Fig. 1.

Apoptotic features of embryonic cells depleted of dUTPase. (A) Electron microscopic (EM) image of control cells, which show normal nuclear structure with dispersed and sparse heterochromatic areas. The embryos were fed with bacteria expressing an empty RNAi vector under inducing conditions. (B) The heterochromatic area shows prominent apoptotic morphology (arrows) in two cells of a dut-1(RNAi) (dUTPase deficient) embryo. Scale bars: 1 μm. (C) Depletion of DUT-1 causes an almost twofold increase in the number of apoptotic cell corpses in embryos. This type of cell death occurs in a CED-3- and CED-4-dependent, but LGG-1-independent manner. Red lines indicate the mean number of cell corpses. The lgg-1-deficient mutant strain is actually of lgg-1(tm3489); Ex[LGG-1::GFP] genotype. The translational fusion LGG-1 reporter is able to rescue viability in lgg-1(−) mutant larvae, but as a result of instability of the extrachromosomal array (Ex), the strain behaves as a genetic mosaic. (D) TUNEL staining of wild-type, nuc-1 mutant (positive control) and dut-1(RNAi) embryos. White dots indicate cells with fragmented DNA. The diagram shows the number of TUNEL-positive cells. Bars are mean ± s.e.m. For dut-1(RNAi) embryos, Student's t-test gives P<0.0001 compared with the control. (E) Depletion of DUT-1 is not accompanied with the activation of CEP-1/p53. Quantitative RT-PCR assay showing a weak induction of two CEP-1 targets, egl-1 and ced-13, in wild-type background (grey bars), clk-2(mn159) mutants (black bars) and ung-1(qa7600) mutants (red bars), following treatment with dut-1 RNAi. This contrasts with the strong induction following treatment with ENU (N-Ethyl-N-nitrosourea). clk-2 encodes a telomere-length-regulating protein that is required for the DNA-damage and S-phase-replication checkpoints (Ahmed et al., 2001), ung-1 encodes a uracil DNA glycosylase, which removes misincorporated uracil from the DNA (Dengg et al., 2006).