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The role of atrioventricular endocardial cushions in the septation of the heart
International Journal of Cardiology, 8 (1985) 25-44
25
Elsevier IJC 00244
The role of atrioventricular endocardial cushions in the septation of the heart A.C.G. Wenink and A.C. Gittenberger-de Department of Anatomy and Embryology, University of Liden,
Groot The Netherlancls
(Received 16 July 1984; revision accepted 8 November 1984)
Wenink ACG, Gittenberger-de Groot AC. The role of atrioventricular cushions in the septation of the heart. Int J Cardiol 1985;8:25-44.
endocardial
The hearts of human embryos, ranging from 3.6 to 25 mM crown-rump length, have been studied in view of the problem of the possible contribution of the atrioventicuhu endocardial cushions to septation. Serial sections and graphic reconstructions were used. It is concluded that the cushions do not materially contribute to the mature muscular septum.
Introduction In classical descriptions of cardiac embryology, the atrioventricular endocardial cushions are said to play an important role. They are derived from the cardiac jelly [l] and it has been argued that they function as valves [2,3], fuse to divide the atrioventricular canal [4] and give rise to the atrioventricular valves [5]. Discussions in this light have then continued on the question as to which part of which cushion would give rise to a particular valve leaflet [6,7]. Recently [8,9], the material contribution of cushion tissue to the atrioventricular valves has been doubted. Instead, valve formation was described as a process of invagination of the atrioventricular sulcus and undermining of ventricular myocardium. These descriptions have been concentrated more on what the cushions do not do, rather than on what they might do. Nonetheless, the process of valve formation is at least topographically related to the sites of the cushions. In this respect, and with regard to septation, the role of the cushions merits further -Correspondence and reprint requests to: Dr. A.C.G. Wenink, Department of Anatomy and Embryology, Wassenaarseweg 62, P.O. Box 9602, 2300 RC Leiden, The Netherlands.
0167-5273/85/$03.30
0 1985 Elsevier Science Publishers B.V. (Biomedical Division)
26 TABLE 1 No.
Crown-rump length (mm)
Plane of sectioning
Section thickness * (cm)
Staining
Graphic reconstruction(s) made
frontal transverse transverse frontal frontal transverse sagittal frontal transverse sag&al transverse transverse transverse transverse transverse transverse transverse transverse transverse frontal transverse transverse transverse transverse transverse transverse frontal transverse
10
+ +
10 10
HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE
transverse transverse transverse transverse transverse transverse transverse transverse transverse transverse transverse transverse transverse sagittaf transverse
6 6 10 10 10 6 10 10 8 10 10 10 10 10 10
HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE
Vienna collection
A8-E AlO-BS All-Fu Al2-F A13-Br AlCDh AlS-Fr AlS-Bp A17-G A17-H A18-Ba A19-Fs A19-Nov.1. A19-Nov.2. A19-FF2 A20-Bd A21-I A22-Bb A23-DC A24-Hw A25-Hi A26Fr A31-J A32-Hm A34-Co B35-H3 B36-Hd B39-Bf Leiden collection S28 S26 s 166 s 31 s 133 s 19 S 16 s164 s 79 S 158 s 168 s 171 s 45 s 90 S40
3.6 4 4.5 4 4 4 4.5 5 5 5 5 6 6 6 6 6 6 6.5 6.5 6.5 7 7 7 7 7.5 7.5 5 5 5 6 6 6 6.5 6.5 7 7 7 9 9 9.5
7
7 10 7
8
8
* These data were only incompletely available to the authors.
+ + +
+
27 TABLE 1 (continued) No.
s 44 S 56 s17 S 106 s 147 s 39 s 88 s 113 s4 s 10 S 36 s 15 s144 s 99 S 136 S 167 s 91 s 131 s9 s 91 S 108 s 20 s 141 s 34 S 29 s 50 s 174 s51 S 83 s 114 s 175 s 30 s 37 s 103 s 54
Crown-rump length (mm)
Plane of sectioning
Section thickness * (am)
Staining
Graphic reconstruction(s) made
9.5 9.5 10 10 10 11 11 11 12 13.5 13.5 14 14 15 15 15 16 16 17 17 17 18 18 19 19.5 20 20 21 22 22 22 23 23 25 25
transverse transverse sag&al transverse transverse transverse sagittal transverse transverse frontal transverse sagittal transverse transverse transverse transverse transverse transverse transv./sagitt. sag&al transverse transverse transverse sag&al transverse transverse transverse transverse transverse sagittal transverse transverse transverse transverse transverse
10 6 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
HE HE Azan HE HE HE HE HE HE HE/Azan HE HE HE HE HE HE HE/Azan HE HE HE/Azan HE HE/Bodian HE HE HE HE HE HE HE HE/Azan HE HE HE HE/Azan l HE
+
+ + +
+
+
+
discussion. With this in mind, we have reinvestigated the human embryonic material which was earlier available to us [lO,ll]. Material and Methods The human embryos described in this study belong to the collections of the Department of Anatomy and Embryology, University of Leiden, and the Department of Histology and Embryology, University of Vienna. The crown-rump lengths of the embryos ranged from 3.6 to 25 mm. Detailed information on the material is
28
given in Table 1. Of 18 embryos, one or more graphic reconstructions [12] were made as indicated in Table 1. This reconstruction technique is able to reveal histological details, the results regarding surface morphology being identical to those obtained by scanning electron microscopy [13]. We are therefore convinced, being well aware of “the bias inherent to studies on serial sections and their reconstruction” [14] that our results remain comparable with those obtained by other investigators. Although to our knowledge none of the hearts had been perfusion fixed (see [15]), the Vienna hearts in particular looked well dilated. Histological preservation of all specimens was excellent. Results The youngest specimen available was an embryo of 3.6 mm crown-rump length, in which the various cardiac segments could be clearly distinguished. The venous sinus (sinus venosus) could be seen to enter the right part of the atrium which itself
Fig. 1. Graphic reconstruction of the heart of a human embryo of 3.6 e crown-rump length, seen from the right. The atrium and atrioventricular canal have be-enopened. Endocardial cushion tissue covers the basal rim of the atrial septum primum (arrows) and fills the atrioventricular canal (c). Cushion tissue also covers the left venous valve (Iv), but not the right venous valve (IV). in = inlet segment; out = outlet segment; ta = distal part of outlet which connects with truncus arteriosus.
