For semi-qRT-PCR analysis (Figure 4B,F,H), the embryos were fixed with RNAlater at room temperature for 10 min, and the heart was dissected using sharp forceps. Each heart was transferred to an individual 1.5-ml tube containing 100 μl of RNAlater, and the samples were stored at −80°C until embryo genotyping was performed. The remaining body was collected into a PCR tube containing 50 μl of DNA extraction buffer and used for PCR genotyping as described above. After genotyping, 15 to 20 pooled hearts of the same genotype were transferred to an individual 1.5-ml tube containing 1 ml of TRIzol and used for semi-qRT-PCR analysis as described subsequently (RT-PCR).