Fig 2: (a) Experimental design and dosimetry. The cumulative dose in Gy are indicated for each developmental stages and sampling time (in red). (b) Detection of ?-H2AX foci in 24 hpf and 48 hpf embryos by whole-mount immunocytochemistry. Standard-error to the mean is indicated for each exposure (Kruskall-wallis test followed by a pairwise Wilcoxon rank sum post-hoc test and adjusted by the Holm method, adjusted p-value: * < 0.05). (c) Examples of positive ?-H2AX foci (green, white arrows) detected in DAPI stained nucleus (blue) by confocal microscopy. (d) Means of body length measured at 48 hpf (in mm) are indicated as white lozenge. Dots represent individual data.

Fig 3: DNA Damage Increases Strongly after Ventricular Cryoinjury in the Absence of Telomerase(A) Representative western blot of ?-H2AX expression in WT and tert-/- hearts without injury and in hearts at 3 dpi.(B) Quantification of western blot signal intensities (n = 9 hearts/condition). Data are means ± SEM. *p < 0.05 (Mann-Whitney test).(C) Representative staining of ?-H2AX foci (green) in cardiac cells in uninjured and 3 dpi WT and tert-/- hearts. Cardiomyocytes are immunostained with anti-MHC (red), and nuclei are counterstained with DAPI (blue). Examples of ?-H2AX+ cardiomyocytes are outlined with green circles. Boxed areas are shown at a higher magnification. Scale bars, 20 µm.(D) Distribution of ?-H2AX-positive cardiomyocytes (green circles) in uninjured and 3 dpi WT and tert-/- hearts. The nuclear area is shown in magenta. The ventricle and injured area are outlined by dotted lines. Scale bars, 100 µm.(E and F) Percentages of (E) ?-H2AX-positive cardiac cells and (F) ?-H2AX-positive cardiomyocytes in uninjured and 3 dpi WT and tert-/- hearts. Data are means ± SEM of cells counted on a minimum of 3 sections/heart in four hearts. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test). See also Figure S7.

Fig 4: Tupgcp3 deficient cells undergo apoptosis and senescence. (A–H) Immunostaining analyses displaying significantly increased TUNEL+ cells in the tubgcp3 mutant retina (D,F,H) at 3 dpf compared to wild-type sibling (C,E,G). Note the increased PH3+ cells in the tubgcp3 mutant retina do not co-localize with the TUNEL+ cells (F,H). (I) Bar chart analyses depicting quantification of TUNEL+ cells in wild-type sibling and the tubgcp3 mutant retinae. (J–Q) Immunostaining analyses exhibiting markedly increased ?-H2AX+ cells in the tubgcp3 mutant retina (M,O,Q) at 3 dpf compared to wild-type sibling (L,N,P). Note that some of the (increased PH3+ cells overlap with the ?-H2AX+ cells in the tubgcp3 mutant (O,Q). Arrows mark the PH3 and ?-H2AX double positive cells in the tubgcp3 mutant retina. Arrowheads indicate the location of RSCs in the CMZ of the retina. (R) Bar chart analyses depicting quantification of ?-H2AX+ and PH3+ cells in wild-type sibling and the tubgcp3 mutant retinae. Data are mean + SEM from 30 sections for each group. Student’s t-test: **P < 0.01. (S–T”) ISH and immunostaining analysis of cell proliferation at the extreme periphery of CMZ at 5 dpf. Zebrafish embryos were incubated in BrdU for 24 h before collected at 5 dpf for the double staining assay. In the wild-type sibling, there are many BrdU+ cells in col15a1b-labeled region (S,S’,S”). In contrast, BrdU+ cells are significantly decreased in this region in the tubgcp3 mutant CMZ (T,T’,T”). (U–X) Senescence-associated ß-galactosidase (SA-ß-gal) staining exhibiting increased ß-galactosidase activity at the CMZ in the tubgcp3 mutant (V) compared to the wild-type sibling (U). Nuclei are stained with DAPI (W,X). Insets indicate high-magnification images of the peripheral edge of CMZ in rectangles in (U–X). (Y) Bar chart analyses depicting quantification of BrdU+ cells in the col15a1b-labeled region in wild-type sibling and the tubgcp3 mutant CMZ. Data are mean + SEM from 36 sections for each group. Student’s t-test: **P < 0.01. Scale bars: 20 µm (A–H); 20 µm (J–Q); 20 µm (S–T”); 50 µm (U–X).

Fig 5: Quantification of senescence markers in gut and testis of older tert-/- zebrafish.(A-B) Western blot analysis of DNA damage and senescence-associated proteins in gut and testis of 3 month (A) or 9-month-old (B) of WT and tert-/- siblings (N >= 5 fish). Representative western blot (left panel) and corresponding quantification (right panel) showing induction of DNA Damage Response (H2A.X-P and p53) in 3-month-old and senescence (p15/16) in 9-month-old tert-/- zebrafish. (C) RT-qPCR analysis of senescence associated genes p15/16 and p21. RT-qPCR graphs are representing mean ± SEM mRNA fold increase after normalisation by rpl13a gene expression levels (* p-value<0.05; ** p-value<0.01, using the Mann-Whitney test).Figure 2—source data 1.Western Blot quantifications, as plotted in Figure 2A-B.Figure 2—source data 2.Real-time qPCR data of p15/16 and p21, as plotted in Figure 2C.