FRAP Analysis of Extracellular Diffusion in Zebrafish Embryos

  • Gary H. Soh
  • Patrick MüllerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1863)


Morphogens are signaling molecules that provide positional information to cells during development. They must move through embryonic tissues in order to coordinate patterning. The rate of a morphogen’s movement through a tissue—its effective diffusivity—affects the morphogen’s distribution and therefore influences patterning. Fluorescence recovery after photobleaching (FRAP) is a powerful method to measure the effective diffusion of molecules through cells and tissues, and has been successfully employed to examine morphogen mobility and gain important insights into embryogenesis. Here, we provide detailed protocols for FRAP assays in vitro and in living zebrafish embryos, and we explain how to analyze FRAP data using the open-source software PyFRAP to determine effective diffusion coefficients.

Key words

Fluorescence Recovery After Photobleaching FRAP Zebrafish Morphogens Extracellular signaling molecules Developmental biology 



We thank Katherine W. Rogers, Alexander Bläßle, David Mörsdorf, and Hannes Preiß for useful discussions. This work was supported by the Max Planck Society and ERC Starting Grant 637840.


  1. 1.
    Wolpert L (1969) Positional information and the spatial pattern of cellular differentiation. J Theor Biol 25(1):1–47. Scholar
  2. 2.
    Müller P et al (2013) Morphogen transport. Development 140(8):1621–1638. Scholar
  3. 3.
    Crick F (1970) Diffusion in embryogenesis. Nature 225(5231):420–422. Scholar
  4. 4.
    Rogers KW, Schier AF (2011) Morphogen gradients: from generation to interpretation. Annu Rev Cell Dev Biol 27:377–407. Scholar
  5. 5.
    Harmansa S et al (2015) Dpp spreading is required for medial but not for lateral wing disc growth. Nature 527(7578):317–322. Scholar
  6. 6.
    Müller P et al (2012) Differential diffusivity of Nodal and Lefty underlies a reaction-diffusion patterning system. Science 336(6082):721–724. Scholar
  7. 7.
    Rogers KW, Müller P (2018) Nodal and BMP dispersal during early zebrafish development. Developmental Biology pii:S0012-1606(17)30925-9.
  8. 8.
    Zinski J et al (2017) Systems biology derived source-sink mechanism of BMP gradient formation. eLife 6:e22199. Scholar
  9. 9.
    Pomreinke AP et al (2017) Dynamics of BMP signaling and distribution during zebrafish dorsal-ventral patterning. eLife 6:e25861. Scholar
  10. 10.
    Poo MM, Cone RA (1973) Lateral diffusion of rhodopsin in Necturus rods. Exp Eye Res 17(6):503–510. Scholar
  11. 11.
    Liebman PA, Entine G (1974) Lateral diffusion of visual pigment in photoreceptor disk membranes. Science 185(4149):457–459. Scholar
  12. 12.
    Lorén N et al (2015) Fluorescence recovery after photobleaching in material and life sciences: putting theory into practice. Q Rev Biophys 48(3):323–387. Scholar
  13. 13.
    Kicheva A et al (2007) Kinetics of morphogen gradient formation. Science 315(5811):521–525. Scholar
  14. 14.
    Gregor T et al (2007) Stability and nuclear dynamics of the Bicoid morphogen gradient. Cell 130(1):141–152. Scholar
  15. 15.
    Umulis DM, Othmer HG (2012) The importance of geometry in mathematical models of developing systems. Curr Opin Genet Dev 22(6):547–552. Scholar
  16. 16.
    Bläßle A et al (2018) Quantitative diffusion measurements using the open-source software PyFRAP. Nature Communications 9(1):1582.
  17. 17.
    Nüsslein-Volhard C, Dahm R (2002) Zebrafish: a practical approach. The practical approach series, vol 261, 1st edn. Oxford University Press, OxfordGoogle Scholar
  18. 18.
    Westerfield M (2007) The zebrafish book: a guide for the laboratory use of zebrafish (Danio rerio). University of Oregon Press, OregonGoogle Scholar
  19. 19.
    Schindelin J et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682. Scholar
  20. 20.
    Rogers KW et al (2015) Measuring protein stability in living zebrafish embryos using Fluorescence Decay After Photoconversion (FDAP). J Vis Exp 95:e52266.
  21. 21.
    Kimmel CB et al (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203(3):253–310. Scholar
  22. 22.
    Bläßle A, Müller P (2015) PyFDAP: automated analysis of Fluorescence Decay After Photoconversion (FDAP) experiments. Bioinformatics 31(6):972–974. Scholar
  23. 23.
    Xu Q (1999) Microinjection into zebrafish embryos. In: Guille M (ed) Molecular methods in developmental biology: xenopus and zebrafish. Humana Press, Totowa, NJ, pp 125–132. Scholar
  24. 24.
    Rosen JN, Sweeney MF, Mably JD (2009) Microinjection of zebrafish embryos to analyze gene function. J Vis Exp 25:e1115.

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Friedrich Miescher Laboratory of the Max Planck SocietyTübingenGermany

Personalised recommendations