The epicardium obscures interpretations on endothelial-to-mesenchymal transition in the mouse atrioventricular canal explant assay

Figures

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  Figure 1. The cellular outgrowth pattern in mouse AVC explants differs depending on the somite-stage of the embryo. (a) Schematic representation of the mouse AVC explant assay. The endocardium from an E9.5 embryo is highlighted in green. The AVC is marked by the “kissing zone” and the cushions. (b–p) AVCs collected from RCEf/0;Tie2CreTg/0 embryos staged between 22 s–34 s were cultured for 48 h, immunostained for GFP, αSMA and CD31 and imaged by confocal microscopy (N = 19 explants). Boxed areas in (b), (g) and (l) are magnified in respectively (c–e), (h–j) and (m–o) and presented as alternating combinations of two channels in magenta and green, overlaid with DAPI in gray. Individual confocal Z-planes are compared with the corresponding phase contrast images (f,k and p). The explant (ex) is delineated by a white loop. The area of cellular outgrowth in panels (b), (g) and (l) is delineated by a gray loop. White dashes in panels (l–p) indicate GFP-negative monolayers. White arrowheads in panels (c–f) indicate αSMA+GFP+CD31− spindle-like shaped mesenchymal cells. White arrowheads in panels (m–o) indicate αSMA+GFP-CD31− cobblestone-shaped cells. The yellow arrows in panels (h–j) indicate an…

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  Figure 2. Epicardial cells contaminate the AVC explant with increasing somite stage which corresponds with the progressive covering of the myocardial wall by epicardial cells. (a–o) AVCs collected from RCEf/0;Tie2CreTg/0 embryos staged between 20 s–36 s (N = 53 explants) were cultured for 48 h and stained for GFP, WT1 and αSMA. Boxed areas in (a), (f) and (k) are magnified in respectively (b–d), (g–i) and (l–n) and presented as alternating combination of 2 channels in magenta and green. Individual confocal Z-planes are compared with the corresponding phase contrast images (e, j and o). The explant (ex) is delineated by a white loop. The area of cellular outgrowth in panels (a), (f) and (k) is delineated by a gray loop. Arrowheads in panels (b–e) indicate αSMA+GFP+WT1− spindle-like shaped mesenchymal cells. Arrows in panels (g–j) indicate epicardial cells undergoing EpiMT. (p) Table representing the numbers of explants isolated per somite stage and the number of explants contaminated with epicardium. NA: not applicable, not dissected in this experiment. (q–u) Parasagittal sections from mouse embryos collected between 16 s–35 s (N = 24 embryos) were stained for GFP, WT1 and MF20. Boxed areas…

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  Figure 3. Inhibition of ALK5 leads to an enrichment in cobblestone-shaped cells. Phase contrast figures of control DMSO (a,b) and SD-208 (c,d) treated explants. Boxed areas in panels (a) and (c) are magnified in panels (b) and (d) respectively. (e) Quantification of the relative amount of cobblestone-shaped cells in the outgrowths. (f) The total number of migrating cells normalized to the explant area (presented per 100 µm2 explant area). The individual datapoints, the mean and standard deviation are presented in panels (e) and (f). The explants (ex) is delineated by black dashes. The area of cellular outgrowth is delineated by a white loop.

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  Figure 4. Inhibition of ALK5 leads to a relative increase in epicardial and endothelial cells. (a–t) 10 pairs of somite matched AVC explants treated with either DMSO or SD-208 in DMSO were stained for GFP, WT1 and αSMA. Boxed areas in panels (a), (f), (k) and (p) are magnified in respectively (b–d), (g–i), (l–n) and (q–s) and presented as alternating combinations of 2 channels in magenta and green. Individual confocal Z-planes are compared with the corresponding phase contrast images (e,j,o and t). The explant (ex) is delineated by a white loop. The area of cellular outgrowth in panels (a), (f), (k) and (p) is delineated by a gray loop. Upon SD-208 treatment, the observed monolayers are either epicardium only (f–j), endothelium only (p–t) or mixed epicardium and endothelium (k–o).

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  Figure 5. Schematic representation of the AVC explant model with and without epicardial cell contamination. (a) Explants collected from embryo between 20 s–23 s contain none to nearly no epicardial cells. Only endothelium-derived mesenchymal cells will grow out of the explant. (b) From 23 s onwards, epicardial contamination increases and becomes apparent as monolayers of epicardium-derived cells capable of undergoing epicardial-to-mesenchymal transitions.