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Atrial septation
DEVELOPMENTAL
BIOLOGY
57, 345-363 (1977)
Atrial Septation I. Scanning MARY Department
of Anatomy,
Electron
Microscopy
J. C. HENDRIX
The George Washington
Received July 8,1976;
in the Chick’
AND DENNIS University
E. MORSE*
School of Medicine,
Washington,
D.C. 20037
accepted in revised form January 26,1977
Chick hearts were prepared for scanning electron microscopy by standard methods, the purpose being to investigate the surface morphology of the developing atria1 septal region. By Day 3, the atria1 septum primum appeared as a sickle-shaped structure. During Day 4, the first representation of the foramina secunda occurred in the mid-dorsal portion of the septum. During Day 5, the septum primum fused with the atrioventricular endocardial cushions, thereby occluding the foramen primum. From Days 5 through 8, the secondary perforations (foramina secunda) multiplied and increased in size. The endocardial-covered cords of cells comprising the septum thickened from Days 9 through 15. This resulted in a marked reduction in the dimensions of the perforations from Day 16 to hatching. The atria1 septum at hatching occasionally contained a small single orifice. At 3 days posthatching, the atria1 septum was a solid sheet covered with flattened endocardial cells. All interatrial communications were occluded. During Days 5 through 9, two distinct cell types became apparent on the endocardialcovered cords. Simultaneously, fenestrations were observed on the cords surrounding the foramina secunda and on the ventral portion of the atria1 septum. The integral role which the fenestrations and cellular types play in the development of the foramina secunda is discussed. INTRODUCTION
Embryologists and cardiologists have striven to give a detailed and accurate description of the normal development of the vertebrate heart for more than a century. In particular, the partitioning of the heart by septa into four chambers has aroused the interest of numerous investigators. However, recent emphasis has been placed on the ventricular region, leaving unsettled problems concerning the development of the atria1 septum. According to classical embryologists (Lindes, 1965; Born, 1889; Hochstetter, 1906; Patten, 1925; Chang, 1931; Bremer, 1932; Quiring, 1933), two distinct septa (atrial and ventricular) are involved in the partitioning of the normal vertebrate heart. Based on observations in the chick ’ This research was aided by a grant from the Washington Heart Association (Washington, D.C.). 2 Present address: Department of Anatomy, Medical College of Ohio, Toledo, Ohio 43614.
embryo, these investigators demonstrated that the atria1 septum primum made its first appearance in the cephalic wall of the atrium between 50 and 53 hr of incubation. Late in the fifth day, this septum has descended sufficiently to contact physically the fused atrioventricular endocardial cushions (septum intermedium). At this time, the area located directly below the descending atria1 septum (foramen primum> is occluded. Secondary communicative openings (foramina secunda) are established in the septum primum at about the time of closure of the foramen primum. The strands of tissue comprising the margins of the foramina secunda have a valvular function which operates accordingly in each phase of the cardiac cycle. This provides the right-to-left interatrial shunt, thus establishing a pulmonary bypass for a portion of the blood entering the heart. Final closure of the foramina secunda occurs in the first few days after birth and 345
Copyright 0 1977by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN
0012-1606
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DEVELOPMENTAL BIOLOGY
coincides with the beginning of pulmonary circulation. It was recognized many years ago that normal embryonic development was accompanied by spontaneous physiological cell death. Glucksmann (1951) pioneered cell death research in developmental biology* Several workers have noted the presence of dead and dying cells in conjunction with cardiac morphogenesis (Manasek, 1969; Krstic’ and Pexieder, 1973; Pexieder, 1975a,b; and Ojeda and Hurle, 1975). Atrial septal defects are among the most common congenital cardiac anomalies occurring in newborns in the United States (Kissane and Smith, 1967). In the interest of studying the etiology of atria1 septal defects, we must first establish the normal sequence of events in the fine structural development of the atria1 septum. Previous work on atria1 septation is limited almost exclusively to light microscopy. Transmission electron microscopic studies are restricted to highly selected stages of development. Scanning electron microscopic studies of atria1 septation are not offered by the literature. Our present research interests concentrate on the formation of the atria1 septum. To accomplish this goal, we employ several microscopic techniques to give a detailed analysis of the morphology and subcellular constituents comprising the atrial septal region. The purpose of this paper is to present our observations of the surface morphology of the atrial septal development in the chick using scanning electron microscopy.
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ries (Hamilton, 1952) after which the hearts were immediately dissected free and were kept in the fixative for a minimum of 2 hrs. Thirty-six chick embryos ranging from hatching through 1 week posthatching were perfused in uiuo using the same fixative plus procaine hydrochloride. In most cases, the lateral atrial walls were removed with the aid of a dissecting microscope. The tissues were then rinsed successively in cacodylate buffer (pH 7.4) and with distilled water. Dehydration was accomplished using a series of ascending grades of ethanol. Final drying of the tissue was achieved via the critical point drying method (Anderson, 1951; Porter et al., 1973). Following this procedure, the atrial septal region was further exposed using microdissecting instruments. The hearts were mounted on aluminum stubs and were coated with gold-palladium to insure electrical conductivity. Specimens were observed in AMR-1000 and JSM-35 scanning electron microscopes. RESULTS
Unless stated otherwise, the results to be described pertain to the atria1 septum during embryonic cardiac morphogenesis. The atria1 septum primum is definitive by Day 3 in the form of a crescentic ridge on the cephalic wall of the atrium, extending caudally and dorsally (Fig. 1). With the final rotation of the U-shaped heart tube to form a loop, the ventricular region constituting the basal portion of the loop swings dorsally. The atria1 and ventricular regions maintain this cephalo-caudal orientation throughout adult life. During the third and fourth days, the truncus arMATERIALS AND METHODS teriosus becomes closely applied to the Two hundred and fifty chick embryos of ventral surface of the atrium (Fig. 1). AS the White Leghorn strain ranging from 2 the atrium grows, it expands on either side through 21 days were incubated with con- of the truncus. These lateral expansions stant temperature, ventilation, and hu- serve as a good external indication of the midity at 38°C. Embryos were removed division of the atrium into right and left from their shells and immersed in cold chambers. Figure 2 illustrates the atria1 septum as cacodylate-buffered Karnovsky’s fixative (Karnovsky, 1965). Specimens were staged it appears on Day 4. Very small perforaaccording to the Hamburger-Hamilton se- tions (averaging 9 pm) first appear in the
