Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with
Silvia Curado1, Didier Y R Stainier, Ryan M Anderson
1Department 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.
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.
Insights
This study introduces a novel hybrid chemical-genetic method for precise cell ablation in zebrafish. This technique offers cell-type specificity, inducibility, and reversibility for studying development and regeneration.
Area of Science:
- Developmental Biology
- Regenerative Medicine
- Zebrafish Models
Background:
- Cell ablation is crucial for understanding embryogenesis and tissue regeneration.
- Existing methods for cell ablation lack specificity, speed, and reversibility.
Purpose of the Study:
- To develop a rapid, specific, and reversible cell ablation method in zebrafish.
- To provide a detailed protocol for a hybrid chemical-genetic approach.
Main Methods:
- Utilized bacterial Nitroreductase (NTR) expressed in transgenic zebrafish under a tissue-specific promoter.
- Administered the prodrug metronidazole (Mtz) to induce cell death specifically in NTR-expressing cells.
- Demonstrated reversibility by removing Mtz to allow for tissue recovery.
Main Results:
- Achieved cell-type specific ablation within 12-72 hours.
- Confirmed rapid induction and reversibility of cell ablation.
- Showcased the method's applicability in regeneration studies.
Conclusions:
- The hybrid chemical-genetic method offers precise spatial and temporal control for cell ablation in zebrafish.
- This approach overcomes limitations of previous methods, enabling robust studies in developmental biology and regeneration.
- The protocol is scalable and facilitates the investigation of tissue recovery mechanisms.
