Abstract-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 (␣MHC-Creϩ), endocardial (Tie2-Creϩ), and neural crest (Wnt1-Creϩ) 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.

Abstract-The venous pole of the mammalian heart is a structurally and electrically complex region, yet the lineage and molecular mechanisms underlying its formation have remained largely unexplored. In contrast to classical studies that attribute the origin of the myocardial sinus horns to the embryonic venous pole, we find that the sinus horns form only after heart looping by differentiation of mesenchymal cells of the septum transversum region into myocardium. The myocardial sinus horns and their mesenchymal precursor cells never express Nkx2-5, a transcription factor critical for heart development. In addition, lineage studies show that the sinus horns do not derive from cells previously positive for Nkx2-5. In contrast, the sinus horns express the T-box transcription factor gene Tbx18. Mice deficient for Tbx18 fail to form sinus horns from the pericardial mesenchyme and have defective caval veins, whereas the pulmonary vein and atrial structures are unaffected. Our studies define a novel heart precursor population that contributes exclusively to the myocardium surrounding the sinus horns or systemic venous tributaries of the developing heart, which are a source of congenital malformation and cardiac arrhythmias. Key Words: sinus horns Ⅲ congenital heart defect Ⅲ Nkx2-5 Ⅲ Tbx18 Ⅲ morphogenesis Ⅲ recruitment T he systemic venous return of the heart consists of multiple anatomical components including the proximal myocardial part of the right superior and inferior caval veins, the coronary sinus (the persisting left caval vein in the mouse), and the sinus venarum. These structures are thought to be the mature counterparts of the right and left sinus horns in the embryo, which are the myocardial parts of the common cardinal veins upstream of the venous valves that bulge into the pericardial cavity. These, in turn, are presumed to derive from the embryonic venous pole or inflow tract of the forming heart and the common cardinal veins. 1,2 Developmental disorders of the heart, which include malformations of the pulmonary and systemic venous returns, 3,4 represent the most common human birth defects. 5,6 In addition, several specific components of the venous returns are found to be the origin of arrhythmias. 7-9 Recent 3D reconstruction and genetic analyses have greatly improved our insight into the morphogenesis of the systemic and pulmonary venous returns. 2,3,10 Nevertheless, the cellular origin of the components of the systemic and pulmonary venous return and the genetic mechanisms underlying their formation are not known.The embryonic heart of amniotes initially represents a tube consisting of precursor cells for most of the left ventricle and a small portion of the atria. Outflow tract, right ventricle, and large portions of the atria are only subsequently recruited from a second lineage of mesenchymal cells. 11-13 Nkx2-5, which encodes a homeobox transcription factor, is expressed in both the first and second lineages of the heart and plays pivotal roles in early and late steps of cardiogenesis. 13,14 Using ...

We identified the T-box transcription factor Tbx3 as a novel and accurate marker for the central conduction system. Our analysis implicates a role for Tbx3 in repressing a chamber-specific program of gene expression in regions from which the components of the central conduction system are subsequently formed.

Increase in cell size and proliferation of myocytes are key processes in cardiac morphogenesis, yet their regionalization during development of the heart has been described only anecdotally. We have made quantitative reconstructions of embryonic chicken hearts ranging in stage from the fusion of the heart-forming fields to early formation of the chambers. These reconstructions reveal that the early heart tube is recruited from a pool of rapidly proliferating cardiac precursor cells. The proliferation of these small precursor cells ceases as they differentiate into overt cardiomyocytes, producing a slowly proliferating straight heart tube composed of cells increasing in size. The largest cells were found at the ventral side of the heart tube, which corresponds to the site of the forming ventricle, as well as the site where proliferation is reinitiated. The significance of these observations is 2-fold. First, they support a model of early cardiac morphogenesis in 2 stages. Second, they demonstrate that regional increase in size of myocytes contributes significantly to chamber formation.

