A pictorial account of the human embryonic heart between 3.5 and 8 weeks of development

Abstract

Heart development is topographically complex and requires visualization to understand its progression. No comprehensive 3-dimensional primer of human cardiac development is currently available. We prepared detailed reconstructions of 12 hearts between 3.5 and 8 weeks post fertilization, using Amira® 3D-reconstruction and Cinema4D®-remodeling software. The models were visualized as calibrated interactive 3D-PDFs. We describe the developmental appearance and subsequent remodeling of 70 different structures incrementally, using sequential segmental analysis. Pictorial timelines of structures highlight age-dependent events, while graphs visualize growth and spiraling of the wall of the heart tube. The basic cardiac layout is established between 3.5 and 4.5 weeks. Septation at the venous pole is completed at 6 weeks. Between 5.5 and 6.5 weeks, as the outflow tract becomes incorporated in the ventricles, the spiraling course of its subaortic and subpulmonary channels is transferred to the intrapericardial arterial trunks. The remodeling of the interventricular foramen is complete at 7 weeks.

Fig. 1. Pictorial timeline of the development of the inflow tract.

Dorsal views of embryonic hearts are shown between CS10 and CS14, with panels a–e emphasizing endocardial continuity and panels f–j the myocardial coat. The panels are aligned relative to the position of the pulmonary vein (black horizontal line). Note that the distance between the arterial and venous poles of the heart loop does not change during cardiac looping (CS10-CS12;^,). The appearance of a myocardial wall indicates the formation of that compartment in the inflow tract. Myocardium appears in the wall of the atrium at CS10 and in the wall of the systemic venous sinus at CS12. The pulmonary vein, along with its flanking atrial ridges, also begins to form at CS12. The sinus node becomes recognizable as a separate structure at CS13. The left and right atriums are already distinguishable at CS10, but the sinuatrial junction does not become a right-sided structure until CS13. The atrial septum appears at CS14. It is identifiable as the “empty” space between left and right atriums in panel e. The hepatocardiac veins are the only source of venous blood for the heart until CS12, when the initially small common cardinal veins appear. All images are also available as preset views in the corresponding 3D-PDFs.

Fig. 2. Looping of the heart tube.

The panels show ventral right and ventral left views of the cardiac lumen and the adjacent vessels in CS10-12 embryos. The panels were aligned on the arterial and venous poles of the heart loop (black horizontal line). The first signs of looping are seen at CS10, when the dorsal mesocardium disappears at the junction of the embryonic ventricle and outflow tract. The center of the heart tube, represented by the yellow wire, bends rightward and ventrally, in particular in its cranial part. At CS11, the loop extends ventrally due to axial growth and becomes the more pronounced “C” loop. The embryonic left ventricle represents the most ventral portion of the heart loop at this stage. The atrioventricular junction has moved leftward, while the common atrium and distal part of the outflow tract remain midline structures. At CS12, endothelial sprouting into the cardiac jelly marks the boundaries of the ballooning apical parts of the ventricles (arrow). The heart loop between the left ventricle and distal outflow tract further increases in length in a rightward and dorsal direction, with the embryonic right ventricle emerging at its apex (see wire loop). Note that looping has induced two helical twists in the heart axis that meet in the right ventricle. All images are also available as preset views in the corresponding 3D-PDFs.

Fig. 3. Pictorial timeline of the changing position of the developing right ventricle.

The figure shows caudal (CS12 and CS13) or ventral views (CS14-23) of the heart lumen between CS12 and CS23. The difference in the viewing angle reflects the changing curvature of the embryonic axis. The position of the right relative to the left ventricle gradually changes over ~60° between CS12 and CS18 (graph; each dot represents a single embryo). The right ventricle is positioned caudally relative to the left ventricle at CS12 and achieves a more cranial position after CS18. The interventricular foramen is relatively long during CS12-14. The wide space between left and right ventricular lumens after CS20 reflects the appearance of compact myocardium and a thick muscular ventricular septum. The ventricular axes are almost sagittal prior to CS20, and become oblique and leftward at CS23, reflecting the changing shape of the rib cage. All images are also available as preset views in the corresponding 3D-PDFs.

Fig. 4. Pictorial timeline of the closure of the interatrial & interventricular foramens.

The panels show right ventral views of the left side of the heart. The left atrial and ventricular cavities, the muscular atrial and ventricular septums, the endocardial cushions, the dorsal mesenchymal protrusion, the secondary atrial septum (CS23 only), and the GlN-positive ring bundle are shown. The superior and inferior endocardial cushions, including their atrial extensions, fuse at CS17. This fusion closes the primary atrial foramen and is accompanied by the appearance of a very wide secondary atrial foramen. The dorsal mesenchymal protrusion acquires a position at the base of the atrial septum due to the expansion of surrounding structures. The protrusion muscularizes, along with the mesenchymal cap, starting at CS18, and concomitant with the proximal endocardial ridges of the outflow tract. The borders of the interventricular foramen remodel as revealed by the course of the GlN-positive ring. As soon as septation of the outflow tract is complete at CS18, the myocardialized part of the fused endocardial ridges and the rightward margins of the atrioventricular endocardial cushions combine to decrease the size of the remaining foramen. The closure is complete at CS20. Gray contours: primary atrial foramen; white contours: secondary atrial foramen; yellow contours: interventricular foramen. All images are also available as preset views in the corresponding 3D-PDFs.

