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Atrial septation
DEVELOPMENTAL
78,25-35
BIOLOGY
(1980)
Atrial II. Formation DENNIS Departments
of Anatomy,
Medical
Received
Septation
of the Foramina
E. MORSE
AND MARY
College of Ohio, School of Medicine,
April
Secunda
Toledo, Ohio Washington,
2, 1979; accepted
in revised
in the Chick
J. C. HENDRIX’ 43699, and George D.C. 20037 form
November
Washington
University
13, 1979
Chick hearts were prepared for light microscopy and transmission electron microscopy by conventional methods, the purpose being to investigate the developing foramina secunda in the atrial septum. As the growing septum approaches and fuses with the endocardial cushions, small perforations (foramina secunda) are formed in the middorsal portion of the septum. During Days 5 and 6 the number and size of these foramina increase significantly. Formation of foramina creates thin cords of endocardium-covered tissue. Two varieties of endocardial cells are associated with the atrial septum during this period. The endocardial cells on the surface of the septum, some distance from the foramina secunda, are flattened and possess long, attenuated processes. Conversely, the endocardium in the rough portion of the septum near the foramina is characterized by the presence of numerous rounded cells which project notably into the atria1 chambers. These rounded endocardial cells resemble phagocytic cells and possess thin cytoplasmic processes which extend into the septal core, gradually separating the cells of the core, resulting in an isolation of a portion of septal tissue. Once a transseptal communication is established the cytoplasmic processes separate, creating a new foramen secundum. The rounded endocardial cells are also responsible for maintaining the integrity of the endocardium-covered septum throughout the remodeling process. The origin of the rounded cells is unknown at this time. The most likely sources are differentiation from existing endocardial cells or circulating phagocytes.
foramen primum by growth of the atrial septum causes an increase in pressure in the right atrium. He further states that the pressure increase causes the septum to break down at its weakest points. Scanning electron microscopy shows the endocardium to remain intact during the formation of foramina secunda (Hendrix and Morse, while hemodynamic 1977). Therefore, forces may influence the formation of these foramina, the septum does not suddenly rupture. Foramen formation appears to be highly organized and to occur at precise locations. Pexieder (1975) has demonstrated convincingly that cell death occurs in the atria1 septum during the period of foramina secunda formation. The cell type or types which are degenerating have not been identified. Likewise, the overall role that cell
INTRODUCTION
Formation of the atrial septum occurs between Days 3 and 5 in the chick embryo (Patten, 1925; Chang, 1931; Bremer, 1932; Quiring, 1933; Her&ix and Morse, 1977). These events are carefully synchronized so that as the growing septum approaches and fuses with the endocardial cushions, anticipating the closure of foramen primum, small perforations (foramina secunda) have already begun to appear in the middorsal portion of the septum. During Days 5 and 6 the number and size of these foramina increase significantly. Several problems need to be addressed regarding foramina secunda formation. Quiring (1933) suggests that obliteration of ’ Present address: vard Medical School, 02115.
Department 25 Shattuck
of Anatomy, St., Boston,
HarMass. 25
0012-1606/80/090025-11$02.00/0 Copyright All rights
0 1980 by Academic Press, of reproduction in any form
Inc. reserved.
26
DEVELOPMENTALBIOLOGY
degeneration plays in atria1 septation is unknown. The role of the extracellular materials, which are integral components in foramen formation, is also still undefined. This study of developing foramina secunda in the chick at the ultrastructural and light microscopic level was undertaken with the above outlined problems in mind. Observations are concentrated on Day 6 since we have previously demonstrated this day to be one of the most prolific periods for atria1 septal development (Hendrix and Morse, 1977). MATERIALS
AND
METHODS
Seventy-five chick embryos of the White Leghorn strain, averaging 6 days in age, were removed from their shells and immersed in cold cacodylate-buffered Karnovsky’s fixative (Karnovsky, 1965). Specimens were staged according to the Hamburger-Hamilton series (Hamilton, 1952) after which the hearts were immediately dissected free and kept in fixative for 1 hr. In most cases, the lateral atrial walls were removed with the aid of a dissecting microscope to facilitate optimum fixation of the atria1 septum. Tissues were postfixed in osmium, stained with 1% uranyl acetate, dehydrated in ascending grades of ethanol, and embedded in Epon/Araldite resin. Ultrathin sections (80-90 nm) were stained with lead citrate and examined in either a Hitachi HU 11 E-l or a JEOL 100-S electron microscope. For light microscopy, l-pm-thick plastic sections were stained with toluidine blue. Other tissues were fixed separately in 10% alcoholic Formalin to preserve the glycogen, embedded in paraffin, and stained with Best’s carmine stain, as quoted by Brown (1969). All sections were observed with a Zeiss photomicroscope. RESULTS
The atria1 septum 6-day chick hearts.
is easily identified in In light microscopic
VOLUME 78,198O
preparations it is characterized by the presence of transseptal communications (foramina secunda) in its middorsal portion in various stages of development (Fig. 1). These stages range from slight dimples in the endocardial surface to complete perforations. Formation of foramina secunda creates thin cords of endocardium-covered tissue, which, when cut in cross section, appear as isolated clusters of cells (Fig. 2). The intact portion of septum primum in the vicinity of forming foramina secunda contains a rough and pitted surface. This is caused by numerous extensions of the endocardium into the core of the septum, which occurs on both right and left sides of the septum and creates a region of irregular thickness. In some areas the septum is so thin that the two endocardial layers appear to be in contact with each other. The endocardium in the rough portion of the septum near the forming foramina is also characterized by the presence of many large rounded cells which project notably into the atrial chambers. Conversely, the endocardial cells on the surface of the septum, some distance from the foramina secunda, are flattened and possess long, attenuated processes which extend considerable distances over the surface of the septum (Fig. 2). A transition from the flattened to the rounded cells can be seen in comparing the perforated and nonperforated portions of the septum. The rounded cells obviously form the only component of the endocardial layer in some instances, whereas, in other cases, they seem to be rather loosely attached to the underlying endocardium. The core of the septum and its associated cords surrounding the foramina secunda are relatively compact at these stages. Portions of the cytoplasm of numerous cells in the septal core stain darkly with toluidine blue. These cells are most conspicuous directly beneath the endocardium (Fig. 2), and they are not seen in the atria1 wall. When the cords of the atria1 septum are
MORSE
AND HENDRIX
Foramina
Secunda Formation
27
FIG. 1. A light microscopic orientation of the atria1 septum (AS) dividing the common atrium into right (RA) and left (LA) chambers. Note the protrusion of the septum toward the left atrium with an interatrial communication located in the middorsal region. x 100. FIG. 2. This preparation is similar to that of Fig. 1, except the place of section lies a few micrometers deeper into the septum. Forming foramina secunda are present in this portion of the septum and transseptal communication is obvious (double-headed arrow). Cords (C) of septal tissue are shown in cross section and longitudinally cut. Numerous large, rounded cells are shown projecting from the endocardial surface (asterisks). x 250. FIG. 3. A cord of the atria1 septum is stained with Best’s carmine for the demonstration of glycogen. Those subendocardial cells, which appear lightly stained with toluidine blue in Fig. 2. are shown here to stain intensely for glycogen content (arrowheads). x 150.
