Autophagy in Zebrafish Extraocular Muscle Regeneration

  • Alfonso Saera-VilaEmail author
  • Phillip E. Kish
  • Alon KahanaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1854)


Zebrafish extraocular muscles regenerate after severe injury. Injured myocytes dedifferentiate to a mesenchymal progenitor state and reenter the cell cycle to proliferate, migrate, and redifferentiate into functional muscles. A dedifferentiation process that begins with a multinucleated syncytial myofiber filled with sarcomeres and ends with proliferating mononucleated myoblasts must include significant remodeling of the protein machinery and organelle content of the cell. It turns out that autophagy plays a key role early in this process, to degrade the sarcomeres as well as the excess nuclei of the syncytial multinucleated myofibers. Because of the robustness of the zebrafish reprogramming process, and its relative synchrony, it can serve as a useful in vivo model for studying the biology of autophagy. In this chapter, we describe the surgical muscle injury model as well as the experimental protocols for assessing and manipulating autophagy activation.


Autolysosome Autophagy Cell reprogramming Dedifferentiation Electron microscopy EOM Extraocular muscle MMT Muscle-to-mesenchymal transition Myectomy Regeneration Stem cell Zebrafish 



This work was funded by R01 EY022633 from the National Eye Institute (A.K.), the Alfred Taubman Medical Research Institute (A.K.), the Alliance for Vision Research (A.K.), and an unrestricted departmental grant from Research to Prevent Blindness, Inc.


  1. 1.
    Becker T, Wullimann MF, Becker CG, Bernhardt RR, Schachner M (1997) Axonal regrowth after spinal cord transection in adult zebrafish. J Comp Neurol 377(4):577–595CrossRefPubMedGoogle Scholar
  2. 2.
    Poss KD, Wilson LG, Keating MT (2002) Heart regeneration in zebrafish. Science 298(5601):2188–2190. CrossRefPubMedGoogle Scholar
  3. 3.
    Raya A, Koth CM, Buscher D, Kawakami Y, Itoh T, Raya RM, Sternik G, Tsai HJ, Rodriguez-Esteban C, Izpisua-Belmonte JC (2003) Activation of Notch signaling pathway precedes heart regeneration in zebrafish. Proc Natl Acad Sci U S A 100(Suppl 1):11889–11895. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Hitchcock PF, Raymond PA (2004) The teleost retina as a model for developmental and regeneration biology. Zebrafish 1(3):257–271. CrossRefPubMedGoogle Scholar
  5. 5.
    Goldman D (2014) Muller glial cell reprogramming and retina regeneration. Nat Rev Neurosci 15(7):431–442. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Poss KD, Shen J, Nechiporuk A, McMahon G, Thisse B, Thisse C, Keating MT (2000) Roles for Fgf signaling during zebrafish fin regeneration. Dev Biol 222(2):347–358. CrossRefPubMedGoogle Scholar
  7. 7.
    Pfefferli C, Jazwinska A (2015) The art of fin regeneration in zebrafish. Regeneration (Oxf) 2(2):72–83. CrossRefGoogle Scholar
  8. 8.
    Kan NG, Junghans D, Izpisua Belmonte JC (2009) Compensatory growth mechanisms regulated by BMP and FGF signaling mediate liver regeneration in zebrafish after partial hepatectomy. FASEB J 23(10):3516–3525. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Berberoglu MA, Gallagher TL, Morrow ZT, Talbot JC, Hromowyk KJ, Tenente IM, Langenau DM, Amacher SL (2017) Satellite-like cells contribute to pax7-dependent skeletal muscle repair in adult zebrafish. Dev Biol 424(2):162–180. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Saera-Vila A, Kasprick DS, Junttila TL, Grzegorski SJ, Louie KW, Chiari EF, Kish PE, Kahana A (2015) Myocyte dedifferentiation drives extraocular muscle regeneration in adult zebrafish. Invest Ophthalmol Vis Sci 56(8):4977–4993. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Louie KW, Saera-Vila A, Kish PE, Colacino JA, Kahana A (2017) Temporally distinct transcriptional regulation of myocyte dedifferentiation and Myofiber growth during muscle regeneration. BMC Genomics 18(1):854. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Saera-Vila A, Kish PE, Kahana A (2016) Fgf regulates dedifferentiation during skeletal muscle regeneration in adult zebrafish. Cell Signal 28(9):1196–1204. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Boya P, Reggiori F, Codogno P (2013) Emerging regulation and functions of autophagy. Nat Cell Biol 15(7):713–720. CrossRefPubMedGoogle Scholar
  14. 14.
    Mizushima N, Levine B (2010) Autophagy in mammalian development and differentiation. Nat Cell Biol 12(9):823–830. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Wong E, Cuervo AM (2010) Autophagy gone awry in neurodegenerative diseases. Nat Neurosci 13(7):805–811. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Eng CH, Abraham RT (2011) The autophagy conundrum in cancer: influence of tumorigenic metabolic reprogramming. Oncogene 30(47):4687–4696. CrossRefPubMedGoogle Scholar
  17. 17.
    Pan H, Cai N, Li M, Liu GH, Izpisua Belmonte JC (2013) Autophagic control of cell ‘stemness’. EMBO Mol Med 5(3):327–331. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Wang S, Xia P, Rehm M, Fan Z (2015) Autophagy and cell reprogramming. Cell Mol Life Sci 72(9):1699–1713. CrossRefPubMedGoogle Scholar
  19. 19.
    Saera-Vila A, Kish PE, Louie KW, Grzegorski SJ, Klionsky DJ, Kahana A (2016) Autophagy regulates cytoplasmic remodeling during cell reprogramming in a zebrafish model of muscle regeneration. Autophagy 12(10):1864–1875. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Saera-Vila A, Louie KW, Sha C, Kelly RM, Kish PE, Kahana A (2018) Extraocular muscle regeneration in zebrafish requires late signals from insulin-like growth factors. PLoS One 13(2):e0192214. CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (2002) Short protocols in molecular biology, 5th edn. Wiley, New YorkGoogle Scholar
  22. 22.
    Hu Z, Zhang J, Zhang Q (2011) Expression pattern and functions of autophagy-related gene atg5 in zebrafish organogenesis. Autophagy 7(12):1514–1527CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Higashijima S, Okamoto H, Ueno N, Hotta Y, Eguchi G (1997) High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin. Dev Biol 192(2):289–299CrossRefPubMedGoogle Scholar
  24. 24.
    He C, Bartholomew CR, Zhou W, Klionsky DJ (2009) Assaying autophagic activity in transgenic GFP-Lc3 and GFP-Gabarap zebrafish embryos. Autophagy 5(4):520–526CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Nüsslein-Volhard C, Dahm R (2002) Zebrafish: a practical approach, vol 975. Oxford University Press, OxfordGoogle Scholar
  26. 26.
    Westerfield M (2007) The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), 5th edn. University of Oregon Press, EugeneGoogle Scholar
  27. 27.
    Fodor E, Sigmond T, Ari E, Lengyel K, Takacs-Vellai K, Varga M, Vellai T (2017) Methods to study autophagy in zebrafish. Methods Enzymol 588:467–496. CrossRefPubMedGoogle Scholar
  28. 28.
    Mathai BJ, Meijer AH, Simonsen A (2017) Studying autophagy in zebrafish. Cell 6(3).

Copyright information

© Springer Science+Business Media New York 2018

Authors and Affiliations

  1. 1.Department of Ophthalmology and Visual Sciences, Kellogg Eye CenterUniversity of MichiganAnn ArborUSA

Personalised recommendations