Endocardial-to-mesenchymal transition underlies cardiac outflow tract septation and bicuspid aortic valve formation in the Syrian hamster model

Figures

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  Fig. 1. (a-c) Schematic representations of an embryonic heart in frontal (a), anatomical position (b) and transverse (c) views during outflow tract (OFT) septation. The frontal view (a) shows conal ridges (CRs) along the OFT, and the distribution of the aortic-pulmonary septation complex (AP) that shows an inverted-U shape (in black), composed of cardiac neural crest (CNC) cells. The schematic representation in (b) shows the orientation of the frontal and transversal sections of the OFT used in the study with respect to a hamster embryo. The transverse views (c) represent consecutive stages of the septation of tricuspid (TAV) and bicuspid aortic valves (BAV). During the normal fusion of the conal ridges (CR), CNC cells (in green), initially distributed in prongs, migrate to the central portion to form the conal septum (CS). A CNC abnormal migration extends the fusion of the posterior margins of the PR and SR in BAV embryos. The CS divides the conus in two aortic (Ao) and pulmonary (Pul) independent OFTs, each containing three (TAV) or two (BAV) valve cushions. (d-k) Transverse (d-f, g-i, ) and frontal (j,k) sections of ED11 (d-f,j,k) and ED12 (g-i) embryonic hearts show the OFT septation…

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  Fig. 2. Transverse sections of the OFT of ED12 (a–i) and ED11 (j–l) hamster embryos with TAV from T and control strains (a–c) and BAV from T strain (d–l), stained with the TUNEL assay (green; a–c, g–k) or with caspase antibodies (red; d–l). Nuclei were stained with DAPI (blue). The red rectangles show magnifications of the thoracic wall (b,e), the CS (c,f) and the area of fusion of the CRs (i,l). The fusion area shown in (j–l) corresponds to the H-E stained section in Fig. 1e,m, and that shown in (g,i) corresponds to Fig. 1i. Arrows in i and l delimit the fusion area. Arrowheads point to apoptotic cells. Scale bar for insets: 100 μm. Scale bar for magnifications: 50 μm.

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  Fig. 3. Frontal semithin sections of the middle OFT of ED11 hamster embryos stained with Toluidine Blue-Basic Fuchsin, showing the fusion area in detail. Each CR was composed of mesenchymal cells covered by a monolayer of endocardial cells (a–c). In the disto-proximal axis of the endocardium covering the CRs (d), three different areas could be distinguished (1–3). In the proximal region (3), septal and parietal CRs were separated, and endocardial cells presented a conventional endothelial morphology (e). In the middle region (2), the two opposite endocardial linings in contact exhibited a similar morphology to that described in e (f). In the most distal region (1), corresponding to the fusion area, endocardial cells from both CR were also in contact, but showed an intermediate phenotype between endothelial and mesenchymal cells. The intermediate phenotype was characterized by an endothelial cell appearance with features similar to those found in mesenchymal cells (g,h). D: distal, P: proximal, PR: parietal ridge, SR: septal ridge. Asterisk: conal myocardium. Black arrows point to endothelial cells, black arrowheads to mesenchymal cells and white arrowheads to intermediate phenotype. Red…

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  Fig. 4. Frontal (a–c) and transverse (d–l) sections of the middle OFT of ED11 hamster embryos. (a) Frontal semithin section of the fusion area stained with Toluidine Blue-Basic Fuchsin. Dashed lines mark the plane of sections shown in (d–f). (b,c,g–l) Histological sections immunostained with CD34 or VE-Cadherin (green) and α-actin (red). White arrowheads point to double stained cells, yellow arrowheads to intracellular VE-Cadherin, and white arrows to forming lamellipodia and filopodia. Nuclei are stained with DAPI (blue). (d–f) Overviews of the OFTs stained with hematoxylin-eosin showing the fusion process transversally. Black rectangles in d–f mark magnified areas shown in g,j; h,k and i,l, respectively. Scale bar for insets: 100 μm. Scale bar for magnifications: 25 μm.

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  Fig. 5. Transverse (a–c, g–l) and frontal (d–f) sections of the middle region of the OFT of ED11 (a–f) and ED12 (g–l) hamster embryos. Normal (TAV) and BAV embryos are shown in (g-i) and (a–f), (j–l), respectively. Overviews in (a,d,g,j) are stained with hematoxylin-eosin. Sections immunostained with CD34 or VE-Cadherin (green) and α-actin (red) are shown in (b,c,e,f,h,i,k,l. Nuclei were stained with DAPI (blue). Black rectangles in a and d indicate magnified areas shown in b,c and e,f respectively. Yellow rectangles in h and k indicate magnified areas shown in i and l, respectively. White arrowheads point to double stained cells, yellow arrowheads to intracellular VE-Cadherin and white arrows to forming lamellipodia and filopodia. Asterisks in a and j indicate fused cushions in a BAV embryo. Scale bar for insets: 100 μm. Scale bar for magnifications: 50 μm.

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  Fig. 6. Schematic representation of EMT process during the fusion of CRs in frontal (a) and transverse (b) views of the OFT during normal (TAV) and bicuspid aortic valve (BAV) development. (a) The EMT process occurs sequentially along the disto-proximal axis of the OFT. Proximal (P) endocardial cells (green) are quiescent. Mid-portion endocardial cells (in red and green) are undergoing EMT, showing an endocardial-mesenchyme mixed morphology. Distal (D) mesenchymal cells (in red) are transformed from endocardial cells. Brown cells are mesenchymal cells that form the core of CRs. (b) Transforming endocardial cells involved in the fusion event are restricted to the center of the CRs (green cells) of ED11 TAV embryos, whereas in BAV embryos the number of transforming cells increase in the posterior (aortic) region of the OFT, continuing at ED12. Therefore, spatial and temporal alteration of the EMT process underlies the development of BAV. Pul: pulmonary artery, Ao: aorta artery.