28
DEVELOPMENTAL BIOLOGY
treated with Best’s carmine histochemical stain, these cells are observed to contain large pools of glycogen (Fig. 3). At the fine-structural level the septum is covered on both surfaces by a continuous endocardium. Portions of the septum where foramina secunda are not forming show the endocardium to be a thin attenuated layer. Some overlapping of the thin endocardial cell processes is common (Fig. 4). The endocardium lacks a continuous basal lamina. The core of the septum is composed almost exclusively of cellular elements (myocytes) . These cells are densely packed and are connected by desmosome-like junctions. A continuous basal lamina lines the subendocardial surface of the myocardium. With the exception of phagocytic cells, all cells of the septal core have myofilaments and myofibrils. The myofilaments are arranged in small organized bundles and as randomly dispersed single units. Within myofibrils, Z lines can be demonstrated but complete sarcomeres are rarely found (Figs. 4 and 5). Mitochondria and lipid droplets are common throughout the cytoplasm. Nuclei of the septal core cells are rarely seen in thin sections. In those tissues stained with uranyl acetate (Figs. 4-9), large areas of glycogen are shown to be extracted (Vye and Fischman, 1971). These cytoplasmic areas typically lie closest to the free edge of the cells and represent the subendocardial areas which stain darkly in light microscopic preparations. The endocardium covering portions of
VOLUME 78,198O
the septum where foramina secunda are forming is considerably different from that described above. The predominant endocardial cell is rounded and projects notably into the atrial cavity. These rounded cells have short cytoplasmic processes which typically communicate with similar extensions from neighboring rounded endocardial cells. Flat, attenuated, lining cells are found infrequently in this region. Characteristics of the rounded endocardial cells include multiple-lobed nuclei, lipid droplets, and a very highly developed Golgi zone (Figs. 5-8). The lipid droplets may be represented as aggregates (Figs. 5 and 6) or occur singularly distributed throughout the cytoplasm (Fig. 7). Inclusions which have contents resembling the cytoplasm of the septal core cells can be demonstrated (Fig. 7). One of the most interesting aspects of the rounded endocardial cells is the presence of thin cytoplasmic processes on their attached surface. These processesextend into the septal core, and appear to separate the cells of the core (Fig. 8). The rounded endocardial cell processesextend into the septum from both surfaces and eventually extend the entire thickness of the septum or contact a cytoplasmic extension from the opposite side (Fig. 9). In either event, the result is an isolation of a portion of septal tissue. Most typically, adjacent rounded cells extend parallel processesinto the septal core. Once a transseptal communication is established the parallel cytoplasmic ex-
FIG. 4. An electron micrograph of a portion of atria1 septum comparable to that of Fig. 3 shows the septum to be covered on both atrial surfaces by a thinly attenuated endocardium (E). The septal core is occupied by densely packed cells which contain myotibrils (F) in various degrees of organization. Large amounts of extracted glycogen cytoplasm in these cells are devoid of organelles. The atria1 chambers occupy the upper left and lower right of this field. x 5950. FIG. 5. The endocardial cells are typically rounded and their processes are short in the portion of the septum where foramina secunda are forming. These cells project notably from the surface of the septum (compare with Figs. 2 and 3) and often possess numerous short microvillus-like extensions. The nuclei (N) are multilobed and the perinuclear zone contains a rich network of organelles. Lipid-like droplets (L) are abundant. The septal core is characteristically thin, often being occupied by only one cell. The atria1 chambers occupy the upper right and lower left of this field. x 7230. FIG. 6. A higher magnification of the lower left-hand portion of Fig. 5 better illustrates the accumulation of lipid-like bodies (L) in the cytoplasm of the rounded endocardial cells. Cytoplasmic filaments are also abundant in these cells. Cell junctions of the desmosome type are common between the endocardial cells. x 14,450.
MORSE
AND HENDRIX
Foramina
FIGS.
4-6
Secunda Formation
29
30
DEVELOPMENTAL
BIOLOGY
FIGS.
