Detection of caspase activation and apoptosis in living embryos of SCAT3 transgenic mice. (A) SCAT3 transgenic mice. (i) Expression cassette for the SCAT3 transgene. A chicken HS4 insulator (2×) was used to stabilize the transgene expression driven by CAG promoter. (ii) SCAT3 fluorescence (Venus and ECFP) in E9.5 transgenic embryos. (B) Detection of caspase activation and apoptosis in living mouse embryos. Caspase activation is represented as the pseudocolors that correspond to V/C (1.0–0.5). Embryos dissected from the uterus at E8.75 were observed by live imaging for 8 h from the dorsal view (hindbrain). In the otic placode (insets) of cultured embryos, both the appearance of cell debris (i.e., dying cells, circled by white lines in the ECFP images; 4–8 h) and the V/C decline (blue in the V/C images) in those cells were completely suppressed by the genetic ablation of apaf-1 or a chemical inhibitor of caspases (200 µM zVAD). wt, wild type. (C) Dependence of SCAT3 cleavage on the intrinsic apoptotic pathway in E8.75 embryos analyzed by Western blotting (WB) using an anti-GFP antibody. The two fragments of cleaved SCAT3 (black arrows) were diminished in SCAT3 transgenic embryos (shown as SCAT3 Tg+) cultured with 200 µM zVAD for 6 h or in apaf-1 (−/−) embryos compared with control littermates (+/+ or +/−). The red arrow indicates full-length SCAT3. Molecular mass is indicated in kilodaltons. (D, left) Detection of caspase activation (decline of V/C) in the MHNP ridge. In control apaf-1^+/− embryos, cells in the MHNP ridge exhibited caspase activation and subsequently became detached (shown by arrowheads). In embryos deficient for apaf-1 (apaf-1^−/−) or treated with caspase inhibitor (apaf-1^+/+; 200 µM zVAD), detaching cells appeared but showed no sign of caspase activation. (right) Time course of caspase activation during imaging. I, intensity. Normalized V/Cs (moving average among three serial time points) are shown by green lines. Bars: (A) 1 mm; (B) 100 µm; (D) 10 µm.