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mid-dorsal portion of the septum primum. These interatrial communications signify the first representation of the foramina secunda. The area located caudal to the lower border of the growing septum primum is the foramen primum. During Day 5, as seen in Figs. 3 and 4, multiple perforations have developed in the dorsal portion of the septum primum, resulting in the enlargement of the entire foramina secunda (average width of foramina: 1.5 pm). By this stage of development, the septum has grown caudally to fuse with the septum intermedium, a partition which developed by the central fusion of the dorsal and ventral atrioventricular endocardial cushions. This fusion of the septum primum with the septum intermedium results in the closure of foramen primum. As seen at higher magnification (Fig. 41, the endocardial cells are arranged in cords serving as boundaries for each perforation. Higher magnifications of atria1 septa representative of 4- and 5-day-old embryonic hearts, respectively, are shown in Figs. 5-8. These figures reveal the different cellular constituents comprising the early developing septum primum. Two distinct endocardial cell types have become apparent during these stages (Figs. 5, 6, and 7). They are the flattened endocardial cell type in comparison with the roundedup cells. Also, small endocardial fenestrations are present in the septum primum in the vicinity of the foramina secunda (Fig. 6). Figure 7 illustrates a developing perforation. Here, an endocardial cord appears to be degenerating to provide a larger interatrial communication. Figure 8 is also an area of developing perforations. Red blood cells can become quite distorted when traveling through these narrow passageways. When in open areas, they resume their normal biconvexed configurations, as observed when traveling through the larger foramina. Days 5 through 8 show the septum primum resembling a very delicate mesh-
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work (“chicken-wire”). The degeneration of the dorsal portion of the septum to form this irregular meshwork is illustrated in Fig. 9. The septum typically bulges into the left atrium. The ventral portion of the septum remains relatively free from interatrial communications. An embryonic heart in its seventh day of development is illustrated in Fig. 10. The endocardial-covered cords of the foramina secunda form a very delicate meshwork. The dimensions of the perforations have continued to increase (averaging 100 Frn in width), thereby allowing a more extensive interatrial shunt. Figure 11 illustrates the porous region of the atria1 septum representative of the embryonic heart late in the first embryonic week. This field is located at the periphery of the endocardial-covered cords. Many fenestrations are outlined with extracellular connective tissue fibrils, obvious in the subendocardial background. Red blood cells can be seen in transit through these fenestrations. It is interesting to note that, although this area is in proximity to the forming cords, large, gapping interatrial communications do not develop. This region remains intact throughout cardiac morphogenesis. By Day 11, the septum primum resembles a coarse meshwork, replacing the fine delicate web seen earlier (Fig. 12). The cords continue to thicken at this stage, thereby causing the overall dimensions of the perforations to decrease. The average dimension of a perforation at this time is approximately 118 pm, considerably less than its maximum value at 9 days (averaging 200 pm). By this stage, the predominant endocardial cell type is flattened. The rounded-up cells which infected the areas surrounding the forming perforations earlier in development are no longer demonstrable. In Fig. 13, a 17-day-old septum illustrates that the cords have progressively thickened (averaging 45 pm), and a very tortuous arrangement is observed. The
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FIGURES
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perforations diminish in size due to the close apposition of the walls of the cords. The cords are observed in multiple convolutions by Day 19 as seen in Fig. 14. They have thickened greatly in diameter and have acquired a flattened appearance (averaging 61 pm). This is the dominant feature observed near hatching. Under higher magnification, Fig. 15 displays the nuclear contours of the flattened endocardial cell types observed surrounding a perforation. This is a portion of the septum primum during the third week of embryonic development. No rounded-up cells are evident. At hatching, as observed in Fig. 16, the endocardial-covered cords fuse. The entire atrial septum is “dimpled” due to the unevenness resulting from the fusion of the cords. In the crevices of the “dimpled” septum, cords in the process of fusing were observed (Fig. 17). Filament-like structures can be seen in the background as the walls surrounding the perforations come into close apposition. By Day 2 of the posthatching period, all of the endocardial-covered cords of the atrial septum have fused as seen in Fig. 18. Perforations of the foramina secunda have been occluded, thus terminating any interatrial communication. DISCUSSION
The establishment of the atrial septum is of primary importance in the separation
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of the circulatory system into systemic and pulmonary pathways. Our study has concentrated on the morphological changes involved in atria1 septation. In the following, our results are correlated with those of previous investigations. On the third day, the septum primum appears as a crescentic ridge growing caudally toward its ultimate fusion with the septum intermedium. The fluid dynamics involved in the formation of the septum primum center around its development across the slowest flowing blood steam from the sinus venosus (Chang, 1931). A large interatrial communication (foramen primum) remains below the lower border of the septum. It is not until Day 5 that the foramen primum is obliterated as a result of the subsequent fusion of the septum primum with the septum intermedium (Quiring, 1933). Before this occlusion occurs, we observe small perforations in the mid-dorsal portion of septum primum during Day 4. This is the first representation of the foramina secunda. It is a well-known fact that a right-to-left atria1 shunt is necessary to sustain embryonic life due to inadequate pulmonary circulation. To compensate for the closure of the foramen primum, new interatrial communications appear in the septum primum to maintain this atria1 shunt. It is suggested by Quiring (1933) that an increase in the blood pressure in the right atrium, due to the closing of the foramen primum, may
FIG. 5. The predominate endocardial cell type during the early stages of atria1 septal formation exists in a rounded-up state and projects noticeably from the surface. These cells have their greatest concentration in the vicinity of developing perforations of the foramina secunda (FS) (1100 x). FIG. 6. The two endocardial cell types of the early atria1 septum are shown. The rounded cells (asterisks) project from the surface, whereas the flattened type (stars) fill in the background. Many of the rounded cells appear to rest on the flattened ones. Higher magnification reveals many small endocardial fenestrations (arrows). These appear both inter- and intracellularly (2000 x). FIG. 7. The cord separating two foramina secunda (FS) in the center of this field has been reduced to a thin cytoplasmic thread. The continuity will soon be lost, leading to a coalescence of the two foramina. The rounded-up cells (asterisks) are numerous in the region of the foramina. In addition, flattened endocardial cells (stars) contribute to the surface lining of the cords (2500 x ). The inset is a low magnification of this 5day septum. The arrow indicates the area of higher magnification (76 x). FIG. 8. Two small newly formed foramina of the foramina secunda (FS) are seen at high magnification. Red blood cells (RBC) shunted from the right to left atrium assume bizarre forms as they traverse the smaller perforations. Endocardial cells border the foramina (5400 x).
