Compromised DNA replication in gut cells underlies tardigrade sensitivity to genotoxic stress

  1. Gonzalo Quiroga-Artigas
  2. Pauline Fontanié
  3. Benjamin Lacroix
  4. Maria Dolores Molina
  5. María Moriel-Carretero

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Abstract

Tardigrades withstand severe DNA insults, including extreme doses of ionizing radiation, through unique protective proteins and strong upregulation of canonical DNA repair pathways. Yet, these extremophile animals are not immortal, and the cellular and organismal processes that ultimately fail under sustained genotoxic stress have not been characterized. Here, we identify DNA replication as the key vulnerability in the tardigrade Hypsibius exemplaris . Using the radiomimetic drug zeocin to induce DNA breaks, we show that continuous exposure progressively kills tardigrades, accompanied by striking body shrinkage and lipid depletion. DNA synthesis labeling reveals that zeocin disrupts replication and triggers de novo reparative synthesis in select non-dividing tissues. Pulse–wash experiments demonstrate that even transient damage to dividing gut cells irreversibly exhausts their replicative capacity, leading to midgut failure and animal death, despite systemic induction of DNA repair genes. Germ cells and embryos, with their high proliferation rates, show heightened sensitivity. Cross-phyla survival assays in the eutelic nematode Caenorhabditis elegans and neoblast-rich planarian Schmidtea mediterranea further link proliferative activity to mortality kinetics under DNA damage. Collectively, our findings pinpoint DNA replication as an Achilles’ heel of organismal survival under genotoxic stress, even in animals renowned for their extraordinary DNA damage tolerance.

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  1. Review Commons

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    Reply to the reviewers

    Since we are at the stage of simply proposing a Revision Plan to an affiliate journal, there is not a revised version of the manuscript yet. But we honestly thank the three reviewers for their important input, which we are taken into consideration very seriously.

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    Referee #3

    Evidence, reproducibility and clarity

    Major Comments:

    It is interesting case study but the main problem with the study is the use of an unsuitable tardigrade model species. It was shown in the past that Hypsibius exemplaris is not a good model species to test tardigrade survival under extreme stress. Of course, results of Hypsibius exemplaris can be published but from the entire manuscript all general comments that tardigrades react in this or in different way need to be removed. This is characteristic only to Hypsibius exemplaris species which is a poor model for studies focused on environmental stressTo present general conclusions use few different tardigrade species or at least a correct tardigrade species with confirmed high resilience for different kind of stress like Milnesium, Ramazzottius, Paramacrobiotus or similar must be tested. Based on present study I can only propose to publish this manuscript as a case study for one poorly stress resistant eutardigrade species, without any general conclusions about other tardigrades. See: Poprawa, I., Bartylak, T., Kulpla, A., Erdmann, W., Roszkowska, M., Chajec, Ł., Kaczmarek, Ł., Karachitos, A. & Kmita, H. (2022) Verification of Hypsibius exemplaris Gąsiorek et al., 2018 (Eutardigrada; Hypsibiidae) application in anhydrobiosis research. PLoS ONE 17(3): e0261485.

    Minor comments:

    1. General comment to entire manuscript. Please do not start sentences with abbreviations, i.e. The DNA instead of DNA, Caenorhabditis instead of C. etc. In bibliography many doin numbers for publications are lacking, you have a different styles of citations, do not use capital letters for words inside the article title e.g. "Tardigrades as a Potential Model Organism in Space Research.", change it to "Tardigrades as a potential model organism in space research." Or use capital letters in all citations. Use italics for Latin names of the species and genera. On figures please try to put all of them like this that specimens ill be situated horizontally and in the middle of figure.
    2. Introduction, Lines 80-96: I do not understand why this section is in Introduction. This is description of the results of the studies could be minimal and details could be moved to proper chapters.
    3. Results: In this section are mixed results with methods. Please put all parts to the correct chapters.
    4. Line 227 and 235: Based on what you interpreted: "fully-grown adults" and "juveniles" that they were adult and fully grown? Please explain in the text.
    5. Line 315: You wrote "These findings demonstrate that even a transient exposure to zeocin causes irreversible DNA damage, leading to delayed mortality." but not to all specimens as you marked above.
    6. Line 461-462: You wrote: "In this study, we probed why tardigrades-despite their impressive DNA repair capacity and extremotolerance-still succumb to genotoxic stress." But only one tardigrade species with poor resilience to stress conditions has been tested in this study. What if more repair mechanisms are activated in tardigrades when tardigrades leaving the state of anhydrobiosis? Authors tested only active animals and in such mechanisms maybe not activated or are activated on lower level. What is even more problematic, and what I marked this in one of the first comments, the species used in study is incorrect because is not very resilient to extreme conditions. This species is also a poor anhydrobiotic species with almost zero ability to anhydrobiosis (during which repair mechanisms are activated).
    7. Line 609: "..actively searching for food.." - How you know that they were looking for food? What was a difference between normal crawling around and looking for food?
    8. Line 635: "In sum, tardigrades illustrate that..." - Only in case of Hypsibius. This is not characteristic for tardigrades. See my previous comments. This conclusion is too strong without adequate proof.
    9. Lines 666-667: "Adults measured {greater than or equal to}240 μm in length, while juveniles ranged between 120-180 μm." - Why such measurements? It was connected with something or is it arbitrary? Please explain.
    10. Lines: 673-677: "For each timepoint, fertility was calculated by dividing the total number of eggs laid by the number of live animals at that time (using the last recorded number of live animals when all animals had died). In Fig. 5A-B, fertility is presented as the mean cumulative number of eggs laid per animal over time; in Fig. S9, it is shown as the mean number of eggs laid per animal at each timepoint." - This method of calculating fertility may be valid only if you know that all the females laid the same number of eggs. It is obvious that some females produced less and some others more eggs. Hence, fertility can not be accurately calculated in this way.

