The roles of NADPH oxidases during adult zebrafish fin regeneration

Background

            Reactive oxygen species (ROS) have been shown to be important signaling molecules during regeneration in a multitude of species. In addition to their role in immune cell recruitment to fight off possible wound site infection (1), ROS are required for the activation of cell proliferation pathways needed for wound healing and tissue outgrowth (2). Non-specific inhibition of NADPH oxidases (NOXes), which are prominent producers of ROS, is the most common method to study the role of ROS during regeneration (3), but little is known about which specific NOX(es) or NOX subunit(s) are the main contributors of ROS production during regeneration. In this preprint, the authors aim to identify the molecular mechanisms of ROS production during regeneration, which may lead to the generation of therapeutic strategies to induce a regenerative response in tissues otherwise lacking regenerative ability.

Figure 1: Methodology and key findings. A) Amputated fins regenerate in four weeks, three weeks post amputation (3wpa) shown for consistency with (C). Methodology for quantification of rate of adult fin regeneration shown under images. B) duox mutants and duox/cyba double mutants show the most significant decrease in regeneration rate compared to controls. C) duox mutants show significantly lower levels of ROS at amputation site. D) Post-amputation ROS levels oscillate with a circadian rhythm. (Adapted from Figs 2, 3, and 5, Chopra et al. 2021)

Key Findings         

            Using CRISPR-Cas9 gene editing technology, Chopra and colleagues generated homozygous mutant zebrafish lines for cyba, the gene that encodes the P22^phox subunit of NOXes 1-4, and for nox5, which encodes the calcium-regulated single subunit NOX enzyme Nox5. In addition, they obtained nonsense mutant allele lines for cyba and duox (another NOX) from the Zebrafish Mutation Project.

First, the authors wanted to assess whether any of the mutants displayed interesting phenotypes. In a previous study, they found that duox mutants had phenotypes consistent with hypothyroidism, such as shorter body length and external goiters (4). Mutations in human CYBA have been linked to chronic granulomatous disease (CGD), where patients experience recurrent bacterial and/or fungal infections due to insufficient ROS production by immune cells, which kill invading pathogens. Interestingly, in this study, the authors found that cyba mutant zebrafish exhibited higher mortality from Aspergillus fumigatus infection, providing evidence for the susceptibility of these lines to fungal infection, and their usefulness as models for human CGD. While no obvious phenotypes were present in nox5 mutants, the authors made the interesting discovery that nox5 mutants show prolonged resistance to anesthesia. Remarkably, there was little phenotypic overlap between the different mutants, highlighting the specific utilization of these NOXes/NOX subunits by diverse biological pathways.

By assessing the regenerative ability of the caudal fin in these mutants, the authors wanted to investigate which NOX or NOX subunit is responsible for ROS production during the process of regeneration. Before moving forward, the authors determined the appropriate age-range of mutants to use in their experiments to limit age biases, since regenerative capacity is known to change over time (5). Based on the results, the authors decided to use six to eight months-old fish for their study. Regeneration rate was determined by calculating the size of the amputated caudal fin each week (zero to four weeks post amputation) and converting that to a percentage of the original fin size (Fig 1A).

When assessing caudal fin regeneration in mutant fish, only the duox nonsense mutant and the duox/cyba double mutants displayed a significantly delayed rate of regeneration, resulting in stunted regeneration over the entire four-weeks (Fig 1B). As mentioned above, the duox mutants displayed phenotypes consistent with hypothyroidism, leading the authors to examine if the reduced regeneration rate in these mutants could be attributed to hypothyroidism. By comparing manet mutant fish, containing a nonsense mutation within the gene coding for the thyroid stimulating hormone receptor (4), with duox mutants, they noted a reduction of the regeneration rate occurred at different times during the four weeks. Thus, they concluded that the effect of duox on regeneration was not solely caused by hypothyroidism.

Baseline ROS levels before and after amputation were assessed using WT animals on a ubb:HyPer background, where HyPer is an oxidative sensitive reporter fluorophore used to measure H₂O₂ level in vivo (6). ROS levels rose significantly after amputation, were highest in the morning and lowest in the afternoon and were sustained for two weeks post amputation. Interestingly, the authors found that ROS levels oscillated with a circadian rhythm (Fig 1D).

Finally, the authors asked whether post-amputation ROS levels were affected in the nox mutants. Cyba mutant animals showed increased ROS post-amputation. These increased levels returned to baseline after one week, quicker than in the controls, and overall ROS levels were lower in this mutant. Duox mutant zebrafish showed consistently lower ROS levels post-amputation, even lower than WT unamputated controls, and nox5 mutants showed increased ROS post-amputation, but levels quickly returned to baseline (Fig 1C). As the duox mutants displayed the most significant effects on tail regeneration and post-amputation ROS production, overall, these data support Duox as being the major NADPH oxidase responsible for ROS production during regeneration.

Why I like this preprint

The importance of metabolic and redox homeostasis during development and regeneration has only recently begun to be appreciated and studied. By using a genetic approach, the authors uncovered duox as the main contributor of ROS during regeneration, a discovery which would not have been possible with the continued use of non-specific NADPH oxidase inhibitors. Moreover, the authors’ finding that post-amputation ROS levels oscillate with a circadian rhythm was completely unexpected and exciting. Future research from this group could further explore this phenomenon and hopefully answer the questions posed in the discussion.

Questions for the authors

1. Did the authors generate multiple double mutant animals? Did any of them display interesting phenotypes?
2. Could the amputated tails of duox or duox/cyba mutants ever reach the size of the original if given more time (past 4 weeks), or was the tail always stunted?
3. While mutations in duox affected regeneration more strongly than mutations in tshr, manet mutants still had decreased rates of regeneration. Do the authors plan to further study the effects of hypothyroidism on regeneration?
4. In the discussion, it was posited that a reason for the stronger effect on regeneration of the double mutant was the lower levels of ROS seen in each individual mutant. Is it possible to assess ROS levels in the double mutant using the reporter line or using some other ROS indicator?

References

1. Yoo S.K., Starnes T.W., Deng Q., Huttenlocher A. 2011. Lyn is a redox sensor that mediates leukocyte wound attraction in vivo. Nature. 480:109–112. https://doi.org/10.1038/nature10632
2. Baddar, N. W. A. H., Chithrala, A. and Voss, S. R. (2019). Amputation-induced reactive oxygen species signaling is required for axolotl tail regeneration. Dev Dynam 248, 189–196.
3. Gauron, C., Rampon, C., Bouzaffour, M., Ipendey, E., Teillon, J., Volovitch, M. and Vriz, S. (2013). Sustained production of ROS triggers compensatory proliferation and is required for regeneration to proceed. Sci Rep 3, 2084.
4. Chopra, K., Ishibashi, S. and Amaya, E. (2019). Zebrafish duox mutations provide a model for human congenital hypothyroidism. Biol Open 8, bio.037655.
5. Yun MH. Changes in Regenerative Capacity through Lifespan. Int J Mol Sci. 2015;16(10):25392-25432. doi:10.3390/ijms161025392
6. Love, N. R., Chen, Y., Ishibashi, S., Kritsiligkou, P., Lea, R., Koh, Y., Gallop, J. L., Dorey, K. and Amaya, E. (2013). Amputation-induced reactive oxygen species are required for successful Xenopus tadpole tail regeneration. Nat Cell Biol 15, 222–228.

Tags: fish

doi: https://doi.org/10.1242/prelights.30240

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