Role of the left interventricular sulcus in formation of the interventricular septum and crista supraventricularis in normal human cardiogenesis

Serial sections of normal human embryos were studied and three-dimensional images reconstructed to determine the early development of the interventricular septum. The position of the interventricular septum is determined in stage 9 of normal development by the formation of the left interventricular sulcus. As a result of unknown properties of the cells of the myocardial layer, the left interventricular sulcus persists while the right disappears, producing the initial lateral asymmetry of the primary heart tube. By stage 14, the left interventricular sulcus forms a spiral which is continuous with the developing interventricular septum. The dorsal limb of the spiral passes to the right between the atrioventricular canal and the origin of the outflow tract, and is lost in the wall of the trabeculated right ventricle. It appears that this dorsal limb of the spiral is the precursor of part of the cirsta supraventricularis. The midportion of the sulcus, the bulboventricular groove, becomes the socalled fibrous continuity between the aortic and mitral valves. The ventral limb of the spiral passes caudally in the anterior interventricular groove and then dorsally and cranially toward the dorsal cushion of the atrioventricular canal. The ventral limb of the spiral is continuous with the crest of the muscular interventricular septum, which develops by apposition of tissue from the expanding right and left ventricles. From stage 14 to stage 19, the muscular interventricular septum, the atrioventricular endocardial cushions, and the ventricular end of the spiral ridges of the outflow tract appose and fuse. Subsequent formation of the membranous interventricular septum completes the physical separation of the right and left ventricles.     

Role of the left interventricular sulcus in formation of interventricular septum and crista supraventricularis in normal human cardiogenesis.

Serial sections of normal human embryos were studied and three-dimensional images reconstructed to determine the early development of the interventricular septum. The position of the interventricular septum is determined in stage 9 of normal development by the formation of the left interventricular sulcus. As a result of unknown properties of the cells of the myocardial layer, the left interventricular sulcus persists while the right disappears, producing the initial lateral asymmetry of the primary heart tube. By stage 14, the left interventricular sulcus forms a spiral which is continuous with the developing interventricular septum. The dorsal limb of the spiral passes to the right between the atrioventricular canal and the origin of the outflow tract, and is lost in the wall of the trabeculated right ventricle. It appears that this dorsal limb of the spiral is the precursor of part of the cirsta supraventricularis. The midportion of the sulcus, the bulboventricular groove, becomes the so-called fibrous continuity between the aortic and mitral valves. The ventral limb of the spiral passes caudally in the anterior interventricular groove and then dorsally and cranially toward the dorsal cushion of the atrioventricular canal. The ventral limb of the spiral is continuous with the crest of the muscular interventricular septum, which develops by apposition of tissue from the expanding right and left ventricles. From stage 14 to stage 19, the muscular interventricular septum, the atrioventricular endocardial cushions, and the ventricular end of the spiral ridges of the outflow tract appose and fuse. Subsequent formation of the membranous interventricular septum completes the physical separation of the right and left ventricles.     

The contribution of the inferior endocardial cushion of the atrioventricular canal to cardiac septation and to the development of the atrioventricular valves: Study in the chick embryo

The contribution of the inferior endocardial cushion of the atrioventricular canal to cardiac septation and to the development of the atrioventricular valves was studied in the chick embryo by in vivo labelling techniques. The study was performed in White Leghorn chick embryos in which the dorsal cushion was labelled at stage 20-21 (Hamburger and Hamilton, 1951), when the endocardial cushions were not yet fused. The embryos were sacrificed at stage 35 (mature heart). These experiments allow us to conclude that the inferior atrioventricular cushion gives origin to: (a) that part of the cardiac septum between the septal insertion of the antero-septal leaflet of the mitral valve and the fibrous ridge which is the equivalent to the human septal leaflet of the tricuspid valve (atrioventricular septum); (b) the region of the interatrial and the interventricular septa adjacent to the atrioventricular septum; (c) the portion of the antero-septal leaflet of the mitral valve which inserts into the septum; (d) the fibrous ridge corresponding to the septal leaflet of the tricuspid valve. Microdissection shows that, when they appear at stage 18, the superior and inferior endocardial cushions of the atrioventricular canal are in continuity, without boundaries, with both the interatrial and interventricular septa. Therefore, each atrioventricular orifice opens into its corresponding ventricle, there being no stage in the development of the chick embryo heart in which the atrioventricular orifices are connected to the left ventricle at the same time. The development of the atrioventricular canal is similar in the chick and human.     

