Rescue of mean telomere length and percentage of short telomeres in late generation telomerase-reconstituted G3 Terc^+/−* keratinocytes. (A) Telomere length histogram obtained by Q-FISH in primary murine skin keratinocytes. The histogram shown is representative of three independent pairs of G3 Terc^−/− and G3 Terc^+/−* littermates (see B). Note a greater abundance of short telomeres in the G3 Terc^−/− mouse compared with the G3 Terc^+/−* littermate. Red lines facilitate visualization of short (<100 a.u.f.) and long (>1,000 a.u.f.) telomeres. The total number of telomere dots used for the quantification and the percentage of short and long telomeres are indicated. (B) Mean telomere length is significantly increased (P < 0.001) in G3 Terc^+/−* compared with G3 Terc^−/− mice. Concomitantly, these mice show a significant (P < 0.001) decrease in the percentage of short telomeres and a significant (P = 0.015) increase in the percentage of long telomeres. Data are mean values ± SEM for three independent pairs of G3 Terc^−/− and G3 Terc^+/−* littermates.

Rescue of HF stem cell mobilization defects, as well as of skin and hair growth defects in late generation telomerase-reconstituted G3 Terc^+/−* mice upon TPA treatment. (A and B) To assay mobilization of LRCs, mice of the indicated genotype were injected with BrdU and after 69 d, whole mounts of tail epidermis were collected from untreated and TPA-treated mice and stained with a BrdU antibody. The number of LRCs at the hair bulge in resting conditions or after stimulation with TPA in littermate G3 Terc^+/−* and G3 Terc^−/− mice are indicated (A). LRC numbers were calculated in at least five mice of each genotype (n), quantifying at least 197 follicles in the control group and at least 190 follicles in the TPA-treated group (A). Note a higher percentage of mobilization of LRCs in TPA-treated G3 Terc^+/−* mice compared with G3 Terc^−/− mice (B). (C) Representative confocal micrographs of tail follicles from G3 Terc^−/− and G3 Terc^+/−* mice were stained for BrdU (green) before (controls) and after (+TPA) TPA treatment. LRCs of all genotypes accumulate in the bulge (Bu) region of the HF. Bar, 80 μm. Note a greater disappearance of LRCs in Terc^+/−* TPA-treated HFs compared with the G3 Terc^−/− littermates. The different compartments (infundibulum, hair bulge, and hair bulb) of the HF are indicated. (D–G) Quantification of HF length from sebaceous glands (SG) to dermal papilla (DP; D and E) and of IFE thickness (F and G) in tail skin from mice of the indicated genotype. Histomorphometry was performed in five mice of each genotype (n), quantifying at least 69 follicles in the control group and at least 76 follicles in the TPA-treated group. Note the increased HF length and IFE thickness in TPA-treated G3 Terc^+/−* mice compared with G3 Terc^−/− littermates (P < 0.05 for both; E and G). (H) Representative tail skin sections from mice of the indicated genotypes before (control) and after (+TPA) TPA treatment. Bracketed continuous black lines mark IFE. Black dashed double-pointed arrows mark HF length from sebaceous glands to dermal papilla. Bar, 150 μm. Error bars in A, B, and D–G represent SEM.

Rescue of proliferative potential of epidermal stem cells ex vivo and of small body-size phenotype in late generation telomerase-reconstituted G3 Terc^+/−* mice. (A) Representative images of number and size of macroscopic colonies obtained from isolated keratinocytes of the indicated genotypes. Note more abundant and larger colonies in G3 Terc^+/−* mice compared with G3 Terc^−/− littermates. (B) Quantification of size and number of macroscopic colonies obtained from isolated keratinocytes of the indicated genotype purified from 2-d-old mice and cultured for 10 d on J2-3T3 mitomycin C–treated feeder fibroblasts. (C) Representative images of newborn (left) and 2-mo-old (right) mice of the indicated genotype. Note a small body size in G3 Terc^−/− mice compared with wild-type controls and that this small body-size phenotype is largely rescued in telomerase-reconstituted G3 Terc^+/−* mice. (D) Quantification of body weight in newborn (left) and 2-mo-old (right) mice of the indicated genotypes. Note significantly reduced body size in newborn G3 Terc^−/− mice compared with both wild-type controls and telomerase-reconstituted G3 Terc^+/−* mice (P < 0.001 for both) and the significantly reduced body size in adult G3 Terc^−/− mice compared with telomerase-reconstituted G3 Terc^+/−* mice (P < 0.001). Error bars in B and D represent SEM.