Fig. 5. Pictorial timeline of the subdivision of the interventricular foramen into the peri-tricuspid inlet, the subaortic outlet, and the membranous septum.

The figure shows the lumens of the atriums, ventricles and outflow tract, and the muscular ventricular septum. Panels a–e show a cranial view, and panels f–j a caudal view. When first formed, the interventricular foramen is a sagittally oriented interventricular conduit, as visualized by the GlN-positive ring. The craniodorsal part of the foramen, from which the GlN fades away at CS16, is indicated by a thinner, hatched line. The tips of the atrial appendages are clipped in the images for CS18 and CS20 (dashed lines) to permit inspection of the atrioventricular junction and outflow tracts. At CS16, the caudal part of the foramen and GlN ring begin to expand in rightward direction, producing a direct connection between the right atrium and ventricle, which is best seen in panels f–j. Meanwhile, the cranial, subaortic part of the foramen, which is best seen in panels a–e, gradually expands craniodorsally. Comparing the arrangements at CS18 and CS20, when the septation of the outflow tract is complete, shows that the subaortic, but not the subpulmonary, ventricular outlet is surrounded by the GlN ring. The remaining connection between the right ventricular cavity and the subaortic channel is still present at CS18. It is obliterated at CS20 by the formation of the membranous septum (not itself visible). All images are also available as preset views in the corresponding 3D-PDFs.

Fig. 6. Changes in size and shape of the myocardial outflow tract and arterial trunks.

The left-sided graph (a) shows the length of the muscular outflow tract between the proximal and distal ends of the endocardial outflow ridges. The green square symbols represent the measurements made in our reconstructions, with the blue triangular symbols taken from measurements made in 14 scanning electron microscopic images, and the red circular symbols representing those made in 18 immunohistochemically stained and partially reconstructed hearts. Each dot in the graphs represents a single embryo. There is axial growth of the muscular outflow tract up to CS16, when its length suddenly declines profoundly, with no resumption up to CS23. The right-sided graph (b) shows the change in volumetric growth rate of the myocardium of the outflow tract (light green triangles), right ventricle (green circles), and left ventricle (dark green squares). The growth rate in all three compartments declines at CS15, with the growth of the outflow tract being practically nonexistent. The lower left graph (c) shows the axial growth of the arterial trunks. The ascending aorta (blue triangles) increases continuously in length between CS14 and 10 weeks of development, whereas axial growth of the pulmonary trunk (red circles) stops after CS17. The distance between the distal myocardial border and the pericardial reflection (green squares) increases little to CS16, indicating that the myocardial jaws of the fishmouth stay close to the pericardial reflection, whereas the myocardial border moves away from the reflection concomitant with the abrupt shortening of the myocardial outflow tract after CS16. Panel d shows cranial views of the myocardial outflow tract, the arterial trunks, and the pericardial reflection (wire loop) to visualize the axial growth of the aortic trunk (dashed line). The images are aligned to the distal myocardial border of the outflow tract (black line), with the fishmouth representing the lateral indentations of the distal myocardium between CS14 and CS16. The slits in the jaws of the myocardial fishmouth are occupied by the non-myocardial mural columns (see Fig. 8a–e). The 6th arch arteries (dark brown) stem from the pulmonary trunk, while the ascending aorta is recognizable by its lateral “horns”. All images are also available as preset views in the corresponding 3D-PDFs.

Fig. 7. Pictorial timeline of the changes in size and shape of the lumen, endocardial ridges and swellings, and neural crest prongs of the outflow tract.

The images are aligned on the location of the developing pulmonary valve (black line). Panels a–g show cranial views of the outflow-tract lumen flanked by the parietal and septal endocardial ridges, and aortic and pulmonary swellings. The viewing angle is the same as for Fig. 6d. The arterial trunks are shown for identification of the subaortic and subpulmonary channels. The ascending aorta is recognizable by its lateral horns. The widening of the distal portions of both outflow ridges during CS16 and CS17 presages their allocation to the right or left semilunar leaflets of the aortic and pulmonary arterial valves at CS18. The valvar portions are marked by a less dark tint of the coding color, and are confined to the distal portion of the myocardial outflow tract. The dashed black line in panels a–g show the axial growth of the intrapericardial component of the aortic trunks. The saddle-shaped wire loop shows the position of the pericardial reflection. Panels h–n show the lumen of the outflow tract as seen from the right side, showing the neural crest cells within the aortopulmonary septum extending as columns of dense mesenchyme into both endocardial outflow ridges. The fusion of these columns creates a temporary “whorl” of neural crest cells between the subaortic and subpulmonary channels. The neural crest cells largely disappear between CS18 and CS23 due to intense apoptosis, with invading cardiomyocytes simultaneously populating the shell of the septum^,. All images are also available as preset views in the corresponding 3D-PDFs.