Septation and valvar formation in the outflow tract of the embryonic chick heart

There is no agreement, in the chick, about the number of the endocardial cushions within the outflow tract or their pattern of fusion. Also, little is known of their relative contributions to the formation of the arterial valves, the subpulmonary infundibulum, and the arterial valvar sinuses. As the chick heart is an important model for studying septation of the outflow tract, our objective was to clarify these issues. Normal septation of the outflow tract was studied in a series of 60 staged chick hearts, by using stained whole-mount preparations, serial sections, and scanning electron microscopy. A further six hearts were examined subsequent to hatching. At stage 21, two pairs of endocardial cushions were seen within the developing outflow tract. One pair was positioned proximally, with the other pair located distally. By stage 25, a third distal cushion had developed. This finding was before the appearance of two further, intercalated, endocardial cushions, also distally positioned, which were first seen at stage 29. In the arterial segment, the aortic and pulmonary channels were separated by the structure known as the aortopulmonary septum. The dorsal limb of this septum penetrated the distal dorsal cushion, whereas the ventral limb grew between the remaining two distal cushions, both of which were positioned ventrally. The three distal endocardial cushions, and the two intercalated endocardial cushions, contributed to the formation of the leaflets and sinuses of the arterial roots. The two proximal cushions gave rise to a transient septum, which later became transformed into the muscular component of the subpulmonary infundibulum. Concomitant with these changes, an extracardiac tissue plane was formed which separated this newly formed structure from the sinuses of the aortic root. Our study confirms that three endocardial cushions are positioned distally, and two proximally, within the developing outflow tract of the chick. The pattern of the distal cushions, and the position of the ventral limb of the aortopulmonary septum, differs significantly from that seen in mammals.     

BMP4 is required in the anterior heart field and its derivatives for endocardial cushion remodeling, outflow tract septation, and semilunar valve development

The endocardial cushions play a critical role in septation of the four-chambered mammalian heart and in the formation of the valve leaflets that control blood flow through the heart. Within the outflow tract (OFT), both cardiac neural crest and endocardial-derived mesenchymal cells contribute to the endocardial cushions. Bone morphogenetic protein 4 (BMP4) is required for endocardial cushion development and for normal septation of the OFT. In the present study, we show that anterior heart field (AHF)-derived myocardium is an essential source of BMP4 required for normal endocardial cushion expansion and remodeling. Loss of BMP4 from the AHF in mice results in an insufficient number of cells in the developing OFT endocardial cushions, defective cushion remodeling, ventricular septal defects, persistent truncus arteriosus, and abnormal semilunar valve formation.     

The position of the left and right ventricular outlets during septation

Summary A comparative study was made of the relative position of the outflow tracts of chicken and rat hearts with respect to the ventricles during septation. For this purpose the position of the left and right ventricular outlet including the aortic and pulmonary valve primordia and the left and right ventricle were established with respect to the midsagittal plane of the embryo, using reconstructions of serial sections of chicken (stage 28–30) and rat (stage 28–30) embryos. In the chicken embryo no rotation of the outflow tract occurs, i.e. the position of the aortic and pulmonary valve primordia with respect to the left and right ventricle remains the same. In the rat embryo a clockwise rotation of the aortic and pulmonary valve primordia with respect to the ventricles does occur. This is in fact a detorsion. The left and right ventricle and the left ventricular outlet do not show change in position with regard to the midsagittal plane. The left ventricular outlet always straddles the interventricular septum, both lying in the midsagittal plane. These interspecies differences in the degree of detorsion of the outflow channels before septation may explain the differences in the extent of the region of contact between the endocardial outflow tract ridges.     

