In normal development pulmonary veins are connected to the sinus venosus segment in the left atrium.код для вставкиСкачать
THE ANATOMICAL RECORD 243:84-92 (1995) In Normal Development Pulmonary Veins Are Connected to the Sinus Venosus Segment in the Left Atrium M.C. DERUITER, A.C. GITTENBERGER-DE GROOT, A.C.G. WENINK, R.E. POELMANN, AND M.M.T. MENTINK ABSTRACT Background: Classic theories describe that the common pulmonary vein develops as an outgrowth from either the sinus venosus or atrial segment. Recent studies show that the pulmonary veins are connected to the sinu-atrial region before its differentiation into a sinus venosus and atrial segment. Methods: The development of the sinu-atrial region with regard to the developing common pulmonary vein and the growth of the atrial septum was investigated in avian embryos, using both scanning electron microscopy and immunohistochemistry. Embryos ranging between stage HH12 and HH28 were incubated with QH-1 that recognizes quail endothelial cells and precursors, HNK-1, that appears in this study to detect the myocardium of the sinus venosus, or with HHF-35,being specific for muscle actins. Also vascular casts of the heart were produced by injecting prepolymerized Mercox into the vascular system. Results: In preseptation stages the common pulmonary vein drains into the left part of the sinus venosus, that is clearly demarcated by the sinuatrial fold and HNK-1expression. During atrial septation the left part of the sinus venosus, in contrast to the right part, loses its HNK-1 antigen from stage HH23 onwards, while at the same time the sinu-atrial fold in the left atrial dorsal wall flattens and disappears. From stage HH25 onwards HNK-1 expression is restricted to the right part of the sinus venosus, which contributes to the right atrium. The myocardial atrial septum never expresses the HNK-1 antigen, suggesting that the septum is of atrial origin. Discussion: It appeared that the sinus venosus does not only contribute to the sinus venarum of the right atrium, but also to the left atrium. 0 1995 Wiley-Liss, Inc. Key words: Atrium, Embryo, HNK-1, Immunohistochemistry, Pulmonary vein, QH-1, Scanning electron microscopy, Sinus venosus Textbooks on human embryology (e.g., Carlson, 1994;Larsen, 1993;Moore, 1982)describe that the embryonic sinus venosus segment is incorporated into the ultimate right atrium. It gives rise to the sinus venarum of the right atrium in which the coronary sinus and the superior and inferior caval veins drain. The appendage of the right atrium develops from the embryonic atrial segment. In contrast, the left atrium is said to develop from the atrial segment without a contribution of the sinus venosus. It is described that the common pulmonary vein develops in a 6 mm human embryo as a lumenized sprout from the dorsal wall of the atrial segment into the developing lungs (Los, 1968, 1978; Neill, 1956). At this time the atrial segment is already divided by the septum primum into a right and left atrium. Although the above theory is generally accepted among embryologists, various other studies contend that the common pulmonary vein drains into the sinus venosus. The difference is often ascribed to species differences comparing birds (Federow, 1910; Squier, 0 1995 WILEY-LISS, INC. 19161, reptiles (Kutsche and VanMierop, 1988) and non-human mammals (Brown, 1913;Flint, 1906).Thus the literature leaves us with two questions. The first is whether the development of the pulmonary veins in the human embryo could be different from that in all other species, including other mammals. If this is not the most probable solution of the problem, the next question arises. What can be the cause that descriptions of the human embryos are incompatible with those of other species? Auer (1941, 1948) studied human embryos, and he described that also in human embryos the common pulmonary vein is initially connected to a cranial appendage at the venous pole, which cannot be defined to belong either to the sinus venosus or to the Received December 12, 1994; accepted March 7, 1995. Address reprint requests to Dr. M.C. DeRuiter, Department of Anatomy and Embryology, University of Leiden, P.O. Box 9602,2300 RC Leide?, The Netherlands. PULMONARY VEIN CONNECTION TO T H E SINUS VENOSUS atrial segment. In those descriptions it remains unsolved how the pulmonary vein becomes