During the period of major organogenesis, the heart and great vessels undergo drastic morphogenetic changes. In the humans, the heart is derived from the anterior splanchnic mesoderm during the third week after fertilization [Carnegie stage (CS) 9]. At first, two crescent-like cardiogenic plates appear in the cardiogenic area and these plates fuse with each other in the midline to form a primary heart tube. The primary heart tube begins to loop (cardiac looping) at the seven-somite stage (CS 10). The heartbeat is assumed to start at CS 10 in the humans. The heart tube balloons and chamber formation begins during the fifth week (CS 12). The endocardial cushion is formed at the atrioventricular canal to separate the primary atrium and ventricle. The left and right chambers are divided by the ventricular and atrial septi during the fifth to sixth weeks (O'Rahilly, 1971; Gittenberger-de Groot et al., 2005). The interventricular septum closes around the sixth week (CS 16) and the interatrial septum just after birth.
The truncus arteriosus, a tube-like structure between the primary ventricle and pharyngeal arch arteries, becomes divided into the ascending aorta and the pulmonary trunk by CS 19 (outflow tract septation) (O'Rahilly, 1971). To form great vessels, the first to sixth pairs of pharyngeal arch arteries develop in turn (the fifth arches remain rudimentary), and major head and neck arteries and the aortic arch arterial system are formed from these arteries by CS 21 (Cooper and O'Rahilly, 1971). Drastic morphogenetic transformation occurs in the pharyngeal arch arterial system, although some of the arteries disappear without forming any definitive blood vessels.
Since the morphogenetic changes of the heart occur three-dimensionally, it is essential to visualize and analyze heart development in three and four dimensions, including the changes occurring with time. Three-dimensional and 4D visualization is also a powerful tool in embryological study and greatly helps students understand the dynamic morphogenetic movements in the embryo (Yamada et al., 2006). From the early days of human embryology, attempts were made to visualize embryonic structure in three dimensions. Traditionally, 3D reconstruction of embryonic structures used to be made from serial histological sections of embryos, often with the wax plate technique (Born, 1883). However, such reconstruction and drawing methods require an enormous time and special skills and cannot be adopted in every laboratory. Recent advancement in computer science has made computer-assisted reconstruction of biological structures more effectively. Various 3D structures have been reconstructed by this method, and the reconstructed images can be manipulated as desired on the viewing screen. In the area of the developmental study of the heart and great vessels, computer-assisted reconstruction and computer graphics (CG) have been used to visualize the developing heart and vessels of the mouse (Smith, 2001; Schneider et al., 2003), chick (Hiruma and Hirakow, 1995), and human (DeGroff et al., 2003; Abdulla et al., 2004). In mice, the 3D sequential images of the developing heart have been made between E8.5 and E14.5 (Soufan et al., 2003). However, no sequential 3D visualization has been reported to date for the cardiac development in staged human embryos.
It is well known that various cardiovascular malformations are often associated with other external anomalies. Holoprosencephaly (HPE) is a spectrum of midline anomalies in central nervous system, produced by impaired or incomplete division of the prosencephalon (embryonic forebrain) into bilateral cerebral hemispheres. There are many associated anomalies in HPE, such as hypotelorism, nasal dysplasia, cleft lip, hypopituitarism, and polydactyly (Cohen, 1989; Yamada et al., 2004). Heart anomalies are often associated with HPE, especially in patients with chromosomal anomalies such as trisomies 13 and 18 (Cohen and Sulik, 1992). However, heart anomalies in HPE embryos have not been examined in detail because many HPE embryos are spontaneously eliminated before birth, and it is difficult to acquire complete serial sections for analyzing the abnormal structures of the embryonic heart.
In the present study, we reconstructed the heart and great vessels of staged human embryos with the aid of computer software and compared their luminal structures in normal and HPE embryos. We show that computer-assisted reconstruction is a useful and powerful tool for analyzing detailed 3D phenotypes in embryos.
MATERIALS AND METHODS
Preparing Human Embryo Specimens
During the past 40 years, a large number of human conceptuses have been collected in Kyoto University with the collaboration of several hundred obstetricians. A great majority of the cases were derived from healthy pregnancies terminated for social reasons during the first trimester of pregnancy (Maternity Protection Law of Japan), and the pregnancies were interrupted mainly by dilatation and curettage. The embryo collection now comprises over 44,000 specimens, and part of them (∼ 20%) are well-preserved, undamaged embryos. The developmental stage of the embryos (Carnegie stage; CS) was determined according to the criteria proposed by O'Rahilly and Müller (1987). Further details of the embryo collection have been described in previous reports (Nishimura et al., 1968; Nishimura, 1975; Shiota, 1991; Yamada et al., 2004, 2005).
