Human Cardiac Development in the First Trimester
2009; Lippincott Williams & Wilkins; Volume: 120; Issue: 4 Linguagem: Inglês
10.1161/circulationaha.108.796698
ISSN1524-4539
AutoresPreeta Dhanantwari, Elaine Lee, Anita Krishnan, Rajeev Samtani, Shigehito Yamada, Stasia A. Anderson, Elizabeth Lockett, Mary T. Donofrio, Kohei Shiota, Linda Leatherbury, Cecilia Lo,
Tópico(s)Genomic variations and chromosomal abnormalities
ResumoHomeCirculationVol. 120, No. 4Human Cardiac Development in the First Trimester Free AccessReview ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissionsDownload Articles + Supplements ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toSupplemental MaterialFree AccessReview ArticlePDF/EPUBHuman Cardiac Development in the First TrimesterA High-Resolution Magnetic Resonance Imaging and Episcopic Fluorescence Image Capture Atlas Preeta Dhanantwari, Elaine Lee, Anita Krishnan, Rajeev Samtani, Shigehito Yamada, Stasia Anderson, Elizabeth Lockett, Mary Donofrio, Kohei Shiota, Linda Leatherbury and Cecilia W. Lo Preeta DhanantwariPreeta Dhanantwari From the Laboratory of Developmental Biology (P.D. E. Lee, A.K., R.S., S.Y., L.L., C.W.L.) and Animal Magnetic Resonance Imaging Core (S.A.), National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Md; Children’s National Heart Institute, Children’s National Medical Center, Washington, DC (P.D., A.K., M.D., L.L.); National Museum of Health and Medicine, Human Developmental Anatomy Center, Washington, DC (E. Lockett): and Congenital Anomaly Research Center, Kyoto University Graduate School of Medicine, Kyoto, Japan (S.Y., K.S.). Search for more papers by this author , Elaine LeeElaine Lee From the Laboratory of Developmental Biology (P.D. E. Lee, A.K., R.S., S.Y., L.L., C.W.L.) and Animal Magnetic Resonance Imaging Core (S.A.), National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Md; Children’s National Heart Institute, Children’s National Medical Center, Washington, DC (P.D., A.K., M.D., L.L.); National Museum of Health and Medicine, Human Developmental Anatomy Center, Washington, DC (E. Lockett): and Congenital Anomaly Research Center, Kyoto University Graduate School of Medicine, Kyoto, Japan (S.Y., K.S.). Search for more papers by this author , Anita KrishnanAnita Krishnan From the Laboratory of Developmental Biology (P.D. E. Lee, A.K., R.S., S.Y., L.L., C.W.L.) and Animal Magnetic Resonance Imaging Core (S.A.), National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Md; Children’s National Heart Institute, Children’s National Medical Center, Washington, DC (P.D., A.K., M.D., L.L.); National Museum of Health and Medicine, Human Developmental Anatomy Center, Washington, DC (E. Lockett): and Congenital Anomaly Research Center, Kyoto University Graduate School of Medicine, Kyoto, Japan (S.Y., K.S.). Search for more papers by this author , Rajeev SamtaniRajeev Samtani From the Laboratory of Developmental Biology (P.D. E. Lee, A.K., R.S., S.Y., L.L., C.W.L.) and Animal Magnetic Resonance Imaging Core (S.A.), National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Md; Children’s National Heart Institute, Children’s National Medical Center, Washington, DC (P.D., A.K., M.D., L.L.); National Museum of Health and Medicine, Human Developmental Anatomy Center, Washington, DC (E. Lockett): and Congenital Anomaly Research Center, Kyoto University Graduate School of Medicine, Kyoto, Japan (S.Y., K.S.). Search for more papers by this author , Shigehito YamadaShigehito Yamada From the Laboratory of Developmental Biology (P.D. E. Lee, A.K., R.S., S.Y., L.L., C.W.L.) and Animal Magnetic Resonance Imaging Core (S.A.), National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Md; Children’s National Heart Institute, Children’s National Medical Center, Washington, DC (P.D., A.K., M.D., L.L.); National Museum of Health and Medicine, Human Developmental Anatomy Center, Washington, DC (E. Lockett): and Congenital Anomaly Research Center, Kyoto University Graduate School of Medicine, Kyoto, Japan (S.Y., K.S.). Search for more papers by this author , Stasia AndersonStasia Anderson From the Laboratory of Developmental Biology (P.D. E. Lee, A.K., R.S., S.Y., L.L., C.W.L.) and Animal Magnetic Resonance Imaging Core (S.A.), National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Md; Children’s