Development of the atrioventricular valve region in the human embryo

Severe cardiac malformations may involve the atrioventricular valve region, but the sequence of embryonic development of this important area has been little studied. In particular, the basis of atrioventricular muscular discontinuity, except at the conduction system, has remained unexplained. To examine this question, serial histologic sections of human embryos from the Carnegie Embryological Collection and from the Hopkins Pathology Collection were studied and six embryos were reconstructed. The atrioventricular sulcus can be identified in Carnegie stage 10 as an indentation or crease on the right side separating the heart tube from the umbilical vein. By stage 12 the sulcus has deepened and rotated anteriorly as the atria appear and the heart tube elongates rapidly within the confining pericardial space. Selective accumulation of cardiac jelly on the endocardial aspect of the constriction of the heart tube produced by the atrioventricular sulcus is pronounced by stage 14. By stage 16 the separation of the atrioventricular orifice into right and left components is well advanced, and by stage 18 the septation of the atria and ventricles is largely complete. The muscular connection between the atria and the ventricles becomes interrupted around most of the artioventricular sulcus, except for the His bundle, during the latter part of the embryonic period. The topography of the original sulcus assumes a catenoidal or saddle-shaped configuration, i.e., convex in one plane and concave in the perpendicular plane. The tension and pressure relationships in such a structure would favor cardiac jelly accumulation and the eventual disintegration of lines of myocyte connections passing across the groove. The preservation of the His bundle connection is explained by the failure of the sulcus to completely encircle the heart.     

The right outflow tract sulcus in embryonic human hearts

The right outflow tract sulcus occurs in a region of complex and rapidly changing embryonic anatomy where cardiac septa and valve rings fuse, but its importance in cardiogenesis is unknown. Regions of the heart which include this sulcus were reconstructed from nine normal human embryos from the Carnegie Embryological Collection using images of serial histological sections and a computer-based three-dimensional (3-D) reconstruction system. The sulcus appears in Carnegie stage 9 as one of the paired cardiogenic folds and persists through stage 19. It maintains a consistent position on the right lateral aspect of the outflow tract, where the outflow tract bends posteriorly. The sulcus elongates in proportion to the increased diameter of the outflow tract to stage 16. With rotation of the outflow tract and division into separate circulations in subsequent stages, the right outflow tract sulcus becomes related only to the left ventricular outflow tract. Between stages 10 and 16, the portion of the outflow tract extending from the right outflow tract sulcus to the aortic sac or great arteries elongates. Subsequent to outflow tract rotation and division, this distance decreases. The right outflow tract sulcus appears to be of importance in contributing to the final cardiac topography where interatrial, atrioventricular, interventricular, and outflow tract sulci meet and where the junction of atrioventricular and semilunar valve rings occurs.     

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 vomeronasal organ in the human embryo, studied by means of three-dimensional computer reconstruction

The human vomeronasal organ is of interest because of its potential role in sex pheromone detection. Due to the scarcity of early human material, studies of its development have concentrated on fetal rather than embryonic stages. The availability of embryonic specimens in the Walmsley Collection has enabled us to study the development of the vomeronasal organ (VNO) in human embryos between Carnegie Stages 17 and 23. Embryos at Carnegie Stage 17 or below showed no evidence of a VNO. One embryo with characteristics intermediate between Carnegie stages 17 and 18 was the earliest to show evidence of a VNO, in the form of a shallow indentation. All embryos at Carnegie Stages 18 or later had VNOs. Three-dimensional computer reconstructions were made of the VNO in each specimen where this was possible. This in part depended on the plane of section. The total volume and lumen volume were measured from these reconstructions and the volume of the vomeronasal epithelium was calculated by subtraction. A generally consistent increase in total volume and epithelial volume was observed with increasing developmental stage. The lumen contributed rather little to the total volume at these stages.     

