A) Representative image of TRAP assay showing that telomerase is not active in the tert^−/− zebrafish, as compared to tert^+/+ siblings. Here shown are caudal fin and skin protein extracts. Hela cell extract is shown as positive control. N = 4. B) Representative image of restriction fragment analysis of caudal fin genomic DNA of 3 different individuals at different ages, by southern blot (random primer-labelled telomeric probe (CCCTAA)₁₂ ³²P-dCTP). tert^+/+ Zebrafish have heterogeneous telomeres, with two distinct peaks of different lengths. In tert^+/+ the highest peak (∼16 Kb, top red arrow) becomes more distinct after 1 months of age and decreases in length over-time (B and D). The lowest peak of telomere intensity also decreases in length (bottom red arrow, B and D). tert^−/− zebrafish have shorter telomeres than tert^+/+ siblings in different tissues (see also Figure S1A and S1B), observed by the decrease in length of the higher TRF peak. The shortest TRF peaks accompany those of tert^+/+ siblings, and decrease over-time at similar rates. C) Testes fractionation in tert^+/+ reveals the two-telomere length populations in whole testes, whereas mature sperm only shows the shorter TRF smear of about 6 Kb, suggesting different telomere lengths in different cells within a tissue. D) TRF mean sizes were calculated as described in [50]. E) Telomere PNA-FISH in 6-month-old gut tissue shows cells with different telomere intensities in the wild type, mainly localizing to the proliferative niche. In contrast tert^−/− mutants display cells with less bright and more homogeneous telomere intensity.

https://doi.org/10.1371/journal.pgen.1006652.g001