VOLUME
7 AND 8
78,1%0
MORSE
AND HENDRIX
Foramina
tensions separate, creating a new foramen secundum. The basal lamina remains continuous on the surface of the newly separated cells of the septal core. During the sixth day the cellular processes extending into the septum become so numerous that it is common to find isolated islands of septal tissue surrounded by endocardium. In the developing foramina secunda region, septal subdivision during the sixth day consists of septal core tissue being isolated into small cords. Each cord is covered by a single layer of endocardium. Between any two cords are the enlarging foramina secunda. The cords of tissue may continue to be cleaved into smaller subunits in a manner similar to that described above (Fig. 10). Many of the cords of tissue are eventually composed of one or two cells of the original septal core surrounded by an endocardial cell. Cell degeneration and death in the vicinity of forming foramina secunda is not abundant. Obvious signs of cell degeneration and phagocytosis are visualized as large cells containing debris-laden bodies. The cytoplasm of the phagocytic cells is similar to that of the rounded endocardial cells with regard to organelle distribution and concentration. Cells containing large amounts of cellular debris are not found on the endocardial surface. DISCUSSION
Statistical evidence indicates that division of the primitive heart into chambers is the cardiovascular developmental event most susceptible to maldevelopment (Warkany, 1971). One of the best examples of a cardiovascular septal development requir-
Secunda
Formation
31
ing precision timing is the formation of foramina secunda. As the atria1 septum primum proliferates and approaches the endocardial cushions, the communication between the right and left atria (foramen primum) progressively narrows. Foramen primum is gradually occluded as the septum primum and endocardial cushions complete their fusion. A right-to-left shunting of blood is essential during fetal life since, for example, in humans the respiratory system remains morphologically incompetent until the third trimester. Likewise, blood flow through the left atrium is necessary for its normal development; prenatal closure of interatrial communication is incompatible with postnatal survival (Wilson et al., 1953). The prenatal interatrial shunt is maintained by the establishment of transseptal perforations (foramina secunda) at a time which parallels the obliteration of foramen primum. Several theories have been proposed regarding the formation of foramina secunda. These include the ideas of Chang (1931) and Quiring (1933) which suggest that pressure increase in the right atrium as the foramen primum narrows causes small points of rupture in the septum primum. Odgers (1935) proposes that foramina in the atria1 septum complex are formed by an incomplete growth of the various components. Thus, he believes that the foramen secundum is always present and is not formed by perforations in the septum primum. Each of these hypotheses is based on the belief that heart septation and remodeling is controlled primarily by hemodynamic forces. A more recent hypothesis suggests that a factor contributing to the
FIG. 7. It is common to find the rounded endocardial cells aggregating into small nodular masses on the endocardial surface. This condition gives the endocardium a stratified appearance. Often the rounded cells exposed to the atria1 cavity (A) are very loosely attached to the other endocardial cells. One of the rounded endocardial cells contains an inclusion (In), the contents of which resemble the extracted glycogen cytoplasm of the septal core cells (S). Note the large number of lipid-like bodies (L) in the rounded cells. x 7230. FIG. 8. The Fist indications of a developing foramen secundum are narrow, shallow indentations on the outer edge (basal lamina surface) of the septal core (arrows). The rounded endocardial cells extend processes (P) into these clefts. The clefts gradually become deeper and wider. x 6470.
32
DEVELOPMENTAL
BIOLOGY
VOLUME
78,198O
MORSE
AND HENDRIX
Foramina
Secunda Formation
33
FIG. 10. As the septum is subdivided by endocardial cells, the smaller units separate to create additional transseptal openings (foramina secunda). The cords of tissue which surround the foramina are composed of a continuous endocardial layer which invests a small number of cells from the septal core (S). As shown in this field, the large cords of tissue may be further subdivided by processes (P) from rounded endocardial cells. x 4930.
formation of interatrial communications is the presence of focal zones of cell death (Pexieder, 1975). Scanning electron microscopy of the development of the atrial septum of the chick heart shows that foramina secunda do indeed form in the intact portion of the septum. However, the foramina do not seem to form as a result of ruptures or “blowouts” in the septum. The endocardial integrity is maintained throughout the period of foramina formation (Hendrix and Morse, 1977).
These observations do not preclude the possibility that hemodynamic forces act in the shaping and thinning of the septum at precise locations, but the effect of blood flow through the heart is not the only mechanism responsible for the formation of foramina secunda. The same is true of cell death in heart septation. While it is undoubtedly an important aspect of cardiac development, cell death represents only one of several mechanisms involved in the construction of the heart (Pexieder, 1977).
FIG. 9. This low-power electron micrograph of the atrial septum in the region of forming foramina secunda shows numerous rounded endocardial cells aggregating in an area where processes (P) are extending into the septum. Cytoplasmic processes can be seen protruding into the septal core (S) from both right and left sides of the septum. Note the various stages of septal subdivision by endocardial cell processes. x 6560.
34
DEVELOPMENTAL BIOLOGY
In this paper we have focused our attention on the large rounded endocardial cells covering the atria1 septum because we believe they represent an important aspect of cardiac remodeling. This conclusion is based partially on the fact that these cells have their greatest density in a zone reported to possesslarge numbers of dying cells (Pexieder, 1975). Relying on previous reports, it could be postulated that these rounded cells may represent (1) phagocytic cells responsible for removing dead cells from cell death foci (Pexieder, 1975; Hendrix and Morse, 1977; Morse, 1978); (2) dying endocardial cells (Ojeda and Hurle, 1975); or (3) dead and dying cells being sloughed into the circulation (Manasek, 1969; Pexieder, 1976). Our study shows that at the ultrastructural level the rounded cells of the endocardium possess numerous characteristics of phagocytic cells. These include a lobulated nucleus, debris-laden vesicles, and highly developed Golgi zones. However, our thin sections of numerous specimens reveal that the number of dead and dying cells in the region of foramina secunda formation is very small. Certainly, their numbers are not great enough to account for the numerous rounded phagocytic cells present. The presence of large numbers of phagocytic cells of the endocardium is better explained when it is realized that they extend processes deep into the septum from both right and left sides. As a result, that portion of the septum which is destined to develop foramina secunda becomes subdivided many times by these rounded cell processes, and numerous islands of septal tissue are formed. Invasion of the cardiac jelly or myocardium by endocardial cells was described earlier in the avian ventricle by Manasek (1970, 1976). He observed that differentiated muscle cells separate as the endocardium rapidly approaches the closest point of possible contact. Fitzharris and Ashcraft (1978) find endocardial processes, called “flutes,” to arch toward the myocardium as a preliminary step to invasion of