connected to the left atrium during atrial septation. The problem was already seen by Quiring (19331, who stated that the sinu-atrial fold is not quite as clearly demarcated in the area of the developing septum primum and the orifice of the pulmonary vein, as the part from which the right and left venous valves develop. This means that, particularly in very early stages, it is difficult to decide whether the primitive pulmonary veins are connected with the sinus venosus or the atrium. Until recently, it was impossible to trace these veins before the stage of lumen formation, because specific endothelial markers were not available. Recent immunohistochemical studies, using an antibody against quail endothelial cells and precursors (DeRuiter, 1992, 19931, showed that a non-lumenized common pulmonary vein is present in the dorsal mesocardium before development of the lungbuds. This strand of endothelial cells, the so-called mid-pharyngeal endothelial strand (MPES), has been separated longitudinally from the endocardial heart tube during the disappearance of part of the dorsal mesocardium. The MPES remains in contact by way of the remaining dorsal mesocardium with the endocardium at the venous pole of the heart. At this site of connection the sinu-atrial region has not differentiated into a sinus venosus and an atrial segment yet. In the present study we reinvestigated the development of the sinus venosus and the atrial segment in relation to the developing pulmonary vein and their contribution to the ultimate left and right atrium in the avian embryo. To detect the sinu-atrial fold we used both immunohistochemistry and scanning electron microscopy. The main questions in this study are: 1)What is the precise position of the sinu-atrial fold, as the junction of the sinus venosus and atrial segment; 2) Is the common pulmonary vein initially connected to the sinus venosus or directly to the atrial segment; and 3) If the common pulmonary vein drains into the sinus venosus, how does the atrial septum primum separate the orifice of the pulmonary vein from the orifices of the systemic veins orifices? 85 conventional methods, mounted on scanning stubs and sputter-coated with gold for 3 min. lmmunohistochemistry Chick and quail embryos, stage HH12 t o HH28, were fixed (24 to 72 h) in 2% acetic acid in 100% ethanol at 4°C. After washing in phosphate buffered saline (PBS; pH 7.3) and dehydration in ethanol the embryos were embedded in paraffin and serially sectioned transversely at 5 pm. The sections were dried overnight at 37°C on albumedglycerin coated slides. The embryos were incubated with the monoclonal antibody HNK-1, a gift of Dr. TM Luider (Rotterdam, The Netherlands), the monoclonal anti-muscle actin antibody HHF-35 (Tsukada et al., 1987a,b), a gift of Dr. AM Gown (Seattle, WA), or with the monoclonal antibody QH-1 (Pardanaud et al., 1987), which detects quail endothelial and hemopoietic cells, diluted in PBS with 0.05% Tween-20 and 1%ovalbumin. After overnight incubation the slides were washed in PBS followed by the second incubation for 2 h with 1:300 diluted rabbit anti-mouse conjugated to horse radish peroxidase (Dako, P260). After washing in PBS, the staining reaction was performed with 0.04% diamino benzidine tetrahydrochloride in 0.05 M tris-maleic acid (pH 7.6) with 0.006% hydrogen peroxide for 10 min, followed by washing in the buffer. Last, the sections were briefly (15 sec) counterstained with Mayer’s hematoxylin. RESULTS Development of the Pulmonary Veins At stage HHlO the heart tube is over its complete length dorsally attached to the foregut. This area is called the dorsal mesocardium. With the increase of cardiac jelly part of the dorsal mesocardium between the arterial and venous pole of the heart narrows and disappears at stage HH12. During this process a strand of endocardial cells remains attached to the foregut. This so-called mid-pharyngeal endothelial strand (MPES) remains connected to the endocardium at the sinu-atrial region of the heart tube (Fig. 1A). At stage HH13 the MPES starts to lumenize in the persistent part of the dorsal mesocardium giving rise to the common pulmonary vein (Fig. 1B). At the peripheral tip of the lumenized pulmonary vein endothelial precursors MATERIALS AND METHODS are present. These cells can be identified as QH-1 posBoth white Leghorn chick embryos (Gallus