Analysis of the proepicardium-epicardium transition during the malformation of the RXRalpha-/- epicardium.

The epicardium of the heart originates from a cluster of mesothelial-derived cells that develop beneath the sinus venosus in the embryonic day (E) 9.0-9.5 mouse. The subsequent proepicardium-epicardium transition that forms the epicardial layer of epithelial cells covering the myocardial surface is nearly complete by E10.0-E10.5 and results in a fully covered heart by E11.0. In this study, we show that an established model of congenital heart disease, the retinoid X receptor alpha knockout (RXRalpha-/-) embryo, displays a malformed epicardium. At E10.0-E10.5, the RXRalpha-/- has several large regions of myocardium that remain bare. Furthermore, by E11.5-E12.5, when a complete epithelial layer is formed in the mutant, large regions of the epicardium become distended from the underlying myocardium. Close examination of the E9.5 mutant revealed an elevated apoptosis level within the proepicardial cluster of mesothelial cells. Additionally, among the extracellular matrix proteins analyzed, expression of fibronectin was elevated in the RXRalpha-/- as assessed by immunostaining in paraffin-embedded sections and proepicardial explants. We propose that these events contribute to a developmental delay in the formation of the epicardium, which leads to an abnormal epicardium and ultimately contributes to the cardiac malformations seen in the RXRalpha-/-.

Elevated transforming growth factor beta2 enhances apoptosis and contributes to abnormal outflow tract and aortic sac development in retinoic X receptor alpha 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 alpha 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 beta2 (TGFbeta2) protein in multiple cell lineages in the heart. Using a whole-mouse-embryo culture system, wild-type E11.5 embryos treated with TGFbeta2 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(-/-). TGFbeta2 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 TGFbeta2 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.