Figure 5. Chromosomal aberrations in ATR/Terc MEFs. (A–C) Quantification of chromosome fusions (A), chromatid breaks and fragments (B), and MTSs (C) in G3 ATR+/+ and ATRS/S MEFs before (nontreated) or after treatment with aphidicolin. n, number of metaphases from two to five independent MEFs per indicated genotype. The number of events out of the total number of telomeres or chromosomes scored is indicated above the bars. Statistical com- parisons using Fisher’s exact test are shown. Error bars represent SEM. Photos show representative images of a metaphase and of individual aberrations. Arrowheads show the chromatid-type fusion (A) and MTSs (C). The boxed area in the larger micrograph in C shows examples of MTSs in detail below. (D) Increased aneuploidy in ATRS/S MEFs. Histograms showing the number of chromosomes per metaphase for each genotype are shown. Aneuploidy is defined as metaphases having other than 40 chromosomes. Statistical comparisons using Fisher’s exact test are shown. Bars, 1 µm. severity of phenotypes associated with ATM deletion (median lifespan and tumorigenesis; Qi et al., 2003; Wong et al., 2003), we were similarly interested in determining the interaction be- tween ATR and Terc at the whole organism level. To study the effect of Terc deficiency on Seckel mouse phenotypes, we first examined the impact on reduced lifespan. Critical telomere shortening leads to premature ageing and shortened lifespan in G3 Terc/ mice, which we confirmed with the observation of significantly reduced survival of ATR+/+/G3 Terc/ mice compared with ATR+/+/G3 Terc+/ mice (Fig. 6 A). Interestingly, we observed a tendency toward a reduced longev- ity in ATRS/S/G3 Terc/ mice compared with ATRS/S/G3 Terc+/ (Fig. 6 A, P = 0.081; almost significant), most likely reflecting the increase in genome instability owed to combined deficien- cies of ATR and Terc (Blasco et al., 1997; Murga et al., 2009). However, Terc deficiency did not contribute to the severe dwarfism of Seckel mice, which is already noticeable at birth, here indi- cated from the measurement of mean body weight at 7 wk of age (Fig. 6 B). Because of severe intrauterine growth defects and developmental abnormalities, Seckel mutant mice were born at reduced non-Mendelian frequencies in comparison with their wild-type and heterozygote littermates, and this was not further aggravated by simultaneous Terc deficiency (Table I; Murga et al., 2009). In line with this, G3 Terc/* mice were born at the same frequency as G3 Terc+/* for all of the ATR genotypes under study (Table I). Some of the most prominent phenotypes attributable to the ATRS/S mutation are severe microcephaly and osteoskeletal and cranial defects. Because telomere dysfunction has previously been associated with osteoporosis (Hofer et al., 2005; Pignolo et al., 2008), we were interested in analyzing the severity of osteoporosis and cranial abnormalities in G3 Terc+/* and G3 Terc/* mice by computed tomography (CT) of 10-wk-old mice. However, the already severe osteoporosis associated with the ATRS/S mutation was not worsened in ATRS/S/G3 Terc/* mice compared with their ATRS/S/G3 Terc+/* siblings (Fig. 6 C). Even though telomere defects have previously been linked to osteoporosis in mice, the severity of this is not apparent until old age (14 mo old) in comparison with the early appearance of osteoporosis in much younger (2 mo old) Seckel mice (Murga 646 JCB • VOLUME 188 • NUMBER 5 • 2010