For the present study, we examined 100 normal human embryos histologically at CS 11 to 20 and selected 8 embryos at CS 11, 12, 13, 14, 15, 16, 18, and 20 for 3D reconstruction. HPE embryos were diagnosed by characteristic external appearances such as severe hypotelorism, proboscis, and narrow head (Yamada et al., 2004). Forty-three HPE embryos were serially sectioned and suitable for observation (Yamada et al., 2004). Two cases had cardiac anomalies histologically: one was malrotation of the heart tube, and the other case ventricular septal defect; they were selected for 3D reconstruction. The remaining 41 HPE embryos were found to have no cardiac anomalies at least by histological examination.
Procedure of 3D Reconstruction
Stained histological sections were digitized with a digital camera (Olympus Camedia C-5050) attached to a microscope (Olympus BH-2), with a normal bright-field illumination. The obtained color images of the serial sections (Fig. 1A) were saved in a TIFF format (1,280 × 960 pixels, 3.5 MB per file) and transferred to a desktop computer. The desktop computer used for the present study was operated using Mac OS X (version 10.3.9; Apple Computer), which contained dual G4 processors (1.25 Ghz CPU) with 2 GB internal memories, a 128 MB GeForce4 Ti 4600 graphic card, and two 80 GB hard disks.
A set of images obtained from serial sections of the embryo under study was aligned with the alignment module contained in DeltaViewer software (http://vivaldi.ics.nara-wu.ac.jp/∼wada/DeltaViewer/), which is specialized for automatic alignment and 3D reconstruction from serial sections. The alignment was first undertaken by the auto-align function of DeltaViewer, and the acquired images were readjusted manually when necessary. These aligned images were segmented by Adobe Photoshop Elements (version 2.0; Adobe). In this procedure, arteries and veins were segmented in red and blue, respectively (Fig. 1B). The four chambers of the heart were distinguished by four colors (Fig. 1C).
To visualize the set of images, it was necessary to reduce the volume of the data for stable function of the software. The TIFF data were reduced to 100–200 KB per file by cutting the blank around the specimen and compressing the image data. The 3D data were obtained by using DeltaViewer.
Heart and Great Vessels in Normal Embryos
The human embryonic heart finishes looping by CS 12 (O'Rahilly, 1971). Our reconstruction from serial histological sections of a CS 12 embryo enabled visualization of the looping heart faithfully (Fig. 2A). The 3D image clearly showed that the heart loop develops from the sinus venosus to form the primordial right and left atria, which continue to the primordial right and left ventricles and the outflow tract. The outflow tract bifurcates into bilateral pharyngeal arch arteries. The primary large veins (posterior cardinal veins) are observed posterior to the heart.
At CS 14, segmentation of the heart tube at the atrioventricular boundary was clearly demonstrated (Fig. 2B). Although embryos at this stage have the looping heart, the first and second pharyngeal arch arteries have disappeared and the outflow tract is found to flow into the third and fourth pharyngeal arch arteries in the reconstructed image. The bilateral dorsal aortae descend and fuse with each other behind the liver. The fused single aorta turns ventrally and bifurcates again in the pelvic region. The bifurcated arteries are found to continue directly to the umbilical arteries, which flow into the umbilical cord. The iliac arteries are not clearly recognized yet.
At CS 15, the septation between the four cardiac chambers is evident (Fig. 2C and D). The ventricles are connected with the outflow tract, which flow into the pharyngeal arch arteries. The sixth pharyngeal arch arteries have been formed below the third and fourth pharyngeal arch arteries. The sixth pharyngeal arch arteries are shown to flow into the left descending aorta to form the future ductus arteriosus. Those pharyngeal arch arteries form aortic arches and flow into the dorsal aortae. The fifth pharyngeal arch arteries are not identified histologically. The bilateral dorsal aortae, accompanied by cardinal veins, descend and fuse with each other to form the single aorta. The bilateral subclavian arteries are found to arise from the descending aortae above the level of fusion between the bilateral aortae. Hepatic vessels are developing and have already begun to form a complex vascular network.
