Primary and immortalized mouse epicardial cells undergo differentiation in response to TGFβ

Abstract

Cells derived from the epicardium are required for coronary vessel development. Transforming growth factor ␤ (TGF␤) induces loss of epithelial character and smooth muscle differentiation in chick epicardial cells. Here, we show that epicardial explants from embryonic day (E) 11.5 mouse embryos incubated with TGF␤1 or TGF␤2 lose epithelial character and undergo smooth muscle differentiation. To further study TGF␤ Signaling, we generated immortalized mouse epicardial cells. Cells from E10.5, 11.5, and 13.5 formed tightly packed epithelium and expressed the epicardial marker Wilm's tumor 1 (WT1). TGF␤ induced the loss of zonula occludens-1 (ZO-1) and the appearance of SM22␣ and calponin consistent with smooth muscle differentiation. Inhibition of activin receptor-like kinase (ALK) 5 or p160 rho kinase activity prevented the effects of TGF␤ while inhibition of p38 mitogen activated protein (MAP) kinase did not. These data demonstrate that TGF␤ induces epicardial cell differentiation and that immortalized epicardial cells provide a suitable model for differentiation. Developmental Dynamics 237:366 -376, 2008.

IGF signaling directs ventricular cardiomyocyte proliferation during embryonic heart development

“…MEC1 cells localized ZO-1 and -catenin (TJP1 and CTNNB1 -Mouse Genome Informatics) in adherens junctions at the cell membrane and organized actin filaments in stress fibers and cortical filaments (Fig. 1C), as seen in prior studies of epicardial mesothelial cells (Dokic and Dettman, 2006;Austin et al, 2008). Furthermore, all MEC1 cells were immunopositive for nuclear WT1 (see Fig.…”

Myocardin-related transcription factors control the motility of epicardium-derived cells and the maturation of coronary vessels

“…To test whether members of the SRF-MRTF signaling axis are expressed in a manner consistent with potentiating epicardial EMT and EPDC differentiation, we isolated epicardial cells from embryonic day (E)11.5 embryos by outgrowth from cardiac explants (Austin et al, 2008). Expression analysis by quantitative real-time RT-PCR (qPCR) revealed robust expression of various epicardial marker genes, including Wt1, Tcf21 and Krt14, and low expression of cardiomyocyte genes (Myh7 and Nkx2-5), confirming the purity of our culture system (Fig.…”

“…Epicardial EMT is regulated by a complex combination of extracellular molecules, including Wnts, platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β, fibroblast growth factor, and retinoic acid (Austin et al, 2008;Kim et al, 2010;Mellgren et al, 2008;Merki et al, 2005;Pennisi and Mikawa, 2009). These upstream signals are thought to impact upon EMT partially through the induction of Snai1 and Snai2 expression (Martinez-Estrada et al, 2010).…”

The fibronectin leucine-rich repeat transmembrane protein Flrt2 is required in the epicardium to promote heart morphogenesis

“…Imaging epicardial explants and whole-mount hearts E10.5 and E11.5 hearts harvested in 1ϫHanks-buffered salt solution, were placed dorsal side downwards on collagen-coated slides (BD Biosciences) and cultured for up to 72 hours in M119 medium containing 5% foetal calf serum (Austin et al, 2008). The outgrowths were fixed in 4% PFA/PBS for 10 minutes and permeabilized with 0.2% TritonX-100 for 5 minutes at room temperature, then blocked in 10% goat serum/1% BSA/PBS for 1 hour at room temperature.…”

“…Epicardial cells were allowed to migrate from the surface of the heart onto a collagen-coated substrate (Austin et al, 2008). The resulting monolayers were stained with antibodies directed against ZO-1 and phalloidin to visualize the plasma membrane and actin cytoskeleton architecture, respectively (Fig.…”