The Use of Complementary Luminescent and Fluorescent Techniques for Imaging Ca2+ Signaling Events During the Early Development of Zebrafish (Danio rerio)

  • Sarah E. WebbEmail author
  • Andrew L. Miller
Part of the Methods in Molecular Biology book series (MIMB, volume 1929)


We have visualized many of the Ca2+ signaling events that occur during the early stages of zebrafish development using complementary luminescent and fluorescent imaging techniques. We initially microinject embryos with the luminescent Ca2+ reporter, f-holo-aequorin, and using a custom-designed luminescent imaging system, we can obtain pan-embryonic visual information continually for up to the first ~24 h postfertilization (hpf). Once we know approximately when and where to look for these Ca2+ signaling events within a complex developing embryo, we then repeat the experiment using a fluorescent Ca2+ reporter such as calcium green-1 dextran and use confocal laser scanning microscopy to provide time-lapse series of higher-resolution images. These protocols allow us to identify the specific cell types and even the particular subcellular domain (e.g., nucleus or cytoplasm) generating the Ca2+ signal. Here, we outline the techniques we use to precisely microinject f-holo-aequorin or calcium green-1 dextran into embryos without affecting their viability or development. We also describe how to inject specific regions of early embryos in order to load localized embryonic domains with a particular Ca2+ reporter. These same techniques can also be used to introduce other membrane-impermeable reagents into embryos, including Ca2+ channel antagonists, Ca2+ chelators, fluorescent dyes, RNA, and DNA.

Key words

Luminescent and fluorescent Ca2+ reporters Microinjection Holo-aequorin Calcium green-1 dextran Zebrafish embryos 



This work was supported by the Hong Kong Research Grants Council (RGC) General Research Fund awards 16101714 and 16100115 and the ANR/RGC joint research scheme award A-HKUST601/13. We also acknowledge funding support from the Hong Kong Innovation and Technology Commission (ITCPD/17-9). We thank Andrew Ho for helping us to photograph the equipment shown in Figs. 2 and 3.