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FIG. 10. This low-power view from the left atria1 cavity shows the atria1 septum and its associated foramina secunda (FS) during the seventh day of development. Many of the foramina are now quite large, and the cords separating them protrude into the left atrium (LA). Note that the ventral-most portion of the septum lacks foramina (also see Figs. 3 and 9). The left (LV) and right (RV) ventricular walls and the aortic arches (AA) are obvious in this specimen (60 x).
cause an erosion of the septum at its weakest points. On the fifth day, we noted an expansion and multiplicity of the perforations in the mid-dorsal portion of the septum primum, resulting in an extensive interatrial communication. Cords of endocardial-covered cells delineate the boundaries of the developing perforations. We encounter two cell types associated with the development of the interatrial communications. One type is the typical flattened endocardial cell, and the second cell type is observed in the raised state.
The rounded-up cells predominate in the mid-dorsal portion of the septum primum during the development of the foramina secunda. These cells line the margins of the foramina, and many appear to be attached to the endocardium by only a slight cytoplasmic stalk. It is evident at this point that the degeneration of a certain portion of the septum primum is of physiological necessity for an alternate route of interatrial circulation following the occlusion of the foramen primum. In the degenerative process involved in the creation of
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FIG. diameter thought
12. During Day of the foramina to be the result
11, the continued secunda (FS). of cord thickening.
thickening The nearly (460 x).
of the cords (C) has led to a decrease in the average occluded perforation to the left of center (arrow) is
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FIG. 13. The cords of the foramina secunda often show a serpentine arrangement in the third week. The cords continue to thicken. In nearly all cases the septum is seen to bulge into the left atrium (300 ~1.
perforations in the septum primum, small fenestrations and rounded-up cells are observed as precursors of this event. Holtfreter (1945) is the first to report that rounded degenerating cells are expelled from normal tissue into the extracellular space. Manasek (1969) believes that rounded-up cells are degenerating myocardial cells involved in programmed cell death in the embryonic chick ventricle. He suggests that the expulsion of these dead cells from normal tissue may facilitate their ingestion by phagocytes. Ojeda and Hurle (1975) show dead and dying endocardial cells that are rounded-up during
fusion of the endocardial tubes in the chick embryo. Pexieder (1975a) demonstrates several death zones in the trabeculae of the dorsal portion of the septum primum using supravital Nile blue sulfate stain. He also states that the dead endocardial cells may be quickly torn off by the action of the blood stream. Johnson (personal communication) reports possible degenerating cells in the lumen of the ventricular region of the stage 11 chick embryo. Cell death as suspected in our preparations would be in agreement with the observations cited previously. Keeping the aforementioned light microscopic and transmis-
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FIG. 14. The thickening cords of the foramina secunda have flattened considerably from earlier stages (compare with Figs. 7 and 11). By Day 19, the tortuous nature of the cords is maintained as hatching approaches (380 x).
sion electron microscopic studies in mind, the rounded-up cells in our scanning electron microscopic study are likely to be associated with the formation of the foram-
ina secunda. Several possibilities must be considered for the presence of these cells. (1) They may be degenerating endocardial cells similar to those observed by
FIG. 15. As evidence here in a tangential view of a cord of the foramina secunda, the rounded-up cell type is no longer demonstrable. A perforation of the foramina secunda (FS) occupies the center of this cord (830 x 1. FIG. 16. The atria1 septal region at hatching presents a very rugged surface. These depressions (Xs) are believed to represent the occluded foramina secunda (590 x). FIG. 17. The occlusion of the foramina secunda at hatching is shown here in various degrees. At the lower right of this field, only a few shallow pita remain of a once patent foramen. In the lower center, a large deep depression remains as evidence of the occluding foramina. At the top, intermediate stages of occlusion are seen. Numerous red blood cells are in the field (640 x). FIG. 18. Day 2 of the posthatching period shows all of the foramina secunda completely occluded and a smooth surface contour of the atria1 septum (AS). The septum is viewed from the left atria1 cavity (315 x ).
FIGURES 15-18 360
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Ojeda and Hurle (1975). (2) Myocardial cells are known to exist in the matrix of the developing atria1 septum. The possibility that these cells are degenerating must not be overlooked. They may be expelled from the septal core into the circulatory system, thus increasing their vulnerability to phagocytosis as suggested by Manasek (1969). (3) Pexieder (1975b) reports seeing macrophages entering the bloodstream from the endocardial surface of the bulbar cushions. (4) Pexieder (1975a) also reports degenerating mesenchymal cells throughout the developing heart. These cells may undergo a fate similar to that of the dying myocardial cells. The possibility that these cells are in the S, G, or M phases of the cell cycle seems unlikely due to the absence of abundant microvilli, blebs, and ruffled cell margins (Porter et al., 1973). The rounded-up cells and the degeneration of the septum primum in our study are restricted to the mid-dorsal region of the septum. The ventral portion of the septum primum remains intact and free of rounded-up cells throughout embryonic development. Also, it is interesting to note that the area immediately in the vicinity of the cords and perforations of septum primum supports only small fenestrations. Large gaping interatrial communications are not formed. It is quite evident that some type of programmed cellular degeneration influences the specific location of the developing foramina secunda. We refer to the appearance of the sep tum primum from Days 5 through 8 as that resembling a “chicken-wire” (Hendrix and Morse, 1976). Progressive enlargement of the perforations forming the foramina secunda is observed during this time leading to the establishment of a very delicate meshwork. In the middle of the ninth day, the cords comprising the septum primum begin to thicken in dimension, and the septum itself appears quite coarse in comparison to its earlier arrangement. An explanation for the deviation of the
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septum primum bulging into the left atria1 cavity is offered by Los (1971/72) in his study of a 5-day embryonic chick heart during diastole and systole. He states that, when a chick embryo is fixed, its heart action stops during the contraction phase of the cardiac cycle. At this time, the perforations are at their widest, and blood from the right atrium is propelled into the left. The complete disappearance of the rounded-up cells is conspicuous during the ninth day, and the predominant endocardial cell from this period through hatching is the flattened type. The endocardial-covered cords become progressively thicker in dimension until hatching. We speculate that this is due to a sudden decrease in the number of dying cells. Cell proliferation without massive cell death would result in a net thickening of the cords associated with the foramina secunda. Thus, by 11 days, we feel that it is safe to assume that perforations in the septum primum are no longer developing for two reasons: (i) There are no rounded-up cells observed in proximity to the opening, which we consider to be forerunners of a developing perforation; and (ii) at this time, the cords of the septum primum are gradually increasing in thickness, causing all perforations to diminish in size. While the endocardial-covered cords of the septum primum continue to thicken in dimension as hatching approaches, they take on a tortuous, almost serpentine appearance. By the nineteenth day, the greatly thickened convoluted cords are arranged in close opposition to one another and ultimately fuse at hatching. Pulmonary circulation functionally begins at hatching and results in an increase in the pulmonary venous return to the left atrium. de la Cruz et al. (1972) state that, at hatching, the pressures tend to equalize permanently in the atria, and the blood circulation through the septal perforations ceases. With the cessation of the blood current, these once-inflated cords surrounding the perforations collapse and soon fuse.
