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.
Highlights
Atrioventricular septal defects often result from impaired endocardial cushion development
Using lineage tracing in combination with marker analyses, we show that cobblestone-shaped morphology in atrioventricular canal (AVC) outgrowths is insufficient to qualify cells as endothelial, and that mesenchymal cells in explants can be obtained via Endothelial-to-mesenchymal transition (EndoMT) and epicardial-to-mesenchymal transition (EpiMT) processes (Fig. 5)
To minimize epicardial cell contamination, the authors scraped the outside wall of 30 s–35 s AVC explants with a tungsten needle, but not those of 20 s–29 s explants
Summary
Atrioventricular septal defects often result from impaired endocardial cushion development. The AVC explant assay, in combination with genetic mouse models, has proven to be a powerful tool to study the molecular mechanisms regulating EndoMT4,8,14–18 While establishing this assay, we observed the typical somite stage-dependent pattern in cellular outgrowth as previously reported[4,5], including the cobblestone-shaped monolayers that were present from 23 s–26 s onwards. Inhibition of TGFβ-signalling resulted in an enrichment in cobblestone-shaped monolayers, identified earlier as endothelium[4], yet the majority of these monolayers were positive for Wilm’s Tumor Protein[1] (WT1), compatible with an epicardial origin
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