Telomere Shortening Exposes Functions for the Mouse Werner and Bloom Syndrome Genes

Mice.

Wrn^+/− Blm^+/− Terc^+/− mice were generated by crossing Wrn^−/− Blm^−/− mice with Terc^+/− mice (7, 50). These were in turn crossed to generate Wrn^−/− Blm^−/− Terc^+/− mice, which were crossed with Wrn^+/− Blm^+/− Terc^+/− mice to generate the different G1 mutant lineages. The frequencies of offspring of the different genotypes were at expected Mendelian ratios. Each G1 lineage was crossed to itself to generate G2 lineages, and so forth, using cousin by cousin matings to avoid generation of substrains. All mice were in a mixed genetic background of 50% C57BL/6, 37.5% 129Svj, and 12.5% BALB/c. For the second set of experiments (“new” crosses), mice carrying the three mutations in heterozygous form were crossed for six generations to homogenize the genetic background prior to the generation of G1 lineages. All mice were weighed weekly from weaning at 3 weeks of age until at least 100 days of age. For reasons of space limitation, studies were restricted to mice under 14 months of age. Mice were examined daily, and those judged to be suffering from ill health were sacrificed for analysis. Mouse experimental protocols were approved by the University of Pennsylvania Institutional Animal Care and Use Committee.

Histology and analysis of intestinal crypt apoptosis.

Tissues were fixed in 10% formalin overnight at 4°C, followed by storage in 70% ethanol at 4°C; 5-μm sections were stained with hematoxylin and eosin. Quantitation of apoptotic cells and mitoses was performed with the observer blind to genotype, and at least 80 crypts were counted per mouse. For terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analyses, 5-μm tissue sections were stained with the ApopTag kit (Intergen) according to the manufacturer's instructions, and at least 50 crypts were counted per mouse.

Bone mineral density measurement.

Dual energy X-ray absorptiometry (DEXA) scans were performed of the vertebrae in the abdominal region of anaesthetized mice with a Lunar PIXImus 2. Scans were calibrated with a phantom model with 0.058 g/cm² bone mineral density.

Wound healing and hair regrowth assays.

After hair removal with electric clippers, full-thickness 3-mm (original crosses) or 4-mm (new crosses) punch biopsies were made through to the panniculus carnosus over each scapula. Two wounds were made per mouse, and five mice per genotype were tested. The long and short axes of each wound were measured daily, and area was calculated with the formula for an ellipse (πab). Hair regrowth assays were done as described (53).

Adult fibroblast culture.

Ears were washed for 10 s in 70% ethanol and then in phosphate-buffered saline containing 250 μg of G418 per ml and then minced and digested four times for 30 min in 0.25% trypsin-EDTA at 37°C. Released cells and partially digested tissue were cultured in a 24-well plate in Dulbecco's modified Eagle's medium with 10% heat-inactivated fetal bovine serum, penicillin-streptomycin, 3% O₂, and 5% CO₂ until confluent, and then split into six-well plates. 3T3 proliferation assays were performed in ambient O₂ and 5% CO₂ as described (6). The usual 3-day passage interval was extended in cases where culture growth slowed substantially; in such cases the cells were refed every 3 days and allowed to become nearly confluent before they were passaged.

Cytogenetic analysis and telomere flow fluorescence in situ hybridization.

