Role of ceramide in CEP-1/p53–mediated germ cell apoptosis. (A) hyl-1 and lagr-1 are epistatic to abl-1 in germ cell apoptosis. Germ cell apoptosis was scored in abl-1(ok171), hyl-1(ok976);abl-1(ok171), lagr-1(gk327);abl-1(ok171), and lagr-1(gk327);hyl-1(ok976);abl-1(ok171) at 36 hours (left) and in lagr-1(gk327);hyl-1(ok976);abl-1(ok171) at the indicated times after exposure to 120 Gy (right). Data (mean ± SEM, represented by error bars) are collated from 10 to 15 worms per group. (B) hyl-1 and lagr-1 deletions do not affect 120-Gy–induced p53-mediated egl-1 (left) and ced-13 (right) up-regulation measured by reverse transcription polymerase chain reaction. Data (mean ± SEM) are compiled from three experiments. (C) Baseline EGL-1 is required for C₁₆-ceramide (∼1 μM gonadal ceramide)–induced germ cell apoptosis. Studies were performed as in Fig. 1B. Data (mean ± SEM) are collated from ≥15 worms per group.

lf hyl-1 and lagr-1 prevent radiation-induced germ cell apoptosis, reversible by C₁₆-ceramide. WT and mutant worms were synchronized at 20°C and irradiated (A, B, and E) or injected with C₁₆-ceramide into the posterior gonad (C to E) at 24 hours after the L4 stage. The posterior gonad distal arm was scored for cell corpses under Nomarski optics. Time dependence of germ cell corpse induction in hyl-1(ok976) (left) and lagr-1(gk327) (right) (A) and in lagr-1(gk327);hyl-1(ok976) (B) after 120 Gy is shown. WT data are identical in (A), left and right; these panels were separated for clarity. C₁₆-ceramide microinjection induces time- (left) and dose-dependent (right, at 36 hours) germ cell apoptosis in WT worms (C) and dose-dependent apoptosis in lagr-1(gk327);hyl-1(ok976) at 36 hours (D). Gonadal ceramide concentration was calculated as described in the SOM. (E) Sublethal C₁₆-ceramide microinjection restores radiation (120 Gy)-induced germ cell apoptosis to lagr-1(gk327);hyl-1(ok976). Data (mean ± SEM, represented by error bars) are collated from ≥15 worms per group in (A) to (E).

Mitochondrial ceramide mediates CED-4 displacement. (A) Gonads were dissected from young adult abl-1(ok171) and lagr-1(gk327);hyl-1(ok976);abl-1(ok171) at 24 hours after 120 Gy and stained with anti–COX-IV antibody (green), anti-ceramide antibody (red), and 4′,6′-diamidino-2-phenylindole (blue). (B and C) Germ cells were released from gonads of young adult abl-1(ok171) and lagr-1(gk327);hyl-1(ok976);abl-1(ok171) at 24 hours after 120 Gy and stained with anti–Ce-lamin (red) and anti–CED-4 (green). CED-4/lamin intensity in individual germ cell nuclei (circles) was measured using Metamorph software. Horizontal bars indicate means from ≥50 nuclei per group. In lagr-1(gk327);hyl-1(ok976);abl-1(ok171), the baseline CED-4/lamin ratio is reduced by 63%, and the post-radiation fold and absolute change are reduced by 40% and 78%, respectively, as compared with abl-1(ok171) animals. (D) L1 larvae of opls219, cultured in Rhodamine B–containing plates until the young adult stage, were exposed to 120 Gy, and GFP (CED-4) and Rhodamine B (mitochondria) signals were imaged at 36 hours post-irradiation. Images represent single confocal planes from the distal gonad of opls219 (upper panels). Boxed insets (lower panels) were enlarged 1.75 times to ease the observation of colocalized CED-4/Rhodamine B mitochondrial yellow signal (top right bottom panel) pre-irradiation and green nuclear CED-4 platformlike structures post-irradiation (white arrows in bottom left lower panel).