Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studies.
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.
Curado
Silvia
S
Department of Biochemistry and Biophysics, Program in Developmental Biology, Liver Center, Diabetes Center and the Cardiovascular Research Institute, University of California, San Francisco, San Francisco, California 94158-2324, USA.
Stainier
Didier Y R
DY
Anderson
Ryan M
RM
D000818:Animals; D030801:Animals, Genetically Modified; D016923:Cell Death / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008795:Metronidazole / Q000378:metabolism / Q000633:toxicity; D008856:Microscopy, Fluorescence; D009601:Nitroreductases / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D011355:Prodrugs / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015027:Zebrafish / Q000235:genetics / Q000254:growth & development* / Q000502:physiology*
C485182:Cyan Fluorescent Protein; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; D008795:Metronidazole; D049452:Green Fluorescent Proteins; D009601:Nitroreductases