Multiple cell types including melanocytes contribute to elastogenesis in the developing murine aortic valve

Figures

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  Fig. 1. ElastinmRNA levels in developing murine aortic valve. (A) Timeline of Eln expression in AoV from early developmental stages to adulthood. qPCR fold change normalized to endogenous Gapdh gene. Mean ± SEM shown; N = 5–9 biological replicate per group; p-value determined by One-way ANOVA followed by Tukey’s multiple comparison test. In situ hybridization RNAscope with Eln probe (red) and nuclei counterstaining for DAPI (blue) of (B) P0 (C) P5 (D) P30. Dotted white line outlines AoV leaflet. Scale bar = 10 μm.

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  Fig. 2. Aortic valve cells clusters separately from other cardiac cells by spatial transcriptomic analysis at P3. (A) Spatial transcriptomics of P3 aortic valve sections. P3 aortic valve H&E image representation with surrounding tissue spots (blue) and aortic valve leaflet spots (red). Scale bar = 1 mm. (B) 2-D t-SNE cluster plot showing aortic valve leaflet tissue cluster (red) and surrounding tissue (blue). (C) List of top ten correlated genes with Eln from spatial transcriptomics data. For each spot (131 total), gene read count values were normalized to the total number of reads for a given spot. (D) Eln expressing cells are positive for alpha smooth muscle actin (αSMA). P0 aortic valve sections assessed by in situ hybridization RNAscope for Eln (red), immunofluorescence for αSMA antibody (green) and nuclei counterstaining for DAPI (blue). Dotted white line outlines aortic valve leaflet. Scale bar = 10 μm. Yellow highlighted box corresponds to the zoom image showing high magnification of αSMA and Eln positive cells in the aortic valve (left). Scalebar = 5 μm. N = 6.

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  Fig. 3. Elastic fiber patterning in developing murine aortic valve. (A) Wholemount elastic (red) and collagen (cyan) fiber staining in WT (C57BL/6J mice), hyperpigmented (K5-Edn3) and hypopigmented (Albino) AoV leaflets at P0 and P7 stages. Leaflets in the light microscope images (inserts) correspond to those in the images of the stained leaflets. P0 images, 60X magnification and P7 images, 20X magnification. (B) Quantification of elastic fiber (left) and collagen fiber (right) staining at P0 (blue circle) and P7 (red circle) stages in WT, K5-Edn3 and Albino. (C) qPCR gene expression for elastin in K5-Edn3 and Albino at P0, P7 and P30 stages. P0 (blue circles), P7 (red circles) and P30 (black circles). Fold change normalized to endogenous Gapdh. Mean ± SEM shown; p-value determined by One-Way ANOVA followed by Tukey’s multiple comparison test; N = 5–9 biological replicates per group.

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  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…

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  Fig. 5. Endothelial and interstitial cells express elastin mRNA except immune cells. 3-D reconstruction of P3 WT aortic valve wholemount leaflet labeled for elastin (Eln, magenta) in situ hybridization RNAscope probe, and antibody staining (green) for (A) VE-Cadherin (VE-Cad), (B) Vimentin (Vim), (C) CD45 and nuclei counterstaining for DAPI (blue). Yellow arrow heads points to 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. Scale bar = 50 μm. Wholemount single channels images are shown for each of the corresponding marker.