Splenocytes were cultured for 48 h in RPMI with 10% heat-inactivated fetal bovine serum, 10 μg of lipopolysaccharide per ml, and 5 μg of concanavalin A per ml and then arrested for 30 min with 0.75 μg of colcemid per ml. Metaphases were fixed, stained with 4′,6′-diamidino-2-phenylindole (DAPI), and analyzed as described (44). For samples from the new crosses, metaphases were scored by an observer blind to genotype. For telomere signal-free end analyses, metaphase spreads were stained with DAPI and a indocarbocyanine-conjugated peptide nucleic acid (PNA) telomere repeat probe, indocarbocyanine-OO-(CCCTAA)₃ (Boston Probes) as described (Unit 18.4, Current Protocols in Cell Biology Online, 2003 [www.interscience.wiley.com]). Telomere flow fluorescence in situ hybridization was performed on bone marrow cells with a fluorescein isothiocyanate-labeled PNA telomere repeat probe as described (2). Telomere fluorescence per cell was calculated for cells gated in the G₁ phase of the cell cycle based on DNA content, and telomere fluorescence for each sample was corrected for background fluorescence by control hybridizations of the same sample without the telomere PNA probe. All samples to be compared directly were processed and measured by flow cytometry on the same days to avoid interrun variability. Repeat measurements of the same sample yielded a coefficient of variation of 6%.

Statistical tests.

Graphpad Prism 3 software was used for statistical analyses. Unpaired two-tailed t tests were used to compare each genotypic group for the data in Fig. 1 to 4 and 6. Kaplan-Meier analyses were performed for the data in Fig. 5, and significance was determined with a log-rank test. Error bars in all figures indicate the standard error of the mean.

Generation of mice with mutations in Wrn, Blm, and Terc and characterization of fertility defects.

Earlier work had demonstrated that mice doubly homozygous for the Blm^M3 hypomorphic allele and a Wrn null allele were viable and fertile (28, 50). Such Wrn^−/− Blm^M3/M3 mice were crossed with Terc^+/− mice to generate Wrn^+/− Blm^+/M3 Terc^+/− triple heterozygotes, which were in turn crossed to generate four lineages: wild type, Wrn^−/− Blm^M3/M3 (wb), Terc^−/− (t), and Wrn^−/− Blm^M3/M3 Terc^−/− (wbt) (see Materials and Methods). Terc^−/− mice must be bred for several generations before telomeres become sufficiently short to elicit pathology (44), and so we carried each lineage through multiple generations.

The first and second generations (G1 and G2) of each lineage were fertile, and no abnormalities were observed for the first year of life in general appearance, body weight, or histologic surveys of the following organs: thymus, lymph nodes, spleen, heart, lung, gastrointestinal tract, liver, pancreas, submandibular gland, kidney, adrenal gland, skeletal muscle, bone marrow, femur, vertebrae, skin, ovaries, and testes (not shown). However, a decline in fertility was evident in G3 wbt mutants, and G4 wbt mutants were sterile; in contrast, the wb and t lineages each generated viable offspring through G7 (Fig. 1A) . Gross and histological examination of gonads from G4 triple mutant mice revealed marked atrophy. In males, this was characterized by a decline in testes mass (Fig. 1B) and by the loss in seminiferous tubules of spermatogenic cells and mature sperm (Fig. 1C). These findings are similar to those observed in later-generation Terc^−/− mutants (17, 26, 44) (Fig. 1B, G6 t mice) but occur approximately two to three generations earlier in wbt mutants.

Although the differences in fertility between the t and wbt mice were large, it was possible that the mixed genetic background of the mice or differences in telomere length among the founders of each lineage might have contributed in a random fashion to the phenotypic differences. To control for these possibilities, we repeated the experiment; hereafter we refer to the first set of crosses as original crosses and the repeat set of crosses as new crosses. Furthermore, prior to beginning the new crosses, we first homogenized the genetic background of the mice through six generations of parent-offspring and intersibling crosses of mice carrying the three mutations in heterozygous form. In addition, in the new crosses, Wrn^−/− (w), Blm^M3/M3 (b), Wrn^−/− Terc^−/− (wt), and Blm^M3/M3 Terc^−/− (bt) lineages were also generated to gauge the individual contributions of Wrn and Blm mutations to the phenotypes. Consistent with results from the original crosses, wbt mice from the new crosses became infertile rapidly, at G3 (one generation earlier than they had in the original crosses), while the other genotypes were fertile through at least G4 (data not shown). Moreover, for the original four genotypes (wild type, wb, t, and wbt), all of the phenotypes tested in both the original and new crosses (fertility, body weight, intestinal pathology, wound healing, and cytogenetics; see below) were affected consistently, indicating that the differences observed between lineages are a consequence of their different Wrn, Blm, and Terc genotypes.