Computer-based reconstructions of the cardiac ventricles of human embryos

The early appearance and relatively large size of the embryonic heart suggest that cardiac function is critical to early development. Previous studies had shown that an index derived from curvature and thickness of the ventricular wall provides an estimate of the pressure generating capacity of the myocardium. To obtain an estimate of the functional capability of the embryonic ventricle, images of serial histologic sections of eight normal human embryos, ranging from stages 9-23, from the Carnegie Embryological Collection were chosen for study. The contours of ventricular components were digitized and entered into a computer, and three-dimensional (3-D) reconstructions were created. Volumes of the components of the ventricles were determined, including the compact or outer portion of the ventricular wall, the cardiac jelly, the overall volume containing trabeculated myocardium in each ventricle, and the proportion of that volume consisting of muscle. The results showed a highly significant increase in overall ventricular size as a function of Carnegie stage and crown-rump length. Cardiac jelly was prominent in the early stages but was progressively replaced by the trabeculated muscle. The volume containing trabeculae had a consistent proportion of muscle, averaging 65%, for all stages after its appearance in stage 13. Curvature and thickness measurements of the compact part of the ventricles were made from images of the reconstructions. The mean curvature-thickness index (CTI) for the embryo hearts ranged from 0.24-0.61, and there was a significant increase in the index as a function of stage and crown-rump length.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histocytochemical and immunohistochemical studies related to the role of glycogen in human developing digestive organs

To elucidate the role of glycogen in the epithelium of developing digestive organs, we investigated the appearance of glycogen and glycogen phosphorylase (GP) in these organs. We studied 64 externally normal human embryos at Carnegie stages 13–23 (5.1–28.0 mm in crown-ramp length, 4–8 weeks of gestation) by histocytochemical staining for glycogen and immunohistochemical staining with antibodies against two isoenzymes of GP: brain-type (BGP) and mucle-brain-type (MBGP) GP. At stage 13, glycogen appeared in the epithelium of the digestive tract and the parenchyma of the pancreas. As development advanced, glycogen granules increased in number and size in these tissues, and they became evenly distributed in the epithelium of the digestive tract as either single particles or aggregates, as deduced by electron microscopy at late embryonic stages. Immunoreactivity specific both for BGP and for MBGP was detected in the digestive tract and the pancreas from stage 13. As development advanced, both BGP- and MBGP-immunoreactive cells increased in number and in immunoreactivity, and the number of MBGP-immunoreactive cells became larger than that of BGP-immunoreactive cells. By contrast, in hepatic cells, which serve as a major storage site for glycogen in adults, glycogen was detected only from stage 20, in smaller amounts, without formation of aggregates, and no immunoreactivity specific for BGP or MBGP was apparent throughout the embryonic stages examined. Thus, in the epithelium of the digestive tract and the parenchyma of the pancreas, but not in hepatic cells, the appearance and localization of GP coincided almost exactly with that of glycogen. These observations suggest that glycogen in the epithelium of the digestive tract and the parenchyma of the pancreas has not only been synthesized but also degraded from an early embryonic period and may, thus, be related to active cellular metabolism that is specific for embryonic development, including proliferation of the epithelium and interactions between epithelium and mesenchyme.     

人胚心静脉窦和传导系统的早期发育

目的 探讨早期人胚心静脉窦及传导系的发生发育机制. 方法 用抗α-平滑肌肌动蛋白(α-SMA)、抗α-横纹肌肌动蛋白(α-SCA)和抗结蛋白(DES)抗体对29例C10～C16期人胚心连续切片行免疫组织化学染色. 结果 人胚发育C12～C13期,系统静脉汇集形成的静脉窦出现于心包腔尾端原始横膈间充质中,静脉窦壁间充质细胞逐渐分化为α-SCA阳性的静脉窦心肌细胞.C14期,心包腔的扩张使静脉窦进入心包腔内,参与了右心房的形成.DES阳性传导系心肌的分化始于C10期心房室管右侧壁,随发育逐渐向室间沟心肌扩展,发育为房室传导系的希氏束、左右束支及心室腔面的小梁心肌.在心房,DES表达首先出现于C11期心房背侧壁,在C13期,可见静脉窦左背侧壁α-SCA、α-SMA、DES阳性心肌带与左心房底部、房室管背侧壁相延续,这条心肌带可能参与了人胚心静脉窦至房室管传导系的发育.C14～C16期,DES强阳性染色从窦房结经左、右静脉瓣及心房的背、腹侧壁延伸至房室管右侧壁,可能是原始的心房传导通路. 结论 心包腔尾端原始横膈间充质是人胚静脉窦心肌发生区,原始横膈间充质细胞逐渐分化为心肌细胞,添加到人胚心管静脉端,形成心静脉窦心肌.人胚心传导系心肌的分化始于房室管,随心管发育逐渐向动、静脉端扩展,在C16期,已分化为形态清晰可辨的DES阳性胚胎心传导系.     

