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Photo-mediated gene activation using caged RNA/DNA in zebrafish embryos

Nature Genetics volume 28, pages 317–325 (2001)Cite this article

Abstract

We report a new and simple technique for photo-mediated temporal and spatial control of gene activation in zebrafish embryos as an alternative to the gene 'knockdown' approach using antisense, morpholino-modified oligonucleotides (morpholinos). The synthetic compound 6-bromo-4-diazomethyl-7-hydroxycoumarin (Bhc-diazo) forms a covalent bond with the phosphate moiety of the sugar-phosphate backbone of RNA, a process known as caging. The 6-bromo-7-hydroxycoumarin-4-ylmethyl (Bhc) group binds to approximately 30 sites on the phosphate moieties per 1 kb of RNA sequence. Bhc-caged mRNA undergoes photolysis (uncaging) when exposed to long-wave ultraviolet light (350 to 365 nm). We show that Bhc-caged green fluorescent protein (Gfp) mRNA has severely reduced translational activity in vitro, whereas illumination of Bhc-caged mRNA with ultraviolet light leads to partial recovery of translational activity. Bhc-caged mRNA is highly stable in zebrafish embryos. In embryos injected with Bhc-caged Gfp mRNA at the one-cell stage, GFP protein expression and fluorescence is specifically induced by ultraviolet light. We also show that, consistent with results obtained using other methods, uncaging eng2a (which encodes the transcription factor Engrailed2a) in the head region during early development causes a severe reduction in the size of the eye and enhanced development of the midbrain and the midbrain-hindbrain boundary at the expense of the forebrain.

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Figure 1: Inactivation of mRNA by caging with Bhc and reactivation by photo-illumination.
Figure 2: Recovery of translational activity of Bhc-caged mRNA by illumination of zebrafish embryos with ultraviolet light.
Figure 3: Ectopic GFP induction by spot illumination with ultraviolet light of embryos injected with Bhc-caged Gfp mRNA at 12 h after fertilization.
Figure 4: Effects of Eng2a overexpression by photoactivation of Bhc-caged eng2a mRNA.
Figure 5: Changes in the patterns of expression of various region-specific genes at 22 h after fertilization in the embryos injected with Bhc-caged eng2a mRNA and spot-illuminated on the head with ultraviolet light at 12 h after fertilization.
Figure 6: Localized activation of caged eng2a mRNA using a sliding screen with a hole on the confocal plane.
Figure 7: Photo-activation of Bhc-caged DNA in vivo.

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Acknowledgements

We thank T. Harada, H. Segawa, S-Y. Yeo and H. Tanaka for technical support, and A. Miyawaki, I. Masai, and A. Thomson for critical comments. We also thank M. Westerfield for the gift of eng2a cDNA, and S. Krauss, T. Jowett, M. Mishina and D. Y. Stainier for providing probes for in situ hybridization. Antibody 4D9 was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by The University of Iowa Department of Biological Sciences. This work was supported in parts by Special Coordination Fund to H.O. and Grants in Aids for Scientific Research on Priority Areas (B) to H.O. and (C) to H.A. from the Ministry of Education, Science, Technology, Culture and Sports of Japan, and grants for Core Research for Evolutional Science and Technology to H.O. and Precursory Research for Embryonic Science and Technology to T.F. from the Japan Science and Technology Cooperation and a grant (NS27177) from the National Institutes of Health to R.Y.T.

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Authors and Affiliations

  1. Laboratory for Developmental Gene Regulation, Brain Science Institute, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, 351-0198, Saitama, Japan

    Hideki Ando & Hitoshi Okamoto

  2. Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, 3-4-5 Nihonbashi, Chuo-ku, 103-0027, Tokyo, Japan

    Hideki Ando & Hitoshi Okamoto

  3. Department of Biomolecular Science, Toho University, 2-2-1 Miyama, Funabashi, 274-8510, Chiba, Japan

    Toshiaki Furuta

  4. Precursory Research for Embryonic Science and Technology, Japan Science and Technology Corporation,

    Toshiaki Furuta

  5. Department of Pharmacology and Howard Hughes Medical Institute, University of California San Diego, La Jolla, 92093, California, USA

    Roger Y. Tsien

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Correspondence to Hitoshi Okamoto.

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Ando, H., Furuta, T., Tsien, R. et al. Photo-mediated gene activation using caged RNA/DNA in zebrafish embryos. Nat Genet 28, 317–325 (2001). https://doi.org/10.1038/ng583

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