Abstract-Recent studies have shown that the primary heart tube continues to grow by addition of cells from the coelomic wall. This growth occurs concomitantly with embryonic folding and formation of the coelomic cavity, making early heart formation morphologically complex. A scarcity of data on localized growth parameters further hampers the understanding of cardiac growth. Therefore, we investigated local proliferation during early heart formation. Firstly, we determined the cell cycle length of primary myocardium of the early heart tube to be 5.5 days, showing that this myocardium is nonproliferating and implying that initial heart formation occurs solely by addition of cells. In line with this, we show that the heart tube rapidly lengthens at its inflow by differentiation of recently divided precursor cells. To track the origin of these cells, we made quantitative 3D reconstructions of proliferation in the forming heart tube and the mesoderm of its flanking coelomic walls. These reconstructions show a single, albeit bilateral, center of rapid proliferation in the caudomedial pericardial back wall. This center expresses Islet1. Cell tracing showed that cells from this caudal growth center, besides feeding into the venous pole of the heart, also move cranially via the dorsal pericardial mesoderm and differentiate into myocardium at the arterial pole. Inhibition of caudal proliferation impairs the formation of both the atria and the right ventricle. These data show how a proliferating growth center in the caudal coelomic wall elongates the heart tube at both its venous and arterial pole, providing a morphological mechanism for early heart formation. Key Words: cardiovascular development Ⅲ proliferation Ⅲ heart fields Ⅲ Islet1 Ⅲ bromodeoxy uridine Ⅲ quantitative 3D reconstruction T he heart is sculpted by precisely orchestrated developmental programs 1,2 that are prone to errors, leading to high incidences of congenital malformations. 3 Proliferation, although not the only mechanism, is an important parameter for the formation of the heart. 4 -8 Research on heart formation was recently revolutionized by the understanding that the initially formed myocardial heart tube continues to grow by recruitment of cells that originate from flanking mesoderm, dubbed the second heart field. 9 -11 This second heart field was originally reserved for cells feeding into the outflow of the primary heart tube to form the right ventricle. 9 Shortly after these findings, cells were also shown to be added to the inflow, 11 and a debate developed regarding the existence of multiple fields of cardiac precursor cells. [12][13][14][15] Limiting factors in this debate are the virtual lack of a 3D context of cardiac growth and the scarcity of data of locally involved parameters, such as proliferation. Early heart formation is of perplexing 3D complexity, because it occurs concomitantly with folding of the embryonic disc and formation of the coelomic cavity. Most likely, this complexity has contributed to the diversity of opinions, because o...

Abstract-Firm knowledge about the formation of the atrial components and of the variations seen in congenital cardiac malformations and abnormal atrial rhythms is fundamental to our understanding of the normal structure of the definitive atrial chambers. The atrial region is relatively inaccessible and has continued to be the source of disagreement. Seeking to resolve these controversies, we made three-dimensional reconstructions of the myocardial components of the developing atrium, identifying domains on the basis of differential expression of myocardial markers, connexin40, and natriuretic precursor peptide A. These reconstructions, made from serial sections of mouse embryos, show that from the outset of atrial development, the systemic and pulmonary veins are directly connected to the atrium. Relative to the systemic junctions, however, the pulmonary venous junction appears later. Our experience shows that three-dimensional reconstructions have three advantages. First, they provide clear access to the combined morphological and molecular data, allowing clarification and verification of morphogenetic concepts for nonmorphological experts and setting the scene for further discussion. Second, they demonstrate that, from the outset, the myocardium surrounding the pulmonary veins is distinct from that clothing the systemic venoatrial junctions. Third, they reveal an anatomical and molecular continuity between the entrance of the systemic venous tributaries, the internodal atrial myocardium, and the atrioventricular region. All these regions are derived from primary myocardium, providing a molecular basis for the observed nonrandom distribution of focal right atrial tachycardias. Key Words: internodal tracts Ⅲ phenotyping Ⅲ dorsal mesocardium Ⅲ conduction system T he atrial chambers represent one of the most complex morphological and electrical areas of the heart. These chambers are the origin of many abnormal rhythms 1 and the seat of many congenital malformations. 2 There are many reasons, therefore, why we need to understand their origin and development. Because of their rapid transformation and dorsal position, these aspects are difficult to study experimentally and many controversies persist regarding their development, ranging from the very existence of a sinus venosus, 3 through whether the pulmonary vein terminates within the sinus venosus 4 -6 or the atrium, 3,7 to the myocardium surrounding the pulmonary veins as a substrate for arrythmogenesis. 8,9 It is our contention that these ongoing issues will only be resolved by making three-dimensional reconstructions of the dynamic and complex structure of the developing heart, associated with the patterns of relevant genes. In the present study, therefore, we used three-dimensional reconstructions of myocardial probes to visualize the atrial components in the mouse. We further subdivided this myocardium into different phenotypes on the basis of differential expression of the markers connexin40 (Cx40) and natriuretic precursor peptide (Nppa, also known as ANF)...