Fourth ventricular floor in human embryos: Scanning electron microscopic observations

The ultrastructural surface features of the normal fourth ventricular floor of seven human embryos ranging from Carnegie stage 14 to stage 19 (crown-rump length: 7.6–16.2 mm) were examined by using scanning electron microscopy (SEM). Low-power SEM views showed the median sulcus, sulcus limitans, and neuromeres, transient structures characteristic of the earlier embryonic period. High-power SEM observation revealed supraependymal cells (SE cells) and supraependymal fibers (SE fibers) which exhibited a characterisitc localization, as well as generalized surface-membrane modifications such as microvilli and cilia. SE cells could be classified into two major groups. The type 1 SE cells seem to possess neuronal functions, as deduced from morphological similarities to their counterparts in adults and the specialized distribution closely related to neuromeres. The type 2 SE cell morphologically resembled the phagocytic SE cell described in related literature. SE fibers ran a course either rostrocaudally in the median sulcus or mediolaterally on the neuromeres, most frequently near the interneuromeric cleft; they made contact with type 1 SE cells and ependymal surface modifications and then penetrated the ependymal layer.     

Development of the outflow tract and closure of the interventricular septum in the normal human heart

The majority of congenital heart malformations in humans involve defects in the atrioventricular valves, the crest of the interventricular septum, and/or the outflow tract, but the position and timing of these structures during cardiac development is controversial. We examined all 622 staged, serially sectioned normal human embryos and fetuses in the Carnegie Embryological Collection, and obtained a statistical tabulation of the appearance of the endocardial cushion components and surrounding structures for 382 embryos in good condition between stages 9 and 23 inclusive, when the heart normally develops. Accurately scaled drawings of ventral and lateral views of the hearts of seven embryos from stage 13 through 22 were prepared from graphic reconstructions in order to visualize the relationships of the structures under consideration. We found that development of the outflow tract septum follows the apparent functional separation of both the left and right ventricles and the blood streams leaving them. Elevations of the endocardial cushion material are continuous throughout the outflow tract and develop as a consequence of the elliptical configuration imposed on the circular cross section of the outflow tract. The membranous interventricular septum is formed of cushion material in the space bounded by the outflow tract septum, interventricular septum, and the fused AV cushion and right outflow tract cushion. The results of this study are consistent with the assertion that functional separation of the aortic and pulmonary outflow tracts precedes anatomic septation, and that anatomic septation is brought about by mechanical modeling of developing myocardium and endocardial cushion material.     

Asplenia and polysplenia malformation complexes explained by abnormal embryonic body curvature

Asplenia and polysplenia malformation complexes characteristically have severe cardiovascular defects and visceral heterotaxy. We examined the hypothesis that the conditions may arise from an altered timing of development of embryonic body curvature: delayed in asplenia, accelerated in polysplenia. The morphologic features of the 25 patients with asplenia and 15 with polysplenia autopsied at The Johns Hopkins Hospital were determined. The time of appearance of various morphologic features and the evolution of body curvature was studied in 351 staged serially sectioned human embryos of The Carnegie Embryological Collection. All asplenia patients had severe atrioventricular canal malformations. Bilateral trilobed lungs were found in 12 patients. The polysplenia patients had severe interatrial septal defects in 10 patients; but ventricular septal defects in only six. Bilateral bilobed lungs were seen in five patients. Comparison of the time of appearance of anatomic structures in normal embryos with the observed malformations suggest that asplenia and polysplenia complexes originate in stages 13 to 15. The observations are consistent with the concept that the malformations in asplenia and polysplenia can be explained by minor alterations in the sequence of development of embryonic body curvature relative to organ maturation.     

The Digestive Tract and Derived Primordia Differentiate by Following a Precise Timeline in Human Embryos Between Carnegie Stages 11 and 13