National Heart Institute, Children’s National Medical Center, Washington, DC (P.D., A.K., M.D., L.L.); National Museum of Health and Medicine, Human Developmental Anatomy Center, Washington, DC (E. Lockett): and Congenital Anomaly Research Center, Kyoto University Graduate School of Medicine, Kyoto, Japan (S.Y., K.S.). Search for more papers by this author , Elizabeth LockettElizabeth Lockett From the Laboratory of Developmental Biology (P.D. E. Lee, A.K., R.S., S.Y., L.L., C.W.L.) and Animal Magnetic Resonance Imaging Core (S.A.), National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Md; Children’s National Heart Institute, Children’s National Medical Center, Washington, DC (P.D., A.K., M.D., L.L.); National Museum of Health and Medicine, Human Developmental Anatomy Center, Washington, DC (E. Lockett): and Congenital Anomaly Research Center, Kyoto University Graduate School of Medicine, Kyoto, Japan (S.Y., K.S.). Search for more papers by this author , Mary DonofrioMary Donofrio From the Laboratory of Developmental Biology (P.D. E. Lee, A.K., R.S., S.Y., L.L., C.W.L.) and Animal Magnetic Resonance Imaging Core (S.A.), National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Md; Children’s National Heart Institute, Children’s National Medical Center, Washington, DC (P.D., A.K., M.D., L.L.); National Museum of Health and Medicine, Human Developmental Anatomy Center, Washington, DC (E. Lockett): and Congenital Anomaly Research Center, Kyoto University Graduate School of Medicine, Kyoto, Japan (S.Y., K.S.). Search for more papers by this author , Kohei ShiotaKohei Shiota From the Laboratory of Developmental Biology (P.D. E. Lee, A.K., R.S., S.Y., L.L., C.W.L.) and Animal Magnetic Resonance Imaging Core (S.A.), National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Md; Children’s National Heart Institute, Children’s National Medical Center, Washington, DC (P.D., A.K., M.D., L.L.); National Museum of Health and Medicine, Human Developmental Anatomy Center, Washington, DC (E. Lockett): and Congenital Anomaly Research Center, Kyoto University Graduate School of Medicine, Kyoto, Japan (S.Y., K.S.). Search for more papers by this author , Linda LeatherburyLinda Leatherbury From the Laboratory of Developmental Biology (P.D. E. Lee, A.K., R.S., S.Y., L.L., C.W.L.) and Animal Magnetic Resonance Imaging Core (S.A.), National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Md; Children’s National Heart Institute, Children’s National Medical Center, Washington, DC (P.D., A.K., M.D., L.L.); National Museum of Health and Medicine, Human Developmental Anatomy Center, Washington, DC (E. Lockett): and Congenital Anomaly Research Center, Kyoto University Graduate School of Medicine, Kyoto, Japan (S.Y., K.S.). Search for more papers by this author and Cecilia W. LoCecilia W. Lo From the Laboratory of Developmental Biology (P.D. E. Lee, A.K., R.S., S.Y., L.L., C.W.L.) and Animal Magnetic Resonance Imaging Core (S.A.), National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Md; Children’s National Heart Institute, Children’s National Medical Center, Washington, DC (P.D., A.K., M.D., L.L.); National Museum of Health and Medicine, Human Developmental Anatomy Center, Washington, DC (E. Lockett): and Congenital Anomaly Research Center, Kyoto University Graduate School of Medicine, Kyoto, Japan (S.Y., K.S.). Search for more papers by this author Originally published28 Jul 2009https://doi.org/10.1161/CIRCULATIONAHA.108.796698Circulation. 2009;120:343–351With rapid advances in medical imaging, fetal diagnosis of human congenital heart disease is now technically feasible in the first trimester. Although the first human embryological studies were recorded by Hippocrates in 300 to 400 BC, present-day knowledge of normal human cardiac development in the first trimester is still limited. In 1886, 2 articles by Dr His described the development of the heart on the basis of dissections of young human embryos. Free-hand wax models were made that illustrated the external developmental anatomy. These wax plate reconstruction methods were used by many other investigators until the early 1900s.1 Subsequently, serial histological sections of human embryos have been used to further investigate human