Age-related molecular variability in Caenorhabditis elegans, T. R. Golden et al. 181 Fig. 2 Accumulation of masses of nucleic acid in aged, wild-type N2 nematodes. (a) 4 days old. (b) 14 days old. (c) 19 days old. Worms were fixed and stained with DAPI to detect DNA. Note the mitotic nuclei at the distal tips of the gonad at each age (arrows). In the 4-day-old individual, nuclei of the developing embryos are visible. In the 14- and 19-day-old individuals, no embryos are present, and the space is filled with amorphous, tumor-like masses (arrowheads). Masses are defined as brightly staining structures not attributable to nuclei of the adult or embryos. These individuals are representative of 10 individuals of each age studied. to published maps of hypodermal nuclei (Altun & Hall, 2005). These nuclei may be lost, or alternatively, the nuclear DNA may be degraded or damaged to such an extent that DAPI staining is lost. This nuclear loss also occurred in long-lived daf-2(e1368) and daf-2(e1370) nematodes, but was delayed relative to the occurrence in N2 (Fig. 1c and data not shown). Masses of DNA accumulate with age in wild-type nematodes The second age-related phenomenon detected via DAPI staining is a massive accumulation of DAPI-stained material in the mid- section of wild-type nematodes of middle age (Fig. 2). These masses of DNA grew to essentially fill the body cavity of the nematode (Fig. 2 and Supplemental movie). Long-lived daf- 2(e1368) nematodes (Supplementary Fig. S1) did accumulate DAPI-reactive masses, but to a lesser extent than N2 and much delayed when compared to N2. The long-lived strain daf-2(e1370) (Fig. 3) rarely accumulated masses. BrdU was incorporated into the region of the masses in 11-day-old N2 nematodes, but not © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2007 No claim to original US government works 9-day-old N2 nematodes (Fig. 4), indicating that their generation requires active DNA synthesis that begins after 9 days of age. The acid treatment necessary for immunostaining with the anti-BrdU antibody interfered with DAPI staining of DNA, preventing costaining of the masses for BrdU and with DAPI. Dramatic changes in nuclear genome copy number occur with age in Caenorhabditis elegans We used quantitative real-time PCR to assay the copies of genomic DNA in individual nematodes. Consistent with the DAPI studies, in N2 worms we detected a dramatic increase in mean nuclear genome copy number at 10 –12 days of age (t-test 4 days vs. 14 days, P < 0.0001) (Fig. 5a), the same point in middle age where the DNA masses were visualized. Quantitation of three single-copy genes, each located on a different C. elegans autosome, gave highly correlated results (R2 = 0.96, see Sup- plementary Fig. S2), indicating that the copy number changes involve the entire genome, and not local amplification of specific loci. The number of genomes per N2 worm was 4228 (3660 ± 1441, 4241 ± 1366, and 4784 ± 1797 for chromosomes II, III and IV, respectively) at 4 days of age, in good agreement with approximately 4000 genomes predicted from what is known about nuclear content and ploidy of young adult worms (Sup- plementary Table S1). In wild-type worms, we also observed a dramatic increase in heterogeneity between individual nematodes with respect to nuclear genome copy number after 10 days of age (P = 0.008) (Fig. 5a). At 14 days of age, the range in genomic DNA copies is tenfold between individuals. We also quantified the same genes in the daf-2(e1370) mutant via qPCR (Fig. 5b). This long-lived strain appears to largely escape the increase in nuclear genome copy number observed in wild-type worms, although interesting age-related changes in nuclear genome copy number occur in this strain as well. We observed a striking heterogeneity between individual daf-2(e1370) nematodes throughout the lifespan; at ages less than 12 days, daf-2 nematodes of the same age are much more heterogeneous than N2 (two-sided P value from variance ratio test for all animals less than 10 days of age is 0). Interestingly, daf-2 nematodes experience an increase in nuclear genome copy number between days 4 and 6. This may reflect an increase in body size and associated endoreduplication, or may be due to an increased amount of embryos contained in the uterus. Mean genomic copy number then falls dramatically after the age of 20 days (t-test 6 days vs. 45 days, P < 0.0001). Similar changes were observed in the strain daf-2(e1368) (Supplementary Fig. S3). To verify that the accumulation of the DNA masses and increased genomic DNA copies required DNA synthesis, we grew worms in the presence of the DNA synthesis inhibitor 5-fluorodeoxyuridine (FUDR). These worms did not develop masses, and did not experience the mid-life increase in copy number (Fig. 5d). In fact, the removal of this massive mid-life proliferation of genomic DNA copies appears to have uncovered