Fig. 4. Programmed cell death in the outflow tract is enhanced in the E12.5 Rxra^–/– embryo. Serial sections from a representative wild-type (A,C,E) and Rxra^–/– (B,D,F) outflow tract showing TUNEL-positive nuclei and co-immunostained with MLC2a to demarcate the OFT myocardium. More cells in the mutant were apoptotic than in the wild type (arrowheads). (G) The relative numbers of cushion cells were counted from the adjacent histological sections and the data are expressed as the relative number of apoptotic cells as a percent of the total cells. Cells from the dextrodorsalconal cushion (DDCC, white bars) and sinistroventralconal cushion (SVCC, black bars) were counted separately. Both OFT cushions in the Rxra^–/– exhibited significantly more apoptosis than in the wild type. *P<0.05, compared with wild type DDCC. **P<0.01, compared with wild type SVCC. Error bars indicate the s.e.m. (H) Caspase activity assays confirmed the results obtained with the TUNEL technique in that mutant hearts demonstrated enhanced apoptosis. Results are expressed as -fold change relative to wild type. Three separate litters were assayed and -fold changes were calculated separately for each litter. Each litter had at least one wild-type and one mutant embryo. *P<0.05, compared with wild type (wt) and heterozygous (het) embryos. Error bars indicate the s.e.m.