Body mass and habitus and bone density.

wbt triple mutants, like the other genotypes, had normal body weight in G1 (data not shown), but G3 and G4 wbt mutants weighed less and were smaller than controls. Weekly measurements revealed differences in the body mass apparent at 3 weeks of age and which increased as the mice grew older (Fig. 2A and 2B). G3 bt mutants also weighed less than controls, but not as little as G3 wbt mutants, indicating that the Blm and Wrn mutations each contribute to low body weight in wbt mutants (Fig. 2C).

G3 and G4 wbt mutants frequently developed a kyphotic (hunched) and scruffy appearance (Fig. 2A). Consistent with the observed kyphosis, DEXA (dual energy X-ray absorptiometry) scans of G3 mice revealed reduced bone density in the vertebral skeleton of wbt mutants compared with controls (Fig. 2D). Although increased hair loss was sometimes observed in aging G3 and G4 wbt mutants, hair regrowth assays did not reveal significant differences between the different mutant lineages (data not shown).

Gastrointestinal pathology of mutant mice.

We noticed that G3 and G4 wbt and bt mutant mice suffered from chronic diarrhea and frequently developed prolapse of the rectum (Fig. 2A). At the histological level, the appearance of the small intestines of all G1 genotypes was normal, but compared with controls, the small intestines of G3 and G4 wbt mice were characterized by distortion of crypt architecture and signs of acute inflammation, including blood vessel dilatation and neutrophil infiltration (Fig. 3A). Under normal conditions, intestinal epithelial cells undergo apoptosis at the end of their developmental cycle as they are sloughed off the tips of villi, but in contrast, epithelial cells in the crypts do not normally undergo apoptosis. However, crypt cells do apoptose in late-generation telomerase mutants (17, 44).

To test if Wrn and Blm mutation influenced the level of crypt apoptosis associated with Terc mutation, we quantified apoptosis by direct histologic examination of hematoxylin- and eosin-stained tissue sections and confirmed these data by quantifying TUNEL-positive nuclei in the tissue sections (Fig. 3B). Levels of apoptosis in intestinal crypts were slightly elevated in G3 t and wt mutants but markedly elevated in G3 and G4 wbt mice compared with controls and reached the levels observed in G7 t mutants (Fig. 3B). G3 bt mutants had levels of apoptosis as high as that of G3 wbt mutants, while G3 wt mutants had levels similar to that of G3 t mutants, indicating that, in this particular tissue, Blm may have a greater role than Wrn in the context of telomerase inactivation. Furthermore, we confirmed through TUNEL analysis that levels of apoptosis in G1 wbt mutants were not elevated (Fig. 3B), indicating that this phenotype depends on telomere loss and not loss of telomerase per se.

Wound healing and skin fibroblast proliferation.

Individuals with Werner syndrome suffer from chronic skin ulcers, particularly around the ankles, and late-generation Terc mutants display slow wound healing (12, 44). We therefore tested our mice for rates of wound healing and observed slow wound healing in G3 wbt mutants compared with controls (new crosses; Fig. 4A). Although the Wrn and Blm mutations each contributed to the slow healing of wbt mutants, the Wrn mutation appeared to contribute more than the Blm mutation because G3 wt but not G3 bt mutants healed wounds more slowly than the controls. Wound healing involves the interplay of several cell types, and we do not yet understand the basis for this defect. However, one cell type important for wound healing is the dermal fibroblast, and we observed significant growth defects in cultured adult skin fibroblasts from wbt mutants compared with controls (Fig. 4B).