At CS 16, the heart appears to undergo further morphological changes (Fig. 3A and B). Septation of the outflow tract is evident, and the pulmonary trunk is recognizable. The heart is being transformed into four chambers. The pulmonary trunk is connected with the descending aorta via the ductus arteriosus. At this stage, the bilateral descending aortae are observed, and the left aorta is found to be larger than the right one, which may indicate the formation of the definitive descending aorta from the left aorta. The carotid arteries have been formed. Since the upper limbs are developing, the subclavian arteries are found to flow into the upper limbs.
By CS 18, the primary segmentation between the four chambers and the septation of the outflow tract has been completed. The left aortic arch and the descending aorta are significantly larger than the right arch. The spiral shape of the outflow tract between the pulmonary artery and the ascending aorta has been completed by this stage (Fig. 3C and D).
At CS 20, the basic plan of the major circulatory system in the embryo has been established (Fig. 3E and F). The pulmonary trunk is found to arise from the right ventricle and the ascending aorta from the left ventricle. At this stage, only one aortic arch and one descending aorta are present, and the right aortic arch and the right descending aorta have disappeared completely. The superior vena cava and the inferior vena cava are formed to flow into the right atrium. Remodeling of the pharyngeal arch arteries is almost completed to form such arteries as the brachiocephalic, left common carotid, and left subclavian arteries. The ductus arteriosus is clearly recognized as the bypass between the pulmonary artery and the descending aorta.
Heart and Great Vessels in HPE Embryos
In an HPE embryo at CS 16, facial anomalies such as hypotelorism, proboscis, and narrow head were clearly observed as compared with normal embryos (Fig. 4A and E). In 3D reconstructed images, malrotation of the heart tube was observed (Fig. 4G and H). At this stage, the four chambers are normally positioned at the same plane and the right and left ventricles are anterior to the right and left atria, respectively (Fig. 4A and B). However, the position of the right and left ventricles in the HPE case was not in the right-left but the craniocaudal sequence (Fig. 4G and H). The distortion of the ventricle seemed to affect the position of the atria. Although these findings suggested malrotation of the primary heart tube, the outflow tract was formed to divide into the ascending aorta and pulmonary trunk as in normal embryos (Fig. 4G and H). The bilateral carotid arteries and the ductus arteriosus flowing into the left descending aorta appeared normal.
An HPE embryo at CS 20 had a characteristic external appearance such as partially fused eyes, single orbit, and a proboscis seen in Figure 5E. In its reconstructed heart, the position of the four chambers appeared normal as compared with that in the normal case, although the left ventricle was hypoplastic (Fig. 5B and F). However, when the images of the bilateral atria were removed to observe the outflow tract, dislocation of the outflow tract was detected. In normal embryos at this stage, the septation of the outflow tract has been finished and the right and left ventricles are connected with the pulmonary trunk and the ascending aorta, respectively (Fig. 5C and D). The pulmonary trunk is formed to flow into the descending aorta (Fig. 5C). In the HPE case, the spatial relationship between the ascending aorta, pulmonary trunk, ductus arteriosus, and descending aorta appeared normal, but both the ascending aorta and the pulmonary trunk were found to emerge from the right ventricle (Fig. 5G and H). The hypoplastic left ventricle communicated with the right ventricle through the ventricular septal defect (VSD; Fig. 5H). Thus, this HPE embryo had the double-outlet right ventricle (DORV) and hypoplasia of the left ventricle. Both atria and other great vessels appeared to be normal.
During embryonic development, dynamic morphogenetic changes occur in a spatially and temporally coordinated manner. The cardiovascular system is one of the organ systems that undergo drastic morphogenetic movements. The sequential changes of the heart and great vessels in human embryos used to be examined by observing histological sections and wax plate models reconstructed from serial sections (Congdon, 1922; Streeter, 1948), which contributed significantly to human embryology and have been well cited by subsequent embryologists (Cooper and O'Rahilly, 1971; O'Rahilly, 1971).