  1. 1.
    Jaffe LF (1999) Organization of early development by calcium patterns. BioEssays 21:657–667CrossRefGoogle Scholar
  2. 2.
    Berridge MJ, Lipp P, Bootman MD (2000) The versatility and universality of calcium signaling. Nature Rev Mol Cell Biol 1:11–21CrossRefGoogle Scholar
  3. 3.
    Webb SE, Miller AL (2003) Calcium signaling during embryonic development. Nature Rev Mol Cell Biol 4:539–551CrossRefGoogle Scholar
  4. 4.
    Porter GA Jr, Makuck RF, Rivkees SA (2003) Intracellular calcium plays an essential role in cardiac development. Dev Dyn 227:280–290CrossRefGoogle Scholar
  5. 5.
    Whitaker M (2006) Calcium at fertilization and in early development. Physiol Rev 86:25–88CrossRefGoogle Scholar
  6. 6.
    Slusarski DC, Pelegri F (2007) Calcium signaling in vertebrate embryonic patterning and morphogenesis. Dev Biol 307:1–13CrossRefGoogle Scholar
  7. 7.
    Rosenberg SS, Spitzer NC (2011) Calcium signaling in neuronal development. Cold Spring Harb Perspec Biol 3(10):a004259. Scholar
  8. 8.
    Zhang J, Webb SE, Ma LH, Chan CM, Miller AL (2011) Necessary role for intracellular Ca2+ transients in initiating the apical-basolateral thinning of enveloping layer cells during the early blastula period of zebrafish development. Develop Growth Diff 53:679–696CrossRefGoogle Scholar
  9. 9.
    Kelu JJ, Webb SE, Parrington J, Galione A, Miller AL (2017) Ca2+ release via two-pore channel type 2 (TPC2) is required for slow muscle cell myofibrillogenesis and myotomal patterning in intact zebrafish embryos. Dev Biol 425:109–129CrossRefGoogle Scholar
  10. 10.
    Brownlee C, Dale B (1990) Temporal and spatial correlation of fertilization current, calcium waves and cytoplasmic contraction in eggs of Ciona intestinalis. Proc R Soc Lond B 239:321–328CrossRefGoogle Scholar
  11. 11.
    Fluck RA, Miller AL, Jaffe LF (1991) Slow calcium waves accompany cytokinesis in medaka fish eggs. J Cell Biol 115:1259–1265CrossRefGoogle Scholar
  12. 12.
    Homa ST, Carroll J, Swann K (1993) Fertilization and early embryology: the role of calcium in mammalian oocyte maturation and egg activation. Hum Reprod 8:1274–1281CrossRefGoogle Scholar
  13. 13.
    Swann K, McDougall A, Whitaker M (1994) Calcium signalling at fertilization. J Mar Biol Assoc UK 74:3–16CrossRefGoogle Scholar
  14. 14.
    Chang DC, Meng C (1995) A localized elevation of cytosolic free calcium is associated with cytokinesis in the zebrafish embryo. J Cell Biol 131:1539–1545CrossRefGoogle Scholar
  15. 15.
    Webb SE, Lee KW, Karplus E, Miller AL (1997) Localized calcium transients accompany furrow positioning, propagation, and deepening during the early cleavage period of zebrafish embryos. Dev Biol 192:78–92CrossRefGoogle Scholar
  16. 16.
    Leung CF, Webb SE, Miller AL (1998) Calcium transients accompany ooplasmic segregation in zebrafish embryos. Develop Growth Differ 40:313–326CrossRefGoogle Scholar
  17. 17.
    Créton R, Kreiling JA, Jaffe LF (2000) Presence and roles of calcium gradients along the dorsal-ventral axis in Drosophila embryos. Dev Biol 217:375–385CrossRefGoogle Scholar
  18. 18.
    Whitaker M (2008) Calcium signalling in early embryos. Phil Trans Royal Soc B 363:1401–1418CrossRefGoogle Scholar
  19. 19.
    Gilland E, Miller AL, Karplus E, Baker R, Webb SE (1999) Imaging of multicellular large-scale rhythmic calcium waves during zebrafish gastrulation. Proc Natl Acad Sci U S A 96:157–161CrossRefGoogle Scholar
  20. 20.
    Leclerc C, Webb SE, Daguzan C, Moreau M, Miller AL (2000) Imaging patterns of calcium transients during neural induction in Xenopus laevis embryos. J Cell Sci 113:3519–3529PubMedGoogle Scholar
  21. 21.
    Tada M, Concha ML (2001) Vertebrate gastrulation: calcium waves orchestrate cell movements. Curr Biol 11:R470–R472CrossRefGoogle Scholar
  22. 22.
    Wallingford JB, Ewald AJ, Harland RM, Fraser SE (2001) Calcium signaling during convergent extension in Xenopus. Curr Biol 11:652–661CrossRefGoogle Scholar
  23. 23.