The development of the arterial outflow tract in the chick embryo heart

Bloodstream flow patterns have been outlined in the arterial outflow tract (ventricular outflow tract and bulbus arteriosus) of the chick embryo heart during the period in which septation takes place, Hemodynamic factors underlying flow changes during this period are discussed. The mapping of flow patterns did not support the concept of a conoventricular flange reported previously. Septation was found to take place between two separate and discrete bloodstreams. The cellular nature of the aorticopulmonary septum has been described. The spiral ridges that form this septum expand by cellular growth, explaining the ability of this septum to develop against the direction of blood flow. The aorticopulmonary septum divides about two-thirds of the arterial outflow tract; the final partitioning of the most proximal portion of the outflow tract was found to take place by means of the apposition of endocardial cushion tissue masses. Failure of aorticopulmonary septum development (truncus arteriosus communis persistens) was found to follow fusion of the bloodstreams in experimental studies. In experimental aortic stenosis the appearance of a small left stream was found to be followed by the development of a stenotic aorta. Thus in the first instance the septum apparently cannot develop unless the streams remain separate and in the second case the size of the prhnordial bloodstreams appears to determine the diameter of the vessel.     

鸡胚心内膜垫及室间隔的发生

本研究用扫描电镜、透射电镜、光学显微镜及组织化学等方法观察了早期鸡胚心脏发生中的心内膜垫及室间隔的发生过程,其结果如下: 1.心内膜垫的发生起始于内膜细胞的增殖,向心胶质内输送内膜垫细胞。内膜垫细胞分裂增殖向肌层方向移动。当出现内膜垫细胞的贮备层以后,表面的内膜细胞停止增殖。融合的内膜垫参与房室口的形成,其右侧结节向尾侧延伸与室间隔融合。 2.早期心内膜垫的心胶质中含有大量透明质酸,以一定的方向排列成“轨道”,内膜垫细胞沿“轨道”向肌层方向迁移并改变基质成分及排列方式。 3.肌性室间隔是心室壁分化形成的小梁在室间沟内侧聚集而成。室间隔背侧部与内膜垫右侧结节的下延部分融合,腹侧部与锥隔相延续。     

Programmed cell death in the developing heart: regulation by BMP4 and FGF2.

Programmed cell death, or apoptosis, plays an important role in embryonic development. To provide new insights into the role of programmed cell death in cardiac development, we examined the hearts of the murine embryos from E9.5 to postnatal day 3. Using terminal transferase-mediated dUTP nick end-labeling assays, apoptosis was detected in the endocardial cushions and myocardium from E11.5 to postnatal day 3 (P3). In the ventricular myocardium, more apoptotic cells were observed in the left than right ventricles throughout embryonic and early postnatal development. Apoptosis was also present in the trabeculae and papillary muscles of the ventricles. In the outflow tract, cell death was present in the endocardial cushions before they fuse to form the conotruncal septum (E11.5-E12. 5) and reached a peak intensity when the conotruncal septum formed (E13.5). In the atrioventricular (AV) endocardial cushions, cell death was detected in the fusion seam of the cushion tissues at E12. 5 and E13.5 during AV septation. When the patterns of apoptosis were compared with patterns of cell division, we found that programmed cell death occurred in the areas in the endocardial cushions and trabeculae where rates of cell proliferation were low. We also found that programmed cell death was regulated by the growth factors, BMP4 and FGF2, in vitro. BMP4 induced, whereas FGF2 inhibited, apoptosis in both endocardial cushions and ventricular myocardium. Overall, our observations show that there is apoptosis in the regions where fusion or remodeling of tissues occurs. We also show that cardiac programmed cell death can be influenced by growth factors.     