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By the second day of the posthatching period, the fusion of the endocardial-covered cords is complete, and an imperforate atria1 septum is observed which is characteristic of the adult heart. The septum primum and the foramen primum are formed in a similar manner in higher vertebrates. However, a single aperture represents the foramen secundum. Also, an additional structure, the septum secundum, develops to the right of the septum primum. The foramen ovale is the open central portion of the septum secundum which has the septum primum as its valve. At birth, this flap-like valve seals shut and occludes the right-to-left atria1 shunt which existed during embryonic development. The septum primum first appears in the human embryo at 12 weeks (Sissman, 19701, and the foramen secundum by the seventeenth week. A correlative relationship can be established by comparing each week of a chicks development with one trimester of a human’s development. The physiological correlates remain the same. The main observations of this study speculate that physiological cell death is a necessity in the normal morphogenesis of the atrial septum primum for the establishment of the foramina secunda. The effect, however, must be controlled or programmed because only the preselected area of the mid-dorsal portion of the septum primum degenerates throughout embryonic life. Saunders (1966) and Saunders and Fallon (1966) suggest that these degenerative events are influenced by genetic programming. We are convinced that the rounded-up cells observed in the septum primum are of a degenerative type and immediately precede the formation of the secondary perforations, collectively known as the foramina secunda. We feel that,. in an “overkill” syndrome, many of these rounded-up cells may slough off and contribute to the excessive enlargement of the foramina secunda. This would represent one of the
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most common atrial septal defects, and its severity would depend on the magnitude of the enlargement. It is our goal to confirm the presence of these degenerating cells using light and transmission electron microscopy. These results will be incorporated in subsequent reports. By comparing the subcellular morphology with our scanning electron microscopic study, a complete picture of normal atrial septation will be realized. It is vital to establish this normal sequential development before experimenting with the mechanisms of teratogenesis. We would like to thank the following people who have kindly given of their time to type the manuscript: Mrs. Charles N. G. Hendrix and Ms. Debra Kornbluh. We are most appreciative to Dr. Frank N. Low and Dr. Richard S. Snell for their critique of the manuscript. REFERENCES ANDERSON, T. F. (1951). Techniques for the preservation of three-dimensional structure in preparing specimens for the electron microscope. Trans. N.Y. Acad. Sci. 13, 130-133. BORN, G. (1889). Beitrage zur Entwicklungsgeshichte des Sauge thierherzens. Arch. Mikrosk. Anat. 33, 284-377. BREMER, J. L. (19321. The presence and influence of two spiral streams in the heart of the chick embryo. Amer. J. Anat. 49, 409-440. CHANG, C. (1931). The interatrial septum in chick embryos. Anat. Rec. 50, 9-22. DE LA CRUZ, M. V., MUNOZ-ARMAS, S., and MUNOZCASTELLANOS, L. (1972). “Development of the Chick Heart.” Johns Hopkins University Press, Baltimore and London. GLUCKSMANN, A. (1951). Cell deaths in normal vertebrate ontogeny. Biol. Rev. 26, 59-86. HAMILTON, H. H. (1952). “Lillie’s Development of the Chick,” 3rd ed. Holt, Rhinehart and Winston, New York. HENDRIX, M. J. C., and MOK~E, D. E. (1976). Development of the foramen secundum in the chick as observed by scanning electron microscopy. In “Proceedings. Electron Microscopy Society of America.” In press. HOCHSTETTER, F. (1906). Die Entwicklung des Blutgefassystems. In “Handbuch vergleichenden und experimentellen Entwicklungslehre der Wirbeltiere,” Vol. 3, pp. 21-157. HOLTFRETER, J. (1945). Neuralization and epidermization of gastrula ectoderm. J. Exp. Zool. 98, 161209.
HENDRIX AND MORSE KARNOVSKY, J. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 137a-138a. KISSANE, J. M., and SMITH, M. G. (1967). “Pathology of Infancy and Childhood.” C. V. Mosby Co., St. Louis, MO. KRSTIC’ , R., and PEXIEDER, T. (1973). Ultrastructure of cell death in bulbar cushions of chick embryo heart. 2. Anat. Entwichlungsgesch. 140, 337-350. Los, J. A. (1971/72). The heart of the 5 day chick embryo during dilation and contraction. Actu Morphol. Neer. Stand. 9, 309-335. MANASEK, F. J. (1969). Myocardial cell death in the embryonic chick ventricle. J. Embryol. Exp. Morphol. 21, 22, 271-284. OJEDA, J. L., and HURLE, J. M. (1975). Cell death during the formation of tubular heart of the chick embryo. J. Embryol. Exp. Morphol. 33,523-534. PATTEN, B. M. (1925). The interatrial septum of the chick heart. Anat. Rec. 30, 53-60. PEXIEDER, T. (1975a). Cell death in the morphogene-
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sis and teratogenesis of the heart. Advan. Anat. Embryol. Cell Biol. 51, 7-99. PEXIEDER, T. (1975b). SEM investigations on physiological cell death in the chick embryo heart. Experientia 31, 745. PORTER, K., PRESCOTT, D., and FRYE, J. (1973). Changes in surface morphology of Chinese Hamster ovary cells during the cell cycle. J. Cell Biol. 57, 815-836. QUIRING, D. P. (1933). The development of the sinoatria1 region of the chick heart. J. Morphol. 55, 81-118. SAUNDERS, J. W., JR. (1966). Death in embryonic systems. Science 154, 604-612. SAUNDERS, J. W., JR., and FALLON, J. F. (1966). Cell death in morphogenesis. In “Major Problems in Developmental Biology” (M. Locke, ed.), pp. 289314. Academic Press, New York. SISSMAN, N. J. (1970). Developmental landmarks in cardiac morphogenesis: Comparative chronology. Amer. J. Cardiol. 25, 204-212.
