Generation of brcc3 knockout zebrafish by using CRISPR/Cas9. (A) Schematic diagram of zebrafish brcc3 gene and CRISPR/Cas9 target site. The blue box represents the exon, and the black line represents the intron. CRISPR/Cas9 gRNA target site locates in the third exon and the target sequence is indicated at the top of the image. Protospacer adjacent motif (PAM) region is shown in red. (B) The generated zebrafish brcc3-null alleles. (C) Schematic diagram of zebrafish Brcc3 domains. The red line indicates the initial abnormal translation site of the mutant brcc3 alleles. (D) The predicted protein sequences of WT and mutant brcc3 alleles are shown. (E) Representative images of WT, MZbrcc3^{−2+3/−2+3}, and MZbrcc3^−7/−7 mutant embryos at 24, 48, and 72 hpf. Lateral views with anterior to the left. Scale bar = 500 μm. (F) The relative mRNA levels of brcc3 in WT and mutant embryos at 24 and 48 hpf, as indicated by qRT-PCR analysis. The results are from three independent replicates. Values are represented as means ± s.d. * p < 0.05; ** p < 0.01 (unpaired two-tailed Student’s t-test). (G) Expression of brcc3 in WT and brcc3 mutant embryos at 24 and 48 hpf. The sense probe of brcc3 was used as a control. Scale bar = 500 μm. The proportion of embryos with the indicated phenotypes is shown in the bottom right corner of each panel.

Depletion of Brcc3 in zebrafish embryos alleviates the resistance to UV radiation and increases the sensitivity to UV radiation-induced DNA damage. (A) Schematic diagram of zebrafish embryos radiated with UV. Embryos were radiated for different times of duration at 24 hpf and then raised to 48 hpf. (B) Representative images of WT and mutant embryos at 48 hpf after UV radiation. WT and mutant embryos at 24 hpf were treated with UV for 0 s, 10 s, and 30 s, and then raised to 48 hpf. hpt, hours-post treatment; Scale bar = 1 mm. (C) Quantitative results from images as shown in (B). WT and mutant embryos at 48 hpf in B were categorized into normal, malformed, and decomposed classes, respectively. Results are from three independent replicates. The total number of embryos in each group is shown above the column. (D) Survival curve of WT and mutant embryos after 30 s UV radiation. WT and mutant embryos at 24 hpf were treated with UV for 30 s and viability was continuously recorded at the indicated timepoint from 8 to 32 h after treatment. The total number of embryos in each group is shown in the upper right corner of the column. Values are represented as means ± s.d. ns, not significant; * p < 0.05; ** p < 0.01 (unpaired two-tailed Student’s t-test). (E) Schematic diagram of UV radiation experiments for immunohistochemical staining analysis. (F) Representative confocal images of WT and brcc3 mutant embryos at 28 hpf which were immunostained with an anti-γ-H2AX antibody after UV radiation. Nuclei were counterstained with DAPI (blue). Somite 16 to 18 and 21 to 23 were selected for confocal imaging analysis, respectively. Scale bar = 10 μm. (G) Quantitative results from images as shown in (F). The γ-H2AX signal was represented by the γ-H2AX positive signal cells/total cells × 1000. Results are from three independent replicates. Values are represented as means ± s.d. ** p < 0.01; *** p < 0.001 (one-way ANOVA followed by Tukey’s post hoc test).

Depletion of Brcc3 in zebrafish embryos amplifies the up-regulatory response of p53 signaling to UV radiation. (A) Schematic diagram of UV radiation experiments for qRT-PCR analysis. (B) The relative mRNA levels of p53 and its target genes in WT and mutant embryos with or without UV radiation are accessed by qRT-PCR. Results are from three independent replicates. Values are represented as means ± s.d. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 (one-way ANOVA followed by Tukey’s post hoc test). (C) Schematic diagram of UV radiation experimental design to access the p53 action after Brcc3 depletion. (D) Survival rate of brcc3 mutant embryos at 48 hpf in the p53^+/M214K or p53^M214K/M214K genetic background. The brcc3^{−2+3bp/−2+3bp}; p53^+/M214K and brcc3^{−2+3bp/−2+3bp}; p53^M214K/M214K were crossed, and the offsprings at 24 hpf were subjected to UV radiation for 30 s. The survival embryos at 48 hpf were subjected to genotyping analysis. Values are represented as means ± s.d. The total number of embryos is shown on each column.

