The epicardium, which is derived from the proepicardial organ (PE) as the third epithelial layer of the developing heart, is crucial for ventricular morphogenesis. An epicardial deficiency leads to a thin compact layer for the developing ventricle; however, the mechanisms leading to the impaired development of the compact layer are not well understood. Using chick embryonic hearts, we produced epicardiumdeficient hearts by surgical ablation or blockade of the migration of PE and examined the mechanisms underlying a thin compact myocardium. Sarcomeric maturation (distance between Z-lines) and cardiomyocyte growth (size) were affected in the thin compact myocardium of epicardium-deficient ventricles, in which the amounts of phospho-smad2 and phospho-ERK as well as expression of transforming growth factor (TGF)β2 and fibroblast growth factor (FGF)2 were reduced. TGFβ and FGF were required for the maturation of sarcomeres and growth of cardiomyocytes in cultured ventricles. In ovo co-transfection of dominant negative (dN)-Alk5 (dN-TGFβ receptor I) and dN-FGF receptor 1 to ventricles caused a thin compact myocardium. Our results suggest that immature sarcomeres and small cardiomyocytes are the causative architectures of an epicardium-deficient thin compact layer and also that epicardium-dependent signaling mediated by TGFβ and FGF plays a role in the development of the ventricular compact layer before the onset of coronary circulation.

BackgroundTransposition of the great arteries is one of the most commonly diagnosed conotruncal heart defects at birth, but its etiology is largely unknown. The anterior heart field (AHF) that resides in the anterior pharyngeal arches contributes to conotruncal development, during which heart progenitors that originated from the left and right AHF migrate to form distinct conotruncal regions. The aim of this study is to identify abnormal AHF development that causes the morphology of transposition of the great arteries.Methods and ResultsWe placed a retinoic acid–soaked bead on the left or the right or on both sides of the AHF of stage 12 to 14 chick embryos and examined the conotruncal heart defect at stage 34. Transposition of the great arteries was diagnosed at high incidence in embryos for which a retinoic acid–soaked bead had been placed in the left AHF at stage 12. Fluorescent dye tracing showed that AHF exposed to retinoic acid failed to contribute to conotruncus development. FGF8 and Isl1 expression were downregulated in retinoic acid–exposed AHF, and differentiation and expansion of cardiomyocytes were suppressed in cultured AHF in medium supplemented with retinoic acid.ConclusionsThe left AHF at the early looped heart stage, corresponding to Carnegie stages 10 to 11 (28 to 29 days after fertilization) in human embryos, is the region of the impediment that causes the morphology of transposition of the great arteries.

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Cushion tissues, the primordia of valves and septa of the adult heart, are formed in the atrioventricular (AV) and outflow tract (OFT) regions of the embryonic heart. The cushion tissues are generated by the endothelial–mesenchymal transition (EMT), involving many soluble factors, extracellular matrix, and transcription factors. Moreover, neural crest‐derived mesenchymal cells also migrate into the OFT cushion. The transcription factor Msx1 is known to be expressed in the endothelial and mesenchymal cells during cushion tissue formation. However, its exact role in EMT during cushion tissue formation is still unknown. In this study, we investigated the expression patterns of Msx1 mRNA and protein during chick heart development. Msx1 mRNA was localized in endothelial cells of the AV region at Stage 14, and its protein was first detected at Stage 15. Thereafter, Msx1 mRNA and protein were observed in the endothelial and mesenchymal cells of the OFT and AV regions. in vitro assays showed that ectopic Msx1 expression in endothelial cells induced p27, a cell‐cycle inhibitor, expression and inhibited fibroblast growth factor 4 (FGF4)‐induced cell proliferation. Although the FGF signal reduced the EMT‐inducing activities of transforming growth factor β (TGFβ), ectopic Msx1 expression in endothelial cells enhanced TGFβ signaling‐induced αSMA, an EMT marker, expression. These results suggest that Msx1 may support the transformation of endothelial cells due to a TGFβ signal in EMT during cushion tissue formation.

Coronary vessel development has been investigated in avian and mouse embryonic hearts. Quail embryos are a useful tool to examine vascular development, particularly because the QH1 antibody and transgenic quail line, Tg (tie1:H2B-eYFP), are useful to trace endothelial cells. However, there are only a few descriptions of the quail coronary vessels. Using ink injection coronary angiography, we examined the course of coronary vessels in the fetal quail heart. The major coronary arteries were the right and left septal arteries, which, respectively, branched off from the right and left coronary stems. The right septal artery ran posteriorly (dorsally) and penetrated the ventricular free wall to distribute to the posterior surface of the ventricles. The left septal artery ran anteriorly (ventrally) and penetrated the ventricular free wall to distribute to the anterior surface of the ventricles. The right and left circumflex arteries were directed posteriorly along the atrioventricular sulci. The cardiac veins consisted of three major tributaries: the middle, great, and anterior cardiac veins. The middle cardiac vein ascended along the posterior interventricular sulcus and emptied into the right atrium. The great cardiac vein ran along the anterior interventricular sulcus, entered the space between the left atrium and conus arteriosus and emptied into the right atrium behind the aortic bulb. The anterior cardiac vein drained the anterior surface of the right ventricle and connected to the anterior base of the right atrium. The course of coronary vessels in the quail heart was basically the same as that observed in chick but was different from those of mouse and human.