Planarian telomere length dynamics. (A) Telomere length in asexual animals increases after both fission and regeneration induced by amputation.UD, undigested genomic DNA; 7 d, animals that underwent fission 7 d previously (mean 28 kb, SD 0.7 kb, n = 10); 3 mo, animals that underwent fission 85–95 d previously (mean 26 kb, SD 0.6 kb, n = 10, P < 0.02, two-tailed t test); Int, intact asexual animals that have not undergone fission for between 85 and 195 d (mean 22.6 kb, SD 1.75 kb, n = 10); Reg, animals that have undergone three rounds of regeneration (mean 26.9 kb, SD 1.48 kb, n = 10, P < 0.04, two-tailed t test). (B) Telomere length in sexual animals decreases with age, with newly born (NB, mean 21.2 kb, SD 1.67 kb, n = 7) animals from 3-y-old parents (3yo, mean 11.1 kb, SD 1.4 kb, n = 6, P < 0.002, two-tailed t test) showing rejuvenated lengths. Serial regeneration of 6- to 12-mo-old animals significantly decreases telomere lengths. Shown are representative animals before and after three rounds of regeneration. Int, sexual animals between 180 and 360 d old (mean 17.5 kb, SD 1.33 kb, n = 10); Reg, animals that have undergone three rounds of regeneration (mean 11.6 kb, SD 1.50 kb, n = 10, P < 0.007, two-tailed t test). (C and D) Graphical representation of asexual and sexual TRF data showing statistically significant comparisons (*P < 0.05, **P< 0.01). (E) TRAP assay indicates that telomerase activity increases at 72 h after regeneration (Reg) in both asexual and sexual animals, with a greater increase visible in asexuals during regeneration compared with intact animals (Int). Hela cells extract (+ve), and heat treated (-ve). (F) γ-Irradiation to remove proliferating pASCs and their recent progeny leads to loss of telomerase activity. IRR, asexual animals 7 d after irradiation; Un-IRR, mock-irradiated animals.

Characterization and RNAi of Smed-TERT, the catalytic subunit of telomerase in S. mediterranea. (A) Smed-tert translation and multispecies alignment reveal that Smed-TERT has the conserved motifs of TERT genes across eukaryotes (Fig. S2). (B) RT-qPCR of Smed-tert in irradiated and mock-irradiated worms. Smed-tert is removed by irradiation (three replicate experiments on batches of five worms, **P < 0.01, two-tailed t test). (C) Smed-tert(RNAi) greatly reduces telomerase activity whereas control DsRed(RNAi) does not, as measured by TRAP assay (three replicate experiments on batches of five worms gave equivalent results). (D) Prolonged Smed-tert(RNAi) (TERT) leads to a reduction of telomere length over time as asexual animals fail to maintain their telomeres; removal of Smed-tert(RNAi) after 18 wk results in the stabilization of telomere length for times 12–33 wk later (r12, r21, r33). (E) Smed-tert(RNAi) leads to telomere erosion of 290 bp/wk in asexual animals. (Error bars are standard deviation of the mean telomere length.)

Whole-mount in situ hybridization of TERT in mature sexual and asexual worms. Expression can be observed in both (A) ovaries and (B) testes in mature sexual samples. (C) A transparenchyma expression concentrating around the pharynx can also be seen in asexual worms, suggesting expression in adult somatic stem cells. (D) Irradiation to remove proliferative stem cells removes Smed-tert expression in the germ line of sexual animals and the proliferative stem cells of asexual animals. (E) Major changes in the localization of Smed-tert expression during regeneration are not detected by in situ hybridization. (F) Controls in situ for irradiation indicating that Smed-H2B expression, a marker of proliferative stem cells and germ line, is removed by irradiation but expression of Smed-GluR, a marker of postmitotic differentiated brain cells, is not affected. All irradiated samples were treated with 85 Gy γ-irradiation. The Smed-tert samples were fixed at 7 d posttreatment, whereas the controls were fixed at 72 h postirradiation. (Scale bars, 1 mm.)

Alternative splicing of Smed-tert. (A) Exonic structure of Smed-tert alternate splicing sites. All four possible transcripts can be detected by PCR. (B) Schematic showing that splicing events that skip exon numbers 4–6 remove the TRBD. (C–F) RT-qPCR analysis of (C) Smed-tert transcript levels; (D) alternate region 1-containing transcripts; (E) alternate region 2-containing transcripts; and (F) the ratio of alternate region 1-positive and -negative transcripts. Errors bars are ± 1 SD of the mean. In each case, transcript levels are significantly increased in regenerating asexual worms (three replicates of batches of five worms each, t test, two-tailed, *P < 0.03).