Jan Schlueter

Popeye domain containing proteins are essential for stress-mediated modulation of cardiac pacemaking in mice

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.

Morphological and molecular left-right asymmetries in the development of the proepicardium: a comparative analysis on mouse and chick embryos.

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. Developmental Dynamics 236:684–695, 2007.

Experimental analyses of the function of the proepicardium using a new microsurgical procedure to induce loss-of-proepicardial-function in chick embryos.

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. Developmental Dynamics 233, 2005. 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) mechanical excision of PE. Both of these techniques, however, have their shortcomings. We have searched, therefore, for new techniques to eliminate the PE. Here, we show that loss‐of‐PE‐function can be induced by photoablation of the PE. Chick embryos were treated in ovo by means of a window in the eggshell at Hamburger and Hamilton (HH) stage 16 (iday 3). The pericardial coelom was opened, and the PE was externally stained with a 1% solution of Rose Bengal by means of a micropipette. Photoactivation of the dye was accomplished by illumination of the operation field with visible light. Examination on postoperative day 1 (iday 4, HH stages 19/20) disclosed complete removal of PE in every experimental embryo. On iday 9 (HH stages 33/34), the survival rate of experimental embryos was 35.7% (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. Developmental Dynamics 233:1454–1463, 2005.

A right-sided pathway involving FGF8/Snai1 controls asymmetric development of the proepicardium in the chick embryo

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.

Development of the proepicardium in Xenopus laevis.

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 (stages 37–46). The PE appears at stage 41 in the form of a cone‐shaped accumulation of mesothelial cells covering the pericardial surface of the right horn of the sinus venosus. No such structure appears on the left sinus horn. At the end of stage 41, the tip of the PE establishes a firm contact with the developing ventricle. A secondary tissue bridge is established facilitating the transfer of PE cells to the heart. During stages 41–46, this tissue bridge is visible in vivo through the transparent body wall. Corresponding to the morphological data, the PE marker gene Tbx18 is expressed only on the right sinus horn suggesting a right‐sided origin of the PE. Left–right lineage tracing has confirmed this idea. These results show that Xenopus PE development proceeds in a bilaterally asymmetric pattern as previously observed in chicks. We speculate that asymmetric PE development is controlled by signals from left–right signaling pathways and that the PE is an indicator for right‐sidedness in Xenopus embryos. Xenopus might be a good model to uncover the role of left–right signaling pathways in the control of asymmetric PE development. Developmental Dynamics 237:3088–3096, 2008.

Epicardial Progenitor Cells in Cardiac Development and Regeneration

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.

Role of fibroblast growth factor signaling during proepicardium formation in the chick embryo

The proepicardium forms at the venous pole of the embryonic heart and gives rise to several cell types of the mature heart. We investigated the role of fibroblast growth factors (FGFs) during proepicardium formation in the chick embryo. Several FGF ligands (Fgf2, Fgf10, and Fgf12) and receptors (Fgfr1, Fgfr2, and Fgfr4) are expressed in the proepicardium. Experimental modulation of FGF signaling in explant cultures affected cell proliferation and survival. In contrast, expression of Tbx18, Wt1, or Tbx5 were unaffected by FGF inhibition. In agreement with the explant data, villous outgrowth of the proepicardium was strongly impaired by FGF inhibition in vivo, however Tbx18 expression was maintained. These data suggest that during proepicardium formation, FGF ligands act as autocrine or paracrine growth factors to prevent apoptosis, maintain proliferation, and to promote villous outgrowth of the proepicardium. However, FGF is not involved in the induction or maintenance of proepicardium‐specific marker gene expression. Developmental Dynamics 239:2393–2403, 2010.

Subpopulation of Proepicardial Cells Is Derived From the Somatic Mesoderm in the Chick Embryo

Rationale: The proepicardium (PE) is a transient structure forming at the venous pole of the heart and gives rise to the epicardium, fibroblasts, and smooth muscle cells. The embryological origin of the PE is presently unclear. Asymmetrical formation of the PE on the right inflow tract is a conserved feature of many vertebrate embryos, and in the chicken is under the control of fibroblast growth factor 8 and snail homolog 1. Objective: To gain further insight into the process of asymmetrical PE formation, we studied the role of TWIST1 during PE formation in the chick embryo. Methods and Results: TWIST1 is asymmetrically expressed on the right side in the somatic mesoderm under the control of snail homolog 1. Fate mapping experiments revealed a contribution of the somatic mesoderm to the PE. After colonization of the heart, this cell lineage gives rise to the epicardium, smooth muscle cells, and potentially fibroblast. Suppression of TWIST1 function in the right coelomic cavity caused a severe disruption of the villous protrusions of the PE and Wilms tumor 1 and transcription factor 21 expression. Rescue with the corresponding mouse cDNA normalized gene expression and PE morphology. Forced expression of TWIST1 on the left side induced ectopic expression domains of Wilms tumor 1 and transcription factor 21. Conclusions: A significant proportion of the PE has its origin outside of the currently proposed domain in the splanchnic layer of the lateral plate mesoderm. The phenotype in embryos subjected to TWIST1 loss- or gain-of-function suggests an important contribution of somatic mesoderm to the mesothelial cell layer of the PE.