Abstract
Ablation studies are used to elucidate cell lineage relationships, developmental roles for specific cells during embryogenesis and mechanisms of tissue regeneration. Previous chemical and genetic approaches to directed cell ablation have been hampered by poor specificity, limited efficacy, irreversibility, hypersensitivity to promoter leakiness, restriction to proliferating cells, slow inducibility or complex genetics. Here, we provide a step-by-step protocol for a hybrid chemical-genetic cell ablation method in zebrafish that, by combining spatial and temporal control, is cell-type specific, inducible, reversible, rapid and scaleable. Bacterial Nitroreductase (NTR) is used to catalyze the reduction of the innocuous prodrug metrodinazole (Mtz), thereby producing a cytotoxic product that induces cell death. Based on this principle, NTR is expressed in transgenic zebrafish using a tissue-specific promoter. Subsequent exposure to Mtz by adding it to the media induces cell death exclusively within NTR+ cells. This approach can be applied to regeneration studies, as removing Mtz by washing permits tissue recovery. Using this protocol, cell ablation can be achieved in 12–72 h, depending on the transgenic line used, and recovery initiates within the following 24 h.
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References
Johnson, S.L. & Weston, J.A. Temperature-sensitive mutations that cause stage-specific defects in Zebrafish fin regeneration. Genetics 141, 1583–1595 (1995).
Bernhardt, R.R., Tongiorgi, E., Anzini, P. & Schachner, M. Increased expression of specific recognition molecules by retinal ganglion cells and by optic pathway glia accompanies the successful regeneration of retinal axons in adult zebrafish. J. Comp. Neurol. 376, 253–264 (1996).
Becker, T., Wullimann, M.F., Becker, C.G., Bernhardt, R.R. & Schachner, M. Axonal regrowth after spinal cord transection in adult zebrafish. J. Comp. Neurol. 377, 577–595 (1997).
Poss, K.D., Wilson, L.G. & Keating, M.T. Heart regeneration in zebrafish. Science 298, 2188–2190 (2002).
Harris, J.A. et al. Neomycin-induced hair cell death and rapid regeneration in the lateral line of zebrafish (Danio rerio). J. Assoc. Res. Otolaryngol. 4, 219–234 (2003).
Yang, C.T., Sengelmann, R.D. & Johnson, S.L. Larval melanocyte regeneration following laser ablation in zebrafish. J. Invest. Dermatol. 123, 924–929 (2004).
Sadler, K.C., Krahn, K.N., Gaur, N.A. & Ukomadu, C. Liver growth in the embryo and during liver regeneration in zebrafish requires the cell cycle regulator, uhrf1. Proc. Natl. Acad. Sci. USA 104, 1570–1575 (2007).
Kurita, R. et al. Suppression of lens growth by alphaA-crystallin promoter-driven expression of diphtheria toxin results in disruption of retinal cell organization in zebrafish. Dev. Biol. 255, 113–127 (2003).
Wan, H. et al. Analyses of pancreas development by generation of gfp transgenic zebrafish using an exocrine pancreas-specific elastaseA gene promoter. Exp. Cell Res. 312, 1526–1539 (2006).
Slanchev, K., Stebler, J., de la Cueva-Mendez, G. & Raz, E. Development without germ cells: the role of the germ line in zebrafish sex differentiation. Proc. Natl. Acad. Sci. USA 102, 4074–4079 (2005).
Jung, J. et al. Ablation of tumor-derived stem cells transplanted to the central nervous system by genetic modification of embryonic stem cells with a suicide gene. Hum. Gene Ther. 18, 1182–1192 (2007).
Chu, Q.D. et al. Rat adenocarcinoma cell line infected with an adenovirus carrying a novel herpes-simplex virus-thymidine kinase suicide gene construct dies by apoptosis upon treatment with ganciclovir. J. Surg. Res. 143, 189–194 (2007).
Springer, C.J. & Niculescu-Duvaz, I. Approaches to gene-directed enzyme prodrug therapy (GDEPT). Adv. Exp. Med. Biol. 465, 403–409 (2000).
Pelengaris, S., Khan, M. & Evan, G.I. Suppression of Myc-induced apoptosis in beta cells exposes multiple oncogenic properties of Myc and triggers carcinogenic progression. Cell 109, 321–334 (2002).
Smith, C.A. et al. Conditional ablation of T-cell development by a novel viral ion channel transgene. Immunology 105, 306–313 (2002).
