Fig. 8. Heterozygosity for Tgfb2 partially restores the outflow tract and apoptotic phenotypes in the Rxra–/–. (A) RT-PCR demonstrating the intermediate expression of TGFß2 message in the E12.5 Tgfb2+/– (HT) embryo when compared with wild type (WT) and knockout (KO) Tgfb2. Numbers represent the -fold change compared with the wild-type sample in the first lane and are normalized to the levels of GAPDH as described in Materials and Methods. Expression levels of TGFß2 in the Tgfb2+/– were intermediate between wild type and knockout. (B) OFT histology of E12.5 wild-type, Rxra–/–/Tgfb2+/+ and Rxra–/–/Tgfb2+/– embryos. The typical malformed AoP septum (aps) is evident in the Rxra–/–/Tgfb2+/+ (arrow) as well as the irregular and hypoplastic endocardial cushions. In the Rxra–/–/Tgfb2+/– embryo, the AoP septum has migrated far enough into the OFT that there is a distinct separation between the aortic outlet and the pulmonary valve primordia (arrow). (C) OFT histology of E13.5 wild-type, Rxra–/–/Tgfb2+/+ and Rxra–/–/Tgfb2+/– embryos. In Rxra–/–/Tgfb2+/– embryos, the AoP septum has descended into the OFT and fused with the SVCC, separating the aortic outlet from the semilunar valve primordia (compare arrows). (D) Quantitation of apoptosis in E12.5 and E13.5 embryos from Rxra/Tgfb2 intercrossed mice. Apoptosis was quantitated as described in Materials and Methods. For E12.5: white bars, dextrodorsalconal cushion; black bars, sinistroventralconal cushion. For E13.5, the single bar represents the total apoptotic nuclei in both cushions as the cushions are fused in the wild type at this age. Rxra wild type (R-WT), Rxra–/– (R-KO), Tgfb2 wild type (T-WT), Tgfb2 heterozygous (T-HT). The levels of apoptosis in both the E12.5 and E13.5 Rxra–/–/Tgfb2+/– embryos are intermediate between the wild-type and Rxra–/–/Tgfb2+/+ embryos. In particular, note that the relative number of apoptotic nuclei in the Rxra–/–/Tgfb2+/– is no longer significantly different from that in Rxra–/–/Tgfb2+/+. Five different litters are represented in each of the two age groups. N, the number of embryos within each genotype combination.
