Cardiac pacemaker cells create rhythmic pulses that control heart rate; pacemaker dysfunction is a prevalent disorder in the elderly, but little is known about the underlying molecular causes. Popeye domain containing (Popdc) genes encode membrane proteins with high expression levels in cardiac myocytes and specifically in the cardiac pacemaking and conduction system. Here, we report the phenotypic analysis of mice deficient in Popdc1 or Popdc2. ECG analysis revealed severe sinus node dysfunction when freely roaming mutant animals were subjected to physical or mental stress. In both mutants, bradyarrhythmia developed in an age-dependent manner. Furthermore, we found that the conserved Popeye domain functioned as a high-affinity cAMP-binding site. Popdc proteins interacted with the potassium channel TREK-1, which led to increased cell surface expression and enhanced current density, both of which were negatively modulated by cAMP. These data indicate that Popdc proteins have an important regulatory function in heart rate dynamics that is mediated, at least in part, through cAMP binding. Mice with mutant Popdc1 and Popdc2 alleles are therefore useful models for the dissection of the mechanisms causing pacemaker dysfunction and could aid in the development of strategies for therapeutic intervention.

The proepicardium (PE) is a transient structure formed by pericardial coelomic mesothelium at the venous pole of the embryonic heart and gives rise to several cell types of the mature heart. In order to study PE development in chick embryos, we have analyzed the expression pattern of the marker genes Tbx18, Wt1, and Cfc. During PE induction, the three marker genes displayed a left-right asymmetric expression pattern. In each case, expression on the right side was stronger than on the left side. The left-right asymmetric gene expression observed here is in accord with the asymmetric formation of the proepicardium in the chick embryo. While initially the marker genes were expressed in the primitive sinus horn, subsequently, expression became confined to the PE mesothelium. In order to search for signaling factors involved in PE development, we studied Bmp2 and Bmp4 expression. Bmp2 was bilaterally expressed in the sinus venosus. In contrast, Bmp4 expression was initially expressed unilaterally in the right sinus horn and subsequently in the PE. In order to assess its functional role, BMP signaling was experimentally modulated by supplying exogenous BMP2 and by inhibiting endogenous BMP signaling through the addition of Noggin. Both supplying BMP and blocking BMP signaling resulted in a loss of PE marker gene expression. Surprisingly, both experimental situations lead to cardiac myocyte formation in the PE cultures. Careful titration experiments with exogenously added BMP2 or Noggin revealed that PE-specific marker gene expression depends on a low level of BMP signaling. Implantation of BMP2-secreting cells or beads filled with Noggin protein into the right sinus horn of HH stage 11 embryos resulted in downregulation of Tbx18 expression, corresponding to the results of the explant assay. Thus, a distinct level of BMP signaling is required for PE formation in the chick embryo.

The proepicardium (PE) is an embryonic progenitor cell population that delivers the epicardium, the majority of the cardiac interstitium, and the coronary vasculature. In the present study, we compared PE development in mouse and chick embryos. In the mouse, a left and a right PE anlage appear simultaneously, which subsequently merge at the embryonic midline to form a single PE. In chick embryos, the right PE anlage appears earlier than the left and only the right anlage acquires the full PE-phenotype. The left anlage remains in a rudimentary state. The expression patterns of PE marker genes (Tbx18, Wt1) correspond to the morphological data, being bilateral in the mouse and unilateral in the chick. Bmp4, which is unilaterally expressed in the right PE of chick embryos, is symmetrically expressed in the sinus venosus wall cranial to the PE in mouse embryos. Asymmetric development of the chicken PE might reflect side-specific differences in topographical relationships to tissues with PE-inducing or repressing activity or might result from the PE-repressing activity of the right PE, which grows earlier. To test these hypotheses, we analyzed PE development in chick embryos, firstly, subsequent to experimentally induced inversion of PE topographical relationships to neighbouring tissues; secondly, in organ cultures; and, thirdly, subsequent to induction of cardia bifida. In all three experiments, only the right PE develops the full PE phenotype. Our results suggest that PE development might be controlled by the L-R pathway in the chick but not in the mouse embryo.

The proepicardium (PE) is a primarily extracardiac progenitor cell population that colonizes the embryonic heart and delivers the epicardium, the subepicardial and intramyocardial fibroblasts, and the coronary vessels. Recent data show that PE-derived cells additionally play important regulatory roles in myocardial development and possibly in the normal morphogenesis of the heart. Research on the latter topics profits from the fact that loss-of-PE-function can be experimentally induced in chick embryos. So far, two microsurgical techniques were used to produce such embryos: (1) blocking of PE cell transfer with pieces of the eggshell membrane, and (2) 7% (15 of 42). Development of the PE-derivatives was compromised in the heart of every survivor. The abnormalities encompassed hydro-or hemopericardium, epicardium-free areas with aneurysmatic outward bulging of the ventricular wall, thin myocardium, defects of the coronary vasculature, and abnormal tissue bridges between the ventricles and the pericardial wall. Our results show that photoablation of the PE is a powerful technique to induce long-lasting loss-of-PE-function in chick embryos. We have additionally obtained new data that suggest that the embryonic epicardium may make important contributions to the passive mechanics of the developing heart.