domesti- itive isolated, non-lumenized endothelial cells (Fig. cus) and Japanese quail embryos (Coturnix coturnix 1B). In the next stages these endothelial precursors japonica) were used. They were staged according to the proliferate and lumenize to form the tributaries of the age-determination criteria of Hamburger and Hamil- common pulmonary vein at stage HH21. ton (1951). Development of the Sinus Venosus Scanning Electron Microscopy When the heart tube is established a t stage HH12 Chick embryos, stage HH16 (28 somites) to HH28, different segments can be recognized. The large left were perfusion fixed in half strength Karnovsky’s fix- and right omphalomesenteric and cardinal veins enter ative (1965). The hearts were sectioned in various the heart laterally by the left and right sinus horns. planes. Hereafter they were rinsed in 0.1 M sodium- These sinus horns together with an undivided common cacodylate buffer (pH 7.2) and postfixed for 1h at 21°C part are defined as the sinus venosus. At this stage, in 1%OsO, in the same buffer, followed by dehydration however, it is not possible to discriminate between the in graded ethanol. The chick hearts were also studied sinus venosus and the atrial segment. Therefore this with the corrosion casting technique, using the pre- bilateral symmetric region is described as the sinupolymerized methacrylate Mercox (CL-2B with cata- atrial region. With the appearance of the sinu-atrial lyst MA; Fa Okenshoji Co, Ltd, Chou-ku, Tokyo 1041, fold the distal border of the sinus venosus segment beas described by DeRuiter and co-workers (1991). All comes demarcated. Proximally the sinus venoms is the preparations were critical point dried over COz by connected to the extra-cardiac tissues at three sites. 86 M.C. DERUITER ET AL. Fig. 1. A and B: Transverse sections of quail embryos incubated with the QH-1 antibody. The mid-pharyngeal endothelial strand (arrow) is connected to the sinu-atrial region (SA). When this strand lumenizes it gives rise to the common pulmonary vein (asterisk). Around the lumenized vein endothelial precursors are present (arrowheads). C and D Transverse sections through the sinus venoms (SV) and the left and right atrial appendages (RA and LA) of the chick embryo. The cushion tissue around the free edge of the atrial septum ( S ) is continuous with the inferior atrio-ventricular cushion (IAV). A) HH12, X 200; B) HH13, X 160; C ) HH17, x 122; and D) HH23, x 77. Atrio-ventricular canal (AV);bronchi (B); dorsal aorta (Dao);foramen primum (Fl); foregut (F);sinu-atrial fold (arrow heads). PULMONARY VEIN CONNECTION TO THE SINUS VENOSUS 87 Fig. 2. A: A dorsal view on a vascular cast of a stage HH17 chick embryo, At the left side of the sinu-atrial region the sinu-atrial fold (white dots) has appeared between the orifice of the pulmonary vein (asterisk) and the left atrial appendage (LA); x 67. B: A cranial view on the same embryo. At the right side the course of the sinu-atrial fold near the atrial septum (black boxes) is still unclear, but the area of the future right atrial appendage (FL4) and sinus venoms (SV) can already be indicated. The atrial septum runs to the right side of the atrio-ventricular canal (AV). The left sinus horn (LS) or the coronary sinus has been shifted to the right side; x 67. Bilaterally the two sinus horns are bordered by the non-myocardial mesenchymal cells around the omphalomesenteric veins. Between these horns the dorsal wall of the common part of the sinus venosus is connected to the lung region by the dorsal mesocardium in which the pulmonary vein runs. At that site the lining of the sinus venosus, which does not express alphaactin until stage HH19 (see Fig. 4B), merges into the mesothelial lining of the lung buds. The symmetry of the sinu-atrial region is lost by the shift of both sinus horns together with the large veins to the right side of the heart (Fig. 2A). Herewith the sinus venoms becomes almost completely situated caudal to the right part of the atrial segment. From stage HH15 onwards the sinu-atrial fold arises between the sinus venoms and the atrial segment (Fig. 3A,B). The first signs of the fold are seen in the left lateral wall of the sinu-atrial region, indicating the incorporation of the left sinus horn in the right atrium. Soon the sinu-atrial fold also extends into the right half of the sinu-atrial region. At