29
was draining into the left-sided inlet segment. The latter opened into the right-sided outlet segment, which gave rise to the arterial component (truncus arteriosus; Figs. 1 and 2). The constricted portion between atrium and inlet segment, the atrioventricular canal, was partly filled by superior and inferior masses of endocardial cushion tissue. Because the atrioventricular canal was completely committed to the inlet segment, it was partly bordered by the fold between inlet and outlet segments. Both cushion masses, therefore, were clearly to the left of this fold. The inferior cushion mass was closely related to an area of very loosely arranged myocardium on the posterior wall of the inlet segment (Figs. 2 and 3). This ridge was judged to indicate already a primitive division between left and right bloodstreams. At the atria1 level, where septation had already started with the formation of a short primary atria1
Fig. 2. Reconstruction of part. The inferior cushion (is) on the posterior wall segment (out). Note that with the groove between
the same heart as in Fig. 1: cranial view after graphic dissection of the superior (c) is seen in the atrioventricular canal. It corresponds with a trabecular mass of the inlet segment. To the right, this segment communicates with the outlet in the inner curvature (arrow) the groove between atrium and inlet coincides inlet and outlet.
Fig. 3. Sections of the same heart as in Figs. 1 and 2, to show the inferior cushion (c) to correspond with a trabecular mass (is) on the posterior inlet wall. In both sections the primary fold (pf) is seen. avc = atrioventricular canal; in = inlet segment; out = outlet segment.
Fig. 4. Section of the same heart, to show the continuity of endocardial cushion tissue (c) with the extracardiac mesenchyme (m) of the mediastinum. la = left atrium; ra = right atrium; rv = right venous valve; Iv = left venous valve (not very prominent in this section, cf. Fig. 1); ta = distal part of outlet which connects with truncus arteriosus.
31
septum, the two endocardial cushion masses appeared to be continuous with each other along the free border of this septum. In addition, this cushion mass also covered the inferior portion of the left venous valve, i.e. the fold between the venous sinus and the atrium (Fig. 1). At the site where the inferior cushion mass covered the primary atria1 septum and the left venous valve, the myocardial wall was interrupted. Thus, at the sinuatrial junction, the endocardial tissue was continuous with the extracardiac mesenchyme of the mediastinum. Because in this young stage the endocardial cushion tissue contained few cells (thus still deserving the name “cardiac jelly”), it could be readily distinguished from the mediastinal mesenchyme, which had a more compact cellular structure (Fig. 4). In later stages, the loose trabeculations on the posterior inlet wall had coalesced to form a more compact structure, which could be called the precursor of the inlet
Fig. 5. Reconstruction of the heart of a 6 mm embryo, anterior view after graphic dissection of the anterior wall. The atrioventricular canal (avc) is seen to drain completely into the inlet segment. The primary fold (pf) forms the border between inlet and outlet segments. Superior (SC) and inferior (ic) endocardial cushions guard the atrioventricular orifice. The inferior cushion borders upon a loose trabecular mass (is) on the posterior inlet wall.
32
septum. The part of the inlet segment to the right of this inlet septum, initially inconspicuous, had started to expand. Consequently, the fold between inlet and outlet segments, which we call the primary fold, showed a curve which surrounded the right part of the inlet segment. As a result of the inlet expansion, the relationship of the inferior cushion with the primary fold disappeared, but this cushion kept its close relationship with the inlet septum. On the other hand, the superior cushion retained its relationship to the left side of the primary fold. Apposition of the two cushions leads to the separation of left and right atrioventricular orifices, the right one coming to drain into the expanded right portion of the inlet segment. The right inlet part was therefore bordered to the left by the inlet septum and to the right by the primary fold. Figs. 5 to 7 show successive developmental stages in which the inlet expansion
i
/ /
‘1 /’ / -
Fig. 6. Reconstruction of the heart of a 7 mm embryo, anterior view after graphic dissection of anterior wall. The primary fold (pf) shows a slight rightward curve. The superior (SC) and inferior endocardial cushions are apposed and to their left and right the mitral (m) and tricuspid (I) orifices seen. The latter is also bordered by the endocardial right posterior outlet ridge (or) which partly covers primary fold.
the (ic) are the
33
can be seen. These stages show the formation of the right ventricle from two sources. Whereas in our 6 mm embryo only the inlet and outlet segments can be recognized, the 7.5 mm embryo (Figs. 7 and 8) already shows the basic morphology of the right ventricle. The inlet portion of this ventricle is the right part of the inlet segment in younger stages, whereas the outlet portion of the right ventricle (including its apical trabecular component) derives from the outlet segment. The curved part of the primary fold between these two portions can be identified as the parietal part of the septomarginal trabecula (moderator band). Figs. 9 to 12 then show further successive developmental stages in which a constant relationship can be seen between the superior atrioventricular cushion and the primary fold. This cushion maintains its position to the left side of the fold. In the 11 mm stage (Figs. 11 and 12) the basal part of the primary fold can be seen to delimit the “aortic” portion of the outlet segment. At this site, it can be seen that the left ventricle is also constituted from dual sources. Its main body (including the
Fig. 7. Reconstruction of the heart of a 7.5 mm embryo, ventral view after graphic dissection of the anterior wall. Note the rightward curve of the primary fold (pf), which is partly covered by the right posterior outlet ridge (or), which is to the right of the tricuspid orifice (t). SC= superior cushion; ic = inferior cushion.
34
apical trabecular component) derives from the inlet segment, whereas the left-posterior part of the outlet segment is also allotted to this ventricle. This is made possible by the development of an endocardial septum within the outlet segment which can be seen to delimit the “aortic” part of the outlet (Fig. 12). In this respect, it should be noted that the interventricular communication of the 11 mm embryo (Figs. 11 and 12) is not comparable at all with the communication between inlet and outlet segments of the 6 mm embryo (Fig. 5). The latter is fully bordered by the primary fold. The interventricular communication of older stages, however, is bordered by the apposed atrioventricular cushions on the one hand and by the endocardial outlet septum on the other. It is this interventricular communication which, between the 16 mm and 18 mm stage, is closed to complete septation (Fig. 13). The morphology of the inlet myocardium merits particular attention. In the 7.5 mm embryo, the trabeculations were obvious. The trabeculations on the posterior inlet wall were clearly related to the inferior atrioventricular cushion (Figs. 7 and S)j whereas trabeculations on the anterior inlet wall showed relations with the superior
Fig. 8. The same heart as in Figure 7. The block shown in Fig. 7 has been graphically dissected further, now in a transverse plane (inset), and the apical part has been tilted forward to show the right portion of the inlet (ri), into which the tricuspid orifice (t) is draining. The right inlet part is delimited by the primary fold (pf) and the inlet septum (is). The latter is contiguous with the right side of the inferior cushion (ic). The arrow indicates the boundary between the inlet septum and the (anterior) primary septum (ps).