The study of the genetic regulation of embryonic development requires the three-dimensional (3D) mapping of gene expression at the microscopic level. Despite the recent burst in the number of methods focusing on 3D reconstruction of embryonic specimens, an adequate and accessible 3D reconstruction protocol for the visualization of patterns of gene expression is lacking. In this communication we describe a protocol that was developed for the 3D visualization of patterns of gene expression determined by in situ hybridization (ISH) on serial sections. The method still requires tissue sectioning, due to penetration limits of the specific staining agents into whole embryo preparations. With regard to expenditure of resources, i.e., hardware, software, and time, the protocol is relatively undemanding. Because the variation between specimens requires the visualization of multiple specimens per stage, it was decided to "do more, less well." The current protocol, therefore, results in reconstructions of sufficient, but not the highest, quality. The use of the protocol is demonstrated on a series of serially sectioned mouse hearts, ranging from embryonic day 8.5 to 14.5. The myocardium of the hearts was identified by ISH using a mixture of specific mRNA probes and reconstructed.

Closure of the primary atrial foramen is achieved by fusion of the atrioventricular cushions with the mesenchymal cap on the leading edge of the muscular primary atrial septum. A fourth component involved is the vestibular spine, originally described by His in 1880 as an intra-cardiac continuation of the extra-cardiac mesenchyme of the dorsal mesocardium. The morphogenesis of this area is of great clinical interest, because of the high incidence of atrial and atrioventricular septal defects. Nonetheless, the origin of the participating components is largely unknown. Here we report that the primary atrial foramen is surrounded in its entirety by mesenchyme derived from endocardium. A second population of mesenchyme not derived from endocardium was observed at the caudal margin of the mesenchymal atrial cap, entirely embedded within the mesenchyme derived from endocardium and contiguous with the mesenchyme of the dorsal mesocardium. Our reconstructions show this second population does indeed take the form of a short spine, albeit that it is the right pulmonary ridge, rather than this spine, that protrudes into the atrial lumen. From the stance of morphological description, therefore, there is little thus far to substantiate the existence of an atrial spine. It is currently thought that the atrioventricular (AV) cushions, along with the primary atrial septum and the mesenchymal cap carried on its leading edge, are the main contributors to the process of atrial septation. A growing body of evidence points to the involvement of a fourth component, namely the vestibular spine ("spina vestibuli"). [1][2][3][4][5][6][7][8] The vestibular spine was initially nominated as playing such a role in 1880, by Wilhelm His the elder. 9 He described the spine as a triangular mesenchymal wedge, which protruded into the lumen of the atrium from a non-muscular area, which he called the "area interposita," in the dorsal wall of the common atrium ( Figure 1, stippled circle). The spine then disappeared from view for more than a century, eventually being retrieved by several workers, 1,2,4 -6,10,11 albeit with disagreements concerning the form and origin of this tissue. To clarify this, we have performed a lineage study using Tie2-Cre mice 12 to label endocardium and endocardium-derived mesenchyme, in combination with threedimensional reconstructions 13 to permit independent evaluation of the structures involved in atrial septation. Materials and MethodsThe Tie2-Cre and R26R transgenic mouse lines have been described previously. 12,14 Detection of ␤-galactosidase activity and immunohistochemistry were performed on 20-m thick cryostat sections. 15 Non-radioactive in situ hybridization analysis was performed on 12-m thick paraffin sections. 16 Three-dimensional visualization and geometry reconstruction of patterns of gene expression was then achieved. 13,17 Movie clips of these reconstructions are available on request. ResultsAt embryonic day (E) 9.5, it is possible to recognize two ridges, of equal size, at the connection of the dorsal m...