For histologic analysis (Figure 4D and G), the upper body of embryos, including the heart, was separated from the lower body at the caudal end of the yolk sac extension using sharp forceps. The upper body was used for analysis, and the lower body was used for genotyping. The upper body of each embryo was transferred to an individual 1.5 ml tube containing 100 μl of 4% paraformaldehyde (PFA) and fixed at room temperature for 60 min, followed by rinsing with fix buffer (100 mM Na2HPO4 [pH 7.4], 4% sucrose, and 0.12 µM CaCl2). The samples were stored in fix buffer at 4°C until embryo genotyping was performed. The lower body was collected into a PCR tube containing 50 μl of DNA extraction buffer (10 mM Tris-Cl [pH 8.0], 2 mM EDTA, 0.2% NP-40, and 200 μg/ml proteinase K) and used for genomic DNA extraction. Genomic DNA was extracted from the collected tissues by incubating the sample tubes at 50°C for 60 min, followed by proteinase inactivation at 95°C for 5 min. One microliter of the DNA solution was used as a PCR template, and genotyping PCR was conducted using a PrimeSTAR 1 GXL kit (Clontech, Mountain View, CA).