Epicardium-derived progenitor cells require β-catenin for coronary artery formation

Abstract

We have previously identified several members of the Wnt/␤-catenin pathway that are differentially expressed in a mouse model with deficient coronary vessel formation. Systemic ablation of ␤-catenin expression affects mouse development at gastrulation with failure of both mesoderm development and axis formation. To circumvent this early embryonic lethality and study the specific role of ␤-catenin in coronary arteriogenesis, we have generated conditional ␤-catenin-deletion mutant animals in the proepicardium by interbreeding with a Cre-expressing mouse that targets coronary progenitor cells in the proepicardium and its derivatives. Ablation of ␤-catenin in the proepicardium results in lethality between embryonic day 15 and birth. Mutant mice display impaired coronary artery formation, whereas the venous system and microvasculature are normal. Analysis of proepicardial ␤-catenin mutant cells in the context of an epicardial tracer mouse reveals that the formation of the proepicardium, the migration of proepicardial cells to the heart, and the formation of the primitive epicardium are unaffected. However, subsequent processes of epicardial development are dramatically impaired in epicardial-␤-catenin mutant mice, including failed expansion of the subepicardial space, blunted invasion of the myocardium, and impaired differentiation of epicardium-derived mesenchymal cells into coronary smooth muscle cells. Our data demonstrate a functional role of the epicardial ␤-catenin pathway in coronary arteriogenesis. The embryonic epicardium originates from a primarily extracardiac primordium, the proepicardum, which is located at the septum transversum near the venous pole of the heart (2, 3). In mouse embryos, proepicardial cells reach the heart predominantly in the form of free-floating vesicles that traverse the pericardial cavity, adhere to the initially naked myocardial surface, and subsequently form the epicardial covering of the heart (reviewed in ref. 1). The recruitment of coronary vessel progenitor cells from the proepicardium and embryonic epicardium involves several steps of epithelial-mesenchymal transition (EMT). As a result of proepicardial EMT, the extracellular matrix of free-floating proepicardial vesicles becomes populated with mesenchymal cells (4-8). Subsequently, the primitive epicardium gives rise to the subepicardial mesenchyme, also by means of EMT.Recent data suggest that the primitive epicardium and epicardium-derived cells (EPDCs) modulate the maturation of other cardiac components, including the embryonic myocardium and the cardiac conduction system (9-14). We have previously shown that the embryonic epicardium is a key signaling tissue responsible for the transmission of the morphogenic signal derived from retinoic acid and identified several components of the Wnt/␤-catenin signaling pathway that are down-regulated upon retinoid signaling deficiency, in particular, ␤-catenin and its activator Wnt9b (15).However, it remains to be shown whether Wnt/␤-catenin signaling plays a specific role in the de...

Endogenous retinoic acid regulates cardiac progenitor differentiation

“…In addition, RXRα mutant epicardial cells have reduced Wnt signaling, rendering the epicardium defective in its cardiac invasive and epithelial-mesenchymal transformation (EMT) ability (11). The Wnt signaling pathway is required for the full contribution of epicardial cells to coronary smooth muscle and coronary fibroblasts (27) and likely is involved in epicardial conversion to cardiomyocytes, although some controversy exists about whether lineage tracing conclusively demonstrates an epicardial origin of cardiomyocytes (42 and references therein).…”

“…S2). Reductions in Wnt signaling produce blunted invasion of epicardial cells into the myocardial space and impaired differentiation into coronary smooth muscle (27), events defective in RXRα mutants as visualized by collagen gel invasion assays (11). Such gel invasion assays performed on E12.5 Raldh2 −/− ventricular epicardium tissue explants revealed normal gel penetration (Fig.…”

MicroRNA-processing Enzyme Dicer Is Required in Epicardium for Coronary Vasculature Development

“…Epicardial Cell Culture-Epicardial cell culture was performed as described previously (24). Briefly, hearts were dissected from E11.5 and E12.5 embryos and ventricles were placed epicardial side down on gelatin-coated chamber slides in epicardial culture medium (1:1 mixture of DMEM and medium 199 supplemented with 100 units/ml penicillin, 100 g/ml streptomycin, 10% FBS, and 2 ng/ml basic fibroblast growth factor (FGF2, PeproTech, NJ)).…”

“…To examine whether coronary vasculature defects seen in Gata5-Cre:Dicer flox/flox hearts were associated with defects in epicardial EMT, a collagen gel invasion assay was performed using epicardial explants from control and Dicer mutants at E11.5 and E12.5. Explants were cultured in EMTinduced medium containing FBS and FGF2 as described previously (24). Epicardium-derived cells lose their epithelial nature, reduce cell-cell contacts, and migrate and invade the collagen gel over time.…”

The Multiple Phases and Faces of Wnt Signaling During Cardiac Differentiation and Development

“…Depletion of ␤-catenin in the proepicardium using a specific GATA5-Cre line resulted in defects in coronary artery formation that finally is lethal. 118 A second study investigated the function of retinoic acid in epicardium development. 119 The loss of retinoic X receptor ␣ (RXR) in the epicardium indicated that RXR is required for EMT of epicardial cells, resulting in damaged myocardial growth and coronary artery formations.…”