The proepicardium (PE) is a transient structure that forms at the venous pole of the embryonic vertebrate heart. This cardiac progenitor cell population gives rise to the epicardium, coronary vasculature, and fibroblasts. In the chicken embryo, the PE displays left-right (L-R) asymmetry and develops only on the right side, while on the left only a vestigial PE is formed, which subsequently gets lost by apoptosis. In this study, we analyzed how the L-R asymmetry pathway affects PE formation. Experimental manipulation of left-side determinants such as Shh, Nodal, and Cfc as well as forced expression of Pitx2 had no effect on the sidedness of PE development. In contrast, inhibition of early-acting regulators of L-R axis formation such as H ؉ /K ؉ -ATPase or primitive streak apoptosis affected the sidedness of PE development. Experimental interference with the right-side determinants Fgf8 or Snai1 prevented PE formation, whereas ectopic left-sided expression of Fgf8 or Snai1 resulted in bilateral PE development. These data provide novel insight into the molecular control of asymmetric morphogenesis suggesting that also the right side harbors an instructive signaling pathway that is involved in the control of PE development. This pathway might be of general relevance for setting up L-R asymmetries at the venous pole of the heart. left-right asymmetry ͉ Tbx18 ͉ Pitx2 ͉ venous pole morphogenesis T he left-right (L-R) axis controls asymmetric organogenesis in vertebrates (1-3). In lower chordates, Xenopus, and chick, breaking of the initial bilateral body symmetry involves the asymmetric distribution of ion channels and transporters, resulting in the development of membrane potential differences between the left and right body side, which may drive an asymmetric transport of small molecules through gap junctions (4, 5). In mammals, Xenopus, and zebrafish, ciliated cells in the organizer generate a fluid flow to the left that transports L-R determinants (6). In mammals, this nodal flow is believed to be the sole mechanism by which L-R asymmetry is initiated (7). In the chicken embryo, asymmetric gene expression in Hensen's node of gastrula stage embryos is an important intermediate step of establishing L-R asymmetry (2). Shh, for example, is asymmetrically expressed on the left side of Hensen's node and induces Nodal expression in a small left-sided domain adjacent to Hensen's node (8). Nodal establishes a large expression domain in the left lateral plate mesoderm (LPM) and induces the expression of Pitx2, a homeobox gene of the bicoid class (7). Pitx2 determines morphological L-R asymmetries in most organ systems (9-12) via modulation of cell-cell adhesion, cell morphology, extracellular matrix composition, spindle orientation, and cell proliferation (13-15).A right-sided regulatory cascade of gene expression is also initiated in Hensen's node including Bmp4 that induces asymmetric expression of Fgf18 and Fgf8 on the right side (16-18). It is believed that the right side does not initiate its own sidespecific morphogene...

The proepicardium (PE) is an embryonic progenitor cell population, which provides the epicardium, the majority of the cardiac interstitium, the coronary vasculature and possibly some cardiomyocytes. Recent studies have documented (1) the presence of bilaterally paired PE anlagen in several vertebrates, and (2) species-specific differences in the fate of the left and right PE anlagen. Here, we document PE development in Xenopus laevis (

Left-right asymmetry of internal organs is widely distributed in the animal kingdom. The chick and mouse embryos have served as important model organisms to analyze the mechanisms underlying the establishment of the left-right axis. In the chick embryo many genes have been found to be asymmetrically expressed in and around the node, while the same genes in the mouse show symmetric expression patterns. In the mouse there is strong evidence for an establishment of left-right asymmetry through nodal cilia. In contrast, in the chick and in many other organisms left-right asymmetry is probably generated by an early-acting event involving membrane depolarization. In both birds and mammals a conserved Nodal-Lefty-Pitx2 module exists that controls many aspects of asymmetric morphogenesis. This review also gives examples of divergent mechanisms of establishing asymmetric organ formation. Thus there is ample evidence for conserved and non-conserved strategies to generate asymmetry in birds and mammals.

The epicardium forms an epithelial layer on the surface of the heart. It is derived from a cluster of mesothelial cells, which is termed the proepicardium. The proepicardium gives rise not only to the epicardium but also to epicardium-derived cells. These cells populate the myocardial wall and differentiate into smooth muscle cells, fibroblast, and possibly endothelial cells. In this review, the formation of the proepicardium is discussed. Marker genes, suitable to identify these cells in the embryo and in the adult, are introduced. Recent evidence suggests that the PE is made up of distinct cell populations. These cell lineages can be distinguished on the basis of marker gene expression and differ in their differentiation potential. The role of the epicardium as a resource for cardiac stem cells and its importance in cardiac regeneration is also discussed.