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NCCs deficient in Alk5 can populate the outflow tract\nNext we used the R26R lineage-tracing assay to determine whether CNCCs could appropriately populate the outflow tract region. Briefly, Alk5Flox/Flox mice were crossed with the ROSA26 Cre reporter mice, and subsequently Alk5Flox/Flox;R26R(+/+) females were crossed with Alk5KO/WT;Wnt1-Cre males. The resulting embryos had the NC-lineage permanently labeled with β-galactosidase expression, and displayed identical phenotypes to those obtained without the R26R reporter. Staining of embryos for β-galactosidase at E8-E11 did not reveal detectable differences in NCC migration between mutants and controls (data not shown). Similarly, serial transverse sectioning of whole mount embryos (E10-E12) and subsequent analysis of positively stained cells in the OFT region demonstrated that CNCCs deficient in Alk5 were capable of populating the PAAs, aortic sac and conotruncal ridges at a level comparable to that of controls (Fig 4). To conclude, the observed phenotypes in Alk5/Wnt1-Cre mutants were certainly not due to defective migration of CNCCs to the pharyngeal and outflow tract regions.\nFigure 4 Normal cardiac NCC migration in Alk5/Wnt1-Cre mutants. The OFT of controls (A, C, E) and Alk5/Wnt1-Cre mutants (B, D, F) display similar staining patterns when analyzed using the R26R lineage tracing assay at E11.0. A-B, whole mount staining (left lateral image); C-F, transverse sections on the level of the 4th (C, D) and 6th (E, F) PAAs. Arrows (A-F) point to the most proximal location staining positive for the β-galactosidase activity."}

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Briefly, Alk5Flox/Flox mice were crossed with the ROSA26 Cre reporter mice, and subsequently Alk5Flox/Flox;R26R(+/+) females were crossed with Alk5KO/WT;Wnt1-Cre males. The resulting embryos had the NC-lineage permanently labeled with β-galactosidase expression, and displayed identical phenotypes to those obtained without the R26R reporter. Staining of embryos for β-galactosidase at E8-E11 did not reveal detectable differences in NCC migration between mutants and controls (data not shown). Similarly, serial transverse sectioning of whole mount embryos (E10-E12) and subsequent analysis of positively stained cells in the OFT region demonstrated that CNCCs deficient in Alk5 were capable of populating the PAAs, aortic sac and conotruncal ridges at a level comparable to that of controls (Fig 4). To conclude, the observed phenotypes in Alk5/Wnt1-Cre mutants were certainly not due to defective migration of CNCCs to the pharyngeal and outflow tract regions.\nFigure 4 Normal cardiac NCC migration in Alk5/Wnt1-Cre mutants. The OFT of controls (A, C, E) and Alk5/Wnt1-Cre mutants (B, D, F) display similar staining patterns when analyzed using the R26R lineage tracing assay at E11.0. A-B, whole mount staining (left lateral image); C-F, transverse sections on the level of the 4th (C, D) and 6th (E, F) PAAs. Arrows (A-F) point to the most proximal location staining positive for the β-galactosidase activity."}

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