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, 8 (1), 4722

The Epicardium Obscures Interpretations on Endothelial-To-Mesenchymal Transition in the Mouse Atrioventricular Canal Explant Assay

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The Epicardium Obscures Interpretations on Endothelial-To-Mesenchymal Transition in the Mouse Atrioventricular Canal Explant Assay

Nathan Criem et al. Sci Rep.

Erratum in

Abstract

Atrioventricular septal defects often result from impaired endocardial cushion development. Endothelial-to-mesenchymal transition (EndoMT) is a critical event in endocardial cushion development that initiates in the atrioventricular canal (AVC). In ex vivo EndoMT studies, mouse AVCs are flat-mounted on a collagen gel. In the explant outgrowths, the ratio of elongated spindle-like mesenchymal cells over cobblestone-shaped cells, generally considered as endothelial cells, reflects EndoMT. Using this method, several key signalling pathways have been attributed important functions during EndoMT. Using genetic lineage tracing and cell-type-specific markers, we show that monolayers of cobblestone-shaped cells are predominantly of epicardial rather than endothelial origin. Furthermore, this epicardium is competent to undergo mesenchymal transition. Contamination by epicardium is common and inherent as this tissue progressively attaches to AVC myocardium. Inhibition of TGFβ signalling, previously shown to blunt EndoMT, caused an enrichment in epicardial monolayers. The presence of epicardium thus confounds interpretations of EndoMT signalling pathways in this assay. We advocate to systematically use lineage tracers and cell-type-specific markers on stage-matched AVC explants. Furthermore, a careful reconsideration of earlier studies on EndoMT using this explant assay may identify unanticipated epicardial effects and/or the presence of epicardial-to-mesenchymal transition (EpiMT), which would alter the interpretation of results on endothelial-to-mesenchymal transition.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
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. (bp) 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 (ce), (hj) and (mo) 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 (lp) 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 αSMA+GFP+CD31+ endothelial cell. The white arrows in panels (h–j) indicate αSMA+GFP-CD31- cobblestone-shaped cells. A: atrium, AVC: atrioventricular canal, EndoMT: endothelial-to-mesenchymal transition, V: ventricle.
Figure 2
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. (ao) 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 (bd), (gi) and (ln) 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. (qu) Parasagittal sections from mouse embryos collected between 16 s–35 s (N = 24 embryos) were stained for GFP, WT1 and MF20. Boxed areas are magnified in (q’-u’). Arrowheads in panels (q’-u’) indicate epicardial cells migrating along the myocardial wall. Asterisks indicate the pro-epicardial organ.
Figure 3
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.
Figure 4
Figure 4
Inhibition of ALK5 leads to a relative increase in epicardial and endothelial cells. (at) 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 (bd), (gi), (ln) and (qs) 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 (fj), endothelium only (pt) or mixed epicardium and endothelium (ko).
Figure 5
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.

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References

    1. Garside VC, Chang AC, Karsan A, Hoodless PA. Co-ordinating Notch, BMP, and TGF-β signaling during heart valve development. Cell. Mol. Life Sci. 2013;70:2899–917. doi: 10.1007/s00018-012-1197-9. - DOI - PMC - PubMed
    1. Ma L. Bmp2 is essential for cardiac cushion epithelial-mesenchymal transition and myocardial patterning. Development. 2005;132:5601–5611. doi: 10.1242/dev.02156. - DOI - PubMed
    1. Sugi Y, Yamamura H, Okagawa H, Markwald RR. Bone morphogenetic protein-2 can mediate myocardial regulation of atrioventricular cushion mesenchymal cell formation in mice. Dev. Biol. 2004;269:505–18. doi: 10.1016/j.ydbio.2004.01.045. - DOI - PubMed
    1. Camenisch TD, et al. Temporal and distinct TGFbeta ligand requirements during mouse and avian endocardial cushion morphogenesis. Dev. Biol. 2002;248:170–81. doi: 10.1006/dbio.2002.0731. - DOI - PubMed
    1. Azhar M, et al. Ligand-specific function of transforming growth factor beta in epithelial-mesenchymal transition in heart development. Dev. Dyn. 2009;238:431–442. doi: 10.1002/dvdy.21854. - DOI - PMC - PubMed

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