Fig. 3

IR-induced p53-Independent Apoptosis after Chk1 Loss Occurs Cell Autonomously and Independently of Caspase-3

(A) Fluorescent images of 25 hpf embryos (anterior, left). TUNEL reactivity after IR (0 or 12.5 Gy) recapitulates live AO labeling (see Figure 2A).

(B) Embryos from the same experiment immunostained with an antiactivated-Caspase-3 antibody. Note the absence of immunoreactivity in the irradiated p53^e7/e7;chk1^MO embryo.

(C) Electron micrographs (sagittal sections) of the CNS in embryos of indicated genotypes after 0 or 12.5 Gy IR. Gö6976 is a specific Chk1 inhibitor (see Figure 5, Figure 6 and Figure 7). Lower row, 2.5x closeups on the areas boxed in yellow in upper panels. Note multiple cells with stereotypical chromatin compaction/segregation in columns 2 and 4, as opposed to healthy nuclei in columns 1 and 3. Organelles and plasma membrane are intact in the shown Chk1-inhibited irradiated p53 mutant cell, as expected from an apoptotic (as opposed to necrotic) event. See Figure S2 for more details. Scale bar, 2 μM.

(D) Experimental procedure for the generation of the genetic chimeras shown in (E).

(E) 5-μm-thick confocal sections of spinal cords in irradiated chimeras. TMR Dextran (red) marks the donor cells. TUNEL shown in green. First row, cells from a p53^e7/e7 embryo that was injected with the chk1 MO at the 1-cell stage (p53^e7/e7;chk1^MO embryo) transplanted into a p53^e7/e7 host. Second row, p53^e7/e7 cells transplanted into a p53^e7/e7;chk1^MO host.