Epicardially derived cells (EPDCs) delaminate from the primitive epicardium through an epithelial-to-mesenchymal transformation (EMT). After this transformation, a subpopulation of cells progressively invades myocardial and valvuloseptal tissues. The first aim of the study was to determine the tissue-specific distribution of two molecules that are thought to play a crucial function in the interaction between EPDCs and other cardiac tissues, namely the Wilms' Tumor transcription factor (WT1) and retinaldehyde-dehydrogenase2 (RALDH2). This study was performed in normal avian and in quail-to-chick chimeric embryos. It was found that EPDCs that maintain the expression of WT1 and RALDH2 initially populate the subepicardial space and subsequently invade the ventricular myocardium. As EPDCs differentiate into the smooth muscle and endothelial cell lineage of the coronary vessels, the expression of WT1 and RALDH2 becomes downregulated. This process is accompanied by the upregulation of lineage-specific markers. We also observed EPDCs that continued to express WT1 (but very little RALDH2) which did not contribute to the formation of the coronary system. A subset of these cells eventually migrates into the atrioventricular (AV) cushions, at which point they no longer express WT1. The WT1/RALDH2-negative EPDCs in the AV cushions do, however, express the smooth muscle cell marker caldesmon. The second aim of this study was to determine the impact of abnormal epicardial growth on cardiac development. Experimental delay of epicardial growth distorted normal epicardial development, reduced the number of invasive WT1/RALDH2-positive EPDCs, and provoked anomalies in the coronary vessels, the ventricular myocardium, and the AV cushions. We suggest that the proper development of ventricular myocardium is dependent on the invasion of undifferentiated, WT1-positive, retinoic acid-synthesizing EPDCs. Furthermore, we propose that an interaction between EPDCs and endocardial (derived) cells is imperative for correct development of the AV cushions.

Previous studies of knock-out mouse embryos have shown that the Wilms' tumor suppressor gene (Wt1) is indispensable for the development of kidneys, gonads, heart, adrenals and spleen. Using OPT (Optical Projection Tomography) we have found a new role for Wt1 in mouse liver development. In the absence of Wt1, the liver is reduced in size, and shows lobing abnormalities. In normal embryos, coelomic cells expressing Wt1, GATA-4, RALDH2 and RXRalpha delaminate from the surface of the liver, intermingle with the hepatoblasts and incorporate to the sinusoidal walls. Some of these cells express desmin, suggesting a contribution to the stellate cell population. Other cells, keeping high levels of RXRalpha immunoreactivity, are negative for stellate or smooth muscle cell markers. However, coelomic cells lining the liver of Wt1-null embryos show decreased or absent RALDH2 expression, the population of cells expressing high levels of RXRalpha is much reduced and the proliferation of hepatoblasts and RXRalpha-positive cells is significantly decreased. On the other hand, the expression of smooth muscle cell specific alpha-actin increases throughout the liver, suggesting an accelerated and probably anomalous differentiation of stellate cell progenitors. We describe a similar retardation of liver growth in RXRalpha-null mice as well as in chick embryos after inhibition of retinoic acid synthesis. We propose that Wt1 expression in cells delaminating from the coelomic epithelium is essential for the expansion of the progenitor population of liver stellate cells and for liver morphogenesis. Mechanistically, at least part of this effect is mediated via the retinoic acid signaling pathway.

Recent reports suggest that mammalian embryonic coronary endothelium (CoE) originates from the sinus venosus and ventricular endocardium. However, the contribution of extracardiac cells to CoE is thought to be minor and nonsignificant for coronary formation. Using classic (Wt1 Cre ) and previously undescribed (G2-Gata4 Cre ) transgenic mouse models for the study of coronary vascular development, we show that extracardiac septum transversum/ proepicardium (ST/PE)-derived endothelial cells are required for the formation of ventricular coronary arterio-venous vascular connections. Our results indicate that at least 20% of embryonic coronary arterial and capillary endothelial cells derive from the ST/PE compartment. Moreover, we show that conditional deletion of the ST/PE lineage-specific Wilms' tumor suppressor gene (Wt1) in the ST/PE ofG2-Gata4 Cre mice and in the endothelium of Tie2 Cre mice disrupts embryonic coronary transmural patterning, leading to embryonic death. Taken together, our results demonstrate that ST/PE-derived endothelial cells contribute significantly to and are required for proper coronary vascular morphogenesis.T he coronary vascular system, whose function is necessary to sustain late embryonic and postnatal cardiac function, is formed by a complex network of blood vessels, including arteries, arterioles, capillaries, venules, and veins (1). Recent reports indicate that various sources of endothelial cells contribute to the mammalian embryonic coronary system (2-4). However, the specific fate and function of these different endothelial cell pools during coronary vascular morphogenesis is the subject of intense controversy (5).Two endocardial populations have been reported to participate in the building of the embryonic coronary vascular system. The first derives from the sinus venosus endocardium, which sprouts to give rise to the nascent Apelin + coronary vasculature (2). A careful analysis of this study suggests that the sinus venosus endocardium, which is able to vascularize subepicardial and myocardial heart layers, mainly provides a cellular scaffold for the development of coronary veins (CoV). Accordingly, a second source of coronary endothelium (CoE) has been identified in the ventricular endocardium (Nfatc1 + lineage), which contributes massively to coronary arterial (CoA) endothelium (3, 6).A third disputed source of CoE is the proepicardium (PE), a structure that comprises epicardial progenitor cells. The PE protrudes from the septum transversum (ST), a folding of the lateral mesoderm that initiates the separation of thoracic and abdominal cavities in mammals (7). Although in vivo cell tracing and in vitro culture of avian PE cells clearly shows that PE cells can differentiate into CoE (8, 9), data from studies in mammals claimed the contribution of PE to CoE is minor (10-12). The so-called "epicardial" Cre constructs used in these studies are based on the expression of genes such as Gata5, Tbx18, or Wilms' tumor suppressor (Wt1) ( Table S1), all of which are expressed by both PE and...