    Rogers KL, Picaud S, Roncali E, Boisgard R, Colasant C, Stinnakre J, Tavatian B, Brûlet P (2007) Non-invasive in vivo imaging of calcium signaling in mice. PLoS One 2:e974CrossRefGoogle Scholar
  24. 24.
    Leung CF, Miller AL, Korzh V, Cong SW, Sleptsova-Friedrich I, Webb SE (2009) Visualization of stochastic Ca2+ signals in the formed somites during the early segmentation period in intact, normally developing zebrafish embryos. Develop Growth Differ 51:617–637CrossRefGoogle Scholar
  25. 25.
    Cheung CY, Webb SE, Love DR, Miller AL (2011) Visualization, characterization and modulation of calcium signaling during the development of slow muscle cells in intact zebrafish embryos. Int J Dev Biol 55:153–174CrossRefGoogle Scholar
  26. 26.
    Shimomura O, Johnson FH, Saiga Y (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 59:223–239CrossRefGoogle Scholar
  27. 27.
    Campbell AK (1974) Extraction, partial purification and properties of obelin, the calcium-activated luminescent protein from the hydroid Obelia geniculata. Biochem J 143:411–418CrossRefGoogle Scholar
  28. 28.
    Markova SV, Vysotski ES, Blinks JR, Burakova LP, Wang B-C, Lee J (2002) Obelin from the bioluminescent marine hydroid Obelia geniculata: cloning, expression, and comparison of some properties with those of other Ca2+-regulated photoproteins. Biochemist 41:2227–2236CrossRefGoogle Scholar
  29. 29.
    Inouye S, Sahara Y (2007) Expression, purification and characterization of a photoprotein, clytin, from Clytia gregarium. Protein Expr Purif 53:384–389CrossRefGoogle Scholar
  30. 30.
    Inouye S, Sahara Y (2009) Expression and purification of the calcium binding photoprotein mitrocomin using ZZ-domain as a soluble partner in E. coli cells. Protein Expr Purif 66:52–57CrossRefGoogle Scholar
  31. 31.
    Burakova LP, Natashin PV, Markova SV, Eremeeva EV, Malikova NP, Cheng C, Liu ZJ, Vysotski ES (2016) Mitrocomin from the jellyfish Mitrocoma cellularia with deleted C-terminal tyrosine reveals a higher bioluminescence activity compared to wild type photoprotein. J Photochem Photobiol B 162:286–297CrossRefGoogle Scholar
  32. 32.
    Shimomura O, Johnson FH (1975) Chemical nature of bioluminescence systems in coelenterates. Proc Natl Acad Sci U S A 72:1546–1549CrossRefGoogle Scholar
  33. 33.
    Shimomura O, Johnson FH (1978) Peroxidized coelenterazine, the active group in the photoprotein aequorin. Proc Natl Acad Sci U S A 75:2611–2615CrossRefGoogle Scholar
  34. 34.
    Shimomura O, Musicki B, Kishi Y (1988) Semi-synthetic aequorin: an improved tool for the measurement of calcium ion concentration. Biochem J 251:405–410CrossRefGoogle Scholar
  35. 35.
    Shimomura O, Musicki B, Kishi Y (1989) Semi-synthetic aequorins with improved sensitivity to Ca2+ ions. Biochem J 261:913–920CrossRefGoogle Scholar
  36. 36.
    Shimomura O, Inouye S, Musicki B, Kishi Y (1990) Recombinant aequorin and recombinant semi-synthetic aequorin. Biochem J 270:309–312CrossRefGoogle Scholar
  37. 37.
    Inouye S, Noguchi M, Sakari Y, Takagi Y, Miyata T, Iwanaga S, Miyata T, Tsuji FI (1985) Cloning and sequence analysis of cDNA for the luminescent protein aequorin. Proc Natl Acad Sci U S A 82:3154–3158CrossRefGoogle Scholar
  38. 38.
    Prasher D, McCann RO, Cormier MJ (1985) Cloning and expression of the cDNA coding for aequorin, a bioluminescent calcium-binding protein. Biochem Biophys Res Comm 126:1259–1268CrossRefGoogle Scholar
  39. 39.
    Shimomura O, Inouye S (1999) The in situ regeneration and extraction of recombinant aequorin from Escherichia coli cells and the purification of extracted aequorin. Prot Express Purific 16:91–95CrossRefGoogle Scholar
  40. 40.
    Higashijima S, Masino MA, Mandel G, Fetcho JR (2003) Imaging neuronal activity during zebrafish behaviour with a genetically encoded calcium indicator. J Neurophysiol 90:3987–3997CrossRefGoogle Scholar
  41. 41.
    Ashworth R, Brennan C (2005) Use of transgenic zebrafish reporter lines to study calcium signalling in development. Brief Funct Gen Proteo 4:186–193CrossRefGoogle Scholar
