MICROSCOPY RESEARCH AND TECHNIQUE 20:305-313 (1992)
Intercellular Junctions in Embryonic Chick Cardiac Muscle Revealed by Rapid Freezing and Freeze-Substitution MASAYUKI SHIOZAKI AND YUTAKA SHIMADA Department of Anatomy and Cell Biology, School of Medicine, Chiba University, Chiba 280, Japan
KEY WORDS
Cryofixation, Development, Fasciae adherentes, Desmosomes, Myocardium
ABSTRACT Using the method of rapid freezing and freeze-substitution, the embryonic chick cardiac muscle was investigated by transmission electron microscopy. Initially, the intercellular junctional complexes (fasciae adherentes and desmosomes) were formed in close proximity to each other along a nearly straight line. Subsequently, the separation of fasciae from desmosomes took place to form intercalated discs. The cell membranes of fasciae adherentes were reinforced with highly interwoven fine fibrils at which myofibrils terminated. The intercellular space of fasciae was bridged with fine fibrillar structures seemingly connected by a thin line at their middle portions. In the intercellular space of desmosomes, central lamina and traversing filaments were clearly observed. The outer and inner leaflets of the desmosomal plasmalemma were asymmetrically differentiated; the outer leaflet was thinner than the inner leaflet. On the inner side of the cell membrane, an electron-lucent layer and a dense desmosomal plaque were observed. The latter structure had protrusions with less electron density towards the cytoplasmic side. Further inside, a meshwork of fine fibrils was seen along and toward which bundles of intermediate filaments ran. The results obtained with freeze-substitution appeared to provide more information than those with thin sections after conventional fixation or with replicas of chemically fixedlglycerinated or physically fixedldeep-etched materials. INTRODUCTION and positioned on a specimen holder such that the The formation and elaboration of the cell iunctions outer surface of the heart faced a copper block cooled by characteristic of cardiac muscle have been studied by liquid helium (about 4°K).The specimens quick-frozen several investigators with the usual chemical fixation with a Polaron Slammer E7200 were placed into 2% methods (Forbes and Sperelakis, 1975; Manasek, 1968, OsO, in acetone at -79°C and substituted for 1.5-2.5 1970; McNutt, 1970; Muir, 1957; Rash et al., 1970; days. They were then warmed in steps; at -20°C for 2 Spira, 1971). Recently, rapid freezing followed by hr, at 4°C for 2 hr, and finally at room temperature for freeze-substitution has proved useful for observing the 2 hr. They were then soaked in 100% methanol. Some biological ultrastructure, since cryofixed materials are preparations were stained en bloc with 2-5% uranyl closer to the native state than those prepared by chem- acetate in 100% methanol (Raviola et al., 1980)for 2 hr ical fixation (Heuser et al., 1979; Hirokawa and Kirino, and then 0.2-1.0% hafnium chloride (Benshalon and Reese, 1985; Tatsuoka and Reese, 1989) in 100%meth1980; Reese and Reese, 1981). The outer myocardial surface of the mature heart is anol for 0.5-3.0 hr. All specimens were passed through covered with epicardial cells and connective tissue, but propylene oxide and embedded in Epon 812. Thin secthat of early embryos is uncovered or covered only with tions were cut parallel to the contact surface. The seca single layer of epicardial cells (Ho and Shimada, tions were double-stained with uranyl acetate and lead 1978; Kurkiewicz, 1909; Manasek, 1969; Shimada and citrate and examined with a JEOL JEM 1200 EX11 Ho, 1980; Shimada et al., 19811, making procedures electron microscope operating at 80 kV. Stereopairs of such as slicing the tissue with a razor blade or a tissue electron micrographs were taken by tilting the grid chopper to expose the concerned areas unnecessary. 27". This material is therefore ideally suited for examinaRESULTS tion with rapid freezing by the metal contact method. Good freezing was obtained to a depth of approxiWe have reinvestigated these structures based on the apparent contribution of the newly developed method mately 15 pm from the metal contact surface of the heart in this study. Since the epicardial cells were eiof physical fixation followed by freeze-substitution. ther absent at most of the surface areas on the myoMATERIALS AND METHODS Fertile white Leghorn eggs were incubated a t 37°C for 2-5 days to yield embryos ranging from stages 12 to 26 of Hamburger and Hamilton (1951). The entire emReceived June 6, 1991; accepted in revised form July 12, 1991. bryos were excised and placed in phosphate-buffered Address reprint requests to Yutaka Shimada, Department of Anatomy, School saline (PBS). Their hearts were quickly dissected out of Medicine, Chiba University, Chiba 280, Japan.
0 1992 WILEY-LISS, INC.