35
cushion (Fig. 10). It would seem that the inlet myocardium was undermined with the exception of the areas covered by cushion tissue. At the atria1 level, the cushion masses completely surrounded the interatrial communication in the 3.6 mm stage (Fig. 1). In addition, they covered part of the left venous valve. In later stages, the initial interatrial communication (ostium primum) diminished and the basal portion of both venous valves became anchored within the cushion mass (Fig. 14). Still later, fusion of the cushion masses had obliterated the ostium primum, whereas a new communication through the septum primum (ostium secundum) had developed (Fig. 15). The continuity of endocardial cushion tissue (particularly the inferior cushion) with extracardiac mesenchyme, which was already clear in the 3.6 mm stage (Fig. 4) had become much more conspicuous in the 9.5 mm embryo (Fig. 16). At this stage
Fig. 9. The same heart as in Fig. 6; posterior view of the graphically dissected anterior part. The primary fold (pf) is partly covered by the superior cushion (SC) and the left anterior outlet ridge (or). The outlet segment (out) is seen to drain into the truncus arteriosus (ta).
36
the base of the atria1 septum had thickened considerably (Figs. 17 and 18). In older stages, proportional changes were most obvious. Thus, the cushion mass remained restricted to the site of the posterior aortic root. Expansion of the inlet part of the ventricles and of the atria had led to production of a greater distance between the cushions and the posterior cardiac wall. Consequently, there was further incorporation of extracardiac tissue in the atrioventricular junction (Fig. 19). In summary, the atrioventricular cushions are considerable masses in young embryonic stages, where they cover the inner wall of the atrioventricular canal. In addition, they protrude into the inlet segment as well as the atrium. In the atrium they cover also the rim of the primary atria1 septum and the basal part of the venous valves. Here, the lower cushion is continuous with the mediastinal mesenchyme. Fusion of the cushion masses leads to obliteration of the interatrial ostium primum. The mass is then found on top of the inlet septum, directly posterior to the aortic root. It borders on the interventricular communication, and its eventual fusion with the endocardial outlet ridges obliterates this communication too. The considerable
Fig. 10. The same heart as in Fig. 7: posterior view of the graphically dissected anterior part. Note the trabeculations on the anterior wall of the inlet segment (in), and the superior cushion (SC) which is to the left of the primary fold (pf). ps = primary septum.
37
Fig. 11. Reconstruction of the heart of an 11 mm embryo, posterior view of the anterior part. Note the primary fold (pf) between inlet (in) and outlet segments (out). A recess (a) leading into the aortic orifice is bordered by the primary fold and by the left anterior outlet ridge (or) and outlet septum (OS).
expansion of the ventricular inlet segment and of the atrium is not accompanied by a corresponding growth of the atrioventricular cushions. Therefore, they finally occupy an inconspicuous area at the site of the future membranous septum. Most of the tissue interposed between atria1 and inlet septa is then no longer cushion tissue but extracardiac mesenchyme. Discussion Our description of the topographic development of the atrioventricular endocardial cushions reveals that these initially massive structures gradually diminish in their relative size. Their volume is relatively large in the early stages, in which the primary interatrial and the interventricular communications also have substantial proportions. After completion of septation, the areas occupied by the cushions are restricted to the sites of final closure of these communications. In the present study, the actual fusion process of endocardial cushions is not described. Although these processes [16-B] are of fundamental importance, for the
38
present purpose it is sufficient to indicate that, on the one hand, the apposition of the superior and inferior cushions leads to the separation of left and right atrioventricular orifices and, on the other hand, that their fusion with the endocardial rim of the primary atrial septum obliterates the ostium primum. The cushions are further involved in the closure of the interventricular communication. In this area they have to fuse with another endocardial structure, namely the outlet septum (Fig. 12). Thus, it may be stated that the endocardial cushions are of prime importance for septational processes. However, the question of their material contribution to the definitive septal structure is not answered by this statement. When their position is extrapolated to the mature heart, they can only contribute to structures near the posterior aortic root and the membranous septum [19], most of the fibrous tissues at the atrioventricular junction being derived from extracardiac tissues. In this respect, the septum of the sinus venosus [20], the central fibrous body and the tendon of Todaro [21] deserve attention.
Fig. 12. The same heart as in Fig. 11: anterior view of the posterior part, to show the posterior part of the outlet septum (OS)and the right posterior outlet ridge (or). These are delimiting the aortic vestibulum (a), which is to the right of the primary fold (pf). The arrows indicate the more apical part of the primary fold, which delimits the right part of the inlet segment. The outlet septum and the superior (SC)and inferior (ic) cushions form the boundaries of the interventricular communication. Fusion of these endocardial structures will eventually obliterate this communication and the aortic vestibule (a) will then be the outlet part of the left ventricle, the right part of the inlet as demarcated by the primary fold (arrows) becoming the inlet part of the right ventricle.
Fig. 13. Transverse section of the heart of an 18 mm embryo, to show the appositton of ngbt postenor outlet ridge (ror), left anterior outlet ridge (for) and inferior cushion (ic). The interventricular communication between aortic vestibule (a) and right ventricle (rv) is closed. The arrow indicates the tricuspid orifice.
100
u
Fig. 14. Reconstruction of the heart of a 6 mm embryo, viewed from the right. The right atnum has been opened graphically, to show that the interatrial ostium primum (op) is completely surrounded by endocardial cushion tissue (c), which is also found at the basis of the right venous valve (TV). The left venous valve cannot be seen (cf. Fig. 1).
De la Cruz et al. [3] have claimed that it is impossible to recognize the real contribution of the cushions to the structures of the mature heart by means of descriptive embryology alone. This statement was made concerning the need to distinguish the inferior and the superior cushion. From their labelling experiments in the chick embryo heart, they concluded that the inferior cushion contributed to the inter-ventricular septum adjacent to the region of the atrioventricular septum. Our observations in the human embryo, in which the atrioventricular conduction axis is always interposed between the cushion tissue and the muscular ventricular (inlet) septum (Fig. 19), together with the notion that in the mature heart the bundle is still on top of this inlet septum, are not in favour of cushion tissue making any contribution to the muscular part of the ventricular septum. Furthermore, the area under discussion has only negligible proportions in the mature heart. Most of the inlet septum is posterior to this area and has never been related to any cushion tissue. Since in the avian heart the bundle is buried within septal musculature [22], our arguments may not hold for the chick as studied by De la Cruz et al. [3]. Similar experiments were described [23] to show the contribution of the superior cushion to the left surface of the anterior basal portion of the ventricular septum.
iooi.4 Fig. 15. Reconstruction of the heart of a 9.5 mm embryo, to show the apposition of the cushion masses (SC and ic) at the basis of the atria1 septum primum (I). The Psterisk indicates the orifice of the left sinus horn. II = interatrial ostium secundum.