stage HH17 an almost complete sinu-atrial ring is present separating the sinus venosus from the atrial segment (Figs. l C , 2A,B). With the appearance of this fold it appears that all veins, including the common pulmonary vein, enter the sinus venoms segment. At the same time the septum primum primordium arises in the dorso-superior wall of the sinu-atrial region and extends t o the right side of the atrio-ventricular canal (Figs. lC, 2A,B, 3A,B). This septum indicates the separation of the future left and right atrium. It is remarkable that with the shift of the left sinus horn a small part of the sinus venosus including the orifice of the common pulmonary vein is still situated at the left side of the atrial septum (Figs. lC, 3A,B). It should be noted that, a t this site, the atrial septum does not consist of myocardium, but of endocardia1 cushion tissue which is continuous with the mesenchyme of the dorsal mesocardium. The remaining part of the sinus venoms is connected to the right atrial appendage a t the right side of the atrial septum. At this time the right atrial appendage is much smaller than the left one (Figs. 2, 3C). The vascular casts (Fig. 2A,B) give the impression that the atrial septum also septates the sinus venosus seg ment. In the next stages the sinu-atrial fold is more pronounced and its position can easily be identified (Fig. 3C,D,E). Starting at the left side of the atrial septum the sinu-atrial fold encircles the pulmonary vein orifice and crosses caudally over to the right half of the sinuatrial region (Fig. 3C,E), just above the entrance of the left sinus horn (Fig. 3C), which will give rise to the coronary sinus. At that point the fold runs along the right lateral and dorsal wall of the right atrium to the atrial septum (Fig. 3C,D). Using scanning electron microscopy or histological sections it is not clear completely how the circle is closed in the area of the dorsal mesocardium. It looks as if all folds (superior and inferior sinu-atrial fold and atrial septum) merge with each other during completion of atrial septation. A continuity of dorsal mesocardial mesenchyme and inferior atrio-ventricular cushion remains obvious. By the growth of the atrial septum and the accumulation of cushion tissue the part of the sinus venosus receiving the common pulmonary vein is separated from the part connected to the systemic veins. The Fig. 3. Scanning electron micrographs of the developing sinu-atrial region in the chick embryo. A Cranial view on a transverse sectioned heart a t stage HH16; x 220. B: Caudal view on the counterpart of the same sectioned heart in Figure 3A. Starting at the left side of the atrial septum (black dots and boxes) the sinu-atrial fold (white dots) runs between the left atrial appendage (LA) and the orifice of the pulmonary vein (star) to the right of the atrio-ventricular canal (AV). At the right side the fold runs cranially towards the atrial septum, where it flattens. The numbers indicate the course of the sinu-atrial fold (1 and 2) and the atrial septum (3) in the counterpart; X 220. C: Ventral view on a frontal sectioned heart a t stage HH19; x 130. D Dorsal view on the counterpart of the same sectioned heart in Figure 3C. The numbers (1 and 2) indicate the course of the sinu-atrial fold in the counterpart; x 130. The sinu-atrial fold encircles the orifice of the pulmonary vein and crosses the midline to the right side. In the midline the fold is covered by cushion tissue. The sinu-atrial fold seems to be divided by the atrial septum (S), but at that place the septum only consists of cushion tissue. E: Cranial view on a transverse sectioned heart through the sinus venoms and the atrial segment at stage HH20. The left atrial appendage (LA) is large, while the left part of the sinus venoms with the pulmonary vein orifice (star) is small. The connection of the left and right part of the sinu-atrial fold is not visible by the overlying cushion tissue of the atrio-ventricular canal (AV), which is continuous with the atrial septum and the mesenchyme of the dorsal mesocardium; x 120. F: A left view on the left sinus horn (LS) or coronary sinus and the atrial appendages (LA and RA) of a frontally sectioned heart (HH25). In the right atrium the left and right sinu-atrial valves (rv and lv) have developed from the sinuatrial fold. Atrial septation has almost