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Figs. 1-3. Intercellular junctions of embryonic chick cardiac muscle at early stages. Initially, fasciae adherentes (black arrows and double arrows) and desmosomes (white arrows) were formed near each other along a nearly straight line. Early fasciae (double arrows
in Figs. 1 and 2) had no relation to myofibrils (Mf), but they later (black arrows in Figs. 2 and 3) became associated with developing myofibrils (arrowheads in Figs. 2 and 3). Fig. 1, ~ 7 3 , 0 0 0 ;Fig. 2, x 78,000; Fig. 3, x 63,000. Bar = 0.2 wm.
CELL JUNCTIONS IN EMBRYONIC CARDIAC MUSCLE
Figs. 4 and 5. Stereopair micrographs of fasciae adherentes (black arrows) and desmosomes (white arrows). Figure 4 is a higher magnification of the rectangle in Figure 3. Small bundles of filaments (arrowheads) separated from myofibrils (Mf) terminated at fasciae at acute angles. Bundles of intermediate filaments (open arrows) were associated with desmosomes. Fig. 4, x 95,000; Fig. 5 , x 50,000. Bar = 0.2 bm.
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Fig. 6. Somewhat matured muscle possessing step-type intercalated discs. Fasciae adherentes (black arrows) situated at the traversing segment and desmosomes (white arrows) on the longitudinal segment of the cells. Myofibrils (Mf) were connected with fasciae almost at right angles (arrowheads). X 37,000. Bar = 0.5 &m.
cardium (at and before stage 17) or still very thin (less than 10 bm at stage 26; Manasek, 1969), both the muscle directly contacted by the metal and that frozen through the epicardium were well preserved morphologically. Wide areas of well-frozen myocytes could be observed by sectioning the tissues tangentially to the surface. During this period, although the development of the myocardium generally proceeded steadily, the myocardial cells at varying stages of differentiation were always encountered in any given heart and, even within a single cell, myofibrils a t various stages of development were also found (see also Manasek, 1968, 1970). In the early myocardium, the cardiac myocytes were joined to each other by fasciae adherentes and desmosomes at points where they came into contact. Initially, these junctional structures were small in size and were formed in close proximity to each other in a nearly straight line along the lateral cell borders (Figs. 1-5). Subsequently, each junctional structure was enlarged and the separation of fasciae from desmosomes took place. Although desmosomes remained a t the lateral cell surface, fasciae became oriented at right angles to the longitudinal axis of the cells and, thus, step-type junctional specializations (intercalated discs) characteristic of the myocardium were formed (Fig. 6). Early fasciae adherentes, which could be recognized by dense material along the inner aspects of the plasma membranes, had no structural relation with nascent myofibrils (Figs. 1, 2). They soon became associated with small bundles of thin filaments, which were separated from developing myofibrils. They approached and terminated a t the fasciae a t an acute angle (Figs. 2-5). As the fasciae changed their position relative to desmosomes, myofibrils came to be connected with the fasciae at oblique or nearly right angles (Fig. 6).
The sarcoplasmic surface of the membranes confronted a t the fasciae adherentes was reinforced with a layer (thickness: 20-60 nm) of interwoven fine fibrils (diameter: 7-15 nm). The thin filaments of the terminal sarcomeres were seen to end in this fibrous mat of apposing cells (Figs. 3 - 5 , 7 , 8 ) . Between thin filaments near their insertion, crosslinking fibrillar structures (diameter: 7-18 nm) were observed. The intercellular space of the junctions was 30-40 nm in width and was bridged by fine fibrils (diameter: less than 4 nm) (Figs. 7, 8). These fine fibrillar bridges were thickened in their middle portion, thus appearing to form a thin line that connected them at their centers (Fig. 8). The opposing cell surfaces a t the desmosomal areas were about 30 nm apart (Figs. 2-5, 9-12). In their intercellular space, the central lamina (thickness: less than 3 nm) and fine traversing filaments (diameter: less than 2 nm) were clearly seen (Figs. 4, 9-11). Intersections of these two structures were thickened (Figs. 4, 9, 10). The external and internal leaflets of the desmosomal plasmalemma were asymmetrically differentiated. The outer leaflet, 2-4 nm thick, was a thin line possessing thickenings to which the traversing filaments appeared to attach. The inner leaflet, 3-5 nm thick, was a continuous dense line. On the inner surface of the desmosomal membrane, an electron-lucent layer 4-8 nm thick was present. On the cytoplasmic side of this layer, an electron-dense, desmosomal plaque 20-30 nm thick was seen. Its outer portion was a uniformly dark layer (thickness: 4-6 nm), while the inner part consisted of small protuberances (height: 15-25 nm) extending from the former and possessing less electron density. Some traversing filaments appeared to reach the desmosomal plaque through the plasmalemma and the electron-lucent layer (Fig. 9).