Heart morphology during the embryonic development of Podocnemis unifilis Trosquel 1948 (Testudines: Podocnemididae)

Cardiogenesis is similar in all vertebrates, but differences in the valvuloseptal morphogenesis among non-crocodilian reptiles, birds, and mammals are noted. The origin of mesenchymal structures such as valves that regulate the passage of blood and the formation of partial septa that prevent the complete mixing of oxygen-rich and low-oxygen blood present in adult chelonians are essential in the evolutionary understanding of complete septation, endothermy and malformations, even in mammals. In this context, this study analyzed the heart morphogenesis of Podocnemis unifilis (Testudines: Podocnemididae) from the 4th to the 60th day of incubation. We identified the tubular heart stage, folding of the cardiac tube and expansion of the atrial and ventricular compartments followed by atrial septation by the septum primum, ventricle septation by partial septa, outflow tract septation and the formation of bicuspid valves with cartilage differentiation at the base. The formation of the first atrial septum with the mesenchymal cap is noted during the development of the atrial septum, joining the atrioventricular cushion on the 17th day and completely dividing the atria. Small secondary perforations appeared in the mid-cranial part, observed up to the 45th day. Partial ventricle septation into the pulmonary, venous, and arterial subcompartments takes place by trabeculae carneae thickening and grouping on the 15th day. The outflow tract forms the aorticopulmonary and interaortic septa on the 16th day and the bicuspid valves, on the 20th day. Therefore, after the first 20 days, the heart exhibits a general anatomical conformation similar to that of adult turtles.     

The distribution and characterization of HNK-1 antigens in the developing avian heart

The heart originates from splanchnic mesoderm and to a lesser extent from neural crest cells. The HNK-1 monoclonal antibody is a marker for early migrating neural crest cells, but reacts also with structures which are not derived from the neural crest. We investigated whether heart structures are HNK-1 positive before neural crest cells colonize these target tissues. To that end, we determined the HNK-1 antigen expression in the developing avian heart on immunohistochemical sections and on Western blots. The HNK-1 immunoreactivity in the developing chick heart is compared with data from literature cm the localization of neural crest cells in chick/quail chimeras. Structures with neural crest contribution, including parts of the early outflow tract and the related endocardial cushions, the primordia of the semilunar valve leaflets and the aorticopulmonary septum were HNK-1 positive. Furthermore, other structures were HNK-1 positive, such as the atrioventricular cushions, the wall of the sinus venosus at stage HH 15 through 21, parts of the endocardium at E3, parts of the myocardium at E6, and the extracellular matrix in the myocardial base of the semilunar valves at E14. HNK-1 expression was particularly observed in morphologically dynamic regions such as the developing valves, the outflow tract cushion, the developing conduction system and the autonomie nervous system of the heart. We observed that atrioventricular endocardial cushions are HNK-1 positive. We conclude that: a HNK-1 immunoreactivity does not always coincide with the presence of neural crest cells or their derivatives; (2) the outflow tract cushions and atrioventricular endocardial cushions are HNK-1 positive before neural crest cells are expected (stage HH 19) to enter the endocardial cushions of the outflow tract; (3) the observed spatio-temporal HNK-1 patterns observed in the developing heart correspond with various HNK-1 antigens. Apart from a constant pattern of HNK-1 antigens during development, stage-dependent HNK-1 antigens were also found.     

Spatial distribution of "tissue-specific" antigens in the developing human heart and skeletal muscle. III. An immunohistochemical analysis of the distribution of the neural tissue antigen G1N2 in the embryonic heart; implications for the development of the atrioventricular conduction system.

A monoclonal antibody raised against an extract from the Ganglion Nodosum of the chick and designated G1N2 proves to bind specifically to a subpopulation of cardiomyocytes in the embryonic human heart. In the youngest stage examined (Carnegie stage 14, i.e., 4 1/2 weeks of development) these G1N2-expressing cells are localized in the myocardium that surrounds the foramen between the embryonic left and right ventricle. In the lesser curvature of the cardiac loop this "primary" ring occupies the lower part of the wall of the atrioventricular canal. During subsequent development, G1N2-expressing cells continue to identify the entrance to the right ventricle, but the shape of the ring changes as a result of the tissue remodelling that underlies cardiac septation. During the initial phases of this process the staining remains recognizable as a continuous band of cells in the myocardium that surrounds the developing right portion of the atrioventricular canal, subendocardially in the developing interventricular septum and around the junction of the embryonic left ventricle with the subaortic portion of the outflow tract. During the later stages of cardiac septation, the latter part of the ring discontinues to express G1N2, while upon the completion of septation, no G1N2-expressing cardiomyocytes can be detected anymore. The topographic distribution pattern of G1N suggests that the definitive ventricular conduction system derives from a ring of cells that initially surrounds the "primary" interventricular foramen. The results indicate that the atrioventricular bundle and bundle branches develop from G1N2-expressing myocytes in the interventricular septum, while the "compact" atrioventricular node develops at the junction of the band of G1N2-positive cells in the right atrioventricular junction (the right atrioventricular ring bundle) and the ("penetrating") atrioventricular bundle. A "dead-end tract" represents remnants of conductive tissue in the anterior part of the top of the interventricular septum. The location of the various components of the avian conduction system is topographically homologous with that of the G1N2-ring in the human embryonic heart, indicating a phylogenetically conserved origin of the conduction system in vertebrates.     