completely finished. The pulmonary vein drains into the left atrium; x 82. PULMONARY VEIN CONNECTION TO T H E SINUS VENOSUS atrial septum grows like a wedge between the left and right atrial appendages. The free edge of the atrial septum is lined by endocardia1 cushion tissue (Fig. 3D,E). The inferior border of the atrial septum is continuous with both the dorsal mesocardium and the inferior atrio-ventricular cushion (Figs. ID, 3E). Herewith the size of the foramen primum is diminished. During atrial septation the sinu-atrial fold in the left atrium (HH23) disappears (Fig. 3F). In the right atrium the sinu-atrial fold is still clearly recognizable and persists as the right and left sinu-atrial valves (Fig. 3F). HNK-1 Expression in the Sinus Venosus At stage HH16 the first HNK-1 expression is seen in the left lateral wall of the sinus venosus, just left of the pulmonary vein orifice. The right side of the sinus venosus is negative. From stage HH17 onwards also the right half of the sinus venosus starts to express the HNK-1 antigen near the sinu-atrial fold. The expression is the highest in the area left to the common pulmonary vein and extends to the sinu-atrial fold (Fig. 4A). Both the myocardium of the atrial appendages and the developing atrial septum are completely negative. At stage HH22 the complete sinus venosus shows HNK-1 expression (Fig. 4C,D). At the left side HNK-1 expression is not only seen in the myocardium lateral to the pulmonary vein, but also between the pulmonary vein entrance and the atrial septum. In the dorsal mesocardium many cells express actin and the HNK-1 antigen. These cells form a layer of loosely connected myocardial cells, which interconnect the myocardium around the pulmonary vein orifice and the left and right sinus horns of the sinus venosus (Fig. 4C,D). From stage HH23 onwards the expression decreases in the left sinus horn and the part of the sinus venoms that contributes to the ultimate left atrium, i.e., around the orifice of the common pulmonary vein (Fig. 4E). Until stage HH25 the loose myocardium in the dorsal mesocardium is still positive for the HNK-1 antibody. In the left and right sinu-atrial valves only the sinus venosus myocardium expresses the antigen (Fig. 4F). At stage HH25 and HH26 the expression is restricted to the part of the sinus venosus, that is connected to the right atrium. Although the scanning electron microscopy did not elucidate the course of the superior part of the sinuatrial fold in the area of the atrial septum, the HNK-1 incubated embryos show that the left sinu-atrial valve is continuous with the myocardium around the pulmonary vein. The results of this study are summarized in the schematic drawings of Figure 5. The common part of the sinus venoms is connected to both the right and left part of the atrial segment (Fig. 5A). The sinu-atrial fold crosses the mid-line of the sinu-atrial region through the foramen primum and below the atrial septum. The common pulmonary vein drains into the sinus venoms just left to the atrial septum. By the growth of the atrial septum the foramen primum closes by the fusion of the free edge of the atrial septum with the atrio-ventricular cushions (Fig. 5B). Leaving the pulmonary vein to drain into the left atrium and the systemic veins and the systemic veins into the right atrium. 89 DISCUSSION Literature on the morphogenesis of the pulmonary veins in various animal species is ambiguous on the question whether these are connected to the sinus venosus or the atrial segment of the heart. Notwithstanding this controversy, it is generally accepted that the pulmonary vein originates from the left atrial segment in human embryos (Los, 1968,1978; Neill, 1956). In reptiles (Kutsche and VanMierop, 1988), birds (Federow, 1910; Squier, 1916), and non-human mammals (Brown, 1913; Flint, 1906), however, the pulmonary vein is supposed to originate from the sinus venoms segment. How in these cases the pulmonary veins later on originate from the left atrium is not discussed in the literature. In the unseptated heart the connection between the sinus venoms and the atrial segment is clearly demarcated by the appearance of the sinu-atrial fold (Quiring, 1933). After septation of the atria by the septum primum the borderline between these segments is still recognizable in the right atrium as the left and right sinu-atrial