The NTR/Prodrug Revolution: Tools for Controlling Cell Loss and Regeneration

Reviewer #1 (Public review):

Summary:

Kim and Parsons present a timely overview of the NTR/prodrug system and its applications in regenerative biology research, with particular emphasis on tissue-specific cell ablation. The system has substantially advanced the field by enabling non-invasive, conditional cell elimination, and has proven especially powerful in zebrafish, though applications in other classical model organisms are also noted. The review covers the historical origins of the NTR system, its use in regeneration studies, small-molecule screening, and genetic and CRISPR-based screening, as well as future directions, including the development of the highly efficient NTR2 enzyme variant.

Strengths:

This is a useful and well-structured contribution. The manuscript is a valuable resource for the regeneration biology community.

Weaknesses:

The impact and scientific value of this paper could be meaningfully enhanced by addressing several points outlined below. The concerns centre on completeness, conceptual precision, and the depth of mechanistic discussion.

(1) Title: Species specificity.

Given that the review's primary focus is the zebrafish model, it would be appropriate to include the species name in the title. This would improve discoverability and accurately set the scope of the article for prospective readers.

(2) Subchapter: Physical injury.

The subchapter enumerates different types of physical injury models but would benefit from a more substantive comparative discussion. In particular, the authors are encouraged to address the following:

(2.1) Outcome comparison: Surgical and other invasive approaches cause damage to entire tissue structures comprising multiple cell types, whereas tissue-specific genetic ablation eliminates a defined cell population while leaving the surrounding architecture largely intact. This fundamental distinction has direct implications for the interpretation of regenerative outcomes and should be clearly articulated.

(2.2) Inflammatory response: Invasive injuries typically trigger a robust inflammatory response, which itself can be a potent driver of regeneration. By contrast, genetic cell ablation may elicit a qualitatively different inflammatory reaction. A comparative discussion of this distinction would help readers appreciate a critical limitation of genetic ablation systems relative to models of natural, accidental tissue damage.

(3) Subchapter: Cell-specific toxins.

This subchapter would benefit from several targeted expansions:

(3.1) Off-target effects: The authors should include evidence that the exemplified drugs have known off-target activities, with a discussion of how these confounded the interpretation of experimental data. At least a few concrete published examples should be cited.

(3.2) Completeness of the toxin list: The current list appears illustrative rather than comprehensive. A more complete enumeration would be valuable, particularly for neurotoxins and drugs targeting sensory cells, as these are highly relevant to the zebrafish regeneration field.

(3.3) Interspecies differences: It would be informative to specify whether drug specificity differs across species, as this is a practical consideration for researchers working in organisms other than zebrafish.

(4) Subchapter: Optogenetic cell ablation.

The authors note that optogenetic cell ablation has not yet been applied in conventional regeneration studies. It would strengthen this section to include a discussion of the underlying reasons for this gap, whether technical or biological, so that readers can appreciate the barriers and potential for future adoption.

(5) Terminology: "Suicide gene".

The use of the term "suicide gene" to nitroreductase is conceptually imprecise and merits reconsideration. Strictly speaking, a suicide gene is one whose expression alone is sufficient to kill the cell, as in the case of genes encoding direct triggers of apoptosis or the catalytic A subunit of diphtheria toxin (DTA). NTR does not meet this criterion: it requires the exogenous administration of a prodrug (e.g., metronidazole) to produce a cytotoxic metabolite, and is therefore only conditionally lethal.

It is worth noting that nitroreductases evolved in bacteria and fungi as enzymes involved in chemoprotection and detoxification, converting potentially toxic and mutagenic nitroaromatic compounds into less harmful metabolites (PMID: 18355273). This biological context further underscores that NTR is not inherently a lethal protein. The authors are encouraged to replace or qualify the term "suicide gene" and instead adopt terminology that more accurately reflects the conditional, prodrug-dependent nature of the system.

(6) NTR/MTZ in regenerative studies: Mechanistic depth.

While the review catalogues several studies employing the NTR/MTZ system, it lacks mechanistic depth regarding the cellular basis of ablation. The following questions should be addressed, where evidence exists in the literature:

(6.1) Temporal dynamics of cell death: What is known about the kinetics of NTR/MTZ-induced lethality across different tissue types in larval and adult zebrafish, as well as other organisms? Are there age- and tissue-specific differences in the speed or completeness of ablation?

(6.2) Mechanism of cell death: What is the cellular basis of NTR/MTZ-induced cytotoxicity in zebrafish? In particular, do the toxic metabolites preferentially cause mitochondrial damage or nuclear DNA damage, and what downstream death pathways are engaged?

(6.3) Proliferative versus post-mitotic cells: Are proliferating and non-proliferating cells equally sensitive to the NTR/MTZ system, or does the proliferative status of a cell influence susceptibility? This is a practically important question for researchers designing ablation experiments in tissues with mixed cell populations.

(6.4) Ablation of progenitor cells: Are there published examples demonstrating that co-ablation of differentiated functional cells and organ-specific progenitor cells abolishes regenerative capacity? Such examples would be highly informative in illustrating the system's power to dissect the cellular requirements for regeneration.

Addressing the points above, particularly the comparative discussion of injury models and inflammatory responses, the clarification of terminology, and the mechanistic discussion of NTR/MTZ-induced cell death would substantially strengthen the review's scientific contribution and utility.

Reviewer #2 (Public review):

Summary:

Kim and Parsons reviewed the nitroreductase (NTR)/prodrug system: when engineered cells expressing the enzyme NTR are treated with prodrug (e.g. metronidazole), NTR converts the prodrug into a cytotoxic compound that kills these cells. The review covers how the system has been developed, spatiotemporal control of targeted cell ablation, and its broad utility to study regenerative mechanisms, model human diseases, and screen chemicals to discover pro-regenerative and protective compounds. They further discussed the newer version of NTR, a more potent prodrug, and experimental design, which not only expands the possible utility of the NTR/prodrug system, but also allows the research community to develop a precise, reproducible and versatile platform.

Strengths:

The review summarized landmark work application of the NTR/prodrug system, and recent studies, with focus on the model organism zebrafish. The review provides a good gateway to understanding the system and considering regenerative studies.

Weaknesses:

No weaknesses were identified by this reviewer.

Reviewer #3 (Public review):

Summary:

This manuscript by Kim and Parsons presents an overview of the nitroreductase/metronidazole (NTR/MTZ) cell ablation system.

Strengths:

This manuscript nicely places the NTR/MTZ system in the context of other cell ablation methods, with a discussion of their respective advantages and disadvantages. This review is particularly useful for highlighting the many ways the NTR/MTZ system has been applied to study the regeneration of multiple cell types and to model different degenerative human diseases. The review concludes with a discussion on recent improvements made to the system and practical considerations and "best practices" for NTR-based experiments. This review could be a helpful resource, especially for researchers new to regeneration or cell ablation studies.

Weaknesses:

Although the NTR/MTZ system has been used in other model organisms, this review is primarily focused on its uses in zebrafish. While this is understandable given the wide adoption of NTR/MTZ in the zebrafish field, discussion of the unique considerations and/or challenges for non-zebrafish systems would be an interesting addition and could broaden the potential audience for this review. Additional minor revisions, as suggested below, could also improve readability.