The Development of Septation in the Four‐Chambered Heart

The past decades have seen immense progress in the understanding of cardiac development. Appreciation of precise details of cardiac anatomy, however, has yet to be fully translated into the more general understanding of the changing structure of the developing heart, particularly with regard to formation of the septal structures. In this review, using images obtained with episcopic microscopy together with scanning electron microscopy, we show that the newly acquired information concerning the anatomic changes occurring during separation of the cardiac chambers in the mouse is able to provide a basis for understanding the morphogenesis of septal defects in the human heart. It is now established that as part of the changes seen when the heart tube changes from a short linear structure to the looped arrangement presaging formation of the ventricles, new material is added at both its venous and arterial poles. The details of these early changes, however, are beyond the scope of our current review. It is during E10.5 in the mouse that the first anatomic features of septation are seen, with formation of the primary atrial septum. This muscular structure grows toward the cushions formed within the atrioventricular canal, carrying on its leading edge a mesenchymal cap. Its cranial attachment breaks down to form the secondary foramen by the time the mesenchymal cap has used with the atrioventricular endocardial cushions, the latter fusion obliterating the primary foramen. Then the cap, along with a mesenchymal protrusion that grows from the mediastinal mesenchyme, muscularizes to form the base of the definitive atrial septum, the primary septum itself forming the floor of the oval foramen. The cranial margin of the foramen is a fold between the attachments of the pulmonary veins to the left atrium and the roof of the right atrium. The apical muscular ventricular septum develops concomitant with the ballooning of the apical components from the inlet and outlet of the ventricular loop. Its apical part is initially trabeculated. The membranous part of the septum is derived from the rightward margins of the atrioventricular cushions, with the muscularizing proximal outflow cushions fusing with the muscular septum and becoming the subpulmonary infundibulum as the aorta is committed to the left ventricle. Perturbations of these processes explain well the phenotypic variants of deficient atrial and ventricular septation. Anat Rec, 297:1414–1429, 2014. © 2014 Wiley Periodicals, Inc.     

Development of aortic and mitral valve continuity in the human embryonic heart

The anatomic relationship of the aortic and mitral valves is a useful landmark in assessing congenital heart malformations. The atrioventricular and semilunar valve regions originate in widely separated parts of the early embryonic heart tube, and the process by which the normal fibrous continuity between the aortic and mitral valves is acquired has not been clearly defined. The development of the aortic and mitral valve relationship was studied in normal human embryos in the Carnegie Embryological Collection, and specimens of Carnegie stages 13, 15, 17, 19, and 23, prepared as serial histologic sections cut in the sagittal plane, were selected for reconstruction. In stage 13, the atrioventricular valve area is separated from the semilunar valve area by the large bend between the atrioventricular and outflow-tract components of the single lumen heart tube created by the left interventricular sulcus. In stages 15 and 17, the aortic valve rotates into a position near the atrioventricular valves with development of four chambers and a double circulation. In stage 19, there is fusion of aortic and mitral endocardial cushion material along the endocardial surface of the interventricular flange, and this relationship is maintained in subsequent stages. Determination of three-dimensional Cartesian coordinates of the midpoints of valve positions shows that, while there is growth of intervalvular distances up to stage 17, the aortic to mitral distance is essentially unchanged thereafter. During the period studied, the left ventricle increases in length over threefold. The relative lack of growth in the saddle-shaped fold between the atrioventricular and outflow tract components of the heart, contrasting with the rapid growth of the outwardly convex components of most of the atrial and ventricular walls, may be attributed to the different mechanical properties of the two configurations. It is postulated that the pathogenesis of congenital heart malformations, which characteristically have failure of development of aortic and mitral valve continuity, may involve abnormalities of rotation of the aortic region or malpositioning of the fold in the heart tube.     