The precise mechanisms through which the digestive tract develops during the somite stage remain undefined. In this study, we examined the morphology and precise timeline of differentiation of digestive tract‐derived primordia in human somite‐stage embryos. We selected 37 human embryos at Carnegie Stage (CS) 11–CS13 (28–33 days after fertilization) and three‐dimensionally analyzed the morphology and positioning of the digestive tract and derived primordia in all samples, using images reconstructed from histological serial sections. The digestive tract was initially formed by a narrowing of the yolk sac, and then several derived primordia such as the pharynx, lung, stomach, liver, and dorsal pancreas primordia differentiated during CS12 (21–29 somites) and CS13 (≥ 30 somites). The differentiation of four pairs of pharyngeal pouches was complete in all CS13 embryos. The respiratory primordium was recognized in ≥ 26‐somite embryos and it flattened and then branched at CS13. The trachea formed and then elongated in ≥ 35‐somite embryos. The stomach adopted a spindle shape in all ≥ 34‐somite embryos, and the liver bud was recognized in ≥ 27‐somite embryos. The dorsal pancreas appeared as definitive buddings in all but three CS13 embryos, and around these buddings, the small intestine bent in ≥ 33‐somite embryos. In ≥ 35‐somite embryos, the small intestine rotated around the cranial‐caudal axis and had begun to form a primitive intestinal loop, which led to umbilical herniation. These data indicate that the digestive tract and derived primordia differentiate by following a precise timeline and exhibit limited individual variations. Anat Rec, 299:439–449, 2016. © 2016 Wiley Periodicals, Inc.     

Embryonic development of the house shrew (Suncus murinus)

Summary The embryonic development during the period from 1 to 12 pairs of somites was observed in an insectivore species, the house shrew (Suncus murinus), which has been bred within a closed colony. Embryos were staged by the number of somite pairs. Each stage was punctuated at every addition of three pairs of somites and numbered after the Carnegie system. The first somite became apparent between 8 and 9.0 days after fertilization, and the 12th somite appeared between 9.5 and 10.0 days. The rate of somite formation was one pair in every 3–4 h on average. The embryonic events during this period were as follows: 1. From the beginning of stage 9, the embryonic body consistently displayed a kyphosis, and as development progressed, the caudal portion of the embryo spiralled clockwise. 2. The first and second pharyngeal arches formed; their development was precocious among mammalian embryos in relation to somitic count. 3. The segmental pattern of the neural fold was similar to that of laboratory rodents and primates. The first fusion of the cranial neural folds took place in the occipital somite region, the second fusion in the diencephalic region, and the third at the end of the neural plate, thus leaving two neuropores in the cephalic region. 4. The timing of appearance of the optic sulcus was similar to that of human embryos but was delayed in comparison with that of laboratory rodents. 5. The heart always showed a more advanced state than that of other mammalian embryos. From the beginning of stage 9, an unpaired endocardial tube was seen in the bulbo-ventricular region, and deflection from a symmetrical appearance soon took place. 6. The differentiation of foregut was also precocious, and the thyroid and respiratory primordia appeared earlier than in other mammals. The present study emphasizes that there are considerable variations in timing and manner of morphogenesis among early mammalian embryos.     

Computer ranking of the sequence of appearance of 100 features of the brain and related structures in staged human embryos during the first 5 weeks of development

The sequence of events in the development of the brain in staged human embryos was investigated in much greater detail than in previous studies by listing 100 features in 165 embryos of the first 5 weeks. Using a computerized bubble-sort algorithm, individual embryos were ranked in ascending order of the features present. This procedure made feasible an appreciation of the slight variation found in the development features. The vast majority of features appeared during either one or two stages (about 2 or 3 days). In general, the soundness of the Carnegie system of embryonic staging was amply confirmed. The rhombencephalon was found to show increasing complexity around stage 13, and the postoptic portion of the diencephalon underwent considerable differentiation by stage 15. The need for similar investigations of other systems of the body is emphasized, and the importance of such studies in assessing the timing of cogenital malformations and in clarifying syndromic clusters is suggested.     

Rotation of the junction of the outflow tract and great arteries in the embryonic human heart

The factors which give rise to the normal relationship between the great arteries and their respective ventricles are unknown. The developmental anatomy of this region was studied by using frontal, sagittal, or transverse serial histologic sections of 17 normal human embryos of Carnegie stages 15-19 from the Carnegie Embryological Collection. Distances and angles between major anatomic landmarks were determined by using computer reconstructions of the serially sectioned embryos, three-dimensional analytic geometry, and Euclidean distance formulas. The findings show that between stages 15 and 19 there is a marked rotation of the axis of the semilunar valves: frontal 121 degrees counterclockwise, sagittal 196 degrees counterclockwise, and transverse 240 degrees clockwise. Simultaneously the great arteries lengthen at a faster rate than the rest of the heart; and there is also an increase in the caliber and wall thickness of the great arteries. These results suggest that the changing rate of growth between the great arteries and the heart is necessary to align the great arteries, the semilunar valves, and the muscular outflow tract septum appropriately with respect to the interventricular septum. Reductions in the rate of growth of the great arteries relative to the heart could, by causing changes in the rotation of great arteries and outflow tract septum, have a role in the pathogenesis of cardiovascular malformations such as tetralogy of Fallot and transposition of the great arteries.     

