Increased Hemodynamic Load in Early Embryonic Stages Alters Endocardial to Mesenchymal Transition

Figures

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  Figure 1. Schematic of outflow tract cushion analysis. The cushions are composed of an outer myocardium layer, a middle cardiac jelly layer, and an inner endocardium monolayer. VE-cadherin junctions connect endocardial cells and are lost when cells delaminate from the endocardium and invade the cardiac jelly region. Cushion cell density is measured using DAPI staining from an area that extends 10 microns into the cushion from the lumen edge, and cell organization is quantified using phalloidin staining from an area that extends across the whole width of the cushion from the lumen edge. Additionally, we measured phalloidin intensity from an area that only includes 25% of the cushion width from the lumen edge. Periostin fibers run through the cardiac jelly, where the fibers concentrate at a front that extends from the myocardium.

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  Figure 2. Example confocal fluorescent image quantifications from control embryos. (A) Phalloidin (red) stain fluorescence intensities from a full cushion, where the average fluorescent intensity at each distance position across the cushion is plotted. (B) Cushion cell density measured from the number of outlined DAPI-stained nuclei (blue) within an area 10 μm into the cushion from the lumen edge. (C) Cell count per endocardium length measured from the number of outlined DAPI-stained nuclei (blue) along with a measured length of the endocardium. (D) Periostin (red) front length measured as a percentage of the length of the full cushion. (E). VE-cadherin (red) junction per endocardium length measured from the number of outlined junctions along a measured length of the endocardium. Scale bar = 50 microns.

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  Figure 3. Phalloidin confocal fluorescent image analysis. Confocal fluorescent images at 20x from whole embryo hearts stained with phalloidin at HH15 (A,D), HH18 (B,E), and HH24 (C,F) at the outflow tract cushions (A–C) and the atrioventricular cushions (D–F). Scale bar = 100 microns. AV, atrioventricular canal; OFT, outflow tract.

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  Figure 4. Confocal fluorescent image analysis with banding. Phalloidin (red) stain at 20x in a control (A) and banded outflow tract with 52% band tightness (B), with phallodin fluorescence intensities quantitated in (C). Phalloidin (red) stain at 63x in a control (D) and banded outflow tract with 52% band tightness (E), with cushion cell density and cell count per endocardium length quantitated from DAPI stain (F). Periostin (red) labeling at 20x in a control (G) and banded outflow tract with 49% band tightness (H), with periostin front length quantitated in (I). VE-cadherin (red) labeling at 63x in a control (J) and banded outflow tract with 41% band tightness (K), with endocardial cell junctions per endocardium length quantitated in (L). Each sample is also stained with DAPI (blue). 0% band tightness refers to the control group. Scale bar = 50 microns.

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  Figure 5. Normal periostin expression in outflow tract cushions. Confocal fluorescent images at 20x from whole embryo hearts labeled with periostin (red) and DAPI (blue) at progressing developmental stages, (A) HH15, (B) HH18, (C) HH21, (D) HH24, and (E) HH25. Scale bar = 100 microns.

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  Figure 6. Quantification comparisons between outer and inner cushions. Phalloidin stain quantifications (A,B), DAPI stain quantifications (C,D), VE-cadherin label quantification (E), and periostin label quantification (F). All control and banded comparisons between outer and inner cushions were not significantly different (p > 0.05).

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  Figure 7. Electron microscopy analysis of endocardium remodeling. Stitched TEM images of the endocardium and adjacent cardiac jelly at 1700x (A,B), and the corresponding 3D FIB-SEM reconstructions of endocardial cell segmentations from image stacks (C–F). Reconstructions display each cell in a different color and are orientated to view the endocardial-lumen edge with translucently colored cells (C,E) and the endocardial-cardiac jelly interface edge with opaquely colored cells (D,F) from an example control embryo (A,C,D) and an example banded embryo (B,E,F). Box plots showing differences in endocardial cell geometry, quantified from 3D segmentations of FIB-SEM images, with surface area-to-volume ratio, SA/V (G), shape factor (H), and elongation factor (I). Boxes mark the upper and lower quartiles and whiskers mark the maximum and minimum values for each parameter. Asterisks denote statistically significant differences between experimental and control samples (p < 0.01). CJ, cardiac jelly; E, endocardium. Scale bar = 5 μm.

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  Figure 8. Fold changes in mass-spectrometry measured protein abundances in banded samples compared to controls that are associated with EMT. Extracellular matrix proteins, cell type markers, EMT regulators, and factors associated with other types of EMT are shown, with positive and negative fold changes representing protein upregulation and downregulation, respectively. Protein abbreviations are listed in Tables 1, 2.

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  Figure 9. Selected network computed by IPA. The network show relationships between transcriptional regulators, molecules affected by mechanotransduction (highlighted with a pink borderline), and molecules related to general epithelial-mesenchymal transition (enclosed in a gray dotted square). The various shapes are associated with different types of molecules; coloring is based on differential expression values from our proteomics dataset: Red, up-regulation; Green, down-regulation. Lines indicate molecular relationships, with arrows indicating the directionality of the interaction.