Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studies

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.

Figure 1

Experimental design for Mtz/NTR tissue-specific ablation. (a) Components of the toxigene cassette: a defined tissue-specific promoter (tsp) is cloned upstream of a cassette comprised of a fluorescent protein (FP) fused to Nitroreductase (NTR) in a transgenesis vector. (b) FP-NTR fusion protein is expressed in a defined population of cells (blue) at the start of ablation (t1). Metronidazole (Mtz) added to the water is converted to a cytotoxin by FP-NTR, which causes apoptosis of the FP-NTR⁺ cells (brown), but not neighboring cells (tan; t2). (c) Samples are divided into four groups for each ablation experiment: DMSO treatment of (A) NTR^- and (B) NTR⁺ embryos/larvae and Mtz treatment of (C) NTR^- embryos/larvae to control for nonspecific effects of Mtz or the transgene, and Mtz treatment of (D) NTR⁺ embryos for tissue ablation. At t1 (start of ablation), the FP-NTR is visible in the tissue to be ablated (blue); at t2 (end of ablation), FP-NTR⁺ cells will be depleted or absent (brown); and at t3 (during recovery after Mtz washout), new FP-NTR⁺ cells may be produced.

Figure 2

Analysis of ablation. Epifluorescent microscopy was used to monitor the expression of CFP (and therefore the progression of ablation) in a target group of cells (β cells, arrows) in individual Tg(ins:CFP-NTR)^s892 larvae throughout their treatment with DMSO or Mtz. Larvae at 80 hpf before treatment (t1; a,b), at 104 hpf, after treatment for 24 h (t2) with DMSO (a’)or 10 mMMtz (b’), and at 128 hpf, after 24 h recovery (t3) from DMSO (a’’) or Mtz (b’’). Loss of CFP during t2 in treated individuals (asterisk in b’) indicates that cells have been successfully ablated; recovery of fluorescence during t3 (b’’) indicates cell recovery. CFP fluorescence is constant in untreated individuals (a,a’ and a’’), indicating that no ablation occurs.

Figure 3

Cell death detection. Detection of apoptosis using (a,a’,b,b’) TUNEL assay or (c,c’) activated Caspase-3 immunostaining in (a,b,c)CFP-NTR^- or (a’,b’,c’) CFP-NTR⁺ larvae after treatment with 10 mM Mtz. Mtz-treated CFP-NTR^- control larvae show no substantial apoptosis in (a)liver, (b) heart or (c) pancreatic islet (activated Caspase-3-stained samples exhibit some nonspecific background staining). In contrast, significant apoptosis was observed in hepatocytes of (a’) Tg(fabp10:CFP-NTR)^s891 larvae, cardiomyocytes of (b’) Tg(cmlc2:CFP-NTR)^s890 larvae (arrows) and (c’) pancreatic β-cells of Tg(ins:CFP-NTR)^s892 larvae (arrows).