Myogenesis pp 245-254 | Cite as

Fluorescence-Activated Cell Sorting of Larval Zebrafish Muscle Stem/Progenitor Cells Following Skeletal Muscle Injury

  • Dhanushika RatnayakeEmail author
  • Peter D. Currie
Part of the Methods in Molecular Biology book series (MIMB, volume 1889)


This chapter describes a protocol for the isolation of larval zebrafish muscle stem/progenitor cells by fluorescence-activated cell sorting (FACS). This method has been successfully applied to isolate pax3a expressing cells 3 days following needle stab skeletal muscle injury. The cell sorting strategy described here can easily be adapted to any cell type at embryonic or larval stages. RNA extracted from the sorted cells can be used for subsequent downstream applications such as quantitative PCR (qPCR), microarrays, or next generation sequencing.

Key words

Zebrafish larvae Skeletal muscle Muscle stem cells Satellite cells Muscle progenitors Needle stab injury Muscle injury Regeneration FACS 


  1. 1.
    Mauro A (1961) Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol 9(2):493–495CrossRefGoogle Scholar
  2. 2.
    Cornelison D, Wold BJ (1997) Single-cell analysis of regulatory gene expression in quiescent and activated mouse skeletal muscle satellite cells. Dev Biol 191(2):270–283CrossRefGoogle Scholar
  3. 3.
    Cooper R, Tajbakhsh S, Mouly V, Cossu G, Buckingham M, Butler-Browne G (1999) In vivo satellite cell activation via Myf5 and MyoD in regenerating mouse skeletal muscle. J Cell Sci 112(17):2895–2901Google Scholar
  4. 4.
    Charge SB, Rudnicki MA (2004) Cellular and molecular regulation of muscle regeneration. Physiol Rev 84(1):209–238CrossRefGoogle Scholar
  5. 5.
    Seger C, Hargrave M, Wang X, Chai RJ, Elworthy S, Ingham PW (2011) Analysis of Pax7 expressing myogenic cells in zebrafish muscle development, injury, and models of disease. Dev Dyn 240(11):2440–2451CrossRefGoogle Scholar
  6. 6.
    Siegel AL, Gurevich DB, Currie PD (2013) A myogenic precursor cell that could contribute to regeneration in zebrafish and its similarity to the satellite cell. FEBS J 280(17):4074–4088CrossRefGoogle Scholar
  7. 7.
    Gurevich DB, Nguyen PD, Siegel AL, Ehrlich OV, Sonntag C, Phan JM, Berger S, Ratnayake D, Hersey L, Berger J (2016) Asymmetric division of clonal muscle stem cells coordinates muscle regeneration in vivo. Science 353(6295):aad9969CrossRefGoogle Scholar
  8. 8.
    Pipalia TG, Koth J, Roy SD, Hammond CL, Kawakami K, Hughes SM (2016) Cellular dynamics of regeneration reveals role of two distinct Pax7 stem cell populations in larval zebrafish muscle repair. Dis Model Mech 9(6):671–684CrossRefGoogle Scholar
  9. 9.
    Chen Y-H, Wang Y-H, Chang M-Y, Lin C-Y, Weng C-W, Westerfield M, Tsai H-J (2007) Multiple upstream modules regulate zebrafish myf5 expression. BMC Dev Biol 7(1):1CrossRefGoogle Scholar
  10. 10.
    Cole NJ, Hall TE, Don EK, Berger S, Boisvert CA, Neyt C, Ericsson R, Joss J, Gurevich DB, Currie PD (2011) Development and evolution of the muscles of the pelvic fin. PLoS Biol 9(10):e1001168CrossRefGoogle Scholar
  11. 11.
    Berger J, Currie PD (2013) 503unc, a small and muscle-specific zebrafish promoter. Genesis 51(6):443–447CrossRefGoogle Scholar
  12. 12.
    Elworthy S, Hargrave M, Knight R, Mebus K, Ingham PW (2008) Expression of multiple slow myosin heavy chain genes reveals a diversity of zebrafish slow twitch muscle fibres with differing requirements for hedgehog and Prdm1 activity. Development 135(12):2115–2126CrossRefGoogle Scholar
  13. 13.
    Nord H, Burguiere A-C, Muck J, Nord C, Ahlgren U, von Hofsten J (2014) Differential regulation of myosin heavy chains defines new muscle domains in zebrafish. Mol Biol Cell 25(8):1384–1395CrossRefGoogle Scholar
  14. 14.
    van Impel A, Zhao Z, Hermkens DM, Roukens MG, Fischer JC, Peterson-Maduro J, Duckers H, Ober EA, Ingham PW, Schulte-Merker S (2014) Divergence of zebrafish and mouse lymphatic cell fate specification pathways. Development 141(6):1228–1238CrossRefGoogle Scholar
  15. 15.
    Elsalini OA, Rohr KB (2003) Phenylthiourea disrupts thyroid function in developing zebrafish. Dev Genes Evol 212(12):593–598PubMedGoogle Scholar
  16. 16.
    Li Z, Ptak D, Zhang L, Walls EK, Zhong W, Leung YF (2012) Phenylthiourea specifically reduces zebrafish eye size. PLoS One 7(6):e40132CrossRefGoogle Scholar
  17. 17.
    Parker MO, Brock AJ, Millington ME, Brennan CH (2013) Behavioral phenotyping of casper mutant and 1-pheny-2-thiourea treated adult zebrafish. Zebrafish 10(4):466–471CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Australian Regenerative Medicine InstituteMonash UniversityClaytonAustralia
  2. 2.European Molecular Biology Laboratory Australia Melbourne NodeMonash UniversityClaytonAustralia

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