BIOLOGY
57, 345-363 (1977)
Atrial Septation I. Scanning MARY Department
of Anatomy,
Electron
Microscopy
J. C. HENDRIX
The George Washington
Received July 8,1976;
in the Chick’
AND DENNIS University
E. MORSE*
School of Medicine,
Washington,
D.C. 20037
accepted in revised form January 26,1977
Chick hearts were prepared for scanning electron microscopy by standard methods, the purpose being to investigate the surface morphology of the developing atria1 septal region. By Day 3, the atria1 septum primum appeared as a sickle-shaped structure. During Day 4, the first representation of the foramina secunda occurred in the mid-dorsal portion of the septum. During Day 5, the septum primum fused with the atrioventricular endocardial cushions, thereby occluding the foramen primum. From Days 5 through 8, the secondary perforations (foramina secunda) multiplied and increased in size. The endocardial-covered cords of cells comprising the septum thickened from Days 9 through 15. This resulted in a marked reduction in the dimensions of the perforations from Day 16 to hatching. The atria1 septum at hatching occasionally contained a small single orifice. At 3 days posthatching, the atria1 septum was a solid sheet covered with flattened endocardial cells. All interatrial communications were occluded. During Days 5 through 9, two distinct cell types became apparent on the endocardialcovered cords. Simultaneously, fenestrations were observed on the cords surrounding the foramina secunda and on the ventral portion of the atria1 septum. The integral role which the fenestrations and cellular types play in the development of the foramina secunda is discussed. INTRODUCTION
Embryologists and cardiologists have striven to give a detailed and accurate description of the normal development of the vertebrate heart for more than a century. In particular, the partitioning of the heart by septa into four chambers has aroused the interest of numerous investigators. However, recent emphasis has been placed on the ventricular region, leaving unsettled problems concerning the development of the atria1 septum. According to classical embryologists (Lindes, 1965; Born, 1889; Hochstetter, 1906; Patten, 1925; Chang, 1931; Bremer, 1932; Quiring, 1933), two distinct septa (atrial and ventricular) are involved in the partitioning of the normal vertebrate heart. Based on observations in the chick ’ This research was aided by a grant from the Washington Heart Association (Washington, D.C.). 2 Present address: Department of Anatomy, Medical College of Ohio, Toledo, Ohio 43614.
embryo, these investigators demonstrated that the atria1 septum primum made its first appearance in the cephalic wall of the atrium between 50 and 53 hr of incubation. Late in the fifth day, this septum has descended sufficiently to contact physically the fused atrioventricular endocardial cushions (septum intermedium). At this time, the area located directly below the descending atria1 septum (foramen primum> is occluded. Secondary communicative openings (foramina secunda) are established in the septum primum at about the time of closure of the foramen primum. The strands of tissue comprising the margins of the foramina secunda have a valvular function which operates accordingly in each phase of the cardiac cycle. This provides the right-to-left interatrial shunt, thus establishing a pulmonary bypass for a portion of the blood entering the heart. Final closure of the foramina secunda occurs in the first few days after birth and 345
Copyright 0 1977by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN
0012-1606
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coincides with the beginning of pulmonary circulation. It was recognized many years ago that normal embryonic development was accompanied by spontaneous physiological cell death. Glucksmann (1951) pioneered cell death research in developmental biology* Several workers have noted the presence of dead and dying cells in conjunction with cardiac morphogenesis (Manasek, 1969; Krstic’ and Pexieder, 1973; Pexieder, 1975a,b; and Ojeda and Hurle, 1975). Atrial septal defects are among the most common congenital cardiac anomalies occurring in newborns in the United States (Kissane and Smith, 1967). In the interest of studying the etiology of atria1 septal defects, we must first establish the normal sequence of events in the fine structural development of the atria1 septum. Previous work on atria1 septation is limited almost exclusively to light microscopy. Transmission electron microscopic studies are restricted to highly selected stages of development. Scanning electron microscopic studies of atria1 septation are not offered by the literature. Our present research interests concentrate on the formation of the atria1 septum. To accomplish this goal, we employ several microscopic techniques to give a detailed analysis of the morphology and subcellular constituents comprising the atrial septal region. The purpose of this paper is to present our observations of the surface morphology of the atrial septal development in the chick using scanning electron microscopy.
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ries (Hamilton, 1952) after which the hearts were immediately dissected free and were kept in the fixative for a minimum of 2 hrs. Thirty-six chick embryos ranging from hatching through 1 week posthatching were perfused in uiuo using the same fixative plus procaine hydrochloride. In most cases, the lateral atrial walls were removed with the aid of a dissecting microscope. The tissues were then rinsed successively in cacodylate buffer (pH 7.4) and with distilled water. Dehydration was accomplished using a series of ascending grades of ethanol. Final drying of the tissue was achieved via the critical point drying method (Anderson, 1951; Porter et al., 1973). Following this procedure, the atrial septal region was further exposed using microdissecting instruments. The hearts were mounted on aluminum stubs and were coated with gold-palladium to insure electrical conductivity. Specimens were observed in AMR-1000 and JSM-35 scanning electron microscopes. RESULTS
Unless stated otherwise, the results to be described pertain to the atria1 septum during embryonic cardiac morphogenesis. The atria1 septum primum is definitive by Day 3 in the form of a crescentic ridge on the cephalic wall of the atrium, extending caudally and dorsally (Fig. 1). With the final rotation of the U-shaped heart tube to form a loop, the ventricular region constituting the basal portion of the loop swings dorsally. The atria1 and ventricular regions maintain this cephalo-caudal orientation throughout adult life. During the third and fourth days, the truncus arMATERIALS AND METHODS teriosus becomes closely applied to the Two hundred and fifty chick embryos of ventral surface of the atrium (Fig. 1). AS the White Leghorn strain ranging from 2 the atrium grows, it expands on either side through 21 days were incubated with con- of the truncus. These lateral expansions stant temperature, ventilation, and hu- serve as a good external indication of the midity at 38°C. Embryos were removed division of the atrium into right and left from their shells and immersed in cold chambers. Figure 2 illustrates the atria1 septum as cacodylate-buffered Karnovsky’s fixative (Karnovsky, 1965). Specimens were staged it appears on Day 4. Very small perforaaccording to the Hamburger-Hamilton se- tions (averaging 9 pm) first appear in the