VOLUME 78,198O
the cushion cardiac jelly by mesenchymal cells. In contrast to flattened endocardial cells which simply line the atria1 cavities, the cytoplasmic processes extending into the septum do not readily develop cell junctions. Thus, two opposing septal processes are easily separated. The separation of two adjacent transseptal processes creates a new interatrial communication. Possible explanations for the mechanisms involved are that (1) the transseptal processes are “pulled” apart due to the continued differential growth and expansion of the atrial walls along with the intact portion of the septum and/or that (2) the processes are “pushed” apart by hemodynamic forces. We have observed two important functions of the rounded endocardial cells in the formation of foramina secunda. First, these cells subdivide the septal tissue and conceivably are the source of hyaluronidase noted to be concentrated in the heart during septation (Orkin and Toole, 1978). Second, these cells are responsible for maintaining the integrity of the endocardium throughout the remodeling process. The origin of the rounded cells is unknown at this time. The most likely sources are differentiation from existing endocardial cells or circulating phagocytes. Evidence exists to support both possibilities. Flattened endocardial cells of the atrium are known to possessphagocytic properties (Ferguson, 1975). The endocardium of the embryonic heart is quite versatile and contributes both cellular and extracellular elements to the cardiac jelly (Markwald and Adams-Smith, 1972; Markwald et al., 1975). Markwald’s laboratory refers to the early endocardial cells as biphasic. An endocardial origin of the rounded cells described herein would suggest that the early endocardium is indeed a multiphasic group of cells. A possible hematogenous origin for these cells is supported by their cytological makeup at the ultrastructural level. The rounded cells are frequently seen to be
MORSE
AND HENDRIX
Foramina
loosely attached to underlying endocardium. This may be the form of a mono- or multicellular aggregate. Further support of the hematogenous theory is gained by the observation of circulating phagocytes in 4day chick embryos (Saunders, 1966). The atrial septum and its developing interatrial communications offer an excellent model for further studies of biochemical, physiological, and morphological cellular and extracellular interactions in a concisely localized area of a developing organ. We are most appreciative to Dr. Elizabeth D. Hay for her helpful suggestions and to Mrs. Charles N. G. Hendrix for typing. This research was supported by a Biomedical Support Grant of Ohio and United States Public Health Service Fellowship F32 HL-05682.
REFERENCES BRAMER, J. L. (1932). The presence and influence of two spiral streams in the heart of the chick embryo. Amer. J. Anat. 49,409-440. BROWN, G. G. (19691. “Primer of Histopathologic Technique,” p. 161. Meredith, New York. CHANG, C. (1931). The interatrial septum in chick embryos. Anat. Rec. 50,9-22. FERGUSON, H. W. (1975). Phagocytosis by the endocardial lining cells of the atrium of Plaica (“Pleuronectes Platessa”). J. Comp. Pathol. 85, 561-569. FITZHARRIS, T. P., and ASHCRAFT, R. L. (1978). Endocardial shape change during cushion tissue mesenchyme formation. J. Cell Biol. 79, 336a. HAMILTON, H. H. (19521. “Lillie’s Development of the Chick,” 3rd ed. Holt, Rhinehart & Winston, New York. HENDRIX, M. J. C., and MORSE, D. E. (1977). Atria1 septation. I. Scanning electron microscopy in the chick. Develop. Biol. 57, 345-363. KARNOVSKY, J. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 137a-138a. KLONER, R. A., FISHBEIN, M. C., MACLEAN, D., BRAUNWALD, E., and MAROKO, P. R. (1977). Effect of hyaluronidase during the early phase of acute myocardial ischemia: An ultrastructural and morphometric analysis. Amer. J. Cardiol. 40, 43-49. MACLEAN, D., FISHBEIN, M. C., MAROKO, P. R., and BRAUNWALD, E. (1976). Hyaluronidase induced reductions in myocardial infarct size. Science 194. 199-200.
Secunda
Formation
35
MANASEK, F. J. (1970). Histogenesis of the embryonic myocardium. Amer. J. Cardiol. 25, 149-168. MANASEK, F. J. (1969). Myocardial cell death in the embryonic chick ventricle. J. Embryol. Exp. Morphol. 21/22,271-284. MANASEK, F. J. (1976). Heart development interactions involved in cardiac morphogenesis. In “The Cell Surface in Animal Embryogenesis and Development” (G. Poste and G. L. Nicolson, eds.). NorthHolland, New York. MARKWALD, R. R., and ADAMS-SMITH, W. N. (1972). Distribution of mucosubstances in the developing rat heart. J. Histochem. Cytochem. 20, X0-180. MARKWALD, R. R., FITZHARRIS, T. P., and ADA~ISSMITH, W. N. (1975). Structural analysis of endocardial cytodifferentiation. Develop. Biol. 42, 160180. MORSE, D. E. (1978). Scanning electron microscopy of the developing septa in the chick heart. Birth Defects: Orig. Article Ser. 14, 91-107. ODGERS, P. N. B. (1935). The formation of the venous valves, the foramen secundum and the septum secundum in the human heart. J. Anat. 69,412-422. 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. ORKIN, R. W., and TOOLE, B. P. (1978). Hyaluronidase activity and hyaluronate content of the developing chick embryo heart. Develop. Biol. 66,308-320. PATTEN, B. M. (1925). The interatrial septum of the chick heart. Anat. Rec. 30,53-60. PEXIEDER, T. (1925). Cell death in the morphogenesis and teratogenesis of the heart. Advan. Anat. Embryol. Cell Biol. 51, 7-99. PEXIEDER, T. (1976). Rasterelektronenmikroskopische Beobachtungen der Obertlache der Henblubuswulste der Huhnerembryonem. Verh. Anat. Ges. 70.747-754. PEXIEDER, T. (1977). SEM observations of the embryonic endocardium under normal and experimental hemodynamic conditions. Bibl. Anat. 15, 531-534. QUIRING, D. P. (1933). The development of the sinoatrial region of the chick heart. J. Morphol. 55, 81118. SAUNDERS, J. W., JR. (1966). Death in embryonic systems. Science 154,604-612. VYE, M. V., and FISCHMAN, D. A. (1971). A comparative study of three methods for the ultrastructural demonstration of glycogen in thin sections. J. Cell Sci. 9, 727-749. WARKANY, J. (1971). “Congenital Malformations,” Chap. 8. Year Book Med. Pub., Chicago. WILSON, J. G., LYON, R. A., and TERRY, R. (1953). Prenatal closure of interatrial foramen. Amer. J. Dis. Child. 85,285-294.
