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Elevated transforming growth factor ß2 enhances apoptosis and contributes to abnormal outflow tract and aortic sac development in retinoic X receptor knockout embryos

Department of Cell Biology and Anatomy, Cardiovascular Developmental Biology Center, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425, USA

View larger version (130K): [in a new window]   Fig. 1. Developmental abnormalities in the Rxra–/– embryo are evident at E11.5 in the aortic sac and at E12.5 in outflow tract cushion tissue. Embryos at various times during embryogenesis were analyzed for morphological malformations of the heart. Transverse sections of E10.5 wild-type (A) and Rxra–/– (B) hearts show that the early stages of epithelial-to-mesenchymal cell transformation in outflow tract cushion tissue occurs in a similar manner between wild type and mutant. Arrows indicate mesenchymal cells that have migrated into the cardiac jelly. (C-H) Transverse sections of E11.5 embryonic hearts. Note throughout the outflow tract (OFT), the extent of mesenchymal seeding in the cushions is similar in the wild type (C,E,G) and Rxra–/– (D,F,H). However, at this age, the aorticopulmonary (AoP) septum in the mutant shows signs of abnormal development, such as incomplete fusion with the sinistroventralconal cushion (F, arrow). (I-L) Transverse sections of E12.5 embryonic hearts. The OFT cushions have begun to fuse in the wild type (K, arrow) while they have become noticeably hypoplastic and malformed in the Rxra–/–. Further evidence of an eventual AoP window is also observed in the Rxra–/– (L, arrow). dd, dextrodorsalconal cushion; sv, sinistroventralconal cushion. (M,N) Quantitation of the relative number of cells was performed by counting nuclei in serial sections 40 µm apart beginning at the most cranial aspects of the OFT cushions and continuing down to the most caudal aspects of the cushions (between broken lines, M). (N) Cells from the dextrodorsalconal cushion (DDCC in M; white bars) and sinistroventralconal cushion (SVCC in M; black bars) were counted separately. Both OFT cushions were significantly smaller in the Rxra–/–. *P<0.05. Error bars represent the s.e.m.

View larger version (153K): [in a new window]   Fig. 2. MLC2v and MLC2a are expressed in a normal manner and mark the developing outflow tract in both wild-type and Rxra–/– embryos. Adjacent transverse sections of E13.5 wild-type (A-C) and Rxra–/– (D-F) embryonic hearts. Both MLC2v (B,E) and MLC2a (C,F) are expressed in their respective chambers in both wild-type and mutant embryos. Note that MLC2a is also expressed in a subset of OFT myocardium (C,F, arrowheads) and that this expression pattern is identical in both wild-type and mutant embryos.

View larger version (109K): [in a new window]   Fig. 3. Delineation of outflow tract proliferation, apoptosis and myocardialization in E13.5 wild-type and Rxra–/– embryos. (A,B) Transverse sections of the outflow tracts of E13.5 wild-type and Rxra–/– embryonic hearts, respectively. Broken line depicts the boundary between endocardial cushion tissue (ec) and myocardializing myocytes. ao, aortic outlet. pa, pulmonary outlet. (C) Adjacent section to A immunostained for MLC2v (red) and BrdU (green). Note less cells are BrdU-positive in the myocardial cuffs (asterisks) and endocardial cushion tissue compared with the ventricular myocardium. Myocardializing myocytes are readily identified (arrowheads) and were more easily visualized at higher magnification (G, arrows). (D) Adjacent section to B immunostained for MLC2a (red) and BrdU (green). Similar to wild-type hearts, less cells are BrdU-positive in the myocardial cuffs (asterisks) and endocardial cushion tissue compared with the ventricular myocardium in the Rxra–/–. (E) Adjacent section to C immunostained for MLC2a and apoptotic cells using the TUNEL assay. Apoptosis was largely confined to the endocardial cushion tissue between the myocardial cuffs (asterisks) in the outflow tract. (F) Adjacent section to D immunostained for MLC2v and apoptotic cells using the TUNEL assay. Apoptosis was again largely confined to the endocardial cushion cells between the myocardial cuffs (asterisks). Myocardializing myocytes were also identifiable in the Rxra–/– (arrowheads) and were more easily visualized at higher magnification (H, arrows).

View larger version (108K): [in a new window]   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.

View larger version (85K): [in a new window]   Fig. 5. TGFß2 mRNA and protein expression is elevated in the Rxra–/– embryo. (A) RNase protection assays showed that message levels for TGFß2 were elevated in the mutant (KO). WT, wild type. (B) Message levels for TGFß1 were not altered while MLC2a was elevated in the mutant. EF1 was used as a loading control. (C) Immunohistochemical analysis on transverse serial sections of E13.5 OFT regions demonstrated that TGFß2 protein was also elevated in the mutant. (D) E12.5 OFT also shows elevated TGFß2 in the mutant heart.

View larger version (19K): [in a new window]   Fig. 6. TGFß2 treatment enhances outflow tract apoptosis in whole mouse embryo culture. Embryonic day 11.5 wild-type embryos were treated with 0.1-30 ng/ml TGFß2 protein and placed in whole embryo culture for 24 hours, then processed as described in Materials and Methods. Wild-type (Control) or mutant (Rxra–/–) E11.5 embryos were also cultured for 24 hours as comparative controls. TGFß2 treatment enhanced outflow tract apoptosis in a concentration-dependent manner. Apoptosis was quantitated using the TUNEL assay as described in Fig. 5. Dextrodorsalconal cushion (white bars). Sinistroventralconal cushion (black bars). *P<0.05, compared with control DDCC. **P<0.05, compared with the respective DDCC and SVCC Control and 0.1 ng/ml TGFß2. Error bars indicate s.e.m.

View larger version (141K): [in a new window]   Fig. 7. TGFß2-treated whole mouse embryos in culture reveal outflow tract malformations that are similar to Rxra–/– embryos. Each group of four images represents serial sections 40 µm apart through the OFT of a representative embryo. (A-D) OFT of a wild-type embryo dissected at E12.5. The orifices of the aortic and pulmonary outlets are well separated. (E-H) OFT of a wild-type embryo dissected at E11.5 and cultured for 24 hours. Similar to the E12.5 wild type, the orifices of the aortic and pulmonary outlets are well separated. (I-L)) OFT of an Rxra–/– embryo dissected at E11.5 and cultured for 24 hours. Rxra–/– embryos in culture retained the malformed outflow tract phenotype. Thus, the AoP septum has not descended into the OFT to the same extent as in the wild type and, as a result, the beginning of an AoP septum defect is apparent (I, arrow). Note the cushions are also underdeveloped and malformed. (M-P) OFT of a wild-type embryo dissected at E11.5 and cultured for 24 hours in the presence of 10 ng/ml TGFß2. The phenotype is similar to the Rxra–/– in that the AoP septum has not formed properly (M, arrow) and the cushions are underdeveloped and malformed. ao, aortic outlet. p, pulmonary outlet.

View larger version (71K): [in a new window]   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.