The NTR/prodrug revolution: Tools for controlling cell loss and regeneration
- Department of Clinical Pharmacy Practice, UC Irvine School of Pharmacy and Pharmaceutical Sciences, United States
- Robert A. Mah Molecular Innovation Center, University of California, Irvine, United States
- Department of Developmental and Cell Biology, School of Biological Sciences, Natural Sciences II, University of California, Irvine, United States
Figures
Figure 1
Commonly targeted cell types for ablation studies in zebrafish.
See Supplementary file 1 for a more complete list. Image of a 5-day post-fertilization (dpf) casper zebrafish larva (White et al., 2008) with approximate position of cell type ablated. Name of cell and transgene provided along with reference. Orange highlights indicate transgenic models used to study human pathologies. *Same line two different tissues (White et al., 2017; Brandt et al., 2021; Niu et al., 2020).
Figure 2
Nitroreductase (NTR)/metronidazole (MTZ)-based screening platforms in zebrafish.
Overview of the integrated chemogenetic screening workflow using NTR-mediated ablation. (A) Experimental design showing transgenic zebrafish expressing NTR in target tissues, baseline imaging, and subsequent MTZ treatment to induce cell-type-specific ablation. The use of parallel transgenic controls and multiwell plate layout enables quantitative assessment of tissue loss and recovery. (B) High-content chemical screening pipeline integrating automated imaging, hit identification, and pathway-level analysis using standardized statistical metrics. (C) Genetic screening framework coupling sgRNA-based mutagenesis with imaging-based phenotype scoring to uncover modifiers of cell loss or regeneration. (D) Behavioral assays to quantify functional recovery or pharmacological response.
Figure 3
Schematic of bipartite systems to drive robust levels of nitroreductase (NTR).
On left, driver lines express (dashed arrows) transactivators (A) Gal4 (blue sphere) or (B) QF2 (purple sphere) under the control of a cis-regulatory element (CRE). These transactivators bind their respective upstream activating sequences (either UAS or QUAS, gray boxes) to achieve controlled and amplified NTR expression (tan spheres) in target cells. NTR expression can be monitored by co-production of mCherry (red spheres) either as a fusion protein or as separate proteins due to P2A-dependent ribosome ‘skipping’ (Provost et al., 2007). (A) Redrawn from Pisharath and Parsons where the CRE was from ptf1a (Pisharath and Parsons, 2009) and (B) redrawn from Lee et al. where the CRE came from mbpa (Lee et al., 2025).
Figure 4
Live imaging of cell-death kinetics.
(A) Schematic of 6-day post-fertilization (dpf) larvae showing position of the pancreatic islet imaged in C-N (red/yellow). (B) Diagram of the two transgenes (ins:Hmgb1-GFP, ins:mCherry-2a-NTR2.0) in the fish. The insulin promoter (gray box) drives expression of the following: (B, above) an Hmgb1-GFP fusion protein and (B, below) NTR2.0 and mCherry (presence of the P2A [blue box] makes separate proteins). (C–N) Confocal images of the islet in three larval fish over a time course from 6 dpf to 7 dpf (times along the X axis). (C–F) Negative control – no MTZ (0). (G–J) Fish treated with high MTZ dose (1 mM). (K–N) Fish treated with a low MTZ dose (10 μM). (G–N) Dying β-cells first lose red fluorescence, revealing green nuclei (arrow heads). A higher dose shows the appearance of green nuclei (H) earlier than the lower dose (N). (J) 24 hr in 1 mM and no debris remains.
Tables
Table 1
Representative pharmacological toxins used for cell-specific ablation in zebrafish.
These compounds enable dose-controlled injury across diverse tissues but often exhibit limited specificity and off-target effects that can complicate interpretation of regenerative responses.
| Toxin | Target Cell Type | Tissue/Organ | Key Limitation | Ref. |
|---|---|---|---|---|
| Acetaminophen | Hepatocytes | Liver | Dose‑dependent toxicity with systemic side effects. | North et al., 2010 |
| Aminoglycosides (neomycin, gentamicin) | Sensory hair cells | Lateral line, inner ear | Differential effects by compound. Incomplete ablation at some doses. | Coffin et al., 2013; Thomas and Raible, 2019; Uribe et al., 2013; Wiedenhoft et al., 2017 |
| Caerulein | Acinar cells | Pancreas | Induces pancreatitis and leads to destruction of adjacent tissue | Falcão et al., 2024; Kim, 2008 |
| Cisplatin | Sensory hair cells | Lateral line, inner ear | Damages support cells and delays regeneration. Nephrotoxicity and ototoxicity. | Lee et al., 2024 |
| Copper sulfate (CuSO4) | Sensory hair cells, support cells | Lateral line | At higher doses also damages support cells and afferent neurons, impairing regeneration. | Holmgren et al., 2021 |
| 6-Hydroxydopamine (6-OHDA) | Dopaminergic neurons | Brain | Broad catecholaminergic toxicity. May require direct injection in some models. | Dovonou et al., 2023 |
| Ouabain | Retinal neurons | Retina | Dose-dependent and can damage multiple retinal cell layers. | Fimbel et al., 2007; Sherpa et al., 2008 |
| MoTP | Melanocytes | Skin (pigment system) | Ablation is developmentally restricted | Yang and Johnson, 2006 |
| MPTP / MPP+ | Dopaminergic neurons | Brain | Species-dependent metabolism. Strict handling required. Off-target effects. | Dovonou et al., 2023 |
| Streptozotocin (STZ) | Pancreatic β-cells | Pancreas | Off-target effects, e.g. hepatotoxicity | Moss et al., 2009 |
Additional files
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Supplementary file 1
Most-cited publications using the nitroreductase (NTR)/prodrug system.
This table lists the most highly cited publications employing the NTR/prodrug system for targeted cell ablation. Candidate studies were identified through a Web of Science search using the query ‘nitroreductase ablation’, and results were manually curated to include only those papers that directly used an NTR-expressing transgenic line or construct together with a prodrug to induce selective cell death. Entries are ranked by citation count at the time of data collection. For each study, the table reports the targeted cell type or tissue, the species and specific NTR transgenic line used, and the primary biological purpose addressed. The columns ad. (adult) and la. (larvae) indicate whether the transgenic system was used in adults, larvae, or both. The transgene shown in green corresponds to a widely used UAS line that drives Gal4-dependent expression of NTR1 and is included here to aid identification of experiments utilizing this common effector line.
- https://cdn.elifesciences.org/articles/110593/elife-110593-supp1-v1.docx
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The NTR/prodrug revolution: Tools for controlling cell loss and regeneration
eLife 15:RP110593.
https://doi.org/10.7554/eLife.110593.3
