Abstract
The epicardium contributes to multiple cardiac lineages and is essential for cardiac development and regeneration. However, the mechanism of epicardium formation is unclear. This study aimed to establish the cellular and molecular mechanisms underlying the dissociation of pro-epicardial cells (PECs) from the pro-epicardium (PE) and their subsequent translocation to the heart to form the epicardium. We used lineage tracing, conditional deletion, mosaic analysis and ligand stimulation in mice to determine that both villous protrusions and floating cysts contribute to PEC translocation to myocardium in a CDC42-dependent manner. We resolved a controversy by demonstrating that physical contact of the PE with the myocardium constitutes a third mechanism for PEC translocation to myocardium, and observed a fourth mechanism in which PECs migrate along the surface of the inflow tract to reach the ventricles. Epicardial-specific Cdc42 deletion disrupted epicardium formation, and Cdc42 null PECs proliferated less, lost polarity and failed to form villous protrusions and floating cysts. FGF signaling promotes epicardium formation in vivo, and biochemical studies demonstrated that CDC42 is involved in the trafficking of FGF receptors to the cell membrane to regulate epicardium formation.
Highlights
CDC42 is an essential protein in the establishment of cell polarity (Etienne-Manneville, 2004), we examined the polarity of control and conditional knockout (CKO) pro-epicardial cells (PECs) by determining the localization of the polarity complex including Par3 and aPKCζ (PRKCZ), a member of the aPKC family, which is required for epicardial development (Christoffels et al, 2009)
The PE extends bleb-like villi that form a transient tissue bridge to reach the heart, and in mouse the PECs are thought to reach the myocardium through the translocation of multicellular cysts across the pericardial cavity (Hirose et al, 2006; Komiyama et al, 1987; Pérez-Pomares et al, 1997; Ratajska et al, 2008; Schulte et al, 2007; Sengbusch et al, 2002; Van den Eijnde et al, 1995)
The PE directly contacted the heart in four out of ten control embryos (Fig. 3A, Fig. S2A-C, Fig. S3A,B) and in three out of six CKO embryos (Fig. 3A,B, Movie 11) examined at E9.5, and in three out of four control hearts at E9.75, validating a previous report of PE direct contact with the myocardium (Movie 12) (Rodgers et al, 2008), which is referred to as the third mechanism in this study. These results indicate that PECs do not translocate to the heart via direct contact at an early stage, but do so at a later stage; the controversy might be due to the different ages of the hearts examined
Summary
The epicardium, which consists of a single layer of squamous epicardial cells (ECs) that covers the heart, is the major source of coronary smooth muscle cells and cardiac fibroblasts A subset of ECs will undergo epicardial-mesenchymal transition (EMT) and differentiate into different cardiac cell types during cardiac development and cardiac regeneration (Cai et al, 2008; Dettman et al, 1998; Mikawa and Fischman, 1992; Mikawa and Gourdie, 1996; Wu et al, 2010; Zhou et al, 2011, 2008)
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