Fig. 8. Pictorial timeline of the appearance of the non-myocardial walls of the arterial trunks.

The panels are aligned on the pericardial reflection, shown by the wire loops, as in Fig. 7. Panels a–e show the lumen of the outflow tract, with the arterial trunks, the columns of neural crest, and the non-myocardial mural columns. The ascending aorta is recognizable by its lateral horns. The neural crest cells and intercalating non-myocardial tissues invade the distal wall of the outflow tract during CS14. The mural cells are first seen as relatively short aortic or cranial, and pulmonary or caudal columns. During CS14 and CS15, the pulmonary column is continuous dorsally with a club-like condensation of peritracheal mesenchyme, which has disappeared at CS16. The endocardial swellings associated with the aortic and pulmonary mural columns are relatively small during CS14 and CS15, but increase in size from CS16 onwards to begin their transformation into the dorsal and ventral semilunar leaflets, respectively, at CS18. As shown by the interrupted line, there is a gradual increase in the distance between the pericardial reflection and the plane of the valvar primordiums (cf. Fig. 7). Panels f–j show the same view of the lumens. Note that the prongs of neural crest mesenchyme mold the subaortic and subpulmonary channels during CS15 and CS16. The fusion of these prongs into a central whorl marks the separation of the subaortic and subpulmonary channels during CS17 and CS18 (the separation of these channels is shown from a different perspective in Fig. 9b). All images are also available as preset views in the corresponding 3D-PDFs.

Fig. 9. Changes in the course of the (sub-)aortic and (sub-)pulmonary channels.

The graph (a) shows the changes in the degree of spiraling of the bloodstreams in time and place. The reference plane is sagittal and each dot represents a single embryo. Axial changes in the position of the components of the outflow tract were measured as described in Supplemental Fig. 18. The line connecting the center of the ridges at their proximal ends is shown in purple solid triangular symbols, with the comparable line at their distal ends shown in red open triangular symbols. The blue solid circular and black open circular symbols identify the line connecting the centers of the orifices of the ascending aorta and pulmonary trunk at their proximal and distal ends, respectively, with the distal end of the ascending aorta measured at the pericardial reflection. The brown square symbols show the movement of the distal endocardial ridges relative to the distal orifices of the subaortic and subpulmonary channels. The green diamond-shaped symbols show the asymmetric development of the horns of the aortic trunk measured as the angle of the lines connecting their junctions with the pharyngeal arch arteries. Comparison of the red (open triangular) and purple (solid triangular) symbols shows that, by CS17, the endocardial ridges have lost the initial spiraling arrangement identifiable at CS14. The change in orientation of the myocardialized proximal ridges between CS20 and CS23, as they transform into the subpulmonary infundibulum, accounts for the decline in the purple symbols (see Fig. 10 for morphological details). The compensatory spiraling of the intrapericardial course of the arterial trunks, as shown by the blue symbols, reflects the oblique ventral extension of the aortopulmonary septum, with the black symbols showing that the change in position of the arterial trunks at their connection with the pharyngeal arch arteries contributes to a much lesser extent. The images in panel b (same viewing angle as Fig. 7h–n) are aligned on the location of the developing aortic valve (black line). The horizontal yellow arrow shows the changing position of the (sub-)aortic and (sub-)pulmonary channels in the middle and distal portions of the outflow tract. These channels separate between CS14 and CS15 in the distal outflow tract, during CS16 and CS17 in the middle outflow tract, and between CS18 and CS20 in the proximal outflow tract. All images are also available as preset views in the corresponding 3D-PDFs.

Fig. 10. Pictorial timeline of the formation of the subpulmonary infundibulum.

Panels a–d show cranial views of the lumens of the left and right ventricles, the muscular ventricular septum, the lumen of the outflow tract with ridges and neural crest, and the arterial trunks, while panels e–h show left lateral views of the same structures. On completion of the fusion of the proximal outflow tract ridges at CS18, the neural crest cells largely disappear and myocardialization begins (Fig. 7h–n). Myocardialization proceeds from proximal to distal, opposite to the direction of septation, reaching the arterial valves at CS23. The myocardial septum, which then separates the subpulmonary and subaortic channels, also known as the embryonic outlet septum, is located on the right side of the muscular ventricular septum and is topographically part of the right ventricle. This right-sided position accounts for subsequent development into the free-standing muscular infundibulum. At CS23, the base of the subaortic channels is on the left side of the muscular ventricular septum, but the aortic root has not yet been incorporated in to the base of the left ventricle. All images are also available as preset views in the corresponding 3D-PDFs.