cardiac development.2–6 Using an analysis of histological sections and scaled reproductions of human embryos, Grant2 showed a large cushion in the developing heart at 6 weeks (Carnegie stage [CS] 14) and separate atrioventricular (AV) valves at 9 weeks (CS 22). At the end of 8 weeks (CS 8), separate aortic and pulmonary outflows were observed. Orts-Llorca et al5 used 3-dimensional (3D) reconstructions of transverse sections of human embryos to define the development of the truncus arteriosus and described completion of septation of the truncus arteriosus in 14- to 16-mm embryos, equivalent to an estimated gestational age (EGA) of 8 weeks (CS 18).Given the complex tissue remodeling associated with cardiac chamber formation and inflow/outflow tract and valvular morphogenesis, the plane of sectioning often limited the information that can be gathered on developing structures in the embryonic heart. These technical limitations, in conjunction with limited access to human embryo specimens, have meant that much of our understanding of early cardiac development in the human embryo is extrapolated from studies in model organisms.7–10 With possible species differences in developmental timing and variation in cardiovascular anatomy, characterization of normal cardiac development in human embryos is necessary for clinical evaluation and diagnosis of congenital heart disease in the first trimester. This will be increasingly important as improvements in medical technology allow earlier access to first-trimester human fetal cardiac imaging and in utero intervention.Recent studies have shown the feasibility of using magnetic resonance imaging (MRI) to obtain information on human embryo tissue structure.11,12 MRI data can be digitally resectioned for viewing of the specimen in any orientation, and 3D renderings can be obtained with ease. Similarly, episcopic fluorescence image capture (EFIC), a novel histological imaging technique, provides registered 2-dimensional (2D) image stacks that can be resectioned in arbitrary planes and rapidly 3D rendered.10 With EFIC imaging, tissue is embedded in paraffin and cut with a sledge microtome. Tissue autofluorescence at the block face is captured and used to generate registered serial 2D images of the specimen with better image resolution than MRI. Data obtained by MRI or EFIC imaging can be easily resectioned digitally or reconstructed in 3D to facilitate the analysis of complex morphological changes in the developing embryonic heart. In this manner, the developing heart in every embryo can be analyzed in its entirety with no loss of information resulting from the plane of sectioning.Using MRI and EFIC imaging, we conducted a systematic analysis of human cardiovascular development in the first trimester. The 2D image stacks and 3D volumes were generated from 52 human embryos from 6 to 9 weeks of EGA, equivalent to CS 13 to 23. These stages encompass the developmental window during which all of the major milestones of cardiac morphogenesis can be observed. Using the MRI and EFIC data, we constructed a digital atlas of human heart development. Data from our atlas were used to generate charts summarizing the major milestones of normal human heart development through the first trimester. MRI and EFIC images obtained as part of this study can be viewed as part of the online Human Embryo Atlas. To view the Human Embryo Atlas content, visit http://apps.nhlbi.nih.gov/ HumanAtlas/home/login.aspx?ReturnUrl=%2fhumanatlas%2fDefault.aspx.SpecimensEmbryos from the Kyoto collection, at the Congenital Anomaly Research Center at the Kyoto University in Japan, were collected after termination of pregnancies for socioeconomic reasons under the Maternity Protection Law of Japan. Embryos were derived from normal pregnancies without any clinical presentations. The specimens were in fixative for an estimated duration of 30 to >40 years, making them unsuitable for immunohistochemistry or any molecular/cellular analysis. This collection represents a random sample of the total intrauterine population of Japan.13–16 During accessioning into the Kyoto collection, the embryos were examined and staged according to the CS criteria proposed by O’Rahilly.17 For this study, 52 embryos from the Kyoto collection (see Table I of the