experiments except for Fig. 4a, e where B.V.F. assayed let-7a and let-7f miRNA levels by RT–PCR.

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Correspondence to Daniel Goldman.

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Ramachandran, R., Fausett, B. & Goldman, D. Ascl1a regulates Müller glia dedifferentiation and retinal regeneration through a Lin-28-dependent, let-7 microRNA signalling pathway. Nat Cell Biol 12, 1101–1107 (2010). https://doi.org/10.1038/ncb2115

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