41
Again, we believe that this possible contribution may be reduced to negligible proportions in the mature heart and may eventually be found in the region of the membranous septum. It should be noted that these workers [3,23] describe their final stages (i.e. 35-37) as “mature heart”, whereas the cavities still have to expand considerably before hatching which takes place in stage 46 [24]. It remains true to state that the question of the role of the atrioventricular cushions cannot be answered by descriptive embryology alone. Nonetheless, it seems justified to conclude that their main action is in keeping together the several muscular septal components, which otherwise would tend to grow far apart [25]. Their role in valve formation is somewhat more speculative. They certainly do not contribute to the material of the valve leaflets [8]. But they might be said to cover
Fig. 16. Transverse section of the same heart as in Fig. 15, to show the continuity of the inferior cushion (ic) with the more compact extracardiac mesenchyme (m). ra = right atrium; rw = right venous valve; sv = sinus venosus; rv = right ventricle; Iv = left ventricle.
Fig. 17. Keconstruction of the same heart as in Fig. 15: anterior view after graphic dissection of the anterior part. Note the apposed superior (SC) and inferior (ic) cushions. The inferior cushion borders on the inlet septum (is).
,
Fig. 18. The same heart as in Fig. 17, after graphic dissection of the endocardial cushions, to show the broad basis of the atria1 septum between the right (ra) and left (la) atrium. The stippled line surrounds the area of extracardiac mesenchyme (m) which is continuous with the inferior cushion.
Fig. 19. Transverse sections of the heart of a 25 mm embryo. a. At the level of the (fused) endocardial cushions (c). Note that the atrioventricular bundle (asterisk) is between the cushions and the ventricular septum (vs). b. At the level of the atrioventricular muscular continuity (av) where no cushion tissue (c) is present on top of the septum (vs) but only to its left and right sides. ra = right atrium; la = left atrium; s = orifice of the left sinus horn; ss = septum of the sinus venosus.
those parts of the inlet myocardium which have to keep together during the undermining process, thus guaranteeing continuous valve leaflets at the end of development. References Davis CL. The cardiac jelly of the chick embryo. Anat Ret 1924;27:201-202. Streeter GL. Developmental horizons in human embryos. Description of age group XIII, embryos of about 4 or 6 millimeters long and age group XIV, period of indentation of lens vesicles. Contr Embryo1 1945;31:27-63. De la Cruz MV, Gimenez-Ribotta M, Saravally 0, Cay& R. The contribution of the inferior endocardial cushion of the atrioventricular canal to cardiac septation and to development of the atrioventricular valves: Study in the chick embryo. Am J Anat 1983;166:63-72. Mall FP. On the development of the human heart. Am J Anat 1912;13:249-298. Odgers PNB. The development of the atrioventricular valves in man. J Anat 1939;73:643-657. Van Mierop LHS. Embryology. In: Yonkman FF, ed. The Ciba collection of medical illustrations. Vol. 5, The heart. New York: Pub1 CIBA, 1969;112-132.
44
7 Ugarte M, Enriquez de Salamanca F, Quero M. Endocardial cushion defects. An anatomical study of 54 specimens. Br Heart J 1976;38:674-682. 8 Van Gils FAW. The formation of the atrioventricular heart valves. Acta Morph01 Neerl-Stand 1978;16:151. 9 Wenink ACG, Van Gils FAW, Gittenberger-de Groot AC, Draulans-Noe HAY, Thiene G. Developmental aspects of atrioventricular septal defects. In: Quero Jimenez M, Anderson RH, eds. Atrioventricular septal defects. Edinburgh: Churchill-Livingstone, 1985;in press. 10 Wenink ACG. Embryology of the ventricular septum. Separate origin of its components. Virchows Arch A 1981;390:71-79. 11 Wenink ACG, Gittenberger-de Groot AC. Left and right ventricular trabecular patterns. Consequence of ventricular septation and valve development. Br Heart J 1982;48:462-468. 12 Tinkelenberg J. Graphic reconstruction, micro-anatomy with a pencil. J Audio+ Media Med 1979;2:102-106. 13 Wenink ACG, Chon Y. The value of graphic reconstructions. Comparison with scanning electron microscopy. Anat Ret 1984;210:537-540. 14 Pexieder T. Changing scene in cardiac embryology. Herr 1979;4:73-77. 15 Pexieder T. Effects of various fixatives and fixation procedures on the shape of the embryonic heart. Eur Heart J 1983;4(suppl E):112. 16 Hay DA, Low FN. The fusion of dorsal and ventral endocardial cushions in the embryonic chick heart: a study in fine structure. Am J Anat 1972;133:1-24. 17 Los JA, Van Eijndthoven E. The fusion of the endocardial cushions in the heart of the chick embryo. Z Anat Entwicklungsgesch 1973;141:55-75. 18 Hay DA. Development and fusion of the endocardial cushions. In: Rosenquist AC, Bergsma D, eds. Morphogenesis and malformation of the cardiovascular system. Birth Defects: Original article Series XIV-7. New York: Alan R Liss, 1978;69-90. 19 Wenink ACG. La formation du septum membranaceum darts le coeur humain. Bull Assoc Anat 1975;58:1127-1132. 20 Los JA. Cardiac septation and development of the aorta, pulmonary trunk and pulmonary veins: previous work in the light of recent observations. In: Rosenquist AC, Bergsma D, eds. Morphogenesis and malformation of the cardiovascular system. Birth Defects: Original article Series XIV-7. New York:Alan R Liss, 1978;109-138. 21 Van Gils FAW. The fibrous skeleton in the human heart: embryological and pathogenetic considerations. Virchows Arch A 1981;393:61-73. 22 Vassall-Adams PR. The development of the atrioventricular bundle and its branches in the avian heart. J Anat 1982;134:169-183. 23 Garcia-Peltiesz J, Diaz-G6ngora G, Arteaga Martinez M. Contribution of the superior atrioventricular cushion to the left ventricular infundibulum. Experimental study in the chick embryo. Acta Anat 1984;118:224-230. 24 Hamburger V, Hamilton HL. A series of normal stages in the development of the chick embryo. J Morph01 1951;88:49-92. 25 Van Mierop LHS. Embryology of the atrioventricular canal region and pathogenesis of endocardial cushion defects. In: Feldt RH, ed. Atrioventricular canal defects. Philadelphia/London/Toronto: W.B. Saunders, 1976;1-12.
