Septation of the outflow tract lumen begins in a cranial-to-caudal direction, starting distally in the aortic sac and proceeding toward the heart [24]. Initially, the condensed mesenchyme derived from the NC forms in the base of the aortic sac between the origins of 4th and 6th PAAs. Subsequently, two prongs of the developing aortico-pulmonary (AP) septum extend into the truncal cushions and the aortico-pulmonary septation complex crosses the aortic sac cranially. In ink-injected control embryos at E11.5, a characteristic conotruncal transition separating the truncus and conus could be seen as a twisted configuration, resulting from a change in orientation of the truncal and conal cushions (Fig. 5). In contrast, in Alk5/Wnt1-Cre mutants the outflow tract appeared unusually straight, failing to demonstrate the distinct conotruncal transition (Fig. 5B,D). This assay also clearly showed a dramatic reduction in the size of the aortic sac. Histological analysis of control samples displayed the characteristic rotation of the aortic sac and truncal OFT at the level where the AP septation takes place and verified the presence of the distinctive condensed AP-septal mesenchyme, which gradually divided the OFT to the aorta and the pulmonary trunk (asterisks in Fig. 6A). R26R lineage tracing showed that this tissue is derived from the NC, while immunostaining for α-SMA showed differentiation into smooth muscle (Fig 6B). In Alk5/Wnt1-Cre mutants the characteristic rotation of the aortic sac and truncal OFT fails to take place (Fig. 6G–L), and a properly formed AP-septum was not detectable (Fig. 6G,H). R26R lineage tracing demonstrated that the defects were not due to failure of NCCs to reach the OFT region. NC-derived cells around the abnomally bifurcated aortic sac, the abnormally large sixth PAAs and the truncus demonstrated strong αSMA staining (Fig. 6H,J,L). Recently, we showed that the NC-specific mutants of the related type I receptor, Alk2, display PTA as well [12]. In Alk2/Wnt1-Cre mutants, the rotation of the aortic sac and truncal OFT failed to occur (Fig. 6M–R) as seen in Alk5/Wnt1-Cre mutants. However, in Alk2 mutants the 6th pair of the PAAs was grossly hypoplastic, and while the Alk2/Wnt1-Cre mutants displayed a noticeable amount of septal tissue between the 4th and 6th PAAs (Fig. 6M,N), the condensed septal mesenchyme lacking Alk2 failed to extend the prongs into the truncal cushions and to form the AP septum. Concurrently, the 6th PAAs were losing their patency, which may have further contributed to the failed AP septation (Fig. 6M,O,Q). While CNCCs managed to migrate to the aortic sac and the truncal cushion level (Fig. 6N,P,R), immunostaining for αSMA appeared much weaker when compared to controls and Alk5 mutants, implying that ALK2-mediated signaling is involved in smooth muscle cell differentiation as previously suggested [12]. To conclude, while both Alk2 and Alk5 mutants demonstrate a failure in both the rotation of the aortic sac and the truncal OFT, and in the formation of the AP septum, the pathogenetic mechanisms behind these defects appear remarkably different.