180 Age-related molecular variability in Caenorhabditis elegans, T. R. Golden et al. molecular heterogeneity in wild-type and long-lived individual nematodes. First, via 4′,6′-diamidino-2-phenylindole hydro- chloride (DAPI) staining of DNA, we observed the systematic loss of nuclei from the tails of individual nematodes. This loss also occurs, but is delayed, in long-lived mutants. This approach also identified the accumulation of abnormal pathological masses of DNA in the uterus of wild-type nematodes with age. These masses are absent or much reduced in long-lived strains. Second, we detected incorporation of the nucleotide analogue 5-bromo- 2-deoxyuridine (BrdU) into the observed DNA masses, demon- strating active replication of DNA in these age-related masses that can expand to occupy the entire diameter of the worm. Third, we measured the copy number of the nuclear genome in individual nematodes of various ages by real-time quantitative polymerase chain reaction (qPCR). We identified a striking heterogeneity among middle-aged nematodes with respect to nuclear genome copy number, as well as a systematic loss of nuclear genome copy number with age in both wild-type and long-lived nematodes. This is the first demonstration of genomic and nuclear variability developing with age between genetically identical individuals. Results Nuclei are systematically lost with age in wild-type and long-lived Caenorhabditis elegans We observed nuclei in aging C. elegans by staining with the DNA-binding dye DAPI. The nematode’s optical transparency allows the visualization of every nuclei in the intact worm. Two dramatic age-related phenomena were revealed by DAPI staining. First, DAPI staining of three nuclei in the tail is lost with age (Fig. 1a,b). These likely are the nuclei of the H9 and H10 hypodermal cells (H10 being binucleate), based on comparison Fig. 1 Loss of hypodermal nuclei with age from the tails of N2 and daf-2 nematodes. (a) Tails of N2 nematodes, stained with DAPI. Worms were picked from a synchronized population initiated with a timed lay. The date of the lay is counted as day 0. Images were examined and scored as to how many nuclei were visible in the tail. In the majority of aged nematodes, the nuclei corresponding to hypodermal nuclei H9 and H10 are lost [(arrowheads, quantified in (b) and (c)]. Although not quantified, we noted the appearance of nuclei with a fragmented or condensed appearance with age (arrows, bottom panel) (top: 4 days old, middle: 14 days old, bottom: 19 days old). (b) Count of nuclei visible in the tails of individual N2 nematodes. (c) Count of nuclei visible in the tails of individual daf-2(e1368) nematodes. Ten individuals were counted per group, except 19-day-old N2 for which nine were counted. © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2007 No claim to original US government works