The localization of fibronectin and 140 Kd fibronectin receptor in the truncus arteriosus of the chick embryonic heart

The distribution of fibronectin and 140 Kd fibronectin-receptor was examined in the truncus arteriosus of the chick embryonic heart during aortico-pulmonary (AP) septation by immunohistochemistry. In 4-day-old chick embryos, immunoreactivity of both anti-fibronectin and anti-140 Kd fibronectin-receptor was seen in the primordia of the AP septum. In 5-day-old embryos, cells stained with anti-140 Kd fibronectin-receptor antibody were rarely found in the AP septum, though strong immunoreactivity of the anti-fibronectin antibody was observed in the AP septum. In 6-day-old embryos, at a late stage of septation, cells stained with anti-140 Kd fibronectin-receptor antibody were not found in the AP septum, and immunoreactivity of the anti-fibronectin antibody had decreased. In the cushion tissue, immunoreactivity of the anti-fibronectin antibody was seen, but that of the anti-140 Kd fibronectin-receptor antibody was recognizable only on a few cushion cells in the 4-day-old embryos. These findings suggest that fibronectin is involved in the formation of the AP septum primordia, that fibronectin does not promote the development of the AP septum, and that the migration mechanism of cushion cells is different from that of neural crest cells.     

Dual role for neural crest cells during outflow tract septation in the neural crest-deficient mutant Splotch2H

Splotch^2H ( Sp^2H ) is a well-recognized mouse model of neural crest cell (NCC) deficiency that develops a spectrum of cardiac outflow tract malformations including common arterial trunk, double outlet right ventricle, ventricular septal defects and pharyngeal arch artery patterning defects, as well as defects in other neural-crest derived organ systems. These defects have been ascribed to reduced NCC in the pharyngeal and outflow regions. Here we provide a detailed map of NCC within the pharyngeal arches and outflow tract of Sp^2H/Sp^2H embryos and fetuses, relating this to the development of the abnormal anatomy of these structures. In the majority of Sp^2H/Sp^2H embryos we show that deficiency of NCC in the pharyngeal region results in a failure to stabilize, and early loss of, posterior pharyngeal arch arteries. Furthermore, marked reduction in the NCC-derived mesenchyme in the dorsal wall of the aortic sac disrupts fusion with the distal outflow tract cushions, preventing the initiation of outflow tract septation and resulting in common arterial trunk. In around 25% of Sp^2H/Sp^2H embryos, posterior arch arteries are stabilized and fusion occurs between the dorsal wall of the aortic sac and the outflow cushions, initiating outflow tract septation; these embryos develop double outlet right ventricle. Thus, NCC are required in the pharyngeal region both for stabilization of posterior arch arteries and initiation of outflow tract septation. Loss of NCC also disrupts the distribution of second heart field cells in the pharyngeal and outflow regions. These secondary effects of NCC deficiency likely contribute to the overall outflow phenotype, suggesting that disrupted interactions between these two cell types may underlie many common outflow defects.     

小鼠胚胎心流出道心内膜垫的形成与融合

目的观察不同发育时期小鼠胚胎心流出道心内膜垫的发育过程。方法对胚龄9-12d小鼠胚胎心脏连续切片进行HE染色和免疫组化染色。结果胚龄10d,流出道远端心胶质内开始观察到间充质细胞。胚龄11d,两侧流出道心内膜垫形成,心内膜垫内部分间充质细胞染色呈α-平滑肌肌动蛋白(α-SMA)阳性。胚龄12d,两侧流出道心内膜垫内部分间充质细胞聚集形成致密漩涡状结构,随着心内膜垫融合,两侧漩涡状结构融合。结论小鼠胚胎流出道心内膜垫形成于胚胎发育第11天,第12天融合,间充质细胞参与心内膜垫融合。