25
Elsevier IJC 00244
The role of atrioventricular endocardial cushions in the septation of the heart A.C.G. Wenink and A.C. Gittenberger-de Department of Anatomy and Embryology, University of Liden,
Groot The Netherlancls
(Received 16 July 1984; revision accepted 8 November 1984)
Wenink ACG, Gittenberger-de Groot AC. The role of atrioventricular cushions in the septation of the heart. Int J Cardiol 1985;8:25-44.
endocardial
The hearts of human embryos, ranging from 3.6 to 25 mM crown-rump length, have been studied in view of the problem of the possible contribution of the atrioventicuhu endocardial cushions to septation. Serial sections and graphic reconstructions were used. It is concluded that the cushions do not materially contribute to the mature muscular septum.
Introduction In classical descriptions of cardiac embryology, the atrioventricular endocardial cushions are said to play an important role. They are derived from the cardiac jelly [l] and it has been argued that they function as valves [2,3], fuse to divide the atrioventricular canal [4] and give rise to the atrioventricular valves [5]. Discussions in this light have then continued on the question as to which part of which cushion would give rise to a particular valve leaflet [6,7]. Recently [8,9], the material contribution of cushion tissue to the atrioventricular valves has been doubted. Instead, valve formation was described as a process of invagination of the atrioventricular sulcus and undermining of ventricular myocardium. These descriptions have been concentrated more on what the cushions do not do, rather than on what they might do. Nonetheless, the process of valve formation is at least topographically related to the sites of the cushions. In this respect, and with regard to septation, the role of the cushions merits further -Correspondence and reprint requests to: Dr. A.C.G. Wenink, Department of Anatomy and Embryology, Wassenaarseweg 62, P.O. Box 9602, 2300 RC Leiden, The Netherlands.
0167-5273/85/$03.30
0 1985 Elsevier Science Publishers B.V. (Biomedical Division)
26 TABLE 1 No.
Crown-rump length (mm)
Plane of sectioning
Section thickness * (cm)
Staining
Graphic reconstruction(s) made
frontal transverse transverse frontal frontal transverse sagittal frontal transverse sag&al transverse transverse transverse transverse transverse transverse transverse transverse transverse frontal transverse transverse transverse transverse transverse transverse frontal transverse
10
+ +
10 10
HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE
transverse transverse transverse transverse transverse transverse transverse transverse transverse transverse transverse transverse transverse sagittaf transverse
6 6 10 10 10 6 10 10 8 10 10 10 10 10 10
HE HE HE HE HE HE HE HE HE HE HE HE HE HE HE
Vienna collection
A8-E AlO-BS All-Fu Al2-F A13-Br AlCDh AlS-Fr AlS-Bp A17-G A17-H A18-Ba A19-Fs A19-Nov.1. A19-Nov.2. A19-FF2 A20-Bd A21-I A22-Bb A23-DC A24-Hw A25-Hi A26Fr A31-J A32-Hm A34-Co B35-H3 B36-Hd B39-Bf Leiden collection S28 S26 s 166 s 31 s 133 s 19 S 16 s164 s 79 S 158 s 168 s 171 s 45 s 90 S40
3.6 4 4.5 4 4 4 4.5 5 5 5 5 6 6 6 6 6 6 6.5 6.5 6.5 7 7 7 7 7.5 7.5 5 5 5 6 6 6 6.5 6.5 7 7 7 9 9 9.5
7
7 10 7
8
8
* These data were only incompletely available to the authors.
+ + +
+
27 TABLE 1 (continued) No.
s 44 S 56 s17 S 106 s 147 s 39 s 88 s 113 s4 s 10 S 36 s 15 s144 s 99 S 136 S 167 s 91 s 131 s9 s 91 S 108 s 20 s 141 s 34 S 29 s 50 s 174 s51 S 83 s 114 s 175 s 30 s 37 s 103 s 54
Crown-rump length (mm)
Plane of sectioning
Section thickness * (am)
Staining
Graphic reconstruction(s) made
9.5 9.5 10 10 10 11 11 11 12 13.5 13.5 14 14 15 15 15 16 16 17 17 17 18 18 19 19.5 20 20 21 22 22 22 23 23 25 25
transverse transverse sag&al transverse transverse transverse sagittal transverse transverse frontal transverse sagittal transverse transverse transverse transverse transverse transverse transv./sagitt. sag&al transverse transverse transverse sag&al transverse transverse transverse transverse transverse sagittal transverse transverse transverse transverse transverse
10 6 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
HE HE Azan HE HE HE HE HE HE HE/Azan HE HE HE HE HE HE HE/Azan HE HE HE/Azan HE HE/Bodian HE HE HE HE HE HE HE HE/Azan HE HE HE HE/Azan l HE
+
+ + +
+
+
+
discussion. With this in mind, we have reinvestigated the human embryonic material which was earlier available to us [lO,ll]. Material and Methods The human embryos described in this study belong to the collections of the Department of Anatomy and Embryology, University of Leiden, and the Department of Histology and Embryology, University of Vienna. The crown-rump lengths of the embryos ranged from 3.6 to 25 mm. Detailed information on the material is
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given in Table 1. Of 18 embryos, one or more graphic reconstructions [12] were made as indicated in Table 1. This reconstruction technique is able to reveal histological details, the results regarding surface morphology being identical to those obtained by scanning electron microscopy [13]. We are therefore convinced, being well aware of “the bias inherent to studies on serial sections and their reconstruction” [14] that our results remain comparable with those obtained by other investigators. Although to our knowledge none of the hearts had been perfusion fixed (see [15]), the Vienna hearts in particular looked well dilated. Histological preservation of all specimens was excellent. Results The youngest specimen available was an embryo of 3.6 mm crown-rump length, in which the various cardiac segments could be clearly distinguished. The venous sinus (sinus venosus) could be seen to enter the right part of the atrium which itself
Fig. 1. Graphic reconstruction of the heart of a human embryo of 3.6 e crown-rump length, seen from the right. The atrium and atrioventricular canal have be-enopened. Endocardial cushion tissue covers the basal rim of the atrial septum primum (arrows) and fills the atrioventricular canal (c). Cushion tissue also covers the left venous valve (Iv), but not the right venous valve (IV). in = inlet segment; out = outlet segment; ta = distal part of outlet which connects with truncus arteriosus.