valves. Ultimately the left sinu-atrial valve contributes to the formation of the atrial septum in the mammalian embryo (Odgers, 1935; Wenink, 1987). By the absence of the septum secundum in the avian embryo the left sinu-atrial valve persists in the right atrium. Although the presence of a demarcation between the sinus venoms and the atrial segment is distinct it has led to many contradicting views concerning the origin of the pulmonary veins. This is probably caused by the difficult identification of the sinu-atrial fold near the developing atrial septum. Even in nonhuman embryos the description of the connection between the left and right sinu-atrial valves near the atrial septum is obscure (Dalgleish, 1976; Quiring, 1933) allowing also for difficulties in determining whether the orifice of the common pulmonary vein is enclosed by the sinu-atrial fold or not. The description for the human embryo by Los (1968,1978)is made from study of hematoxilin-eosin stained human embryos of 4.4 mm and 6 mm. Without the special immunohistochemical techniques and detection of endothelial precursors, a decision on a possible contribution of the sinus venosus to the left atrium is not allowed from this material. In the present study it was possible to detect the pulmonary vein in the avian embryo before it achieves a lumen. The pulmonary vein develops from the midpharyngeal endothelial strand (MPES) which is part of the splanchnic vascular plexus around the developing foregut and lungbuds. From its onset this plexus is connected with both the arterial and venous poles of the heart. The pulmonary vein develops from this splanchnic plexus by developing a lumen, and not as an outgrowth from the endocardium of the sinu-atrial region of the heart (DeRuiter et al., 19931, as described in the classic studies. From stage HH15 onwards the sinu-atrial region differentiates into a sinus venosus and atrial segment. It is remarkable that the first signs of the fold are seen in the left lateral wall of the sinu-atrial region just left to the orifice of the common pulmonary vein. Thereafter, the fold becomes prominant in the right half of the sinu-atrial region a t stage HH17. The scanning elec- 90 M.C. DERUITER ET AL. Fig. 4. Transverse sections through chick embryos of various stages. A The myocardium of the sinus venosus (SV) left to the common pulmonary vein (*) at stage HH18 express the HNK-1 antigen. At the right side of the sinus venosus only few cells express the HNK-1 antigen. The arrow heads indicate the sinu-atrial fold; x 160. B: Adjacent section to that of Figure 4A, incubated with HHF-35. HNK-1 expression seems to be restricted to sinus venosus myocardium which contains actin filaments; x 160. C: At stage HH22 the complete sinus venosus is positive for the HNK-1 antibody. The atrial myocardium and the atrial septum (S) are negative; x 102. D The adjacent section to that of Figure 4C incubated with HHF-35 shows the differentiated myocardium of the sinus venosus and the inferior border of the atrial segment. In the dorsal mesocardium, dorsal to the atrial septum, myocardial cells (arrows) are present which are both positive for HHF-35 and HNK-1; x 102. E and F The myocardium left to the common pulmonary vein looses its HNK-1 expression at stage HH23, but in the dorsal mesocardium cells are still positive for both actin and HNK-1 (arrow). This loose myocardium interconnect the left (LS) and right sinus horn with the myocardium around the pulmonary vein orifice. In the left and right sinu-atrial valves (lvv and rvv) HNK-1 expression is restricted to the myocardium contribution of the sinus venosus. Left atrial appendage (LA); right atrial appendage (RA); x 102. tron microscopic findings are supported by the immunohistochemistry. The pulmonary vein is encircled by myocardium, which expresses the HNK-1 antigen. This myocardium is continuous with the HNK-1 posi- tive left sinu-atrial valve, at the right side of the atrial septum. Although the superior sinu-atrial fold is not distinct in the area of the atrial septum, the continuity of HNK-1 positive myocardial cells suggests that the PULMONARY VEIN CONNECTION TO THE SINUS VENOSUS 91 U Fig. 5. Schematic representations of two stages of atrial septation and the contribution of the sinus venosus to the left atrium. A The common part of the sinus venosus (SV) is connected to the