Brcc3-depleted zebrafish embryos exhibit increased sensitivity to ETO treatment. (A) Schematic diagram of ETO treatment of WT and brcc3 mutant embryos for immunohistochemical staining and qRT-PCR analysis. (B) The various degrees of apoptosis of embryos after treatment with ETO. Scale bar = 500 μm. (C) Quantitative results from images as shown in (B). WT and brcc3 mutant embryos at 28 hpf in (B) were categorized into each indicated group. Results are from three independent replicates. Values are represented as means ± s.d. The total number of embryos is shown at the top of each column. (D) Acridine orange staining of WT and brcc3 mutant embryos at 28 hpf after ETO treatment with various doses. Scale bar = 500 μm. (E) Quantitative results from images of head (upper panel) or 16–21 somite (lower panel) region as shown in (D). The total number of embryos is shown on each column. Values are represented as means ± s.d. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 (one-way ANOVA followed by Tukey’s post hoc test). (F) The relative mRNA levels of p53 and indicated genes in WT and brcc3 mutant embryos at 48 hpf with/without ETO treatment. Results are from three independent replicates. Values are represented as means ± s.d. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001 (one-way ANOVA followed by Tukey’s post hoc test).

Pharmacological inhibition of ATM activation mitigates the enhanced UV radiation-induced effects after Brcc3 depletion. (A) Model for ATM or ATR in Response to DNA Damage. (B) Schematic diagram of qRT-PCR or phenotypic analysis of embryos after UV radiation with/without ATM inhibitors. WT and brcc3 mutant embryos at 22 hpf with/without ATM inhibitors treated were raised to 24 hpf, treated with UV radiation for 30 s, and then subjected for qRT-PCR or phenotypic analysis at indicated timepoint. (C) Representative images of UV radiation-treated WT and brcc3 mutant embryos at 48 hpf in addition of different doses of ATM/ATR inhibitors. Scale bar = 1 mm. (D) The viability of WT and brcc3 mutant embryos in (B). Values are represented as means ± s.d. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. (one-way ANOVA followed by Tukey’s post hoc test). (E) The relative mRNA levels of p53 and its target genes in UV radiation-treated WT and brcc3 mutant embryos at 28 hpf with/without KU60019 inhibitor. Results are from three independent replicates. Values are represented as means ± s.d. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. (one-way ANOVA followed by Tukey’s post hoc test).

Pharmacological inhibition of ATM activation counteracts the elevated ETO-induced effects in brcc3 mutant embryos. (A) Schematic diagram of WT and brcc3 mutant embryos treated with ETO or ETO plus an ATM inhibitor. (B) Addition of an ATM inhibitor KU60019 rescued the phenotypic abnormalities caused by ETO treatment. The proportion of embryos with the indicated phenotypes is shown in the bottom right corner of each panel. Scale bar = 500 μm. (C) Quantitative results of embryos with morphological normal as shown in (B). The total number of embryos is shown below the column. Values are represented as means ± s.d. *** p < 0.001; **** p < 0.0001 (one-way ANOVA followed by Tukey’s post hoc test). (D) Acridine orange staining of WT and brcc3 mutant embryos at 28 hpf after ETO or ETO plus KU60019 treatment. White boxes indicate local magnification. Scale bar = 500 μm. (E) Quantitative results from images of head or 16–21 somite regions as shown in (D). The total number of embryos is shown above the column. Results are from three independent replicates. Values are represented as means ± s.d. ns, not significant; * p < 0.05; ** p < 0.01; **** p < 0.0001 (one-way ANOVA followed by Tukey’s post hoc test).