Our study using computer-assisted reconstruction of the heart and great vessels of externally normal embryos largely confirmed the results of those classical reports, although some discrepancies were noted. For example, it was previously shown that the first and second pharyngeal arch arteries develop (Davis, 1927) and the third pairs of pharyngeal arch arteries begin to form (DeVries and Saunders, 1962) by CS 12, but in our constructed models, only one pair of pharyngeal arch arteries could be observed at CS 12 (Fig. 2A), which was confirmed by reexamining the relevant histological sections. Such a discrepancy may partly be due to the individual difference in the timing of morphogenesis of specific structures.
The embryonic venous system was also reconstructed in this study, including the hepatic sinusoidal connection. Although there have been some classic studies on venous development in human embryos (Streeter, 1942, 1945; Licata, 1954; O'Rahilly, 1971), it seems worthwhile to undertake a systematic study on the development of the venous system in staged human embryos by employing modern morphological techniques. The development of the coronary circulation, outflow tract, and craniofacial arteries as well as the hepatic vascular system is now under investigation in our laboratory and will be described in detail in subsequent papers.
HPE is one of the most common developmental disorders of the brain associated with specific craniofacial dysmorphogenesis. Recent genetic studies have revealed the mutations of many genes in HPE patients, such as SHH, ZIC2, SIX3, TGIF, TDGF1, FAST1, PTCH, GLI2, and DHCR7 (Edison and Muenke, 2003; Cohen, 2004), but the relationship between the genotype and the phenotype has not been well elucidated. Various anomalies have been reported to be associated with HPE (Cohen, 1989), and congenital heart anomalies are often encountered in some syndromic HPE (Cohen and Sulik, 1992). Although the HPE–heart anomalies–polydactyly sequence has been described (Hennekam et al., 1991), embryonic HPE cases with heart anomalies have rarely been reported. Since the shape and size of the heart and great vessels undergo dramatic changes during development, it is sometimes difficult to understand the detailed 3D anatomy of heart anomalies. For example, the outflow tract is separated into the ascending aorta and pulmonary trunk, and its septum is not simple but spiral in shape. Therefore, 3D analysis is important and powerful as shown in the present study.
For this study, two HPE cases associated with heart anomalies were selected. Based on histological observation, one case (case 25556) was diagnosed to have malrotation of the primary heart tube, and the other case (case 28200) had VSD. By 3D analysis, DORV (with subpulmonic VSD) and hypoplastic LV were detected in the latter. There have been only two reports of postnatal HPE cases associated with DORV (Bollmann et al., 1989; van Essen et al., 1993), but no embryo cases of HPE with DORV have been reported so far. HPE with DORV or other congenital heart anomalies may have been overlooked because many HPE embryos have not been examined in detail except for selected cases (Müller and O'Rahilly, 1989).
In one of our HPE cases, malrotation of the heart tube was noted. Although some gene mutations that affect cardiac looping have been identified in experimental animals and humans (Kathiriya and Srivastava, 2000), HPE genes directly causing failure of cardiac looping have not been identified. However, cardiac looping is closely related to left-right patterning, in which Shh, an HPE gene, plays an important role (Maclean and Dunwoodie, 2004). This warrants further study to identify malrotations of the heart in HPE patients. Some Shh null mutant mice display congenital heart anomalies, including those of the outflow tract (Washington Smoak et al., 2005). Their heart defects resembled the tetralogy of Fallot (TOF), which consists of pulmonary valve stenosis, VSD, overriding aorta, and RV hypertrophy. One of our HPE cases had DORV, in which both the aorta and pulmonary artery emerged from the right ventricle and VSD was also associated. Although TOF and DORV are not the same diseases, they both result from the failure of remodeling of the outflow tract (Sakabe et al., 2005). Outflow tract malformations are considered to result from failure of the function of neural crest cells (Sugishita et al., 2004), and DORV has been reported to be induced in diabetic rats (Siman et al., 2000) or mutant mice (Campione et al., 2001). It will be of interest to study the etiology and pathogenesis of outflow tract anomalies, including DORV associated with HPE.
This is the first report describing the 3D analysis of the developing heart and great vessels of normal and HPE human embryos. Various softwares are now available for computer-assisted 3D reconstruction of biological structures and some of them are rather easy to use. Three-dimensional reconstruction of embryonic structures could be utilized not only in human embryology but also for morphological analysis of gene knockout and transgenic animals.
The authors thank Mr. Yoshihiro Ueda for his technical assistance. Supported by the BIRD grant from the Japan Science and Technology Agency (to K.S.).