right and left part of the atrial segment. The sinus venosus just below the free edge of the atrial septum (S), facing the foramen primum (F1) is covered by cushion tissue. B: The pulmonary vein is separated from the systemic orifices by the atrial septum when its free edge fuses with the atrio-ventricular cushions. Only the inferior atrio-ventricular cushion is drawn. Part of the sinus venosus receiving the common pulmonary vein (PV) is connected to the left atrial appendage (LA). Foramen secundum (F2); right atrial appendage (RA). myocardium around the pulmonary vein belongs to the sinus venosus and not to the atrial segment, in which otherwise no HNK-1 positivity was noted in any stage. When the atrial septum primordium descends into the sinu-atrial region the common pulmonary vein is separated from the orifices of the systemic veins and enters the left atrium. At that time the sinu-atrial fold flanking the orifice of the common pulmonary vein flattens and disappears. The significance of the HNK-1 antigen expression by the myocardium of the sinus venosus is not clear. Luider and co-workers (1993) demonstrated that the HNK-1 antigen, which is originally described as a marker for premigratory and migrating neural crest cells (Bronner-Fraser, 1987), can also be seen in morphologically active regions, that are not neural crest related. For instance the myocardium of the outflow tract expresses the antigen during coronary artery development. Recently it is described that the future areas of the conduction system in the rat are also positive for the HNK-1 antibody (Sakai et al., 1994). In contrast to earlier studies (Dalgleish, 1976; Quiring, 1933) we were able with the used techniques to determine the sinu-atrial border even in the area of the atrial septum. The inferior border is clearly demarcated by both a pronounced sinu-atrial fold and the expression pattern of the HNK-1 antigen. The superior border near the atrial septum is less distinct, because the connecting part of the fold runs as a thin sheet of loosely connected myocardial cells in the dorsal myocardium between the left sinu-atrial valve and the orifice of the common pulmonary vein. Some of the scan- ning microscopical pictures give the impression that the sinu-atrial fold is crossed by the atrial septum. This would imply that the atrial septum consists of a sinus venosus and an atrial part. However, the superior sinuatrial border runs just below the inferior border of the myocardial atrial septum. Since the inferior rim of the atrial septum is covered with endocardia1 cushion tissue, which itself is continuous with the atrioventricular cushions as well as the mesenchyme of the dorsal mesocardium, it remains difficult to completely delineate all components of the mature atrial septum. Since this study shows that the left (“pulmonary”)portion of the sinus venosus ends up to the left of the atrial septum, it can be inferred that the septum of the sinus venosus contributes to the atrial septum. This contribution can well be derived from the mesenchyme of the dorsal mesocardium. Our conclusions about the bounderies between the sinus venosus and the atrium have still to be corroborated by a detailed study of the relationship between myocardium and mesenchyme in this area. It will be of value to restudy mammalian embryos with the new findings in chick in mind. Recent data show, that similar to our findings, pulmonary veins are seen to originate from a sinus venosus segment in the left atrium in mouse embryos (Tasaka et al., 1995). This emphasises the need to also restudy the human embryo with the use of early immunohistochemical markers. The knowledge that the left atrial part of the sinu-atrial junctions disappears during development as a recognizable borderline should be kept in mind with the study of older embryos. 92 M.C. DERUITER ET AL. LITERATURE CITED Auer, J . 1941 The early development of the sinu-atrial region in the heart. Acta Neerlandica Morphologiae, 4t214-232. Auer, J . 1948 The development of the human pulmonary vein and its major variations. Anat. Rec., 101:581-594. Bronner-Fraser, M. 1987 Perturbation of cranial neural crest migration by the HNK-1 antibody. Dev. Biol., 123t321-331. Brown, A.J. 1913 The development of the pulmonary vein in the domestic cat. Anat. Rec., 7:299-329. 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