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Cell tracking using a photoconvertible fluorescent protein

Nature Protocols volume 1, pages 960–967 (2006)Cite this article

Abstract

The tracking of cell fate, shape and migration is an essential component in the study of the development of multicellular organisms. Here we report a protocol that uses the protein Kaede, which is fluorescent green after synthesis but can be photoconverted red by violet or UV light. We have used Kaede along with confocal laser scanning microscopy to track labeled cells in a pattern of interest in zebrafish embryos. This technique allows the visualization of cell movements and the tracing of neuronal shapes. We provide illustrative examples of expression by mRNA injection, mosaic expression by DNA injection, and the creation of permanent transgenic fish with the UAS-Gal4 system to visualize morphogenetic processes such as neurulation, placode formation and navigation of early commissural axons in the hindbrain. The procedure can be adapted to other photoconvertible and reversible fluorescent molecules, including KikGR and Dronpa; these molecules can be used in combination with two-photon confocal microscopy to specifically highlight cells buried in tissues. The total time needed to carry out the protocol involving transient expression of Kaede by injection of mRNA or DNA, photoconversion and imaging is 2–8 d.

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Figure 1: Arranging eggs for microinjection.
Figure 2: Mounting embryos for photoconversion and observation.
Figure 3: Photoconversion in desired patterns.
Figure 4: Four-dimensional imaging of convergent-extension movement of neural plate and organogenesis derived from placodes in the zebrafish embryo.
Figure 5: Tracing neurons and other cell types using Kaede and KikGR combined with the Gal4-UAS system in either transient or permanent transgenic fish.
Figure 6: Tracking some of the earliest commissural axons navigating in the hindbrain in the cross between Tg(deltaD:Gal4) and Tg(UAS:Kaede), dorsal view.

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Acknowledgements

We thank S. Aizawa for support; S. Hayashi for encouragement; S. Aramaki, H. Togashi, S. Yonemura, M. Hibi and K. Agata for help; M. Royle for comments and J. Clarke for encouraging us to publish this protocol. pKaede1 and pKikGR3 were kindly provided by A. Miyawaki; pEF:Gal4VP16, pUlyn and pEF:Gal4VP16-UlynU2B17 by R. Köster and S. Fraser; pDeltaD:Gal4 (ref. 11) by N. Sheer and J. Campos-Ortega; the HuC promoter20,21 by S. Higashijima; the HSP promoter12 by W. Shoji; vectors for T2 transposon–mediated gene transfer22,23 by K. Kawakami and Tg(deltaD:Gal4)11 and Tg(hsp:Gal4)14 by the Zebrafish International Resource Center. K.H. received grants-in-aid for Scientific Research from The Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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  1. Laboratory for Vertebrate Body Plan, Center for Developmental Biology, RIKEN Kobe 2-2-3 Minatojima Minamimachi, Chuo-ku, 650-0047, Kobe, Japan

    Kohei Hatta, Hitomi Tsujii & Tomomi Omura

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  1. Kohei Hatta
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Correspondence to Kohei Hatta.

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Hatta, K., Tsujii, H. & Omura, T. Cell tracking using a photoconvertible fluorescent protein. Nat Protoc 1, 960–967 (2006). https://doi.org/10.1038/nprot.2006.96

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