Fig. 4.

Aberrant neural crest and SHF lineage distribution in Nos3^−/− embryos. (A) Cell lineage analysis in Nos3^−/− embryos showed that significantly fewer neural-crest-derived cells contribute to the right coronary (RC) leaflet in Nos3^−/− than in wild-type embryos at E12.5 (*P<0.05). (B) Increased contribution of NKX2.5⁺/TM⁻ SHF cells was observed in the left coronary (LC) leaflet in Nos3^−/− embryos as compared to age-matched wild-type embryos (*P<0.05). (C) The NC leaflet of Nos3^−/− contains a significantly larger NKX2.5⁺/TM⁻ SHF population than those found in wild-type embryos at E12.5 (P<0.05). (D-F) Immunofluorescent images of Wnt1-Cre;mTmG tricuspid aortic valve (TAV) wild-type (D), Nos3^−/− TAV (E) and bicuspid aortic valve (BAV) Nos3^−/− (F) embryos at E12.5 showed diminished neural crest populations (green) in the leaflets of the aortic valve in Nos3^−/− embryos. (G-I) Immunofluorescent images of wild-type TAV (G), Nos3^−/− TAV (H) and Nos3^−/− BAV (I) show increased NKX2.5⁺/TM⁻ SHF cells in the aortic leaflets of Nos3^−/− embryos. Colour scheme: anti-PECAM1 (red), anti-tropomyosin (TM; grey), lineage marker (green), nuclei were stained with DAPI (blue). (J-L) 3D reconstructions showed that the NKX2.5⁺/TM⁻ SHF cells (orange) primarily localize in the NC leaflet in wild-type TAV and Nos3^−/− TAV mice, whereas, in Nos3^−/− BAV mice, the majority of SHF cells were located in the right (R) leaflet. BAV embryos developed the position of the left commissure (arrowheads) more posteriorly than wild-type TAV and Nos3^−/− TAV mice. Data are mean±s.d. for n≥4 mice per group. *P<0.05 determined by two-tailed Student's t-test. RC, right coronary leaflet; LC, left coronary leaflet; NC, non-coronary leaflet. Scale bars: 50 µm.