    Significance

    Studies described in the manuscript are very interesting for many potential readers, however manuscript need to be modified as case study for one tardigrades species without generalization of the results for all tardigrades. It is very important to not suggest that all tardigrades react in the same way especially that species used is not a good candidate for this type of studies (see my major comments).

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    This manuscript studies the effects of genotoxic stress using zeocin, a bleomycin-family drug, in the tardigrade species H. exemplaris. In a first experimental set, the authors evaluate the survival of the organisms as well as the levels of DNA damage.

    A RT-qPCR analysis of a set of DNA repair genes identified in a previous study by another group (Clark-Hachtel, Courtney M. et al.; Curr Biol, Vol. 34, Issue 9, 1819-1830.e6) and a comet assay reveal the damage observed during treatment.

    Experiments on fasting animals show variations in animal size that overlap with those seen in groups of animals treated with the genotoxic drug. Physiological variations are also observed, such as lipid loss and cuticle alteration.

    In a subsequent experimental set, the authors indicate that the genotoxic drug blocks DNA replication and activates DNA repair systems in various tissues, particularly the digestive tissue, which appears to be specifically targeted in terms of its replicative capacity following DNA damage caused by the drug. A sensitivity study of tardigrade embryo development then shows that their proliferative capacity, which is highly dependent on replication, mobilizes different sets of DNA repair genes that may be more closely associated with replication than in adults.

    Finally, a comparative study of the development of two organisms (C. elegans and planarian) also shows sensitivity to drugs that disrupt the replication process during development.

    The authors conclude from all of this work that the cells of the animals' intestines are the main target of the genotoxic stress induced by the drug. The effects of disruption of the normal replication process in intestinal cells are thought to be the cause of the observed loss of tissue homeostasis (loss of lipids and tissue renewal capacity).

    Major comments:

    1. Zeocin is a drug derived from bleomycin but has not yet been extensively studied. Could you give examples of the use/validation of zeocin as a radiomimetic in other biological systems?

    2. Similarities in transcriptional responses between UV and dehydration genotoxic stresses have already been observed (Yoshida et al., 2022; BMC Genomics 23, 405) in a tardigrade species closely related to H. exemplaris (R. varieornatus). However, no correlation in transcriptional responses could be observed after treating H. exemplaris with genotoxic stresses such as desiccation and 500 Gy gamma ray irradiation (Clark-Hachtel, Courtney M. et al.; Curr Biol, Vol 34, Issue 9, 1819 - 1830.e6). These results indicate that, depending on the type of genotoxic stress, transcriptomic responses can appear to be very different and sometimes uncorrelated, particularly in the species H. exemplaris. Bleomycin has been studied in previous reports (refs Yoshida Y, et al. Proc Jpn Acad Ser B Phys Biol Sci. 2024 100(7):414-428; Clark-Hachtel, Courtney M. et al.; Curr Biol, Vol 34, Issue 9, 1819 - 1830.e6; Marwan Anoud et al., 2024, eLife 13:RP92621), which used a transcriptomic study to confirm that it behaves as a radiomimetic for the species H. exemplaris.

