A pictorial account of heart development: spatial and temporal aspects of 1 the human embryonic heart between 3.5 and 8 weeks of development 2

Heart development is topographically complex and requires visualization to understand its 21 progression. No comprehensive 3-dimensional primer of human cardiac development is 22 currently available. We prepared detailed reconstructions of 12 hearts between 3.5 and 8 weeks 23 post fertilization, using Amira® 3D-reconstruction and Cinema4D®-remodeling software. The 24 models were visualized as calibrated interactive 3D-PDFs. We describe the developmental 25 appearance and subsequent remodeling of 70 different structures incrementally, using 26 sequential segmental analysis. Pictorial timelines of structures highlight age-dependent events, 27 while graphs visualize growth and spiraling of the wall of the heart tube. The basic cardiac 28 layout is established between 3.5 and 4.5 weeks. Septation at the venous pole is completed at 29 6 weeks. Between 5.5 and 6.5 weeks, as the outflow tract becomes incorporated in the 30 ventricles, the spiraling course of its subaortic and subpulmonary channels is transferred to the 31 intrapericardial arterial trunks. The remodeling of the interventricular foramen is complete at 32 7 weeks.


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Embryology is a visual discipline. Many aspects of embryonic development are 35 topographically complex, such that 3-dimensional (3D) models are exceedingly helpful in fully 36 understanding the temporal events. Examples of often used or cited classical models are Born's 37 "Plattenmodellen", and Ziegler's freehand models of embryos, which were studied and 38 described by His 1,2 . Other examples are Blechschmidt's models and drawings of human 39 embryos 3,4 , and van Mierop's images of the developing heart 5 , which were redrawn by Netter 40 6 . All these successful approaches have in common that medical artists collaborated with 41 embryologists who had artistic capacities themselves. Because the methods used to make the 42 models were labor-intensive, existing illustrations were often modified rather than new 43 versions being created. An example, documented in detail 7 , is the frequently cited treatise of 44 Kramer on the septation of the outflow tract 8 . Such serial modifications, however, tend to 45 propagate concepts rather than observations, and need to be assessed with caution. 46 The advent of computer-aided reconstruction methods has significantly decreased the time 47 necessary for reconstruction of sectioned bodies. A recent example is the digital atlas of human   being oriented transversely at CS12 to achieving a frontal position at CS13, this change also reflecting 180 the descent of the heart. All images are also available as preset views in the corresponding 3D-PDFs.  its flanking atrial ridges, also begins to form at CS12. The sinus node becomes recognizable as a 198 separate structure at CS13. The left and right atriums are already distinguishable at CS10, but the 199 sinuatrial junction does not become a right-sided structure until CS13. The atrial septum appears at 200 CS14. It is identifiable as the "empty" space between left and right atriums in the upper panel). The 201 hepatocardiac veins are the only source of venous blood for the heart until CS12, when the initially 202 small common cardinal veins appear. All images are also available as preset views in the corresponding 203 3D-PDFs.  lumen and the adjacent vessels in CS10-12 embryos. The panels were aligned on the arterial and venous 222 poles of the heart loop (black horizontal line). The first signs of looping are seen at CS10, when the 223 dorsal mesocardium disappears at the junction of the embryonic ventricle and outflow tract. The center 224 of the heart tube, represented by the yellow wire, bends leftward and ventrally, in particular in its cranial 225 part. At CS11, the loop extends ventrally due to axial growth and becomes more pronounced, producing 226 the so-called "C"-loop. The embryonic left ventricle represents the most ventral portion of the heart 227 loop at this stage. The atrioventricular junction has moved leftward, while the common atrium and distal 228 part of the outflow tract remain midline structures. At CS12, endothelial sprouting into the cardiac jelly 229 marks the boundaries of the ballooning apical parts of the ventricles (arrow). The heart loop between 230 the left ventricle and distal outflow tract further increases in length in a rightward and dorsal direction, 231 with the embryonic right ventricle emerging at its apex (see wire loop). Note that looping has induced 232 two helical twists in the heart axis that meet in the right ventricle. All images are also available as preset 233 views in the corresponding 3D-PDFs. while the caudal neuropore is now distal to somite #23, which is equivalent to vertebral level 237 T12. The fore-and hindgut have further elongated. The first 2 pharyngeal pouches have formed recognized penetrating the dorsal mesocardium between the arms of the inflow tract ( Figure   246 2). The atrial margin of the mesocardium is now flanked by paired mesenchymal ridges ( Figure   247 2, lower panel). Growth of the right-sided ridge, and its fusion with the primary atrial septum,  shows caudal (CS12 and CS13) or ventral views (CS14-23) of the heart lumen between CS12 and CS23.