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mid-dorsal portion of the septum primum. These interatrial communications signify the first representation of the foramina secunda. The area located caudal to the lower border of the growing septum primum is the foramen primum. During Day 5, as seen in Figs. 3 and 4, multiple perforations have developed in the dorsal portion of the septum primum, resulting in the enlargement of the entire foramina secunda (average width of foramina: 1.5 pm). By this stage of development, the septum has grown caudally to fuse with the septum intermedium, a partition which developed by the central fusion of the dorsal and ventral atrioventricular endocardial cushions. This fusion of the septum primum with the septum intermedium results in the closure of foramen primum. As seen at higher magnification (Fig. 41, the endocardial cells are arranged in cords serving as boundaries for each perforation. Higher magnifications of atria1 septa representative of 4- and 5-day-old embryonic hearts, respectively, are shown in Figs. 5-8. These figures reveal the different cellular constituents comprising the early developing septum primum. Two distinct endocardial cell types have become apparent during these stages (Figs. 5, 6, and 7). They are the flattened endocardial cell type in comparison with the roundedup cells. Also, small endocardial fenestrations are present in the septum primum in the vicinity of the foramina secunda (Fig. 6). Figure 7 illustrates a developing perforation. Here, an endocardial cord appears to be degenerating to provide a larger interatrial communication. Figure 8 is also an area of developing perforations. Red blood cells can become quite distorted when traveling through these narrow passageways. When in open areas, they resume their normal biconvexed configurations, as observed when traveling through the larger foramina. Days 5 through 8 show the septum primum resembling a very delicate mesh-
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work (“chicken-wire”). The degeneration of the dorsal portion of the septum to form this irregular meshwork is illustrated in Fig. 9. The septum typically bulges into the left atrium. The ventral portion of the septum remains relatively free from interatrial communications. An embryonic heart in its seventh day of development is illustrated in Fig. 10. The endocardial-covered cords of the foramina secunda form a very delicate meshwork. The dimensions of the perforations have continued to increase (averaging 100 Frn in width), thereby allowing a more extensive interatrial shunt. Figure 11 illustrates the porous region of the atria1 septum representative of the embryonic heart late in the first embryonic week. This field is located at the periphery of the endocardial-covered cords. Many fenestrations are outlined with extracellular connective tissue fibrils, obvious in the subendocardial background. Red blood cells can be seen in transit through these fenestrations. It is interesting to note that, although this area is in proximity to the forming cords, large, gapping interatrial communications do not develop. This region remains intact throughout cardiac morphogenesis. By Day 11, the septum primum resembles a coarse meshwork, replacing the fine delicate web seen earlier (Fig. 12). The cords continue to thicken at this stage, thereby causing the overall dimensions of the perforations to decrease. The average dimension of a perforation at this time is approximately 118 pm, considerably less than its maximum value at 9 days (averaging 200 pm). By this stage, the predominant endocardial cell type is flattened. The rounded-up cells which infected the areas surrounding the forming perforations earlier in development are no longer demonstrable. In Fig. 13, a 17-day-old septum illustrates that the cords have progressively thickened (averaging 45 pm), and a very tortuous arrangement is observed. The
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perforations diminish in size due to the close apposition of the walls of the cords. The cords are observed in multiple convolutions by Day 19 as seen in Fig. 14. They have thickened greatly in diameter and have acquired a flattened appearance (averaging 61 pm). This is the dominant feature observed near hatching. Under higher magnification, Fig. 15 displays the nuclear contours of the flattened endocardial cell types observed surrounding a perforation. This is a portion of the septum primum during the third week of embryonic development. No rounded-up cells are evident. At hatching, as observed in Fig. 16, the endocardial-covered cords fuse. The entire atrial septum is “dimpled” due to the unevenness resulting from the fusion of the cords. In the crevices of the “dimpled” septum, cords in the process of fusing were observed (Fig. 17). Filament-like structures can be seen in the background as the walls surrounding the perforations come into close apposition. By Day 2 of the posthatching period, all of the endocardial-covered cords of the atrial septum have fused as seen in Fig. 18. Perforations of the foramina secunda have been occluded, thus terminating any interatrial communication. DISCUSSION
The establishment of the atrial septum is of primary importance in the separation
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of the circulatory system into systemic and pulmonary pathways. Our study has concentrated on the morphological changes involved in atria1 septation. In the following, our results are correlated with those of previous investigations. On the third day, the septum primum appears as a crescentic ridge growing caudally toward its ultimate fusion with the septum intermedium. The fluid dynamics involved in the formation of the septum primum center around its development across the slowest flowing blood steam from the sinus venosus (Chang, 1931). A large interatrial communication (foramen primum) remains below the lower border of the septum. It is not until Day 5 that the foramen primum is obliterated as a result of the subsequent fusion of the septum primum with the septum intermedium (Quiring, 1933). Before this occlusion occurs, we observe small perforations in the mid-dorsal portion of septum primum during Day 4. This is the first representation of the foramina secunda. It is a well-known fact that a right-to-left atria1 shunt is necessary to sustain embryonic life due to inadequate pulmonary circulation. To compensate for the closure of the foramen primum, new interatrial communications appear in the septum primum to maintain this atria1 shunt. It is suggested by Quiring (1933) that an increase in the blood pressure in the right atrium, due to the closing of the foramen primum, may
FIG. 5. The predominate endocardial cell type during the early stages of atria1 septal formation exists in a rounded-up state and projects noticeably from the surface. These cells have their greatest concentration in the vicinity of developing perforations of the foramina secunda (FS) (1100 x). FIG. 6. The two endocardial cell types of the early atria1 septum are shown. The rounded cells (asterisks) project from the surface, whereas the flattened type (stars) fill in the background. Many of the rounded cells appear to rest on the flattened ones. Higher magnification reveals many small endocardial fenestrations (arrows). These appear both inter- and intracellularly (2000 x). FIG. 7. The cord separating two foramina secunda (FS) in the center of this field has been reduced to a thin cytoplasmic thread. The continuity will soon be lost, leading to a coalescence of the two foramina. The rounded-up cells (asterisks) are numerous in the region of the foramina. In addition, flattened endocardial cells (stars) contribute to the surface lining of the cords (2500 x ). The inset is a low magnification of this 5day septum. The arrow indicates the area of higher magnification (76 x). FIG. 8. Two small newly formed foramina of the foramina secunda (FS) are seen at high magnification. Red blood cells (RBC) shunted from the right to left atrium assume bizarre forms as they traverse the smaller perforations. Endocardial cells border the foramina (5400 x).