    On the other hand, since zeocin is a bleomycin-family drug, it is possible that its effects may differ slightly from those of bleomycin, exhibiting specific effects as observed by comparison of chemical radiomimetic and radiation treatments.

    A control experiment comparing the effects of bleomycin and zeocin using RNAseq would validate that their use is equivalent.

    1. A major conclusion of the manuscript is that DNA damage induced by the genotoxic drug disrupts replication mechanisms and leads to the observed effects. Are RT-qPCR analyses on a subset of drug-induced repair genes induced solely by the drug itself or by its indirect effect on replication?

    It would be interesting to block replication in embryos and assess whether the same sets of DNA repair genes are induced when compared with treatment with zeocin only. Additionally, it will be interesting to redo the same DNA replication block experiments with additional treatment to compare the induced sets of DNA reparation genes. This will help to understand the true effect that will be directly imputable to zeocin.

    Minor comments:

    The data are well presented, and the experiments are well described for general understanding. Previous studies in this field have been well referenced. However, the link between DNA damage caused by the drug and its impact on replication needs to be better explained.

    Finally, the use of the drug zeocin should be validated in this system by comparison with bleomycin.

    Significance

    This study evaluates the resistance of a species of tardigrades to genotoxic stress. Several previous studies have conducted this type of experiment using the same species with consistent results and using the same type of genotoxic chemical drug : bleomycin. In this study, a new genotoxic drug is evaluated for its effects on DNA damage as well as on the survival of organisms and their embryonic development. Definitive validation experiments of this new genotoxic chemical tool are necessary to determine its similarities with drugs already known for their effects in the literature.

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    Referee #1

    Evidence, reproducibility and clarity

    This manuscript concerns tardigrade sensitivity to genotoxic stress. Using the radiomimetic drug Zeocin to induce DNA breaks, authors show that continuous exposure progressively kills tardigrades, accompanied by striking body shrinkage and lipid depletion. Authors show that germ cells and embryos, with their high proliferation rates, show heightened sensitivity. To resume, their findings pinpoint DNA replication as an Achilles' heel of organismal survival under genotoxic stress.

    Major comments:

    The claims and conclusions in this article are not sufficiently supported by the data. They require additional experiments or analyses.

    The fundamental problem with this paper is the use of a single molecule, Zeocin, as a radiomimetic. It is absolutely essential to compare the results obtained with radiation. In the bibliography, researchers compare a drug with radiation. Bob Goldstein, for example, in his 2024 Current Biology paper uses radiation and bleomycin. The same is true for Concordet in his 2024 elife paper. Zeocin has been used very little on tardigrades. It cannot be used alone to draw conclusions from this study.

    Additionally, at the beginning of the paper, the authors tested different concentrations of Zeocin. They showed results at two concentrations : 100ug/ml and 1mg/ml. In the remainder of the paper, only the latter concentration is used. This is not sufficient. The analyses should have been conducted in parallel on several concentrations in order to compare and analyze a potential dose-dependent effect.

    Finally, the authors focused on two types of cells that have the particularity of replicating themselves: gut cells and storage cells. It would have been necessary to work on other cell types to compare the results.

    The realization of these additional experiences are completely realistic.

    The data and methods are presented in a reproducible manner. But experiments sometimes lack independent replicates and need to be reproduced.

    The legend to Figure 1, for example, indicates that the experiment was conducted with 3 to 7 biological replicates and 60 to 120 animals. These are still very different numbers. And this can lead to significant bias.

    For the other figures, no biological replicates were indicated and the numbers « n » are sometimes very different, as in Figure 4 with n=107 and n=166. A little more homogenization allows for better robustness of the results. And biological replicates are essential.

    Sometimes there are some unclear elements in the figures. In Figure 3, if I understand correctly, A and B show the gut cells (adult) and C and D the storage cells (juvenile). The size difference is not very clear in this image. How old is the juvenile compared to this adult?

    Significance

    This study, if confirmed by additional experiments that are absolutely essential to validate these conclusions, will be interesting for the community of researchers working on tardigrades, even if the effects of genotoxic stress on tardigrades are already widely studied.

    This study is relatively complete on only one molecule, Zeocin, at a concentration of 1 mg/ml. To be relevant, another genotoxic stress should be included in the study. And the study should also be conducted at the concentration of 100 ug/ml, which did show effects but was abandoned for the rest of the study. Similarly, only storage cells and gut cells were studied given their replication capacity. Other cell types should have been included in the study for comparison.

  5. Version published to 10.1101/2025.07.23.666276 on bioRxiv