online-only Data Supplement) were donated to the Carnegie collection of normal human embryos archived at the National Museum of Health and Medicine of the Armed Forces Institute of Pathology (http://nmhm.washingtondc.museum/collections/hdac/Carnegie_collection.htm). Each embryo’s age was determined through the use of previously reported postconceptional ages,14 which were then converted to EGA or menstrual age by adding 14 days and reported in weeks.MRI and EFICHigh-resolution MRI and EFIC images were obtained from 52 human embryos from 6 to 9 weeks of gestation (CS 13 to 23). These specimens from the Kyoto collection were imaged by MRI and EFIC during preparation for accessioning into the Carnegie collection (Table I of the online-only Data Supplement). Human embryos in formalin were treated with 1:20 Magnevist (Berlex, Montville, NJ)/10% formalin solution for ≥3 days and then rinsed and prepared in 5- to 30-mm tubes, depending on embryo size, with fixative or low-melting agar. Samples that diffused gadolinium into the media were further soaked in plain fixative for ≥2 days and reimaged. Imaging was performed at the National Institutes of Health Mouse Imaging Facility on a 7.0-T Bruker vertical-bore MRI system with 150-G/cm gradients (Bruker, Billerica, Mass) and 5- to 30-mm microimaging birdcage coils (Bruker). Some larger samples also were imaged on a 7.0-T, 16-mm horizontal-bore Bruker Paravision system with 39-G/cm gradients and a 38-mm birdcage coil. MRI was acquired with Paravision 3.0.2 operating systems. Samples were imaged with a 3D rapid gradient echo (SNAP) sequence with a repetition time of 30 to 40 ms, an echo time of 3.3 to 4.0 ms, 20 to 90 averages, an acquisition time of ≈12 to 50 hours, and matrixes of 256×128×128 to 512×512×512 (see Table II of the online-only Data Supplement). Over the whole collection, MRI resolution ranged from 29×35×35 to 117×105×105 μm3. Resolution was proportionate to the sample size, with the smallest embryos having the highest-resolution data sets. Individual image data sets are 3D and nearly isotropic, with all 3 voxel dimensions being within 10 μm of each other in an individual data set. Most data sets are in the range of 35×35×35 to 60×60×60 μm3. The resolution of each data set is listed in Table II of the online-only Data Supplement.In preparation for EFIC, embryos stored in 10% phosphate-buffered formalin were dehydrated and embedded in a mixture of paraffin wax (70.4%), Vybar (24.9%), stearic acid (4.4%), and red aniline dye Sudan IV (0.4%) using techniques previously described.10,18 The embedded embryos were then sectioned with a sliding microtome (Leica SM 2500) to obtain 5- to 8-μm-thick sections. The block face was sequentially photographed using epifluorescent illumination with a 100-W mercury lamp and a Leica MZ16A stereomicroscope equipped with 425/480-nm excitation/emission filters. Images were captured with an ORCA-ER digital camera (Hamamatsu, Shizuoka, Japan).Offline Image Processing and Data AnalysisMRI images originally recorded in DICOM were converted into TIFF format with ImageJ (http://rsb.info.nih.gov/ij/). The EFIC 2D image stacks were captured and exported as TIFF files. Both the EFIC and MRI data were processed with OpenLab (Improvision Inc, Waltham, Mass). We generated 3D reconstructions and QuickTime virtual-reality movies with Volocity (Improvision Inc). The 2D image stacks also were digitally resectioned with Volocity to view internal and external cardiac structures in planes similar to standard echocardiographic imaging planes used clinically. In EFIC images, each pixel was a square with length dimensions ranging from 2.34 to 13.4 μm per pixel edge. Thus, pixel dimensions ranged from 5.48 to 179.56 μm2. For each embryo, we generated serial 2D image stacks and 3D reconstructions. From this analysis, we were able to delineate all of the major milestones of human heart development, including chamber formation; septation of the atria, ventricles, and truncus arteriosus; and valvular morphogenesis.Cardiac LoopThe cardiac loop or looped heart tube is observed from EGA 6 to 7 weeks (CS 13 to 17). A 3D reconstruction of the heart at 7 weeks (CS 17) reveals internal structures of the cardiac loop (Figure 1). The only exit for blood from the left-sided inflow