78,25-35
BIOLOGY
(1980)
Atrial II. Formation DENNIS Departments
of Anatomy,
Medical
Received
Septation
of the Foramina
E. MORSE
AND MARY
College of Ohio, School of Medicine,
April
Secunda
Toledo, Ohio Washington,
2, 1979; accepted
in revised
in the Chick
J. C. HENDRIX’ 43699, and George D.C. 20037 form
November
Washington
University
13, 1979
Chick hearts were prepared for light microscopy and transmission electron microscopy by conventional methods, the purpose being to investigate the developing foramina secunda in the atrial septum. As the growing septum approaches and fuses with the endocardial cushions, small perforations (foramina secunda) are formed in the middorsal portion of the septum. During Days 5 and 6 the number and size of these foramina increase significantly. Formation of foramina creates thin cords of endocardium-covered tissue. Two varieties of endocardial cells are associated with the atrial septum during this period. The endocardial cells on the surface of the septum, some distance from the foramina secunda, are flattened and possess long, attenuated processes. Conversely, the endocardium in the rough portion of the septum near the foramina is characterized by the presence of numerous rounded cells which project notably into the atria1 chambers. These rounded endocardial cells resemble phagocytic cells and possess thin cytoplasmic processes which extend into the septal core, gradually separating the cells of the core, resulting in an isolation of a portion of septal tissue. Once a transseptal communication is established the cytoplasmic processes separate, creating a new foramen secundum. The rounded endocardial cells are also responsible for maintaining the integrity of the endocardium-covered septum throughout the remodeling process. The origin of the rounded cells is unknown at this time. The most likely sources are differentiation from existing endocardial cells or circulating phagocytes.
foramen primum by growth of the atrial septum causes an increase in pressure in the right atrium. He further states that the pressure increase causes the septum to break down at its weakest points. Scanning electron microscopy shows the endocardium to remain intact during the formation of foramina secunda (Hendrix and Morse, while hemodynamic 1977). Therefore, forces may influence the formation of these foramina, the septum does not suddenly rupture. Foramen formation appears to be highly organized and to occur at precise locations. Pexieder (1975) has demonstrated convincingly that cell death occurs in the atria1 septum during the period of foramina secunda formation. The cell type or types which are degenerating have not been identified. Likewise, the overall role that cell
INTRODUCTION
Formation of the atrial septum occurs between Days 3 and 5 in the chick embryo (Patten, 1925; Chang, 1931; Bremer, 1932; Quiring, 1933; Her&ix and Morse, 1977). These events are carefully synchronized so that as the growing septum approaches and fuses with the endocardial cushions, anticipating the closure of foramen primum, small perforations (foramina secunda) have already begun to appear in the middorsal portion of the septum. During Days 5 and 6 the number and size of these foramina increase significantly. Several problems need to be addressed regarding foramina secunda formation. Quiring (1933) suggests that obliteration of ’ Present address: vard Medical School, 02115.
Department 25 Shattuck
of Anatomy, St., Boston,
HarMass. 25
0012-1606/80/090025-11$02.00/0 Copyright All rights
0 1980 by Academic Press, of reproduction in any form
Inc. reserved.
26
DEVELOPMENTALBIOLOGY
degeneration plays in atria1 septation is unknown. The role of the extracellular materials, which are integral components in foramen formation, is also still undefined. This study of developing foramina secunda in the chick at the ultrastructural and light microscopic level was undertaken with the above outlined problems in mind. Observations are concentrated on Day 6 since we have previously demonstrated this day to be one of the most prolific periods for atria1 septal development (Hendrix and Morse, 1977). MATERIALS
AND
METHODS
Seventy-five chick embryos of the White Leghorn strain, averaging 6 days in age, were removed from their shells and immersed in cold cacodylate-buffered Karnovsky’s fixative (Karnovsky, 1965). Specimens were staged according to the Hamburger-Hamilton series (Hamilton, 1952) after which the hearts were immediately dissected free and kept in fixative for 1 hr. In most cases, the lateral atrial walls were removed with the aid of a dissecting microscope to facilitate optimum fixation of the atria1 septum. Tissues were postfixed in osmium, stained with 1% uranyl acetate, dehydrated in ascending grades of ethanol, and embedded in Epon/Araldite resin. Ultrathin sections (80-90 nm) were stained with lead citrate and examined in either a Hitachi HU 11 E-l or a JEOL 100-S electron microscope. For light microscopy, l-pm-thick plastic sections were stained with toluidine blue. Other tissues were fixed separately in 10% alcoholic Formalin to preserve the glycogen, embedded in paraffin, and stained with Best’s carmine stain, as quoted by Brown (1969). All sections were observed with a Zeiss photomicroscope. RESULTS
The atria1 septum 6-day chick hearts.