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FIG. 10. This low-power view from the left atria1 cavity shows the atria1 septum and its associated foramina secunda (FS) during the seventh day of development. Many of the foramina are now quite large, and the cords separating them protrude into the left atrium (LA). Note that the ventral-most portion of the septum lacks foramina (also see Figs. 3 and 9). The left (LV) and right (RV) ventricular walls and the aortic arches (AA) are obvious in this specimen (60 x).
cause an erosion of the septum at its weakest points. On the fifth day, we noted an expansion and multiplicity of the perforations in the mid-dorsal portion of the septum primum, resulting in an extensive interatrial communication. Cords of endocardial-covered cells delineate the boundaries of the developing perforations. We encounter two cell types associated with the development of the interatrial communications. One type is the typical flattened endocardial cell, and the second cell type is observed in the raised state.
The rounded-up cells predominate in the mid-dorsal portion of the septum primum during the development of the foramina secunda. These cells line the margins of the foramina, and many appear to be attached to the endocardium by only a slight cytoplasmic stalk. It is evident at this point that the degeneration of a certain portion of the septum primum is of physiological necessity for an alternate route of interatrial circulation following the occlusion of the foramen primum. In the degenerative process involved in the creation of
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FIG. diameter thought
12. During Day of the foramina to be the result
11, the continued secunda (FS). of cord thickening.
thickening The nearly (460 x).
of the cords (C) has led to a decrease in the average occluded perforation to the left of center (arrow) is
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FIG. 13. The cords of the foramina secunda often show a serpentine arrangement in the third week. The cords continue to thicken. In nearly all cases the septum is seen to bulge into the left atrium (300 ~1.
perforations in the septum primum, small fenestrations and rounded-up cells are observed as precursors of this event. Holtfreter (1945) is the first to report that rounded degenerating cells are expelled from normal tissue into the extracellular space. Manasek (1969) believes that rounded-up cells are degenerating myocardial cells involved in programmed cell death in the embryonic chick ventricle. He suggests that the expulsion of these dead cells from normal tissue may facilitate their ingestion by phagocytes. Ojeda and Hurle (1975) show dead and dying endocardial cells that are rounded-up during
fusion of the endocardial tubes in the chick embryo. Pexieder (1975a) demonstrates several death zones in the trabeculae of the dorsal portion of the septum primum using supravital Nile blue sulfate stain. He also states that the dead endocardial cells may be quickly torn off by the action of the blood stream. Johnson (personal communication) reports possible degenerating cells in the lumen of the ventricular region of the stage 11 chick embryo. Cell death as suspected in our preparations would be in agreement with the observations cited previously. Keeping the aforementioned light microscopic and transmis-
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FIG. 14. The thickening cords of the foramina secunda have flattened considerably from earlier stages (compare with Figs. 7 and 11). By Day 19, the tortuous nature of the cords is maintained as hatching approaches (380 x).
sion electron microscopic studies in mind, the rounded-up cells in our scanning electron microscopic study are likely to be associated with the formation of the foram-
ina secunda. Several possibilities must be considered for the presence of these cells. (1) They may be degenerating endocardial cells similar to those observed by
FIG. 15. As evidence here in a tangential view of a cord of the foramina secunda, the rounded-up cell type is no longer demonstrable. A perforation of the foramina secunda (FS) occupies the center of this cord (830 x 1. FIG. 16. The atria1 septal region at hatching presents a very rugged surface. These depressions (Xs) are believed to represent the occluded foramina secunda (590 x). FIG. 17. The occlusion of the foramina secunda at hatching is shown here in various degrees. At the lower right of this field, only a few shallow pita remain of a once patent foramen. In the lower center, a large deep depression remains as evidence of the occluding foramina. At the top, intermediate stages of occlusion are seen. Numerous red blood cells are in the field (640 x). FIG. 18. Day 2 of the posthatching period shows all of the foramina secunda completely occluded and a smooth surface contour of the atria1 septum (AS). The septum is viewed from the left atria1 cavity (315 x ).
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Ojeda and Hurle (1975). (2) Myocardial cells are known to exist in the matrix of the developing atria1 septum. The possibility that these cells are degenerating must not be overlooked. They may be expelled from the septal core into the circulatory system, thus increasing their vulnerability to phagocytosis as suggested by Manasek (1969). (3) Pexieder (1975b) reports seeing macrophages entering the bloodstream from the endocardial surface of the bulbar cushions. (4) Pexieder (1975a) also reports degenerating mesenchymal cells throughout the developing heart. These cells may undergo a fate similar to that of the dying myocardial cells. The possibility that these cells are in the S, G, or M phases of the cell cycle seems unlikely due to the absence of abundant microvilli, blebs, and ruffled cell margins (Porter et al., 1973). The rounded-up cells and the degeneration of the septum primum in our study are restricted to the mid-dorsal region of the septum. The ventral portion of the septum primum remains intact and free of rounded-up cells throughout embryonic development. Also, it is interesting to note that the area immediately in the vicinity of the cords and perforations of septum primum supports only small fenestrations. Large gaping interatrial communications are not formed. It is quite evident that some type of programmed cellular degeneration influences the specific location of the developing foramina secunda. We refer to the appearance of the sep tum primum from Days 5 through 8 as that resembling a “chicken-wire” (Hendrix and Morse, 1976). Progressive enlargement of the perforations forming the foramina secunda is observed during this time leading to the establishment of a very delicate meshwork. In the middle of the ninth day, the cords comprising the septum primum begin to thicken in dimension, and the septum itself appears quite coarse in comparison to its earlier arrangement. An explanation for the deviation of the
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septum primum bulging into the left atria1 cavity is offered by Los (1971/72) in his study of a 5-day embryonic chick heart during diastole and systole. He states that, when a chick embryo is fixed, its heart action stops during the contraction phase of the cardiac cycle. At this time, the perforations are at their widest, and blood from the right atrium is propelled into the left. The complete disappearance of the rounded-up cells is conspicuous during the ninth day, and the predominant endocardial cell from this period through hatching is the flattened type. The endocardial-covered cords become progressively thicker in dimension until hatching. We speculate that this is due to a sudden decrease in the number of dying cells. Cell proliferation without massive cell death would result in a net thickening of the cords associated with the foramina secunda. Thus, by 11 days, we feel that it is safe to assume that perforations in the septum primum are no longer developing for two reasons: (i) There are no rounded-up cells observed in proximity to the opening, which we consider to be forerunners of a developing perforation; and (ii) at this time, the cords of the septum primum are gradually increasing in thickness, causing all perforations to diminish in size. While the endocardial-covered cords of the septum primum continue to thicken in dimension as hatching approaches, they take on a tortuous, almost serpentine appearance. By the nineteenth day, the greatly thickened convoluted cords are arranged in close opposition to one another and ultimately fuse at hatching. Pulmonary circulation functionally begins at hatching and results in an increase in the pulmonary venous return to the left atrium. de la Cruz et al. (1972) state that, at hatching, the pressures tend to equalize permanently in the atria, and the blood circulation through the septal perforations ceases. With the cessation of the blood current, these once-inflated cords surrounding the perforations collapse and soon fuse.
