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Autophagy in Zebrafish Extraocular Muscle Regeneration
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.
KeywordsAutolysosome 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.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):577–595Google Scholar
- 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. https://doi.org/10.1073/pnas.1834204100 CrossRefGoogle Scholar
- 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.Hu Z, Zhang J, Zhang Q (2011) Expression pattern and functions of autophagy-related gene atg5 in zebrafish organogenesis. Autophagy 7(12):1514–1527Google Scholar
- 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):289–299Google Scholar
- 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):520–526Google Scholar
- 25.Nüsslein-Volhard C, Dahm R (2002) Zebrafish: a practical approach, vol 975. Oxford University Press, OxfordGoogle Scholar
- 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