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First published online 28 April 2004
doi: 10.1242/dev.01136

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A Werner syndrome protein homolog affects C. elegans development, growth rate, life span and sensitivity to DNA damage by acting at a DNA damage checkpoint

Department of Biochemistry, College of Science, Yonsei University, Seoul 120-749, Korea

View larger version (9K): [in a new window]   Fig. 1. Comparison of C. elegans WRN-1 with human Werner syndrome protein. WRN-1 shares 43% identity in amino acid sequence in the helicase motif and 25% identity in the RQC-HRDC region with human WRN. RQC, RecQ conserved domain; HRDC, helicase RNase D C-terminal conserved domain; NLS, nuclear localization signal.

View larger version (55K): [in a new window]   Fig. 2. Immunolocalization of WRN-1 in C. elegans at various developmental stages. C. elegans embryos or worms were reacted with a mouse antiserum against WRN-1 (green) and then with an FITC-conjugated secondary antibody, followed by nuclear staining with DAPI (blue). (A) C. elegans embryos. Mitotic prophase nuclei, two nuclei in the 6-cell embryo and one nucleus in the 8-cell embryo, are marked with arrowheads; a metaphase nucleus in the 8-cell embryo is marked with an arrow. (B) L1 larval and adult stage worms. (C) Intestine and gonad from an L4 stage larva. (D) An oocyte and an embryonic metaphase cell. Scale bars: A, 10 µm; B, 50 µm; C, 50 µm; D, 5 µm for the oocyte and 1 µm for the metaphase cell.

View larger version (12K): [in a new window]   Fig. 3. Life span of F1 progeny produced by P0 C. elegans worms microinjected with double-stranded RNA of wrn-1. The life span of wrn-1(RNAi) worms was reduced by 2.6 (P<0.001) days compared with wild-type N2 strain at 25°C. wrn(RNAi), 11.0±0.2 (s.e.m.) days; N2(control), 13.6±0.1 days. Over 100 F1 worms were used per single data point, and each experiment was repeated three times.

View larger version (44K): [in a new window]   Fig. 4. Premature aging of wrn-1(RNAi) worms. Wild-type N2 and wrn-1(RNAi) worms at 1 to 7 days of adulthood were photographed on the same day under identical conditions. wrn-1(RNAi) worms were prepared by feeding on E. coli cells producing dsRNA of wrn-1, and growth temperature was shifted from 20°C to 25°C at the L4 stage. (A) Accumulation of lipofuscin autofluorescence. Ten worms of each strain were photographed every day, and one worm with an averaged intensity of autofluorescence is shown. (B) C. elegans heads were photographed with a Nomarski optics microscope. Photographs of heads were given a score of 1-5, with 1 representing a youthful appearance and 2-5 denoting increasing orders of overall deterioration in the tissue. (C) Scatter diagram of the values assigned to tissue deterioration in the head. Each dot corresponds to a single animal. Scale bars: A, 500 µm; B, 50 µm.

View larger version (26K): [in a new window]   Fig. 5. Developmental abnormalities of wrn-1(RNAi) worms and their penetrance influenced by growth temperature and ionizing radiation. (A) Morphological phenotypes of F1 progeny produced by P0 C. elegans worms microinjected with dsRNA of wrn-1. Small body, shorter than 70% of the wild-type body length and thin; dumpy, short and fat; ruptured body, gonad and intestine bursting out of the worm; bag of worms, hatched worms inside the adult worm due to a blockage of egg-laying; growth arrest, at various larval stages. Scale bars: 100 µm. (B-D) Frequency of wrn-1(RNAi) phenotypes. Wild-type N2 and F1 wrn-1(RNAi) worms were grown at (B) 20°C and (C) 25°C, and phenotypes were scored up to the 8-day adult stage. (D) Wild-type N2 and F1 wrn-1(RNAi) worms were -irradiated at 60 Gy and 20°C and their phenotypes scored after 3 days. Over 500 F1 worms were used in each measurement for B and C, and over 200 worms for D. Each experiment was repeated three times and standard errors (s.e.m.) are shown by error bars. Note that the % phenotype scale in D is different from that in B and C.

View larger version (19K): [in a new window]   Fig. 6. The rapid growth to L4 stage of wrn-1(RNAi) worms is unaffected by -irradiation. Wild-type N2 worms were microinjected with dsDNA derived from Ce-wrn-1 cDNA. F1 progeny were irradiated with different dose (0, 10, 20 Gy) of -rays at the L1 larval stage and their growth to L4 stage was measured every twelve hours at 20°C. Over 150 F1 worms were used per measurement with two additional repetitions, and standard errors (s.e.m.) are shown by error bars.

View larger version (83K): [in a new window]   Fig. 7. Reduction of S-phase duration by wrn-1(RNAi) during embryogenesis. (A) Accelerated cell division of a wrn-1(RNAi) embryo observed with Nomarski optics at 25°C. (B) Time-lapse DIC microscopy of 2- to 4-cell embryos. At 0 minutes, AB (right) and P1 (left) cells of a wild-type N2 embryo immediately after cytokinesis are shown. Nuclear envelope breakdown (NEBD) of AB and P1 cells occurs at 12 and 14 minutes, respectively. (C) Average duration of S phase in AB and P1 determined by timing cytokinesis and NEBD. The bar graphs correspond to the time separating cytokinesis of P0 from NEBD of either the AB or the P1 cell. Standard errors (s.e.m.) are shown by error bars. Ten embryos were observed for each estimate of S-phase duration. Scale bars: A, 20 µm; B, 10 µm.

View larger version (27K): [in a new window]   Fig. 8. Morphological changes of pre-meiotic germ cell nuclei induced by hydroxyurea. Images are DAPI-stained nuclei in untreated (–HU) gonads and in those exposed to hydroxyurea (+HU). Upon HU treatment, pre-meiotic nuclei in the wild-type N2 gonad were substantially enlarged and reduced in number, but those in the wrn-1(RNAi) gonad were much smaller. Scale bar: 50 µm.

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