Duox is the primary NADPH oxidase responsible for ROS production during adult caudal fin regeneration in zebrafish

Abstract

Sustained elevated levels of reactive oxygen species (ROS) have been shown to be essential for regeneration in many organisms. This has been shown primarily via the use of pharmacological inhibitors targeting the family of NADPH oxidases (NOXes). To identify the specific NOXes involved in ROS production during adult caudal fin regeneration in zebrafish, we generated nox mutants for duox, nox5 and cyba (a key subunit of NOXes 1-4) and crossed these lines with a transgenic line ubiquitously expressing HyPer, which permits the measurement of ROS levels. Homozygous duox mutants had the greatest effect on ROS levels and rate of fin regeneration among the single mutants. However, duox:cyba double mutants showed a greater effect on fin regeneration than the single duox mutants, suggesting that Nox1-4 also play a role during regeneration. This work also serendipitously found that ROS levels in amputated adult zebrafish fins oscillate with a circadian rhythm.

Keywords: Cell biology; Ichthyology; Molecular genetics.

Graphical abstract

Figure 1

The family of NADPH oxidases (A–E) All NOXes are transmembrane proteins that transport oxygen across biological membranes, reducing oxygen to superoxide, which can then undergo dismutation to generate H₂O₂. NOXes are multi-componential proteins, and p22phox (the product of the cyba gene) is a common subunit to NOX1-4. Zebrafish lack Nox3 and have a single Duox isoform. Mutants for cyba (B) and nox5 (C) were generated using CRISPR, resulting in a 5bp deletion (cyba^umc403) and a 4bp indel (nox5^umc402), respectively. The mutations led to the removal of restriction enzyme sites, providing an easy diagnostic method for screening animals. Characterization of cyba^sa11798 (D) and duox^sa9892 (E) via Sanger sequencing shows the single nucleotide changes T>A and C>T, respectively, in contrast to a WT reference sequence. Panel A courtesy Dr. Kalin Narov, https://kalinnarov.wixsite.com/embryosafari. See also Figures S1 and S2.

Figure 2

Methodology for quantifying rate of adult fin regeneration (A–F) Amputated adult caudal fins in zebrafish achieve complete, scar-free regeneration by 4 weeks post-amputation (wpa). In all cases, the fin was imaged prior to amputation (A), immediately following amputation (B) and then once weekly at 1wpa (C), 2wpa (D), 3wpa (E), and 4wpa (F). The distinctive stripe and pigment patterns on the body and anal fin enabled identification of individual animals. Fins were amputated midway, and the caudal peduncle was used to demarcate the proximal extent. Scale bar = 5 mm. (G) Repeat (triplicate) measurements of caudal fin areas in 18 adult fish (n = 18). See also Figures S3, S4, Tables S1, and S2.

Figure 3

duox mutants regenerate the caudal fin slower than WTs (A–C) Significant differences in regeneration were observed between WT and duox mutants during 0-1wpa (A) and 1-2wpa (B). By 2-3wpa (C) these differences were resolved, with only the double mutant cyba:duox continuing to significantly trail behind. (D) An overall view across 0-3wpa highlights how duox^sa9892 and cyba:duox animals are significantly slower than every other group. Asterisks denote statistically significant differences (Bonferroni’s multiple comparisons test, ∗p < 0.5, ∗∗p < 0.01, ∗∗∗∗p < 0.0001). WT (n = 16); cyba^umc403 (n = 5); cyba^sa11798 (n = 9); nox5^umc402 (n = 9); duox^sa9892 (n = 9); cyba:duox (n = 7). See also Figures S5–S8.

Figure 4

Thyroid hormone deficiency slows down caudal fin regeneration but only during the late phases of regeneration (A–C) The impact of duox^sa9892, but not tshr^wpr23e1, on the rate of regeneration is significant during 0-1wpa. By 2wpa (B) the effect of tshr^wpr23e1 becomes significant, and this effect continues into 2-3wpa (C). (D) An overall view across 0-3wpa indicates duox^sa9892 and tshr^wpr23e1 animals are significantly slower than WTs during regeneration. Asterisks denote statistically significant differences (Bonferroni’s multiple comparisons test, ∗p < 0.5, ∗∗p < 0.01, ∗∗∗∗p < 0.0001). WT (n = 25); duox^sa9892 (n = 12); tshr^wpr23e1 (n = 3). See also Figure S9.

Figure 5

HyPer ratios in WT animals and nox mutants WT animals and nox mutants were generated in a Tg(ubb:HyPer)umc400 transgenic background, which permits in vivo assessment of ROS levels. (A) ROS Hyper ratios in individual WT animals (n = 8) observed at three time points during the day over three days post-amputation reveal an oscillatory pattern of ROS levels. (B) ROS levels over three consecutive days post-amputation were significantly higher in the mornings (7a.m.) than in the afternoons (3 p.m.) or nights (10p.m.). (C and D) Long-Scargle periodogram reveals that the ROS oscillations follow a daytime-dependent trend, with a periodicity of around 0.4 (∗ on graph), indicative of a ∼24 h circadian cycle (D). (E) Graph of ROS levels measured at the same time of day (3p.m.) in WT (unamputated), WT (amputated), cyba^umc403, duox^sa9892, and nox5^umc402 animals. (F) Graph of mean Hyper ratios measured over three consecutive days at 3p.m. reveals significantly attenuated post-amputation ROS levels among the duox^sa9892 mutants relative to the WT amputated fins and the other nox mutants. Indeed, ROS levels in the duox^sa9892 fins are significantly lower than even those found in unamputated WT fins. ROS levels in the amputated cyba mutant fins are significantly lower than those present in WT amputated fins but not as low as in the duox^sa9892 fins. ROS levels in the amputated nox5^umc402 mutant fins are not significantly different from those present in amputated WT fins. (Bonferroni’s multiple comparisons test, ∗p < 0.5, ∗∗p < 0.01, ∗∗∗∗p < 0.0001). For A-D, WT (n = 8). For E-F, WT unamputated (n = 7); WT amputated (n = 8); cyba^umc403 (n = 10); duox^sa9892 (n = 7); nox5^umc402 (n = 7). See also Figures S10 and S11.