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was draining into the left-sided inlet segment. The latter opened into the right-sided outlet segment, which gave rise to the arterial component (truncus arteriosus; Figs. 1 and 2). The constricted portion between atrium and inlet segment, the atrioventricular canal, was partly filled by superior and inferior masses of endocardial cushion tissue. Because the atrioventricular canal was completely committed to the inlet segment, it was partly bordered by the fold between inlet and outlet segments. Both cushion masses, therefore, were clearly to the left of this fold. The inferior cushion mass was closely related to an area of very loosely arranged myocardium on the posterior wall of the inlet segment (Figs. 2 and 3). This ridge was judged to indicate already a primitive division between left and right bloodstreams. At the atria1 level, where septation had already started with the formation of a short primary atria1
Fig. 2. Reconstruction of part. The inferior cushion (is) on the posterior wall segment (out). Note that with the groove between
the same heart as in Fig. 1: cranial view after graphic dissection of the superior (c) is seen in the atrioventricular canal. It corresponds with a trabecular mass of the inlet segment. To the right, this segment communicates with the outlet in the inner curvature (arrow) the groove between atrium and inlet coincides inlet and outlet.
Fig. 3. Sections of the same heart as in Figs. 1 and 2, to show the inferior cushion (c) to correspond with a trabecular mass (is) on the posterior inlet wall. In both sections the primary fold (pf) is seen. avc = atrioventricular canal; in = inlet segment; out = outlet segment.
Fig. 4. Section of the same heart, to show the continuity of endocardial cushion tissue (c) with the extracardiac mesenchyme (m) of the mediastinum. la = left atrium; ra = right atrium; rv = right venous valve; Iv = left venous valve (not very prominent in this section, cf. Fig. 1); ta = distal part of outlet which connects with truncus arteriosus.
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septum, the two endocardial cushion masses appeared to be continuous with each other along the free border of this septum. In addition, this cushion mass also covered the inferior portion of the left venous valve, i.e. the fold between the venous sinus and the atrium (Fig. 1). At the site where the inferior cushion mass covered the primary atria1 septum and the left venous valve, the myocardial wall was interrupted. Thus, at the sinuatrial junction, the endocardial tissue was continuous with the extracardiac mesenchyme of the mediastinum. Because in this young stage the endocardial cushion tissue contained few cells (thus still deserving the name “cardiac jelly”), it could be readily distinguished from the mediastinal mesenchyme, which had a more compact cellular structure (Fig. 4). In later stages, the loose trabeculations on the posterior inlet wall had coalesced to form a more compact structure, which could be called the precursor of the inlet
Fig. 5. Reconstruction of the heart of a 6 mm embryo, anterior view after graphic dissection of the anterior wall. The atrioventricular canal (avc) is seen to drain completely into the inlet segment. The primary fold (pf) forms the border between inlet and outlet segments. Superior (SC) and inferior (ic) endocardial cushions guard the atrioventricular orifice. The inferior cushion borders upon a loose trabecular mass (is) on the posterior inlet wall.
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septum. The part of the inlet segment to the right of this inlet septum, initially inconspicuous, had started to expand. Consequently, the fold between inlet and outlet segments, which we call the primary fold, showed a curve which surrounded the right part of the inlet segment. As a result of the inlet expansion, the relationship of the inferior cushion with the primary fold disappeared, but this cushion kept its close relationship with the inlet septum. On the other hand, the superior cushion retained its relationship to the left side of the primary fold. Apposition of the two cushions leads to the separation of left and right atrioventricular orifices, the right one coming to drain into the expanded right portion of the inlet segment. The right inlet part was therefore bordered to the left by the inlet septum and to the right by the primary fold. Figs. 5 to 7 show successive developmental stages in which the inlet expansion
i
/ /
‘1 /’ / -
Fig. 6. Reconstruction of the heart of a 7 mm embryo, anterior view after graphic dissection of anterior wall. The primary fold (pf) shows a slight rightward curve. The superior (SC) and inferior endocardial cushions are apposed and to their left and right the mitral (m) and tricuspid (I) orifices seen. The latter is also bordered by the endocardial right posterior outlet ridge (or) which partly covers primary fold.
the (ic) are the
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can be seen. These stages show the formation of the right ventricle from two sources. Whereas in our 6 mm embryo only the inlet and outlet segments can be recognized, the 7.5 mm embryo (Figs. 7 and 8) already shows the basic morphology of the right ventricle. The inlet portion of this ventricle is the right part of the inlet segment in younger stages, whereas the outlet portion of the right ventricle (including its apical trabecular component) derives from the outlet segment. The curved part of the primary fold between these two portions can be identified as the parietal part of the septomarginal trabecula (moderator band). Figs. 9 to 12 then show further successive developmental stages in which a constant relationship can be seen between the superior atrioventricular cushion and the primary fold. This cushion maintains its position to the left side of the fold. In the 11 mm stage (Figs. 11 and 12) the basal part of the primary fold can be seen to delimit the “aortic” portion of the outlet segment. At this site, it can be seen that the left ventricle is also constituted from dual sources. Its main body (including the
Fig. 7. Reconstruction of the heart of a 7.5 mm embryo, ventral view after graphic dissection of the anterior wall. Note the rightward curve of the primary fold (pf), which is partly covered by the right posterior outlet ridge (or), which is to the right of the tricuspid orifice (t). SC= superior cushion; ic = inferior cushion.
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apical trabecular component) derives from the inlet segment, whereas the left-posterior part of the outlet segment is also allotted to this ventricle. This is made possible by the development of an endocardial septum within the outlet segment which can be seen to delimit the “aortic” part of the outlet (Fig. 12). In this respect, it should be noted that the interventricular communication of the 11 mm embryo (Figs. 11 and 12) is not comparable at all with the communication between inlet and outlet segments of the 6 mm embryo (Fig. 5). The latter is fully bordered by the primary fold. The interventricular communication of older stages, however, is bordered by the apposed atrioventricular cushions on the one hand and by the endocardial outlet septum on the other. It is this interventricular communication which, between the 16 mm and 18 mm stage, is closed to complete septation (Fig. 13). The morphology of the inlet myocardium merits particular attention. In the 7.5 mm embryo, the trabeculations were obvious. The trabeculations on the posterior inlet wall were clearly related to the inferior atrioventricular cushion (Figs. 7 and S)j whereas trabeculations on the anterior inlet wall showed relations with the superior
Fig. 8. The same heart as in Figure 7. The block shown in Fig. 7 has been graphically dissected further, now in a transverse plane (inset), and the apical part has been tilted forward to show the right portion of the inlet (ri), into which the tricuspid orifice (t) is draining. The right inlet part is delimited by the primary fold (pf) and the inlet septum (is). The latter is contiguous with the right side of the inferior cushion (ic). The arrow indicates the boundary between the inlet septum and the (anterior) primary septum (ps).