is easily identified in In light microscopic
VOLUME 78,198O
preparations it is characterized by the presence of transseptal communications (foramina secunda) in its middorsal portion in various stages of development (Fig. 1). These stages range from slight dimples in the endocardial surface to complete perforations. Formation of foramina secunda creates thin cords of endocardium-covered tissue, which, when cut in cross section, appear as isolated clusters of cells (Fig. 2). The intact portion of septum primum in the vicinity of forming foramina secunda contains a rough and pitted surface. This is caused by numerous extensions of the endocardium into the core of the septum, which occurs on both right and left sides of the septum and creates a region of irregular thickness. In some areas the septum is so thin that the two endocardial layers appear to be in contact with each other. The endocardium in the rough portion of the septum near the forming foramina is also characterized by the presence of many large rounded cells which project notably into the atrial chambers. Conversely, the endocardial cells on the surface of the septum, some distance from the foramina secunda, are flattened and possess long, attenuated processes which extend considerable distances over the surface of the septum (Fig. 2). A transition from the flattened to the rounded cells can be seen in comparing the perforated and nonperforated portions of the septum. The rounded cells obviously form the only component of the endocardial layer in some instances, whereas, in other cases, they seem to be rather loosely attached to the underlying endocardium. The core of the septum and its associated cords surrounding the foramina secunda are relatively compact at these stages. Portions of the cytoplasm of numerous cells in the septal core stain darkly with toluidine blue. These cells are most conspicuous directly beneath the endocardium (Fig. 2), and they are not seen in the atria1 wall. When the cords of the atria1 septum are
MORSE
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Foramina
Secunda Formation
27
FIG. 1. A light microscopic orientation of the atria1 septum (AS) dividing the common atrium into right (RA) and left (LA) chambers. Note the protrusion of the septum toward the left atrium with an interatrial communication located in the middorsal region. x 100. FIG. 2. This preparation is similar to that of Fig. 1, except the place of section lies a few micrometers deeper into the septum. Forming foramina secunda are present in this portion of the septum and transseptal communication is obvious (double-headed arrow). Cords (C) of septal tissue are shown in cross section and longitudinally cut. Numerous large, rounded cells are shown projecting from the endocardial surface (asterisks). x 250. FIG. 3. A cord of the atria1 septum is stained with Best’s carmine for the demonstration of glycogen. Those subendocardial cells, which appear lightly stained with toluidine blue in Fig. 2. are shown here to stain intensely for glycogen content (arrowheads). x 150.
28
DEVELOPMENTAL BIOLOGY
treated with Best’s carmine histochemical stain, these cells are observed to contain large pools of glycogen (Fig. 3). At the fine-structural level the septum is covered on both surfaces by a continuous endocardium. Portions of the septum where foramina secunda are not forming show the endocardium to be a thin attenuated layer. Some overlapping of the thin endocardial cell processes is common (Fig. 4). The endocardium lacks a continuous basal lamina. The core of the septum is composed almost exclusively of cellular elements (myocytes) . These cells are densely packed and are connected by desmosome-like junctions. A continuous basal lamina lines the subendocardial surface of the myocardium. With the exception of phagocytic cells, all cells of the septal core have myofilaments and myofibrils. The myofilaments are arranged in small organized bundles and as randomly dispersed single units. Within myofibrils, Z lines can be demonstrated but complete sarcomeres are rarely found (Figs. 4 and 5). Mitochondria and lipid droplets are common throughout the cytoplasm. Nuclei of the septal core cells are rarely seen in thin sections. In those tissues stained with uranyl acetate (Figs. 4-9), large areas of glycogen are shown to be extracted (Vye and Fischman, 1971). These cytoplasmic areas typically lie closest to the free edge of the cells and represent the subendocardial areas which stain darkly in light microscopic preparations. The endocardium covering portions of
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the septum where foramina secunda are forming is considerably different from that described above. The predominant endocardial cell is rounded and projects notably into the atrial cavity. These rounded cells have short cytoplasmic processes which typically communicate with similar extensions from neighboring rounded endocardial cells. Flat, attenuated, lining cells are found infrequently in this region. Characteristics of the rounded endocardial cells include multiple-lobed nuclei, lipid droplets, and a very highly developed Golgi zone (Figs. 5-8). The lipid droplets may be represented as aggregates (Figs. 5 and 6) or occur singularly distributed throughout the cytoplasm (Fig. 7). Inclusions which have contents resembling the cytoplasm of the septal core cells can be demonstrated (Fig. 7). One of the most interesting aspects of the rounded endocardial cells is the presence of thin cytoplasmic processes on their attached surface. These processesextend into the septal core, and appear to separate the cells of the core (Fig. 8). The rounded endocardial cell processesextend into the septum from both surfaces and eventually extend the entire thickness of the septum or contact a cytoplasmic extension from the opposite side (Fig. 9). In either event, the result is an isolation of a portion of septal tissue. Most typically, adjacent rounded cells extend parallel processesinto the septal core. Once a transseptal communication is established the parallel cytoplasmic ex-
FIG. 4. An electron micrograph of a portion of atria1 septum comparable to that of Fig. 3 shows the septum to be covered on both atrial surfaces by a thinly attenuated endocardium (E). The septal core is occupied by densely packed cells which contain myotibrils (F) in various degrees of organization. Large amounts of extracted glycogen cytoplasm in these cells are devoid of organelles. The atria1 chambers occupy the upper left and lower right of this field. x 5950. FIG. 5. The endocardial cells are typically rounded and their processes are short in the portion of the septum where foramina secunda are forming. These cells project notably from the surface of the septum (compare with Figs. 2 and 3) and often possess numerous short microvillus-like extensions. The nuclei (N) are multilobed and the perinuclear zone contains a rich network of organelles. Lipid-like droplets (L) are abundant. The septal core is characteristically thin, often being occupied by only one cell. The atria1 chambers occupy the upper right and lower left of this field. x 7230. FIG. 6. A higher magnification of the lower left-hand portion of Fig. 5 better illustrates the accumulation of lipid-like bodies (L) in the cytoplasm of the rounded endocardial cells. Cytoplasmic filaments are also abundant in these cells. Cell junctions of the desmosome type are common between the endocardial cells. x 14,450.
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Foramina
FIGS.
4-6
Secunda Formation
29
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FIGS.