  42. 42.
    Horikawa K, Yamada Y, Matsuda T, Kobayashi K, Hashimoto M, Matsu-ura T, Miyawaki A, Michikawa T, Mikoshiba K, Nagai T (2010) Spontaneous network activity visualized by ultrasensitive Ca2+ indicators, yellow Cameleon-Nano. Nature Meth 7:729–732CrossRefGoogle Scholar
  43. 43.
    Muto A, Kawakami K (2011) Imaging functional neural circuits in zebrafish with a new GCaMP and the Gal4FF-UAS system. Commun Integr Biol 4:566–568CrossRefGoogle Scholar
  44. 44.
    Chen J, Xia L, Bruchas MR, Solnica-Krezel L (2017) Imaging early embryonic calcium activity with GCaMP6s transgenic zebrafish. Dev Biol 430:385–396CrossRefGoogle Scholar
  45. 45.
    Yuen MYF, Webb SE, Chan CM, Thisse B, Thisse C, Miller AL (2013) Characterization of Ca2+ signaling in the external yolk syncytial layer during the late blastula and early gastrula periods of zebrafish development. Biochim Biophys Acta 1833:1641–1656CrossRefGoogle Scholar
  46. 46.
    Ma LH, Webb SE, Chan CM, Zhang J, Miller AL (2009) Establishment of a transitory dorsal-biased window of localized Ca2+ signaling in the superficial epithelium following the mid-blastula transition in zebrafish embryos. Dev Biol 327:143–157CrossRefGoogle Scholar
  47. 47.
    Webb SE, Miller AL (2003) Imaging intercellular calcium waves during late epiboly in intact zebrafish embryos. Zygote 11:175–182CrossRefGoogle Scholar
  48. 48.
    Roosen-Runge EC (1938) On the early development-bipolar differentiation and cleavage of the zebrafish, Brachydanio rerio. Biol Bull 75:119–133CrossRefGoogle Scholar
  49. 49.
    Beams HW, Kessel RG, Shim CY, Tung HN (1985) Scanning electron microscope studies on blastodisc formation in zebrafish, Brachydanio rerio. J Morphol 189:41–49CrossRefGoogle Scholar
  50. 50.
    Cheung CY, Webb SE, Meng A, Miller AL (2006) Transient expression of apoaequorin in zebrafish embryos: extending the ability to image calcium transients during later stages of development. Int J Dev Biol 50:561–569CrossRefGoogle Scholar
  51. 51.
    Webb SE, Miller AL (2007) Ca2+ signalling during embryonic cytokinesis in animal systems. In: Krebs J, Michalak M (eds) Calcium: a matter of life and death, vol 17. Elsevier, Amsterdam, Netherlands, pp 443–468Google Scholar
  52. 52.
    Lee KW, Webb SE, Miller AL (2003) Ca2+ released via IP3 receptors is required for furrow deepening during cytokinesis in zebrafish embryos. Int J Dev Biol 47:411–421PubMedGoogle Scholar
  53. 53.
    Kelu JJ, Chan HLH, Webb SE, Cheng AHH, Ruas M, Parrington J, Galione A, Miller AL (2015) Two-pore channel 2 activity is required for slow muscle cell-generated Ca2+ signaling during myogenesis in intact zebrafish. Int J Dev Biol 59:313–325CrossRefGoogle Scholar
  54. 54.
    Kelu JJ, Webb SE, Galione A, Miller AL (2018) TPC2-mediated Ca2+ signalling is required for the establishment of synchronized activity in developing zebrafish primary motor neurons. Dev Biol 438(1):57–68CrossRefGoogle Scholar
  55. 55.
    Bonsignorio D, Perego L, Del Giacco L, Cotelli F (1996) Structure and macromolecular composition of the zebrafish egg chorion. Zygote 4:101–108CrossRefGoogle Scholar
  56. 56.
    Miller AL, Karplus E, Jaffe LF (1994) Imaging Ca2+ with aequorin using a photon imaging detector. In: Nuccitelli R (ed) Methods in cell biology: a practical guide to the study of calcium in living cells, vol 40. Academic Press Inc., San Diego, CA, pp 305–338CrossRefGoogle Scholar
  57. 57.
    Webb SE, Rogers KL, Karplus E, Miller AL (2010) The use of aequorins to record and visualize Ca2+ dynamics: from subcellular microdomains to whole organisms. In: Whitaker M (ed) Methods in cell biology: calcium in living cells, vol 99. Academic Press Inc., San Diego, CA, pp 263–300CrossRefGoogle Scholar

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

Authors and Affiliations

  1. 1.Division of Life Science and State Key Laboratory of Molecular NeuroscienceHKUSTClear Water BayPeople’s Republic of China

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