Smith, S.J., Kotecha, S., Towers, N. & Mohun, T.J. Targeted cell-ablation in Xenopus embryos using the conditional, toxic viral protein M2(H37A). Dev. Dyn. 236, 2159–2171 (2007).
Curado, S. et al. Conditional targeted cell ablation in zebrafish: a new tool for regeneration studies. Dev. Dyn. 236, 1025–1035 (2007).
Pisharath, H., Rhee, J.M., Swanson, M.A., Leach, S.D. & Parsons, M.J. Targeted ablation of beta cells in the embryonic zebrafish pancreas using E. coli nitroreductase. Mech. Dev. 124, 218–229 (2007).
Bridgewater, J.A. et al. Expression of the bacterial nitroreductase enzyme in mammalian cells renders them selectively sensitive to killing by the prodrug CB1954. Eur. J. Cancer 31A, 2362–2370 (1995).
Lindmark, D.G. & Muller, M. Antitrichomonad action, mutagenicity, and reduction of metronidazole and other nitroimidazoles. Antimicrob. Agents Chemother. 10, 476–482 (1976).
Anlezark, G.M. et al. The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)—I. Purification and properties of a nitroreductase enzyme from Escherichia coli—a potential enzyme for antibody-directed enzyme prodrug therapy (ADEPT). Biochem. Pharmacol. 44, 2289–2295 (1992).
Edwards, D.I. Nitroimidazole drugs—action and resistance mechanisms. II. Mechanisms of resistance. J. Antimicrob. Chemother. 31, 201–210 (1993).
Bridgewater, J.A., Knox, R.J., Pitts, J.D., Collins, M.K. & Springer, C.J. The bystander effect of the nitroreductase/CB1954 enzyme/prodrug system is due to a cell-permeable metabolite. Hum. Gene Ther. 8, 709–717 (1997).
Kawakami, K. et al. A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. Dev. Cell 7, 133–144 (2004).
Grabher, C., Joly, J.S. & Wittbrodt, J. Highly efficient zebrafish transgenesis mediated by the meganuclease I-SceI. Methods Cell Biol. 77, 381–401 (2004).
Huang, C.J., Tu, C.T., Hsiao, C.D., Hsieh, F.J. & Tsai, H.J. Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish. Dev. Dyn. 228, 30–40 (2003).
Her, G.M., Chiang, C.C., Chen, W.Y. & Wu, J.L. In vivo studies of liver-type fatty acid binding protein (L-FABP) gene expression in liver of transgenic zebrafish (Danio rerio). FEBS Lett. 538, 125–133 (2003).
Isles, A.R. et al. Conditional ablation of neurones in transgenic mice. J. Neurobiol. 47, 183–193 (2001).
Cui, W., Allen, N.D., Skynner, M., Gusterson, B. & Clark, A.J. Inducible ablation of astrocytes shows that these cells are required for neuronal survival in the adult brain. Glia 34, 272–282 (2001).
Felmer, R., Cui, W. & Clark, A.J. Inducible ablation of adipocytes in adult transgenic mice expressing the E. coli nitroreductase gene. J. Endocrinol. 175, 487–498 (2002).
Searle, P.F. et al. Nitroreductase: a prodrug-activating enzyme for cancer gene therapy. Clin. Exp. Pharmacol. Physiol. 31, 811–816 (2004).
Westerfield, M. The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish(Danio rerio). 4th edn, (University of Oregon Press, Eugene, Oregon, 2000).
Cole, L.K. & Ross, L.S. Apoptosis in the developing zebrafish embryo. Dev. Biol. 240, 123–142 (2001).
Acknowledgements
We thank Justin Bosch for comments on the manuscript and help in testing the protocol for adult ablations, Stephen J. Johnson (Washington University) for his suggestion to use the NTR/Mtz system in zebrafish, Jeff Mumm and Eric Schroeter (Washington University) for providing the CFP-NTR construct and Ana Ayala and Koroboshka Brand for expert help maintaining the fish. R.M.A. was supported by a postdoctoral fellowship from the JDRF. This work was supported in part by grants from the NIH (NHLBI and NIDDK) and the Packard Foundation to D.Y.R.S.
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Curado, S., Stainier, D. & Anderson, R. Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studies. Nat Protoc 3, 948–954 (2008). https://doi.org/10.1038/nprot.2008.58
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DOI: https://doi.org/10.1038/nprot.2008.58
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