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78,1%0
MORSE
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tensions separate, creating a new foramen secundum. The basal lamina remains continuous on the surface of the newly separated cells of the septal core. During the sixth day the cellular processes extending into the septum become so numerous that it is common to find isolated islands of septal tissue surrounded by endocardium. In the developing foramina secunda region, septal subdivision during the sixth day consists of septal core tissue being isolated into small cords. Each cord is covered by a single layer of endocardium. Between any two cords are the enlarging foramina secunda. The cords of tissue may continue to be cleaved into smaller subunits in a manner similar to that described above (Fig. 10). Many of the cords of tissue are eventually composed of one or two cells of the original septal core surrounded by an endocardial cell. Cell degeneration and death in the vicinity of forming foramina secunda is not abundant. Obvious signs of cell degeneration and phagocytosis are visualized as large cells containing debris-laden bodies. The cytoplasm of the phagocytic cells is similar to that of the rounded endocardial cells with regard to organelle distribution and concentration. Cells containing large amounts of cellular debris are not found on the endocardial surface. DISCUSSION
Statistical evidence indicates that division of the primitive heart into chambers is the cardiovascular developmental event most susceptible to maldevelopment (Warkany, 1971). One of the best examples of a cardiovascular septal development requir-
Secunda
Formation
31
ing precision timing is the formation of foramina secunda. As the atria1 septum primum proliferates and approaches the endocardial cushions, the communication between the right and left atria (foramen primum) progressively narrows. Foramen primum is gradually occluded as the septum primum and endocardial cushions complete their fusion. A right-to-left shunting of blood is essential during fetal life since, for example, in humans the respiratory system remains morphologically incompetent until the third trimester. Likewise, blood flow through the left atrium is necessary for its normal development; prenatal closure of interatrial communication is incompatible with postnatal survival (Wilson et al., 1953). The prenatal interatrial shunt is maintained by the establishment of transseptal perforations (foramina secunda) at a time which parallels the obliteration of foramen primum. Several theories have been proposed regarding the formation of foramina secunda. These include the ideas of Chang (1931) and Quiring (1933) which suggest that pressure increase in the right atrium as the foramen primum narrows causes small points of rupture in the septum primum. Odgers (1935) proposes that foramina in the atria1 septum complex are formed by an incomplete growth of the various components. Thus, he believes that the foramen secundum is always present and is not formed by perforations in the septum primum. Each of these hypotheses is based on the belief that heart septation and remodeling is controlled primarily by hemodynamic forces. A more recent hypothesis suggests that a factor contributing to the
FIG. 7. It is common to find the rounded endocardial cells aggregating into small nodular masses on the endocardial surface. This condition gives the endocardium a stratified appearance. Often the rounded cells exposed to the atria1 cavity (A) are very loosely attached to the other endocardial cells. One of the rounded endocardial cells contains an inclusion (In), the contents of which resemble the extracted glycogen cytoplasm of the septal core cells (S). Note the large number of lipid-like bodies (L) in the rounded cells. x 7230. FIG. 8. The Fist indications of a developing foramen secundum are narrow, shallow indentations on the outer edge (basal lamina surface) of the septal core (arrows). The rounded endocardial cells extend processes (P) into these clefts. The clefts gradually become deeper and wider. x 6470.
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DEVELOPMENTAL
BIOLOGY
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Foramina
Secunda Formation
33
FIG. 10. As the septum is subdivided by endocardial cells, the smaller units separate to create additional transseptal openings (foramina secunda). The cords of tissue which surround the foramina are composed of a continuous endocardial layer which invests a small number of cells from the septal core (S). As shown in this field, the large cords of tissue may be further subdivided by processes (P) from rounded endocardial cells. x 4930.
formation of interatrial communications is the presence of focal zones of cell death (Pexieder, 1975). Scanning electron microscopy of the development of the atrial septum of the chick heart shows that foramina secunda do indeed form in the intact portion of the septum. However, the foramina do not seem to form as a result of ruptures or “blowouts” in the septum. The endocardial integrity is maintained throughout the period of foramina formation (Hendrix and Morse, 1977).
These observations do not preclude the possibility that hemodynamic forces act in the shaping and thinning of the septum at precise locations, but the effect of blood flow through the heart is not the only mechanism responsible for the formation of foramina secunda. The same is true of cell death in heart septation. While it is undoubtedly an important aspect of cardiac development, cell death represents only one of several mechanisms involved in the construction of the heart (Pexieder, 1977).
FIG. 9. This low-power electron micrograph of the atrial septum in the region of forming foramina secunda shows numerous rounded endocardial cells aggregating in an area where processes (P) are extending into the septum. Cytoplasmic processes can be seen protruding into the septal core (S) from both right and left sides of the septum. Note the various stages of septal subdivision by endocardial cell processes. x 6560.
34
DEVELOPMENTAL BIOLOGY
In this paper we have focused our attention on the large rounded endocardial cells covering the atria1 septum because we believe they represent an important aspect of cardiac remodeling. This conclusion is based partially on the fact that these cells have their greatest density in a zone reported to possesslarge numbers of dying cells (Pexieder, 1975). Relying on previous reports, it could be postulated that these rounded cells may represent (1) phagocytic cells responsible for removing dead cells from cell death foci (Pexieder, 1975; Hendrix and Morse, 1977; Morse, 1978); (2) dying endocardial cells (Ojeda and Hurle, 1975); or (3) dead and dying cells being sloughed into the circulation (Manasek, 1969; Pexieder, 1976). Our study shows that at the ultrastructural level the rounded cells of the endocardium possess numerous characteristics of phagocytic cells. These include a lobulated nucleus, debris-laden vesicles, and highly developed Golgi zones. However, our thin sections of numerous specimens reveal that the number of dead and dying cells in the region of foramina secunda formation is very small. Certainly, their numbers are not great enough to account for the numerous rounded phagocytic cells present. The presence of large numbers of phagocytic cells of the endocardium is better explained when it is realized that they extend processes deep into the septum from both right and left sides. As a result, that portion of the septum which is destined to develop foramina secunda becomes subdivided many times by these rounded cell processes, and numerous islands of septal tissue are formed. Invasion of the cardiac jelly or myocardium by endocardial cells was described earlier in the avian ventricle by Manasek (1970, 1976). He observed that differentiated muscle cells separate as the endocardium rapidly approaches the closest point of possible contact. Fitzharris and Ashcraft (1978) find endocardial processes, called “flutes,” to arch toward the myocardium as a preliminary step to invasion of
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the cushion cardiac jelly by mesenchymal cells. In contrast to flattened endocardial cells which simply line the atria1 cavities, the cytoplasmic processes extending into the septum do not readily develop cell junctions. Thus, two opposing septal processes are easily separated. The separation of two adjacent transseptal processes creates a new interatrial communication. Possible explanations for the mechanisms involved are that (1) the transseptal processes are “pulled” apart due to the continued differential growth and expansion of the atrial walls along with the intact portion of the septum and/or that (2) the processes are “pushed” apart by hemodynamic forces. We have observed two important functions of the rounded endocardial cells in the formation of foramina secunda. First, these cells subdivide the septal tissue and conceivably are the source of hyaluronidase noted to be concentrated in the heart during septation (Orkin and Toole, 1978). Second, these cells are responsible for maintaining the integrity of the endocardium throughout the remodeling process. The origin of the rounded cells is unknown at this time. The most likely sources are differentiation from existing endocardial cells or circulating phagocytes. Evidence exists to support both possibilities. Flattened endocardial cells of the atrium are known to possessphagocytic properties (Ferguson, 1975). The endocardium of the embryonic heart is quite versatile and contributes both cellular and extracellular elements to the cardiac jelly (Markwald and Adams-Smith, 1972; Markwald et al., 1975). Markwald’s laboratory refers to the early endocardial cells as biphasic. An endocardial origin of the rounded cells described herein would suggest that the early endocardium is indeed a multiphasic group of cells. A possible hematogenous origin for these cells is supported by their cytological makeup at the ultrastructural level. The rounded cells are frequently seen to be
MORSE
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Foramina
loosely attached to underlying endocardium. This may be the form of a mono- or multicellular aggregate. Further support of the hematogenous theory is gained by the observation of circulating phagocytes in 4day chick embryos (Saunders, 1966). The atrial septum and its developing interatrial communications offer an excellent model for further studies of biochemical, physiological, and morphological cellular and extracellular interactions in a concisely localized area of a developing organ. We are most appreciative to Dr. Elizabeth D. Hay for her helpful suggestions and to Mrs. Charles N. G. Hendrix for typing. This research was supported by a Biomedical Support Grant of Ohio and United States Public Health Service Fellowship F32 HL-05682.
