Jörg Männer

Lineage and Morphogenetic Analysis of the Cardiac Valves

We used a genetic lineage-labeling system to establish the material contributions of the progeny of 3 specific cell types to the cardiac valves. Thus, we labeled irreversibly the myocardial (&agr;MHC-Cre+), endocardial (Tie2-Cre+), and neural crest (Wnt1-Cre+) cells during development and assessed their eventual contribution to the definitive valvar complexes. The leaflets and tendinous cords of the mitral and tricuspid valves, the atrioventricular fibrous continuity, and the leaflets of the outflow tract valves were all found to be generated from mesenchyme derived from the endocardium, with no substantial contribution from cells of the myocardial and neural crest lineages. Analysis of chicken-quail chimeras revealed absence of any substantial contribution from proepicardially derived cells. Molecular and morphogenetic analysis revealed several new aspects of atrioventricular valvar formation. Marked similarities are seen during the formation of the mural leaflets of the mitral and tricuspid valves. These leaflets form by protrusion and growth of a sheet of atrioventricular myocardium into the ventricular lumen, with subsequent formation of valvar mesenchyme on its surface rather than by delamination of lateral cushions from the ventricular myocardial wall. The myocardial layer is subsequently removed by the process of apoptosis. In contrast, the aortic leaflet of the mitral valve, the septal leaflet of the tricuspid valve, and the atrioventricular fibrous continuity between these valves develop from the mesenchyme of the inferior and superior atrioventricular cushions. The tricuspid septal leaflet then delaminates from the muscular ventricular septum late in development.

The Origin, Formation and Developmental Significance of the Epicardium: A Review

Questions on the embryonic origin and developmental significance of the epicardium did not receive much recognition for more than a century. It was generally thought that the epicardium was derived from the outermost layer of the primitive myocardium of the early embryonic heart tube. During the past few years, however, there has been an increasing interest in the development of the epicardium. This was caused by a series of new embryological data. The first data showed that the epicardium did not derive from the primitive myocardium but from a primarily extracardiac primordium, called the proepicardial serosa. Subsequent data then suggested that the proepicardial serosa and the newly formed epicardium provided nearly all cellular elements of the subepicardial and intermyocardial connective tissue, and of the coronary vasculature. Recent data even suggest important modulatory roles of the epicardium and of other proepicardium-derived cells in the differentiation of the embryonic myocardium and cardiac conduction system. The present paper reviews our current knowledge on the origin and embryonic development of the epicardium.

DOES THE SUBEPICARDIAL MESENCHYME CONTRIBUTE MYOCARDIOBLASTS TO THE MYOCARDIUM OF THE CHICK EMBRYO HEART? A QUAIL-CHICK CHIMERA STUDY TRACING THE FATE OF THE EPICARDIAL PRIMORDIUM

Morris (J. Anat., 1976;121:47–64) proposed that the subepicardial mesenchyme might represent a continuing source of myocardioblasts during embryonic and fetal development. Recent studies have shown that the epicardium and subepicardial mesenchyme, and the coronary vasculature are all derived from a region of the pericardial wall, called the proepicardial serosa. In avian embryos, the cells from the proepicardial serosa colonize the heart via a secondary tissue bridge formed by attachment of proepicardial villi to the heart. In the present study, Morriss hypothesis was tested by tracing the fate of the proepicardial serosa. This was achieved by constructing quail‐chick chimeras. The proepicardial serosa was transplanted from HH16/17 quail embryos to HH16/17 chick embryos (ED3). A new transplantation technique facilitated an orthotopic attachment of the quail proepicardial villi to the chicken heart, and prevented the attachment of the chicken proepicardial villi to the heart. The fate of the grafted quail cells was traced in chimeras from ED4 to ED18 with immunohistochemistry, using quail‐specific antibodies (QCPN, QH‐1). From ED4 onward, the transplant was connected to the dorsal heart wall via its proepicardial villi. Starting from the point of attachment of the quail proepicardial villi to the heart, the originally naked myocardium became almost completely covered by quail‐derived epicardium, and quail mesenchymal cells populated the subepicardial, myocardial, and subendocardial layers including the av‐endocardial cushions. Quail cells formed the endothelial and smooth muscles cells of the coronary vessels, and the perivascular and intramyocardial fibroblasts. Quail myocardial cells were never found in the subepicardial, myocardial, and subendocardial layers. This suggests that the subepicardial mesenchyme normally does not contribute a substantial number of myocardioblasts to the developing avian heart. The new transplantation technique presented facilitates the production of chimeric hearts in which the derivatives of the proepicardial serosa are almost completely of donor origin. Thistechnique might be useful for future studies analyzing the role of certain genes in cardiac development by the creation of somatic transgenics. Anat Rec 255:212–226, 1999.