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The difference in the viewing angle reflects the changing curvature of the embryonic axis. The position 272 of the right relative to the left ventricle gradually changes over ~60° between CS12 and CS18 (graph).

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The right ventricle is positioned caudally relative to the left ventricle at CS12 and achieves a more 274 cranial position after CS18. The interventricular foramen is relatively long during CS12-14. The wide 275 space between left and right ventricular lumens after CS20 reflects the appearance of compact 276 myocardium and a thick muscular ventricular septum. The ventricular axes are almost sagittal prior to 277 CS20, and become oblique and leftward at CS23, reflecting the changing shape of the rib cage 58 . All 278 images are also available as preset views in the corresponding 3D-PDFs. 279 The appearance of endocardial sprouting into the cardiac jelly marks the morphological  When the second heart field starts to contribute cells to the arterial pole of the heart 32,63 , the 292 walls of the loop take a helical path between the atrioventricular canal and distal outflow tract structures that form loops, such as the intestines 69 , follow strikingly similar courses. During 297 this phase of looping, the elongating muscular outflow tract forms an acute bend between its 298 transversely oriented proximal part, which is also known as the "conus", and its ventrodorsally 299 oriented distal part, also known as the "truncus" 8 . The pronounced "bayonet" 70 or "dog-leg" 300 bend 71 between these parts marks the junction. This bend may be a critical structural element 301 for effective valveless pumping in these early hearts 72 . The presence of the bend permits the 302 outflow tract to be described as having proximal and middle parts, which are myocardial, with 303 the non-myocardial distal part being added when the arterial trunks begin to form in CS15 304 embryos (Table 1).

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By this stage, it is possible to recognize the first two pharyngeal arches, along with their 306 accompanying arteries. The vascular space within the ventral pharyngeal mesenchyme that 307 connects the outflow tract with the arteries of the pharyngeal arches is known as the aortic sac.

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The endothelium of the first two pharyngeal arches shares its lineage with that of the dorsal   Figure 6. Due to dorsal growth in its sacral region, first noticeable at CS12, the 316 embryonic body axis assumes a helical shape, with the tail region typically on the right side of 317 the body 74 . The heart, within its pericardial cavity, remains surrounded by the transverse 318 septum, the pharynx, and the forebrain. Due to the rapid growth of the brain and foregut 319 between CS9 and CS14, the transverse septum gradually changes in orientation from frontal at 320 CS9 to near-transverse at CS11 (Figure 1). It also "descends" from ~6 somite lengths cranial  The protrusion muscularizes, along with the mesenchymal cap, starting at CS18, and concomitant with 369 the proximal endocardial ridges of the outflow tract. The borders of the interventricular foramen 370 remodel as revealed by the course of the GlN-positive ring. As soon as septation of the outflow tract is 371 complete at CS18, the myocardialized part of the fused endocardial ridges and the rightward margins 372 of the atrioventricular endocardial cushions combine to decrease the size of the remaining foramen. 373 Closure is complete at CS20. Gray contours: primary atrial foramen; white contours: secondary atrial 374 foramen; yellow contours: interventricular foramen. All images are also available as preset views in the 375 corresponding 3D-PDFs. 376 The sinuatrial connection, now narrow, is guarded by the venous valves. These valves merge 377 into the spurious septum craniodorsally, and attach in the primary myocardium of the atrial The tips of the atrial appendages are clipped in the images for CS18 and CS20 (dashed lines) to permit 420 inspection of the atrioventricular junction and outflow tracts. At CS16, the caudal part of the foramen 421 and GlN ring begin to expand in rightward direction, producing a direct connection between the right 422 atrium and ventricle, which is best seen in the lower panel. Meanwhile, the cranial, subaortic part of the 423 foramen, which is best seen in upper panel, gradually expands craniodorsally. Comparing the 424 arrangements at CS18 and CS20, when the septation of the outflow tract is complete, shows that the 425 subaortic, but not the subpulmonary, ventricular outlet is surrounded by the GlN ring. The remaining 426 connection between right ventricular cavity and the subaortic channel is still present at CS18. It is 427 obliterated at CS20 by formation of the membranous septum (not itself visible). All images are also 428 available as preset views in the corresponding 3D-PDFs. and partially reconstructed hearts 107 . There is axial growth of the muscular outflow tract up to CS16, 436 when its length suddenly declines profoundly, with no resumption up to CS23. The right-sided graph 437 shows the axial growth of the arterial trunks. The ascending aorta (blue) increases continuously in length 438 between CS14 and 10 weeks of development, whereas axial growth of the pulmonary trunk (red) stops 439 after CS17. The distance between the distal myocardial border and the pericardial reflection (green) 440 increases little to CS16, indicating that the myocardial jaws of the fishmouth stay close to the 441 pericardial reflection. Concomitant with the abrupt shortening after CS16, the myocardial border moves 442 away from the reflection. The lower panel shows cranial views of the myocardial outflow tract, the 443 arterial trunks, with 6 th arch and pulmonary arteries, and the pericardial reflection (wire loop). The 444 images are aligned to the distal myocardial border of the outflow tract (black line). The dashed black 445 line shows the axial growth of the aortic trunk (also shown in Figure 8). The slits in the jaws of the 446 myocardial fishmouth are occupied by the non-myocardial mural tissues (not shown, but see Figure 9, 447 upper row). All images are also available as preset views in the corresponding 3D-PDFs. 448 Septation proceeds centripetally from the venous and arterial poles towards the interventricular