REFERENCES BRAMER, J. L. (1932). The presence and influence of two spiral streams in the heart of the chick embryo. Amer. J. Anat. 49,409-440. BROWN, G. G. (19691. “Primer of Histopathologic Technique,” p. 161. Meredith, New York. CHANG, C. (1931). The interatrial septum in chick embryos. Anat. Rec. 50,9-22. FERGUSON, H. W. (1975). Phagocytosis by the endocardial lining cells of the atrium of Plaica (“Pleuronectes Platessa”). J. Comp. Pathol. 85, 561-569. FITZHARRIS, T. P., and ASHCRAFT, R. L. (1978). Endocardial shape change during cushion tissue mesenchyme formation. J. Cell Biol. 79, 336a. HAMILTON, H. H. (19521. “Lillie’s Development of the Chick,” 3rd ed. Holt, Rhinehart & Winston, New York. HENDRIX, M. J. C., and MORSE, D. E. (1977). Atria1 septation. I. Scanning electron microscopy in the chick. Develop. Biol. 57, 345-363. KARNOVSKY, J. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 137a-138a. KLONER, R. A., FISHBEIN, M. C., MACLEAN, D., BRAUNWALD, E., and MAROKO, P. R. (1977). Effect of hyaluronidase during the early phase of acute myocardial ischemia: An ultrastructural and morphometric analysis. Amer. J. Cardiol. 40, 43-49. MACLEAN, D., FISHBEIN, M. C., MAROKO, P. R., and BRAUNWALD, E. (1976). Hyaluronidase induced reductions in myocardial infarct size. Science 194. 199-200.
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MANASEK, F. J. (1970). Histogenesis of the embryonic myocardium. Amer. J. Cardiol. 25, 149-168. MANASEK, F. J. (1969). Myocardial cell death in the embryonic chick ventricle. J. Embryol. Exp. Morphol. 21/22,271-284. MANASEK, F. J. (1976). Heart development interactions involved in cardiac morphogenesis. In “The Cell Surface in Animal Embryogenesis and Development” (G. Poste and G. L. Nicolson, eds.). NorthHolland, New York. MARKWALD, R. R., and ADAMS-SMITH, W. N. (1972). Distribution of mucosubstances in the developing rat heart. J. Histochem. Cytochem. 20, X0-180. MARKWALD, R. R., FITZHARRIS, T. P., and ADA~ISSMITH, W. N. (1975). Structural analysis of endocardial cytodifferentiation. Develop. Biol. 42, 160180. MORSE, D. E. (1978). Scanning electron microscopy of the developing septa in the chick heart. Birth Defects: Orig. Article Ser. 14, 91-107. ODGERS, P. N. B. (1935). The formation of the venous valves, the foramen secundum and the septum secundum in the human heart. J. Anat. 69,412-422. 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. ORKIN, R. W., and TOOLE, B. P. (1978). Hyaluronidase activity and hyaluronate content of the developing chick embryo heart. Develop. Biol. 66,308-320. PATTEN, B. M. (1925). The interatrial septum of the chick heart. Anat. Rec. 30,53-60. PEXIEDER, T. (1925). Cell death in the morphogenesis and teratogenesis of the heart. Advan. Anat. Embryol. Cell Biol. 51, 7-99. PEXIEDER, T. (1976). Rasterelektronenmikroskopische Beobachtungen der Obertlache der Henblubuswulste der Huhnerembryonem. Verh. Anat. Ges. 70.747-754. PEXIEDER, T. (1977). SEM observations of the embryonic endocardium under normal and experimental hemodynamic conditions. Bibl. Anat. 15, 531-534. QUIRING, D. P. (1933). The development of the sinoatrial region of the chick heart. J. Morphol. 55, 81118. SAUNDERS, J. W., JR. (1966). Death in embryonic systems. Science 154,604-612. VYE, M. V., and FISCHMAN, D. A. (1971). A comparative study of three methods for the ultrastructural demonstration of glycogen in thin sections. J. Cell Sci. 9, 727-749. WARKANY, J. (1971). “Congenital Malformations,” Chap. 8. Year Book Med. Pub., Chicago. WILSON, J. G., LYON, R. A., and TERRY, R. (1953). Prenatal closure of interatrial foramen. Amer. J. Dis. Child. 85,285-294.