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cushion (Fig. 10). It would seem that the inlet myocardium was undermined with the exception of the areas covered by cushion tissue. At the atria1 level, the cushion masses completely surrounded the interatrial communication in the 3.6 mm stage (Fig. 1). In addition, they covered part of the left venous valve. In later stages, the initial interatrial communication (ostium primum) diminished and the basal portion of both venous valves became anchored within the cushion mass (Fig. 14). Still later, fusion of the cushion masses had obliterated the ostium primum, whereas a new communication through the septum primum (ostium secundum) had developed (Fig. 15). The continuity of endocardial cushion tissue (particularly the inferior cushion) with extracardiac mesenchyme, which was already clear in the 3.6 mm stage (Fig. 4) had become much more conspicuous in the 9.5 mm embryo (Fig. 16). At this stage
Fig. 9. The same heart as in Fig. 6; posterior view of the graphically dissected anterior part. The primary fold (pf) is partly covered by the superior cushion (SC) and the left anterior outlet ridge (or). The outlet segment (out) is seen to drain into the truncus arteriosus (ta).
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the base of the atria1 septum had thickened considerably (Figs. 17 and 18). In older stages, proportional changes were most obvious. Thus, the cushion mass remained restricted to the site of the posterior aortic root. Expansion of the inlet part of the ventricles and of the atria had led to production of a greater distance between the cushions and the posterior cardiac wall. Consequently, there was further incorporation of extracardiac tissue in the atrioventricular junction (Fig. 19). In summary, the atrioventricular cushions are considerable masses in young embryonic stages, where they cover the inner wall of the atrioventricular canal. In addition, they protrude into the inlet segment as well as the atrium. In the atrium they cover also the rim of the primary atria1 septum and the basal part of the venous valves. Here, the lower cushion is continuous with the mediastinal mesenchyme. Fusion of the cushion masses leads to obliteration of the interatrial ostium primum. The mass is then found on top of the inlet septum, directly posterior to the aortic root. It borders on the interventricular communication, and its eventual fusion with the endocardial outlet ridges obliterates this communication too. The considerable
Fig. 10. The same heart as in Fig. 7: posterior view of the graphically dissected anterior part. Note the trabeculations on the anterior wall of the inlet segment (in), and the superior cushion (SC) which is to the left of the primary fold (pf). ps = primary septum.
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Fig. 11. Reconstruction of the heart of an 11 mm embryo, posterior view of the anterior part. Note the primary fold (pf) between inlet (in) and outlet segments (out). A recess (a) leading into the aortic orifice is bordered by the primary fold and by the left anterior outlet ridge (or) and outlet septum (OS).
expansion of the ventricular inlet segment and of the atrium is not accompanied by a corresponding growth of the atrioventricular cushions. Therefore, they finally occupy an inconspicuous area at the site of the future membranous septum. Most of the tissue interposed between atria1 and inlet septa is then no longer cushion tissue but extracardiac mesenchyme. Discussion Our description of the topographic development of the atrioventricular endocardial cushions reveals that these initially massive structures gradually diminish in their relative size. Their volume is relatively large in the early stages, in which the primary interatrial and the interventricular communications also have substantial proportions. After completion of septation, the areas occupied by the cushions are restricted to the sites of final closure of these communications. In the present study, the actual fusion process of endocardial cushions is not described. Although these processes [16-B] are of fundamental importance, for the
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present purpose it is sufficient to indicate that, on the one hand, the apposition of the superior and inferior cushions leads to the separation of left and right atrioventricular orifices and, on the other hand, that their fusion with the endocardial rim of the primary atrial septum obliterates the ostium primum. The cushions are further involved in the closure of the interventricular communication. In this area they have to fuse with another endocardial structure, namely the outlet septum (Fig. 12). Thus, it may be stated that the endocardial cushions are of prime importance for septational processes. However, the question of their material contribution to the definitive septal structure is not answered by this statement. When their position is extrapolated to the mature heart, they can only contribute to structures near the posterior aortic root and the membranous septum [19], most of the fibrous tissues at the atrioventricular junction being derived from extracardiac tissues. In this respect, the septum of the sinus venosus [20], the central fibrous body and the tendon of Todaro [21] deserve attention.
Fig. 12. The same heart as in Fig. 11: anterior view of the posterior part, to show the posterior part of the outlet septum (OS)and the right posterior outlet ridge (or). These are delimiting the aortic vestibulum (a), which is to the right of the primary fold (pf). The arrows indicate the more apical part of the primary fold, which delimits the right part of the inlet segment. The outlet septum and the superior (SC)and inferior (ic) cushions form the boundaries of the interventricular communication. Fusion of these endocardial structures will eventually obliterate this communication and the aortic vestibule (a) will then be the outlet part of the left ventricle, the right part of the inlet as demarcated by the primary fold (arrows) becoming the inlet part of the right ventricle.
Fig. 13. Transverse section of the heart of an 18 mm embryo, to show the appositton of ngbt postenor outlet ridge (ror), left anterior outlet ridge (for) and inferior cushion (ic). The interventricular communication between aortic vestibule (a) and right ventricle (rv) is closed. The arrow indicates the tricuspid orifice.
100
u
Fig. 14. Reconstruction of the heart of a 6 mm embryo, viewed from the right. The right atnum has been opened graphically, to show that the interatrial ostium primum (op) is completely surrounded by endocardial cushion tissue (c), which is also found at the basis of the right venous valve (TV). The left venous valve cannot be seen (cf. Fig. 1).
De la Cruz et al. [3] have claimed that it is impossible to recognize the real contribution of the cushions to the structures of the mature heart by means of descriptive embryology alone. This statement was made concerning the need to distinguish the inferior and the superior cushion. From their labelling experiments in the chick embryo heart, they concluded that the inferior cushion contributed to the inter-ventricular septum adjacent to the region of the atrioventricular septum. Our observations in the human embryo, in which the atrioventricular conduction axis is always interposed between the cushion tissue and the muscular ventricular (inlet) septum (Fig. 19), together with the notion that in the mature heart the bundle is still on top of this inlet septum, are not in favour of cushion tissue making any contribution to the muscular part of the ventricular septum. Furthermore, the area under discussion has only negligible proportions in the mature heart. Most of the inlet septum is posterior to this area and has never been related to any cushion tissue. Since in the avian heart the bundle is buried within septal musculature [22], our arguments may not hold for the chick as studied by De la Cruz et al. [3]. Similar experiments were described [23] to show the contribution of the superior cushion to the left surface of the anterior basal portion of the ventricular septum.
iooi.4 Fig. 15. Reconstruction of the heart of a 9.5 mm embryo, to show the apposition of the cushion masses (SC and ic) at the basis of the atria1 septum primum (I). The Psterisk indicates the orifice of the left sinus horn. II = interatrial ostium secundum.
