Elevated transforming growth factor β2 enhances apoptosis and contributes to abnormal outflow tract and aortic sac development in retinoic X receptor α knockout embryos

Septation of the single tubular embryonic outflow tract into two outlet segments in the heart requires the precise integration of proliferation, differentiation and apoptosis during remodeling. Lack of proper coordination between these processes would result in a variety of congenital cardiac defects such as those seen in the retinoid X receptor α knockout (Rxra^–/–) mouse. Rxra^–/– embryos exhibit lethality between embryonic day (E) 13.5 and 15.5 and harbor a variety of conotruncal and aortic sac defects making it an excellent system to investigate the molecular and morphogenic causes of these cardiac malformations. At E12.5, before the embryonic lethality, we found no qualitative difference between wild type and Rxra^–/– proliferation (BrdU incorporation) in outflow tract cushion tissue but a significant increase in apoptosis as assessed by both TUNEL labeling in paraffin sections and caspase activity in trypsin-dispersed hearts. Additionally, E12.5 embryos demonstrated elevated levels of transforming growth factor β2 (TGFβ2) protein in multiple cell lineages in the heart. Using a whole-mouse-embryo culture system, wild-type E11.5 embryos treated with TGFβ2 protein for 24 hours displayed enhanced apoptosis in both the sinistroventralconal cushion and dextrodorsalconal cushion in a manner analogous to that observed in the Rxra^–/–. TGFβ2 protein treatment also led to malformations in both the outflow tract and aortic sac. Importantly, Rxra^–/– embryos that were heterozygous for a null mutation in the Tgfb2 allele exhibited a partial restoration of the elevated apoptosis and of the malformations. This was evident at both E12.5 and E13.5. The data suggests that elevated levels of TGFβ2 can (1) contribute to abnormal outflow tract morphogenesis by enhancing apoptosis in the endocardial cushions and (2) promote aortic sac malformations by interfering with the normal development of the aorticopulmonary septum.