Cardiac looping in the chick embryo: A morphological review with special reference to terminological and biomechanical aspects of the looping process †

Understanding early cardiac morphogenesis, especially the process of cardiac looping, is of fundamental interest for diverse biomedical disciplines. During the past few years, remarkable progress has been made in identifying molecular signaling cascades involved in the control of cardiac looping. Given the rapid accumulation of new data on genetic, molecular, and cellular aspects of early cardiac morphogenesis, and given the widespread interest in cardiac looping, it seems worth reviewing those aspects of the looping process that have received less attention during the past few years. These are terminological problems, the “gross” morphological aspects, and the biomechanical concepts of cardiac looping. With respect to terminology, emphasis is given to the unperceived fact that different viewpoints exist as to which part of the normal sequence of morphogenetic events should be called cardiac looping. In a short‐term version, which is preferred by developmental biologists, cardiac looping is also called dextral‐ or rightward‐looping. Dextral‐looping comprises only those morphogenetic events leading to the transformation of the originally straight heart tube into a c‐shaped loop, whose convexity is normally directed toward the right of the body. Cardioembryologists, however, regard cardiac looping merely as a long‐term process that may continue until the subdivisions of the heart tube and vessel primordia have approximately reached their definitive topographical relationship to each other. Among cardioembryologists, therefore, three other definitions are used. Taking into account the existence of four different definitions of the term cardiac looping will prevent some confusion in communications on early cardiac morphogenesis. With respect to the gross morphological aspects, emphasis is given to the following points. First, the straight heart tube does not consist of all future regions of the mature heart but only of the primordia of the apical trabeculated regions of the future right and left ventricles, and possibly a part of the primitive conus (outflow tract). The remaining part of the primitive conus and the primordia of the great arteries (truncus arteriosus), the inflow of both ventricles, the primitive atria, and the sinus venosus only appear during looping at the arterial (truncus arteriosus) and venous pole (other primordia). Second, dextral‐looping is not simply a bending of the straight heart tube toward the right of the body, as it has frequently been misinterpreted. It results from three different morphogenetic events: (a) bending of the primitive ventricular region of the straight heart tube toward its original ventral side; (b) rotation or torsion of the bending ventricular region around a craniocaudal axis to the right of the body, so that the original ventral side of the heart tube finally forms the right convex curvature and the original dorsal side forms the left concave curvature of the c‐shaped heart loop; (c) displacement of the primitive conus to the right of the body by kinking with respect to the arterial pole. Third, dextral‐looping does not bring the subdivisions of the heart tube and vessel primordia approximately into their definitive topographical relationship to each other. This is achieved by the morphogenetic events following dextral‐looping. This review seeks to bring together data from the diverse disciplines working on the developing heart. Anat Rec 259:248–262, 2000.

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

We have previously identified several members of the Wnt/β-catenin pathway that are differentially expressed in a mouse model with deficient coronary vessel formation. Systemic ablation of β-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 β-catenin in coronary arteriogenesis, we have generated conditional β-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 β-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 β-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-β-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 β-catenin pathway in coronary arteriogenesis.

Experimental study on the formation of the epicardium in chick embryos

In chick embryos, the formation of the epicardium proceeds from the attachment of a secondary sinuventricular mesocardium. This mesocardium is formed by the adhesion of pericardial villi with the dorsal surface of the heart. It was the aim of this study to clarify the role of the pericardial villi in the formation of the epicardium. For this purpose, the contact between the pericardial villi and the heart was prevented by placement of a piece of the shell membrane between them. After re-incubation, the hearts of the experimental embryos could be assigned to one of two different groups: hearts completely lacking a secondary mesocardium (Group A), and hearts without the sinu-ventricular but with a dystopic secondary mesocardium (Group B). In Group A, the formation of the epicardium and subepicardial mesenchyme was found to be severely disturbed. In Group B, the formation of the epicardium proceeded from the point of attachment of a dystopic secondary mesocardium; defective development of the subepicardial mesenchyme was not encountered. These results support the view that the epicardium is derived from the pericardial epithelium.

The development of pericardial villi in the chick embryo

SummaryThe development of pericardial villi and their relation to the development of the cardiac surface was studied in chick embryos from the 3rd to 10th day of incubation by scanning electron microscopy. During the 3rd day of incubation (stage 14–17 HH) the coelomic epithelium covering the ventral wall of the sinus venosus forms villous protrusions. By the end of the 3rd day (stage 17 HH) these protrusions contact the dorsal wall of the heart, so that a secondary dorsal mesocardium is formed. This bridges the pericardial cavity between the ventral wall of the sinus venosus and the dorsal base of the ventricles. This sinu-ventricular mesocardium exists only temporarily, as on the 8th day of incubation it becomes a part of the cardiac wall due to fusion with the epicardium of the coronary sulcus. During the 4th and 5th day of incubation (stage 17 – 25 HH), the formation of the epicardium proceeds from the point of attachment of the sinu-ventricular mesocardium. Although these findings suggest that the epithelium of the villous protrusions spreads over the surface of the embryonic heart, one cannot exclude other hypotheses on epicardial origin. The impression of a spreading epicardium could also be created if epicardial cells were to delaminate from a local epithelium in a temporally and spatially organized pattern.

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.

The anatomy of cardiac looping: A step towards the understanding of the morphogenesis of several forms of congenital cardiac malformations

The early embryonic heart of vertebrates is a simple tubular pump. During the early phases of its development, the initially straight embryonic heart tube becomes transformed into a helically wound loop that is normally seen with a counterclockwise winding. This process is named cardiac looping. Such looping not only establishes the basic type of topological left‐right asymmetry of the ventricular chambers but, additionally, is also said to bring the segments of the heart tube and the developing great vessels into an approximation of their definitive topographical relationships. Cardiac looping is, therefore, regarded as the key process in cardiac morphogenesis and pathologists have speculated since the beginning of the 20th century that several forms of congenital cardiac malformations (e.g., with mirror‐imaged arrangement of the ventricular chambers) might result from disturbances in looping morphogenesis. In this article a review is given on (1) differences in the usage of the term cardiac looping; (2) our current knowledge of the dynamically changing anatomy of the looping embryonic heart; and (3) our current knowledge of the role of looping anomalies in the morphogenesis of congenital cardiac malformations. Clin. Anat. 22:21–35, 2009.

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.