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Again, we believe that this possible contribution may be reduced to negligible proportions in the mature heart and may eventually be found in the region of the membranous septum. It should be noted that these workers [3,23] describe their final stages (i.e. 35-37) as “mature heart”, whereas the cavities still have to expand considerably before hatching which takes place in stage 46 [24]. It remains true to state that the question of the role of the atrioventricular cushions cannot be answered by descriptive embryology alone. Nonetheless, it seems justified to conclude that their main action is in keeping together the several muscular septal components, which otherwise would tend to grow far apart [25]. Their role in valve formation is somewhat more speculative. They certainly do not contribute to the material of the valve leaflets [8]. But they might be said to cover
Fig. 16. Transverse section of the same heart as in Fig. 15, to show the continuity of the inferior cushion (ic) with the more compact extracardiac mesenchyme (m). ra = right atrium; rw = right venous valve; sv = sinus venosus; rv = right ventricle; Iv = left ventricle.
Fig. 17. Keconstruction of the same heart as in Fig. 15: anterior view after graphic dissection of the anterior part. Note the apposed superior (SC) and inferior (ic) cushions. The inferior cushion borders on the inlet septum (is).
,
Fig. 18. The same heart as in Fig. 17, after graphic dissection of the endocardial cushions, to show the broad basis of the atria1 septum between the right (ra) and left (la) atrium. The stippled line surrounds the area of extracardiac mesenchyme (m) which is continuous with the inferior cushion.
Fig. 19. Transverse sections of the heart of a 25 mm embryo. a. At the level of the (fused) endocardial cushions (c). Note that the atrioventricular bundle (asterisk) is between the cushions and the ventricular septum (vs). b. At the level of the atrioventricular muscular continuity (av) where no cushion tissue (c) is present on top of the septum (vs) but only to its left and right sides. ra = right atrium; la = left atrium; s = orifice of the left sinus horn; ss = septum of the sinus venosus.
those parts of the inlet myocardium which have to keep together during the undermining process, thus guaranteeing continuous valve leaflets at the end of development. References Davis CL. The cardiac jelly of the chick embryo. Anat Ret 1924;27:201-202. Streeter GL. Developmental horizons in human embryos. Description of age group XIII, embryos of about 4 or 6 millimeters long and age group XIV, period of indentation of lens vesicles. Contr Embryo1 1945;31:27-63. De la Cruz MV, Gimenez-Ribotta M, Saravally 0, Cay& R. The contribution of the inferior endocardial cushion of the atrioventricular canal to cardiac septation and to development of the atrioventricular valves: Study in the chick embryo. Am J Anat 1983;166:63-72. Mall FP. On the development of the human heart. Am J Anat 1912;13:249-298. Odgers PNB. The development of the atrioventricular valves in man. J Anat 1939;73:643-657. Van Mierop LHS. Embryology. In: Yonkman FF, ed. The Ciba collection of medical illustrations. Vol. 5, The heart. New York: Pub1 CIBA, 1969;112-132.
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7 Ugarte M, Enriquez de Salamanca F, Quero M. Endocardial cushion defects. An anatomical study of 54 specimens. Br Heart J 1976;38:674-682. 8 Van Gils FAW. The formation of the atrioventricular heart valves. Acta Morph01 Neerl-Stand 1978;16:151. 9 Wenink ACG, Van Gils FAW, Gittenberger-de Groot AC, Draulans-Noe HAY, Thiene G. Developmental aspects of atrioventricular septal defects. In: Quero Jimenez M, Anderson RH, eds. Atrioventricular septal defects. Edinburgh: Churchill-Livingstone, 1985;in press. 10 Wenink ACG. Embryology of the ventricular septum. Separate origin of its components. Virchows Arch A 1981;390:71-79. 11 Wenink ACG, Gittenberger-de Groot AC. Left and right ventricular trabecular patterns. Consequence of ventricular septation and valve development. Br Heart J 1982;48:462-468. 12 Tinkelenberg J. Graphic reconstruction, micro-anatomy with a pencil. J Audio+ Media Med 1979;2:102-106. 13 Wenink ACG, Chon Y. The value of graphic reconstructions. Comparison with scanning electron microscopy. Anat Ret 1984;210:537-540. 14 Pexieder T. Changing scene in cardiac embryology. Herr 1979;4:73-77. 15 Pexieder T. Effects of various fixatives and fixation procedures on the shape of the embryonic heart. Eur Heart J 1983;4(suppl E):112. 16 Hay DA, Low FN. The fusion of dorsal and ventral endocardial cushions in the embryonic chick heart: a study in fine structure. Am J Anat 1972;133:1-24. 17 Los JA, Van Eijndthoven E. The fusion of the endocardial cushions in the heart of the chick embryo. Z Anat Entwicklungsgesch 1973;141:55-75. 18 Hay DA. Development and fusion of the endocardial cushions. In: Rosenquist AC, Bergsma D, eds. Morphogenesis and malformation of the cardiovascular system. Birth Defects: Original article Series XIV-7. New York: Alan R Liss, 1978;69-90. 19 Wenink ACG. La formation du septum membranaceum darts le coeur humain. Bull Assoc Anat 1975;58:1127-1132. 20 Los JA. Cardiac septation and development of the aorta, pulmonary trunk and pulmonary veins: previous work in the light of recent observations. In: Rosenquist AC, Bergsma D, eds. Morphogenesis and malformation of the cardiovascular system. Birth Defects: Original article Series XIV-7. New York:Alan R Liss, 1978;109-138. 21 Van Gils FAW. The fibrous skeleton in the human heart: embryological and pathogenetic considerations. Virchows Arch A 1981;393:61-73. 22 Vassall-Adams PR. The development of the atrioventricular bundle and its branches in the avian heart. J Anat 1982;134:169-183. 23 Garcia-Peltiesz J, Diaz-G6ngora G, Arteaga Martinez M. Contribution of the superior atrioventricular cushion to the left ventricular infundibulum. Experimental study in the chick embryo. Acta Anat 1984;118:224-230. 24 Hamburger V, Hamilton HL. A series of normal stages in the development of the chick embryo. J Morph01 1951;88:49-92. 25 Van Mierop LHS. Embryology of the atrioventricular canal region and pathogenesis of endocardial cushion defects. In: Feldt RH, ed. Atrioventricular canal defects. Philadelphia/London/Toronto: W.B. Saunders, 1976;1-12.