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The progenitor cells in the club converge and extend into a procession of cells that moves 500 towards, and then into the relatively narrow outflow tract before locally differentiating  In contrast to the neural crest cells, the cells of aortic and pulmonary mural columns do not 509 penetrate the distal endocardial jelly, but maintain an oblique lateral-to medial zone of 510 apposition. Following Tandler 124 and Kramer 8 , we will name these endocardial structures 511 "swellings". The cranial, or aortic, swelling differs from the caudal, or pulmonary swelling in 512 that it is invaded by some neural crest cells 113 . The swellings differ from the ridges in that they  ridges. The fusion of these columns creates a temporary "whorl" of neural crest cells between the 532 subaortic and subpulmonary channels. The neural crest cells largely disappear between CS18 and CS23 533 due to intense apoptosis 125 , with invading cardiomyocytes simultaneously populating the shell of the 534 septum 68,126 . All images are also available as preset views in the corresponding 3D-PDFs. 535 536 Figure 9: Pictorial timeline of the appearance of the non-myocardial walls of the arterial trunks. 537 The panels are aligned on the pericardial reflection, shown by the wire loops, as in Figure 8. The upper 538 panel shows the lumen of the outflow tract, with the arterial trunks, the columns of neural crest, and the 539 non-myocardial mural columns. The neural crest cells and intercalating non-myocardial tissues invade 540 the distal wall of the outflow tract during CS14. The mural cells are first seen as relatively short aortic 541 or cranial, and pulmonary or caudal columns. During CS14 and CS15, the pulmonary column is 542 continuous dorsally with a club-like condensation of peritracheal mesenchyme, which has disappeared 543 at CS16. The endocardial swellings associated with the aortic and pulmonary mural columns are 544 relatively small during CS14 and CS15, but increase in size from CS16 onwards to begin their 545 transformation into the dorsal and ventral semilunar leaflets, respectively, at CS18. As shown by the 546 interrupted line, there is a gradual increase in the distance between the pericardial reflection and the 547 plane of the valvar primordiums (cf. Figure 8). The lower panel shows the same view of the lumens. 548 Note that the prongs of neural crest mesenchyme mold the subaortic and subpulmonary channels during 549 CS15 and CS16. The fusion of these prongs into a central whorl marks the separation of the subaortic 550 and subpulmonary channels during CS17 and CS18. All images are also available as preset views in the 551 corresponding 3D-PDFs.  These channels separate between CS14 and CS15 in the distal outflow tract, during CS16 and CS17 in 613 the middle outflow tract, and between CS18 and CS20 in the proximal outflow tract. All images are 614 also available as preset views in the corresponding 3D-PDFs.    (Figures 5 and 6). In the inner curvature of 643 the heart, the right wall of the atrioventricular canal continues into the caudal part of the 644 interventricular foramen. Rightward expansion of the confluent part of these structures across 645 the muscular ventricular septum has produced a direct connection between the right atrium and 646 the right ventricle ( Figure 6, lower panel; 135 ). Subsequently, the cranial portion of the interventricular foramen will evolve into the channel between the left ventricle and the 648 subaortic outlet (Figure 6, upper panel). At this latter location, however, the primary ring 649 (hatched section) has lost its GlN expression 88 , but remains identifiable as part of the central  images are also available as preset views in the corresponding 3D-PDFs. 781 While the structural components that are responsible for ventricular septation become 782 morphologically identifiable, the right ventricle gradually begins to occupy a more cranial reaching the arterial valves at CS23. The myocardial septum, which then separates the subpulmonary 832 and subaortic channels, also known as the embryonic outlet septum, is located on the right side of the 833 muscular ventricular septum and is topographically part of the right ventricle. This right-sided position 834 accounts for subsequent development into the free-standing muscular infundibulum. At CS23, the base 835 of the subaortic channels is on the left side of the muscular ventricular septum, but the aortic root has 836 not yet been incorporated in to the base of the left ventricle. All images are also available as preset 837 views in the corresponding 3D-PDFs. 838 Cup-shaped arterial valvar leaflets have formed in the distal part of the muscular outflow tract.

