CMV Promoter-Driven Expression and Visualization of Tagged Proteins in Live and Fixed Zebrafish Embryonic Epidermis

  • Kirti Gupta
  • Mahendra Sonawane
Part of the Springer Protocols Handbooks book series (SPH)


In the recent past, zebrafish has emerged as a highly useful vertebrate model for biomedical research. Owing to its easy handling, suitability for high-throughput genetic and chemical screens, and tractability by high-resolution microscopy, it is possible to study molecular mechanisms of vertebrate development and disease biology using zebrafish. This chapter introduces zebrafish epidermis as a model for studying cell biology of epithelial tissues in vivo and describes the technique of protein expression using plasmid vectors having cytomegalovirus (CMV) promoter. It details protocols for microinjection of plasmids into fertilized zebrafish oocytes, screening of the epidermal clones expressing the tagged protein, immunostaining, and mounting of embryos for both fixed and live imaging by confocal microscopy. This collection of protocols allows for analysis of localization of a wide range of proteins including those involved in intracellular transport, cell polarity, cell adhesion, and so on, during early developmental stages of zebrafish embryos and larvae.


Zebrafish Microinjections CMV promoter–based expression Live imaging Confocal microscopy 



We would like to thank Sumit Sen for the illustrations, Dr. Clyde Pinto for contributing to Fig. 5, Geetika Chouhan and Mandar Phatak for critically reading the manuscript, and Kalidas Kohale for fish maintenance. This work was supported by funding from TIFR-DAE (12P-121).


  1. 1.
    Le Guellec D, Morvan-Dubois G, Sire JY (2004) Skin development in bony fish with particular emphasis on collagen deposition in the dermis of the zebrafish. Int J Dev Biol 48:217–231CrossRefGoogle Scholar
  2. 2.
    Sonawane M (2005) Zebrafish penner/lethal giant larvae 2 functions in hemidesmosome formation, maintenance of cellular morphology and growth regulation in the developing basal epidermis. Development 132:3255–3265CrossRefGoogle Scholar
  3. 3.
    Lee RTH, Asharani PV, Carney TJ (2014) Basal keratinocytes contribute to all strata of the adult zebrafish epidermis. PLoS One 9(1):e84858CrossRefGoogle Scholar
  4. 4.
    Sonawane M, Martin-Maischein H, Schwarz H et al (2009) Lgl2 and E-cadherin act antagonistically to regulate hemidesmosome formation during epidermal development in zebrafish. Development 136:1231–1240CrossRefGoogle Scholar
  5. 5.
    Reischauer S, Levesque MP, Nüsslein-Volhard C et al (2009) Lgl2 executes its function as a tumor suppressor by regulating ErbB signaling in the zebrafish epidermis. PLoS Genet 5(11):e1000720CrossRefGoogle Scholar
  6. 6.
    Morris JL, Cross SJ, Lu Y et al (2018) Live imaging of collagen deposition during skin development and repair in a collagen I—GFP fusion transgenic zebrafish line. Dev Biol 441(1):4–11CrossRefGoogle Scholar
  7. 7.
    Raman R, Damle I, Rote R et al (2016) APKC regulates apical localization of Lgl to restrict elongation of microridges in developing zebrafish epidermis. Nat Commun 7:11643CrossRefGoogle Scholar
  8. 8.
    Pinto CS, Khandekar A, Bhavna R et al (2019) Microridges are apical epithelial projections formed of F-actin networks that organize the glycan layer. Sci Rep 9:12191CrossRefGoogle Scholar
  9. 9.
    Slanchev K, Carney TJ, Stemmler MP et al (2009) The epithelial cell adhesion molecule EpCAM is required for epithelial morphogenesis and integrity during zebrafish epiboly and skin development. PLoS Genet 5:e1000563CrossRefGoogle Scholar
  10. 10.
    Webb AE, Driever W, Kimelman D (2008) Psoriasis regulates epidermal development in Zebrafish. Dev Dyn 237:1153–1164CrossRefGoogle Scholar
  11. 11.
    Morita H, Grigolon S, Bock M et al (2017) The physical basis of coordinated tissue spreading in Zebrafish gastrulation. Dev Cell 40:354–366CrossRefGoogle Scholar
  12. 12.
    Haas P, Gilmour D (2006) Chemokine signaling mediates self-organizing tissue migration in the zebrafish lateral line. Dev Cell 10:673–680CrossRefGoogle Scholar
  13. 13.
    Eisenhoffer GT, Slattum G, Ruiz OE et al (2017) A toolbox to study epidermal cell types in zebrafish. J Cell Sci 130:269–277CrossRefGoogle Scholar
  14. 14.
    Gong Z, Ju B, Wang X et al (2002) Green fluorescent protein expression in germ-line transmitted transgenic zebrafish under a stratified epithelial promoter from keratin8. Dev Dyn 223:204–215CrossRefGoogle Scholar
  15. 15.
    Clark BS, Winter M, Cohen AR et al (2011) Generation of Rab-based transgenic lines for in vivo studies of endosome biology in zebrafish. Dev Dyn 240:245–265CrossRefGoogle Scholar
  16. 16.
    Asakawa K, Kawakami K (2010) A transgenic zebrafish for monitoring in vivo microtubule structures. Dev Dyn 239:2695–2699CrossRefGoogle Scholar
  17. 17.
    Miyoshi H, Blömer U, Takahashi M et al (1998) Development of a self-inactivating lentivirus vector. J Virol 72:8150–5157CrossRefGoogle Scholar
  18. 18.
    Turner DL, Weintraub H (1994) Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate. Genes Dev 8:1434–1447CrossRefGoogle Scholar
  19. 19.
    Gökirmak T, Campanale JP, Shipp LE et al (2012) Localization and substrate selectivity of seaurchin multidrug (MDR) efflux transporters. J Biol Chem 287:43876–43883CrossRefGoogle Scholar
  20. 20.
    Nusslein-Volhard C, Dahm R (2002) Zebrafish: a practical approach. Oxford University Press, New YorkGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Kirti Gupta
    • 1
  • Mahendra Sonawane
    • 1
  1. 1.Department of Biological SciencesTata Institute of Fundamental ResearchMumbaiIndia

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