FIGURE 5.

Apoptosis signaling pathway components are highly activated in stac mutants. A–C, quantification of apoptosis and cell cycle regulator expression levels by real time RT-PCR at different stages. The tested genes were selected from cDNA microarray assay. D, bioluminescent assay of caspase 8, 9, and 3/7 activities. Twenty stac and wild type (WT) sibling embryos at 30 hpf were treated with corresponding Caspase-Glo reagents (Promega), and luminescence was measured 2 and 3 h after treatment. The p value was 0.0847, 0.0273, or 0.0014 for 2-h treated caspase 8, 9, or 3/7 activity, respectively, and 0.0445, 0.0165, or 0.0004 for 3-h treatments. Significance of differences is as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001 (by Student's t test). Error bars indicate S.D. E, immunostaining of activated caspase 3 using an antibody against active caspase 3. F, excessive apoptosis in stac mutant embryos was inhibited by injection of p53 morpholino (p53-MO). One-cell embryos derived from stac heterozygote intercrosses were injected. The mutants were sorted based on GFP expression level around 24 hpf and were observed directly by bright field microscopy or observed by fluorescent microscopy after staining for apoptotic cells by TUNEL assay at indicated stages.