Fig. 4

Alpha smooth muscle actin expressing cells that are positive for melanocyte specific markers express elastin in aortic valve leaflets. (A) Eln expressing cells are positive for smooth muscle actin and melanocyte markers. 3-D reconstruction of P0 aortic valve wholemount leaflet labeled with elastin (Eln, gray) and Tyrosinase (Tyr, magenta) RNAscope probes, and alpha-smooth muscle actin (αSMA, green) immunolabeling. XZ and YZ orthogonal views for merged and separate channels are shown in the bottom and side panels, respectively. N = 6. (B) In situ hybridization with Eln and Tyr probe and immunofluorescence with αSMA immunolabeling of P0 aortic valve section. Dotted white line outlines aortic valve leaflet. Scale bar = 10 μm. N = 4. In situ hybridization of wholemount P3 WT aortic valve labeled for Eln (gray) and antibody staining (magenta) for (C) Tyrosine related protein 1 (Trp1), (D) Microphthalmia-associated transcription factor (Mitf) and nuclei counterstaining for DAPI (blue). Scale bar = 50 μm. Yellow arrow heads point to markers co-localization. The yellow line shows the cross section corresponding to the orthogonal views. The XZ and YZ orthogonal views are shown in the bottom and side panels with only magenta and gray channel, respectively. N = 6. Wholemount single channels images are shown for each of the corresponding marker.