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By the second day of the posthatching period, the fusion of the endocardial-covered cords is complete, and an imperforate atria1 septum is observed which is characteristic of the adult heart. The septum primum and the foramen primum are formed in a similar manner in higher vertebrates. However, a single aperture represents the foramen secundum. Also, an additional structure, the septum secundum, develops to the right of the septum primum. The foramen ovale is the open central portion of the septum secundum which has the septum primum as its valve. At birth, this flap-like valve seals shut and occludes the right-to-left atria1 shunt which existed during embryonic development. The septum primum first appears in the human embryo at 12 weeks (Sissman, 19701, and the foramen secundum by the seventeenth week. A correlative relationship can be established by comparing each week of a chicks development with one trimester of a human’s development. The physiological correlates remain the same. The main observations of this study speculate that physiological cell death is a necessity in the normal morphogenesis of the atrial septum primum for the establishment of the foramina secunda. The effect, however, must be controlled or programmed because only the preselected area of the mid-dorsal portion of the septum primum degenerates throughout embryonic life. Saunders (1966) and Saunders and Fallon (1966) suggest that these degenerative events are influenced by genetic programming. We are convinced that the rounded-up cells observed in the septum primum are of a degenerative type and immediately precede the formation of the secondary perforations, collectively known as the foramina secunda. We feel that,. in an “overkill” syndrome, many of these rounded-up cells may slough off and contribute to the excessive enlargement of the foramina secunda. This would represent one of the
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most common atrial septal defects, and its severity would depend on the magnitude of the enlargement. It is our goal to confirm the presence of these degenerating cells using light and transmission electron microscopy. These results will be incorporated in subsequent reports. By comparing the subcellular morphology with our scanning electron microscopic study, a complete picture of normal atrial septation will be realized. It is vital to establish this normal sequential development before experimenting with the mechanisms of teratogenesis. We would like to thank the following people who have kindly given of their time to type the manuscript: Mrs. Charles N. G. Hendrix and Ms. Debra Kornbluh. We are most appreciative to Dr. Frank N. Low and Dr. Richard S. Snell for their critique of the manuscript. REFERENCES ANDERSON, T. F. (1951). Techniques for the preservation of three-dimensional structure in preparing specimens for the electron microscope. Trans. N.Y. Acad. Sci. 13, 130-133. BORN, G. (1889). Beitrage zur Entwicklungsgeshichte des Sauge thierherzens. Arch. Mikrosk. Anat. 33, 284-377. BREMER, J. L. (19321. The presence and influence of two spiral streams in the heart of the chick embryo. Amer. J. Anat. 49, 409-440. CHANG, C. (1931). The interatrial septum in chick embryos. Anat. Rec. 50, 9-22. DE LA CRUZ, M. V., MUNOZ-ARMAS, S., and MUNOZCASTELLANOS, L. (1972). “Development of the Chick Heart.” Johns Hopkins University Press, Baltimore and London. GLUCKSMANN, A. (1951). Cell deaths in normal vertebrate ontogeny. Biol. Rev. 26, 59-86. HAMILTON, H. H. (1952). “Lillie’s Development of the Chick,” 3rd ed. Holt, Rhinehart and Winston, New York. HENDRIX, M. J. C., and MOK~E, D. E. (1976). Development of the foramen secundum in the chick as observed by scanning electron microscopy. In “Proceedings. Electron Microscopy Society of America.” In press. HOCHSTETTER, F. (1906). Die Entwicklung des Blutgefassystems. In “Handbuch vergleichenden und experimentellen Entwicklungslehre der Wirbeltiere,” Vol. 3, pp. 21-157. HOLTFRETER, J. (1945). Neuralization and epidermization of gastrula ectoderm. J. Exp. Zool. 98, 161209.
HENDRIX AND MORSE KARNOVSKY, J. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 137a-138a. KISSANE, J. M., and SMITH, M. G. (1967). “Pathology of Infancy and Childhood.” C. V. Mosby Co., St. Louis, MO. KRSTIC’ , R., and PEXIEDER, T. (1973). Ultrastructure of cell death in bulbar cushions of chick embryo heart. 2. Anat. Entwichlungsgesch. 140, 337-350. Los, J. A. (1971/72). The heart of the 5 day chick embryo during dilation and contraction. Actu Morphol. Neer. Stand. 9, 309-335. MANASEK, F. J. (1969). Myocardial cell death in the embryonic chick ventricle. J. Embryol. Exp. Morphol. 21, 22, 271-284. OJEDA, J. L., and HURLE, J. M. (1975). Cell death during the formation of tubular heart of the chick embryo. J. Embryol. Exp. Morphol. 33,523-534. PATTEN, B. M. (1925). The interatrial septum of the chick heart. Anat. Rec. 30, 53-60. PEXIEDER, T. (1975a). Cell death in the morphogene-
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sis and teratogenesis of the heart. Advan. Anat. Embryol. Cell Biol. 51, 7-99. PEXIEDER, T. (1975b). SEM investigations on physiological cell death in the chick embryo heart. Experientia 31, 745. PORTER, K., PRESCOTT, D., and FRYE, J. (1973). Changes in surface morphology of Chinese Hamster ovary cells during the cell cycle. J. Cell Biol. 57, 815-836. QUIRING, D. P. (1933). The development of the sinoatria1 region of the chick heart. J. Morphol. 55, 81-118. SAUNDERS, J. W., JR. (1966). Death in embryonic systems. Science 154, 604-612. SAUNDERS, J. W., JR., and FALLON, J. F. (1966). Cell death in morphogenesis. In “Major Problems in Developmental Biology” (M. Locke, ed.), pp. 289314. Academic Press, New York. SISSMAN, N. J. (1970). Developmental landmarks in cardiac morphogenesis: Comparative chronology. Amer. J. Cardiol. 25, 204-212.