Pronounced rescue of short telomeres in Terc-reconstituted and Terc-deficient keratinocytes and splenocytes. (A) Telomere length histograms obtained by Q-FISH on tail skin sections. The histogram shown is representative of three independent age-matched (10 mo old) pairs of Terc^+/− and telomerase-reconstituted G3 Terc^+/−* mice (see B). Red lines facilitate visualization of short (<60 a.u.f.) and long (>600 a.u.f.) telomeres. The total number of telomere dots used for the quantification and the percentage of short and long telomeres are indicated. Note very similar telomere length distributions in age-matched Terc^+/− and telomerase-reconstituted G3 Terc^+/−* mice. (B) Mean telomere length is indistinguishable between age-matched Terc^+/− and telomerase-reconstituted G3 Terc^+/−* mice (P = 0.98). Concomitantly, these mice show similar percentages of short telomeres (P = 0.22) and long telomeres (P = 0.70). Data are mean values ± SEM for three independent pairs of G3 Terc^−/− and G3 Terc^+/−* mice. (C) Telomere length histograms obtained by Q-FISH directly on metaphases of freshly isolated splenocytes. The histogram shown is representative of three age-matched (1–2 yr old) pairs of Terc^+/− and telomerase-reconstituted G4 Terc^+/−* mice. In this case, arbitrary units of fluorescence were converted into kilobases as described in Materials and methods. Red lines facilitate visualization of short (<10 Kb) and long (>50 Kb) telomeres. The total number of individual telomeres used for the quantification and the percentage of short and long telomeres are indicated. Note very similar telomere length distributions in age-matched Terc^+/− and telomerase-reconstituted G4 Terc^+/−* mice. (D) Mean telomere length is indistinguishable between age-matched Terc^+/− and telomerase-reconstituted G4 Terc^+/−* mice (P = 0.31). Concomitantly, these mice show similar percentages of signal-free ends (P = 0.22), short telomeres (<10 Kb; P = 0.59), and long telomeres (>50 Kb; P = 0.75). Data are mean values ± SEM for three independent Terc^+/− and three independent G4 Terc^+/−* mice.

Reconstitution of telomerase activity in G3 Terc^+/−* mice compared with their G3 Terc^−/− siblings. Telomerase is successfully reconstituted in two independent MEF cultures from G3 Terc^+/−* mice compared with their telomerase-deficient G3 Terc^−/− littermates. Two wild-type MEFs are also included as a positive control for telomerase activity. The protein concentration used is indicated on top of each lane. Extracts were treated (+) or not treated (−) with RNase as a negative control. An internal control (IC) for PCR efficiency was also included.

Significant rescue of signal-free ends and end-to-end chromosomal fusions in G3 Terc^+/−* keratinocytes. (A) Quantification of the frequency of the indicated chromosomal aberrations in primary keratinocytes from G3 Terc^−/− and G3 Terc^+/−* mice. The total number of chromosomes scored for the analysis is indicated on top of each bar. Statistical comparisons using the χ² test are shown. (B) Representative examples of the indicated chromosomal aberrations are shown below the graph. Note that signal-free ends and end-to-end fusions are significantly rescued in Terc-reconstituted G3 mice compared with the G3 Terc-deficient littermates, which is in agreement with the fact that telomerase elongates short telomeres, thus preventing telomere dysfunction. Bar, 0.4 μm.

Rescue of skin and hair growth defects in late generation telomerase-reconstituted G3 Terc^+/−* mice upon hair plucking of back skin. (A–D) Quantification of HF length from IFE to dermal papilla (A and B) and of dermis thickness (C and D) in back skin from mice of the indicated genotype. Histomorphometry was performed in five mice of each genotype, quantifying at least 55 follicles in the control groups and at least 87 follicles in the TPA-treated groups. Note the increased HF length and dermis thickness in G3 Terc^+/−* mice compared with G3 Terc^−/− littermates after plucking (B and D). Error bars represent SEM. (E) Representative back skin sections from mice of the indicated genotypes before (control) and after (pluck) plucking. Black double-pointed arrows mark dermis thickness. Arrowheads mark HF length from IFE to dermal papilla. Bar, 480 μm.

Rescue of organismal life span in late generation telomerase-reconstituted G3 Terc^+/−* mice. (A) Kaplan-Meyer survival curve of mice of the indicated genotype. n is the number of mice of each genotype. Statistical comparisons using the log rank test are also shown. Note the significant rescue in survival of telomerase-reconstituted G3 Terc^+/−* mice compared with G3 Terc^−/− mice (P < 0.001). (B) Median survival obtained from Kaplan-Meyer plots of mice of the indicated genotypes. Statistical comparisons using the log rank test are also shown. Note the significantly reduced median survival of G3 Terc^−/− mice compared with the other genotypes (P < 0.001). (C) Maximal lifespan of mice of the indicated genotypes. Note the decreased maximal life span of G3 Terc^−/− mice and how this is corrected in telomerase-reconstituted G3 Terc^+/−* mice. (D) Percentage of mice of the indicated genotype showing malignant tumors (lymphoma, histiocytic sarcoma, or sarcoma/carcinoma) at the time of death. Statistical comparisons using the χ² test are also shown. Note the decreased incidence of malignant tumors in G3 and G4 Terc^−/− mice in agreement with a tumor-suppressor role associated with telomerase deficiency and short telomeres. Telomerase-reconstituted G4 Terc^+/−* mice show a similar tumor incidence to that of wild-type controls. (E) Percentage of mice of the indicated genotype showing degenerative pathologies (atrophies) in the indicated tissues at the time of death. Statistical comparisons using the χ² test are also shown. Note the decreased incidence of intestinal atrophies in telomerase-reconstituted G4 Terc^+/−* mice compared with G3 and G4 Terc^−/− mice.