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The myocardial support provided to the still plump leaflets of the arterial valves may assist  (Figure 5; 133 ). It is against this fold, which is incorrectly known as the secondary atrial 915 "septum", that the primary septum will eventually rest to close the oval foramen.  The muscular septum in the proximal outflow tract, also known as outlet septum, is normally 933 a temporary embryological structure. It changes in shape and orientation from a dumbbell-like 934 structure perpendicular to the muscular ventricular septum at CS20 to a flat blade almost 935 parallel to the muscular ventricular septum at CS23 (Figure 12). Extension of its 936 myocardialization towards the developing arterial roots underlies this change in orientation. We have described the morphological development of the human heart between its first 959 appearance at CS9 up to CS23, when almost all structures of the definitive heart have formed, 960 although at this stage several have still to reach their relative sizes and definitive positions.

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Because we used embryos that had been carefully staged at the Carnegie Institution without 962 exclusive attention to heart development, we were able to assign critical events in heart 963 development to specific stages of human embryonic development.

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The first heart field produces the embryonic left ventricle, which contributes eventually to no 965 more than parts of the definitive left ventricle to the formed heart 16,19,20 . Ongoing addition of 966 cardiomyocytes from the second heart field to the venous and arterial poles of the embryonic 967 left ventricle, and differentiation into cardiac compartments, is therefore necessary to form the 968 definitive heart 24,48,111 . Accordingly, the atriums form at CS10-11, and the systemic venous sinus at CS12. The embryonic right ventricle forms at CS10, while the myocardial outflow 970 tract forms at CS11-12. The pharyngeal arch arteries are successively added between CS12 and 971 CS14, and the non-myocardial distal portion of the outflow tract begins to appear at CS15.

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Furthermore, endocardial cushions and ridges form at CS14 to allow for separate blood flows.

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Development progresses, therefore, by addition of cardiomyocytes at the venous and arterial 974 periphery of the heart tube (Table 1). This "peripheral growth" model of heart development 975 ends when the building plan of the heart has been established at the phylotypic stage (CS13). 976 Subsequently, central structures in the heart, such as the atrioventricular canal, inner heart 977 curvature, and muscular outflow tract temporarily retain their relatively undifferentiated status 978 as remnants of the primary heart tube. They contribute to the internal remodeling that is 979 necessary to achieve septation 108 . Septation is associated temporally with the appearance of show a near identical morphology with respect to its cardiovascular system 9,24 , we assume that 1062 the reconstruction represents a normal stage of heart development. Our findings in human 1063 embryos fall in line with earlier observations in mice, revealing that heart and early somite 1064 development do not proceed strictly synchronously 43,188 . Another example is the appearance 1065 of the pulmonary arteries in our model embryo at CS15, whereas Sizarov and colleagues 1066 associated their appearance with CS14 39 . In the Carnegie collection, the pulmonary arteries 1067 make their appearance in ~75% of the 44 embryos at CS14 and in the remainder at CS15 11 . 1068 Such data indicate that small interindividual differences exist in the developmental timing of