limb, consisting of the atrial cavity, AV junction, and the presumptive left ventricle, is the interventricular foramen (also known as primary foramen, primary interventricular foramen, bulboventricular foramen, or embryonic interventricular foramen; double arrow in Figure 1); the only exit for blood from the right-sided outflow limb, consisting of the presumptive right ventricle, is the truncus arteriosus (arrowhead in Figure 1). Also of note, the AV junction (Figure 1) is surrounded by endocardial cushion tissue, which is contiguous with the truncus arteriosus. Download figureDownload PowerPointFigure 1. A 3D view of the cardiac loop in an embryo at EGA of 7 weeks (CS 17). The 2D EFIC image stacks were reconstructed in 3D to show the looped heart tube in an embryo at EGA of 7 weeks (CS 17). The double-headed arrow indicates the interventricular foramen. The orifice of the developing AV junction is seen as a horizontal line above the AV label. The truncus arteriosus (arrowhead) is also seen. Endocardial cushion tissue surrounding the AV junction is adjacent to the truncus arteriosus. Scale bar=0.6 mm. A indicates anterior; P, posterior; R, right; L, left; Cr, cranial; Ca, caudal; RA, right atrium; LA, left atrium; RV, presumptive right ventricle; and LV, presumptive left ventricle.The developmental changes seen in the cardiac loop are shown in more detail in Figure 2, with images from embryos at 6 weeks (CS 13) (Figure 2A through 2E) and 7 weeks (CS 17) (Figure 2F through 2I). As the looped heart tube matures, the atrial and ventricular chambers expand in size, giving rise to distinct subdivisions recognizable as the primitive left and right atria and presumptive left and right ventricles (Figure 2H). At 6 weeks (CS 13), the endocardial cushions seen lining the AV junction appear thin with little apparent cellular content. As development progresses, they become filled with dense material (Figure 2B, 2E, and 2G and Movie VIIIa and VIIIb of the online-only Data Supplement). The interventricular foramen also shows striking changes during this developmental period. It is a wide and open communication at 6 weeks (CS 13) (asterisk in Figure 2C), but as the chambers grow, it becomes a narrow and more distinct opening (foramen) by 7 to 7 weeks (CS 16 to 17) (asterisk in Figures 2H and 3C). The superior AV cushion can be seen (Figure 2G). The inflow, consisting of the venous confluence or primitive atrium (Figure 2A), is observed to communicate with the ventricular chamber via the AV junction (Figure 2B, 2E, and 2G). The presumptive left ventricle communicates with the presumptive right ventricle via the interventricular foramen (asterisk in Figure 2C and 2H). The outflow from the cardiac loop comprises the as-yet undivided truncus arteriosus (T in Figure 2D and 2I) arising from the presumptive right ventricle. Download figureDownload PowerPointFigure 2. Defining structures of the cardiac loop. A through E, EFIC and MRI images of embryos at EGA of 6 weeks (CS 13) shown in various imaging planes. Imaging in the frontal plane (A) shows the common cardinal veins or the open venous confluence (arrow); the sagittal view (B) shows primitive endocardial cushions at the AV junction (arrowhead). A 3D model of the same embryo (E) shows the extent of the interventricular foramen and the contour of the endocardial cushions. MRI of another embryo in the sagittal plane (C) shows the presumptive right ventricle (RV), atrial chamber (A), and a nondistinct interventricular foramen (*), whereas the ventricular chamber (V) and a single, undivided truncus arteriosus (T) can be seen in an frontal section of a third embryo (D). Scale bar=0.4 mm (A through D) and 0.25 mm (E). F through I, MRIs of an embryo at EGA of 7 weeks (CS 17). Image from an oblique transverse plane (F) shows the right (RA) and left atrial (LA) chambers as septation progresses (arrowhead). The developing ventricle (V) is seen. Viewed in the transverse plane (G), well-formed dense endocardial cushion tissue is seen at the AV junction (arrowhead). Another section in the transverse plane (H) shows the right and left ventricular cavities with a more distinct interventricular foramen (*). Septum primum can be seen as atrial septation progresses (arrowhead). The single undivided truncus