Skin fibroblasts were obtained from the ears of 10-month-old G1 siblings (new crosses) and cultured serially in 3T3 assays. No significant differences in the rates of culture doubling were observed between wild-type and t cells, consistent with earlier studies (6), while wb and wbt cultures doubled more slowly but similarly to each other in early passages. However, after seven population doublings, the wbt cells slowed and failed to proliferate further for over 1 month. Based on the sloughing of cells that failed to exclude trypan blue from the plate into the medium and based on the enlarged and flattened appearance of the remaining adherent cells, a mixture of cell death and senescence might be responsible for the poor growth of the triple mutant cultures. It is possible that the impaired growth of wbt cells from G1 mice after serial culture under the damaging conditions of standard cell culture (41) reflects defects that also occur at later generations in vivo in wbt mice. Regardless, these observations provide another example of how mutations in Wrn, Blm, and Terc synergize to affect cell function.

Reduced life span of later-generation wbt mice.

Consistent with their frail appearance, we observed a dramatically reduced life span in G3 wbt mutants versus controls. As shown in Fig. 5, the median age of death for G3 wbt mutants was 7 months, compared with greater than 10 months for all other genotypes (P < 0.0001 compared with all other genotypes by log-rank test). In addition, G3 bt mice also have a significantly reduced life span, with a median age of death of 11 months (P ≤ 0.002 compared with all other genotypes by log-rank test). Because the G3 wbt life span is significantly shorter than the G3 bt life span, the Wrn mutation contributes to the mortality of the wbt mutants, even though it has no effect on life span in the presence of active telomerase and also does not appear to shorten life span dramatically in G3 wt mice (Fig. 5) (28). There was no increased mortality for any of the genotypes in G1 animals during the first year of life (data not shown), indicating that the rapid mortality of G3 wbt and bt mutants apparently depends on telomere shortening.

Telomere function and length.

The pathology observed in wbt mutants could arise from direct exacerbation by Wrn and Blm mutation of telomere dysfunction initiated by loss of telomerase, or it might instead reflect the summation of genomewide instability caused by mutation of Wrn and Blm combined with genome instability caused by loss of telomerase. To begin to address these possibilities, we tested aspects of telomere function. A cardinal indicator of telomere dysfunction is end-to-end chromosomal fusion, which becomes more frequent in later-generation Terc^−/− mice (15). Fusion levels in metaphase spreads from splenocytes from G1 mice were not elevated for any genotype, but were significantly elevated in G3 and G4 wbt mutants (Fig. 6A and 6C). Neither G3 bt nor wt mutants had significant elevations in fusions, indicating that the Wrn and Blm mutations each contribute to this wbt phenotype.

Telomere dysfunction in wbt triple mutants could reflect a propensity of telomeres, at a given degree of shortening, to malfunction in the absence of WRN and BLM or could reflect an elevated loss of telomere repeat DNA in wbt compared with Terc^−/− mutants or both. Flow fluorescence in situ hybridization analysis of bone marrow cells indicated no differences in telomere length among G1 genotypes but revealed a significantly shorter mean cellular telomere length in G3 and G4 wbt samples compared with t samples (Fig. 6B). Consistent with these measurements, the frequency of chromatid termini without detectable telomere repeat DNA in metaphase chromosome spreads (signal-free ends) was elevated in samples from G3 wbt compared with G3 t samples (Fig. 6C and 6D). Therefore, at least one contributor to the severe phenotypes of late-generation wbt mutants appears to be accentuated loss of telomere repeat DNA in this lineage compared with the t lineage. Furthermore, the occurrence of signal-free ends was not increased in wb mutants, indicating that, at least in the presence of telomerase, the Wrn and Blm mutations do not themselves cause significant telomere repeat loss. This contrasts with a recent demonstration of stochastic loss of complete telomere repeat arrays at chromosome ends in a telomerase-positive human tumor cell line overexpressing a dominant-negative form of WRN (3) and suggests that, in murine splenocytes, the Wrn and Blm mutations either do not cause such complete loss events or that telomerase is sufficient to repair such events.