The bronchial tree of the human embryo: an analysis of variations in the bronchial segments

A classical study has revealed the general growth of the bronchial tree and its variations up to Carnegie stage (CS) 19. In the present study, we extended the morphological analysis CS by CS until the end of the embryonic period (CS23). A total of 48 samples between CS15 and CS23 belonging to the Kyoto Collection were used to acquire imaging data by performing phase-contrast X-ray computed tomography. Three-dimensionally reconstructed bronchial trees revealed the timeline of morphogenesis during the embryonic period. Structures of the trachea and lobar bronchus showed no individual difference during the analyzed stages. The right superior lobar bronchus was formed after the generation of both the right middle lobar bronchus and the left superior lobar bronchus. The speed of formation of the segmental bronchi, sub-segmental bronchi, and further generation seemed to vary among individual samples. The distribution of the end-branch generation among five lobes was significantly different. The median branching generation value in the right middle lobe was significantly low compared with that of the other four lobes, whereas that of the right inferior lobe was significantly larger than that of both the right and left superior lobes. Variations found between CS20 and CS23 were all described in the human adult lung, indicating that variation in the bronchial tree may well arise during the embryonic period and continue throughout life. The data provided may contribute to a better understanding of bronchial tree formation during the human embryonic period.     

Spatiotemporal and tissue specific distribution of apoptosis in the developing chick heart.

To investigate spatial and temporal distributions of apoptosis in the embryonic chick heart and its relation to different tissue types, we examined apoptosis in the embryonic chick heart from Hamburger and Hamilton stage 17 through 3 days after hatching. MF20 antibody, alpha-smooth muscle actin (SMA) antibody and EAP-300 antibody were applied to delineate specific cell types. During early development of the embryonic chick heart, very few apoptotic cells were detected. The first distinctive zone of apoptosis was observed in the outflow tract at stage 25. This focus was most prominent during septation of the pulmonary artery from the aorta (i.e., between stages 28 and 29), and diminished to virtually background level by stage 32, except in the subconal regions. Subsequently, remarkable apoptosis appeared in the atrioventricular cushions by stage 26, peaked at stages 29-31, and dropped significantly thereafter. Characteristic distribution patterns of apoptotic cells were also detected in the cardiac conduction tissues, including the His bundle, the bundle branches, and the ventricular trabeculae. After stage 36, cell death dropped to background level, except in developing coronary vessels. MF20 and TUNEL double staining revealed that apoptosis in cardiomyocytes was limited to a few specific regions, much less than in cushion tissues. SMA and TUNEL double staining demonstrated that vascular structures were the major foci of apoptosis from stage 40 to 44, whereas adjacent perivascular Purkinje cells displayed significantly less cell death at these stages. The characteristic spatiotemporal locations of apoptosis parallel the morphologic changes and tissue differentiation during heart development, suggesting that apoptosis is crucial to the transformation of the heart from a simple tube to a complex multichambered pump.     

Atrioventricular canal malformation interpreted as secondary to reduced compression upon the developing heart.

This study was undertaken to evaluate the nature and pathogenesis of malformations of the atrioventricular canal in relation to normal cardiogenesis. Serial histologic sections of normal human embryos and fetuses were made, from which three-dimensional images were reconstructed to show the relationship between the developing heart and its surrounding structures, and the course of development of the atrial septum and atrioventricular valves. Based on these reconstructions and on examination of the hearts of 59 patients with atrioventricular canal malformations, it is suggested that the spectrum of atrioventricular malformations may arise as a result of reduced compression of the developing atria by surrounding structures during embryonic Stages 13 through 18. Comparison of hearts with atrioventricular canal defects with normal embryos indicated that the malformations may be classified as primitive canals, complete canals, or partial canals, corresponding to failure of completion of normal development in Stages 14 through 18. In primitive canal the atrial septum was absent or had only a portion of septum primum. In complete canal both atrial septums were present, but the atrioventricular valve material was not subdivided and the four chambers were in communication. In partial canal, the atrioventricular valve was divided, but atrial and ventricular septal defects and valve clefts were present in varying degrees of severity. It is proposed that the spectrum of cardiac abnormalities which constitutes atrioventricular canal malformations may be understood as arising from varying degrees of lack of normal compression of the developing heart by surrounding structures. (Am J Pathol 95.579-598, 1979)     

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.