arteriosus (T) and interventricular foramen communicating with the presumptive left ventricle (V) can be seen in an oblique transverse plane (I). Scale bar=1.250 mm (E through H). A indicates anterior; P, posterior; R, right; L, left; Cr, cranial; Ca, caudal; LV, presumptive left ventricular chamber; and T, truncus arteriosus.Download figureDownload PowerPointFigure 3. Major events of atrial and ventricular septation. A and B, EFIC image an embryo at EGA of 6 weeks (CS 14) in the transverse plane (A) shows the atrial spine (arrowhead) attached to the inferior cushion (*). A 3D reconstruction (B) highlights the endocardial cushions and trabeculation in the ventricular chamber. Scale bar=0.515 mm (A) and 0.272 (B). C and D, An EFIC image of an embryo at EGA of 7 weeks (CS 16) in the oblique plane (C) showing right and left ventricular chambers connected by an interventricular foramen (*). A 3D reconstruction of the same embryo (D) delineates the contour of the interventricular foramen and the orifices of the AV canal and the truncus arteriosus. Scale bar=0.5 mm (C) and 0.900 mm (D). E, MRI of an embryo at EGA of 7 weeks (CS 16) also in the transverse plane. It shows the formation of septum primum (*) between the right (RA) and left atria (LA). Scale bar=0.5 mm. F and G, MRI of an embryo at EGA of 8 weeks (CS 18) in an oblique transverse plane (F) shows a complete atrial septum (*). The most caudal portion of the septum primum, the mesenchymal cap, has fused to the superior cushion. The growth of the muscular ventricular septum into the ventricular cavity also is shown. The crest of the muscular interventricular septum is present with an incomplete inlet ventricular septum (arrowhead) immediately above it. G, Another MRI of the same embryo in an oblique coronal plane shows the formed outlet ventricular septum (arrowhead). Together, these 2 images show that outlet ventricular septation is completed before inlet ventricular septation. Scale bar=1.5 mm (F and G). H, MRI of an embryo at EGA of 9 weeks (CS 22) in an oblique coronal plane shows a completed inlet ventricular septum (arrowhead). Scale bar=2 mm. A (on compass) indicates anterior; P, posterior; R, right; L, left; Cr, cranial; Ca, caudal; A, primitive atrium/venous confluence; RA, right atrium; LA, left atrium; V, ventricular chamber; LV, left ventricular chamber; and RV, right ventricular chamber.Atrial Septation (EGA 6 to 8 Weeks)The process of atrial septation is thought to begin with a thin septum primum growing from the posterior wall of the atrium, from a location cranial to the pulmonary vein orifice. It grows toward and eventually fuses with the endocardial cushions.19 At 6 weeks of gestation (CS 14), the mesenchymal cap of the primary atrial septum can be seen in contact with the superior AV cushion. The atrial spine, a mesenchymal structure, also was observed. The atrial spine fuses with the inferior AV cushion (6 weeks [CS 14]) (Figure 3A and 3B and Movie VIIIa and VIIIb) and plays an important role in closure of the primary foramen. Although the pulmonary vein orifice was not seen by our imaging, we can infer from previous studies that it lies to the left of the atrial spine.20 Septum primum can be observed at 6 weeks of gestation, and its developmental progression through 7 weeks can be seen in Figures 2H, 3A, 3B, and 3E and Movies I, VIIIa, and VIIIb). Later, septum secundum develops as an infolding of the dorsal wall of the right atrium, completing atrial septation with fenestrations forming the foramen ovale. Both atrial septum primum and secundum were present by 8 weeks (CS 18) (Movie II of the online-only Data Supplement). At this stage, the mesenchymal cap can be seen fused with the now divided superior AV cushion (Figure 3F). This is consistent with developmental timing suggested by others.21,22Ventricular Septation (EGA 7 to 9 Weeks)Toward the end of the looped heart tube stages of development (7 and 7 weeks, CS 16 and 17), distinct separation of presumptive left and right ventricular chambers is evident. The beginning of the muscular interventricular septum can be seen at these stages, but ventricular septation is not yet complete (Figures 2H, 3C, and 3D and Movie IVa and IVb of the online-only Data Supplement). By 8 weeks (CS 18), the muscular ventricular septum can be seen extending from the floor of the ventricular chamber toward the crux of the heart (Figure 3F). This leaves open a relatively large interventricular foramen that allows communication between the ventricles (Movies I and II of the online-only Data Supplement). Recent lineage tracing experiments in mice have suggested that the muscular interventricular septum is made up of cells originating from the ventral aspect of the primitive ventricle, with closure of the ventricular foramen mediated by dorsal migration of this precursor cell population; these cells likely represent a subpopulation of cells derived from the secondary heart field.23 Immunohistochemical analysis of human fetal cardiac tissue showed myocytes expressing the G1N2 antigen localized in a ring around the junction between the future right and left ventricles.24 In later developmental stages, G1N2-expressing cells are found in the area clinically called the inlet ventricular septum but not in the subaortic outflow septum.At 8 weeks (CS 18), the ventricular septum at the level of the left ventricular outflow is closed (Figure 3G), but part of the inlet ventricular septum at the level of the AV valves remains open (arrowhead in Figure 3F and Movies III, IV, and VI of the online-only Data Supplement). The inlet and membranous portions of the ventricular septum are fully closed at 9 weeks (CS 22), completing ventricular septation (Figure 3H and Movie V of the online-only Data Supplement). The area clinically called the inlet ventricular septum has been shown in prior studies to originate from the embryonic right ventricle.25 In agreement with previous reports on human development, our data showed that the final portion of the ventricular septum to close included what likely makes up a combination of the membranous and inlet ventricular septum. These findings suggest that an arrest in the development of the ventricular septum could result in ventricular septal defects similar to those observed clinically.Formation of the AV Valves (EGA 7 to 8 Weeks)AV valve morphogenesis begins at the looped heart tube stages, with large endocardial cushions seen prominently at the center of the cardiac loop (asterisks in Figures 3A and 4A). The AV canal is divided by the endocardial cushions, which form on the posterior (dorsal) and anterior (ventral) walls of the AV canal. These cushions eventually divide the AV canal into right and left AV orifices.2,19 A well-delineated AV junction can be seen at 7 weeks of gestation (CS 16) (Figure 4B and 4C and Movie Xa and Xb of the online-only Data Supplement). At 7 and 7 weeks (CS 16 and 17), the AV junction was still undivided. A few days later, by 8 weeks of gestation (CS 18), separate AV valves can be seen (arrowheads in Figure 4D and 4E), with left-sided mitral and right-sided tricuspid valves forming. An embryo at 8 weeks (CS 18) is ≈10 mm in size, correlating well with the embryonic stage at which fusion of the endocardial cushions is thought to occur.26 The valve leaflets, however, appear thick at this stage (see Movie IV of the online-only Data Supplement). By 9 weeks (CS 22), the AV valve leaflets are thinner and more mature in appearance (Figure 3H and Movie V of the online-only Data Supplement). At 7 weeks (CS 16), distinct posterior and anterior cushions are not observed and the inferior AV cushion is observed; this timing is consistent with previous reports of human embryonic development.22Download figureDownload PowerPointFigure 4. Major milestones of AV valve morphogenesis. A, EFIC image of an embryo at EGA of 6 weeks (CS 14) in the transverse plane shows a large endocardial cushion (*) in the center of the cardiac loop. Scale bar=0.515 mm. B and C, EFIC image of an embryo at EGA of 7 weeks (CS 16) in a sagittal plane (B) shows a tight, well-formed AV junction (arrowhead) and the truncus arteriosus (T). Three-dimensional volume of the same embryo (C) shows exquisite detail of the contour and shape of the endocardial cushions and the truncus arteriosus. Scale bar=0.389 mm (B and C). D and E, MRI in an oblique transverse plane (D) of an embryo at EGA 8 weeks (CS 18) shows separate AV valves. The valve leaflets appear thick at this stage. Note the right a
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