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Figs. 7 and 8. Higher magnification of fascia adherens. On the cytoplasmic side of the plasmalemma, a fibrous mat (*I was seen, at which thin filaments (large arrowheads) of myofibrils (Mf) terminated. Between these thin filaments, crosslinking fibrils (black arrows) were visible. In the intercellular space of the fascia, bridgingstructures (small arrowheads) were seen. When properly oriented and cut, thickenings of bridges at their middle portions were visible (small white arrowheads in Fig. 8). Fig. 7, x 98,000. Bar = 0.2 km; Fig. 8, x 140,000.Bar = 0.1 km.
Further into the cytoplasmic side of the plaque, a meshwork of fine fibrils (diameter: about 5 nm) was present. This meshwork was 70-90 nm thick and became more evident in en bloc preparations stained with uranyl acetate and hafnium chloride (Figs. 4, 10-12). Intermediate filaments in the cytoplasm converged near the desmosomes to form bundles. These bundles ran parallel to the desmosomal plaque and associated laterally with the fine fibrillar lattice. Occasionally, intermediate filaments bridging neighboring desmosomes were seen where a series of desmosomes were formed (Fig. 12). Figure 13 shows a structural model of a fascia adherens and desmosomes based on the present observations by rapid freezing and freeze-substitution of embryonic cardiac muscle. DISCUSSION In support of the assumptions of previous studies with chemical fixation (Forbes and Sperelakis, 1975; Manasek, 1968,1970; McNutt, 1970; Muir, 1957; Rash et al., 1970;Spira, 1971), the present study (by physical fixation followed by freeze-substitution) has demonstrated that membranes of myocytes are initially closely apposed where minute amounts of dense materials are associated. In these areas, fasciae adherentes and desmosomes can be distinguished and are seen to be formed side by side along the lateral cell boundaries. Cells with more extensive contacts demonstrate that
Fig. 8.
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Figs. 9 and 10. Higher magnification of desmosomes without (Fig. 9) and with (Fig. 10)en bloc staining. In the intercellular space, central laminae (small white arrowheads) and traversing filaments (small black arrowheads) were discernible. The external leaflet (1) of the desmosomal plasmalemma was thinner than the internal leaflet (2). Traversing filaments reached the desmosomal plaque through the plasmalemma and the electron-lucent layer (3). From the uniformly
dark, outer part (4) of the desmosomal plaque, processes with less electron density (5) extended. On the cytoplasmic side of the desmosoma1 plaque, a meshwork of fine fibrils (*) was seen. Bundles of intermediate filaments (open arrows) converged on the cytoplasmic side of the meshwork. Fig. 9, x 290,000; Fig. 10, x 120,000. Bar = 0.1 Fm.
sarcomeric thin and intermediate filaments attach to the respective junctional structures. Later, fasciae adherentes are oriented at the transverse segments and desmosomes on the longitudinal segments of the intercalated discs. Initially, myofibrils insert into developing fasciae adherentes at acute angles, but during development reorientation occurs so that fibril insertion becomes approximately 90". It appears likely that contractile forces, which are transmitted from one cell to another via the fasciae adherentes while cells are attached to each other at their lateral sides with desmosomes, are responsible for this reorientation. A detail of the region a t the fasciae adherentes,
which does not seem to have been noticed before by the usual chemical fixation (Fawcett and McNutt, 1969; McNutt and Fawcett, 19691, was the occurrence of bridging structures between the membranes confronted, with a thin line connecting the center of these bridges. In quick-freeze, deep-etch replicas of an adherens junction of mouse intestinal epithelial cells, similar bridging structures have been observed (Hirokawa and Heuser, 19811, but the central lines have not been noted. It is necessary to clarify whether these structures are associated with cell-adhesion-relatedproteins such as A-CAM (Volk and Geiger, 19861 or cadherins (Shimoyama et al., 1989; Takeichi, 1988). Furthermore, the nature of the crosslinking fibrils between
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traversing filaments in the intercellular space, the electron-lucent layer between the sarcolemma and the electron-dense desmosomal plaque, and protrusions of desmosomal plaques with less electron density. The extension of traversing filaments in the intercellular space to the desmosomal plaque through the sarcolemma and the electron-lucent layer has not been reported before. Freeze-fracture or freeze-fractureldeepetch methods (Hirokawa and Heuser, 1981; Kelly and Kuda, 19811, although revealing the traversing filaments in desmosomes, failed to demonstrate the central lamella, which is one of the features that has been used to distinguish this type of junction in thin-sectioned materials. Thus, by using the method of rapid freezing and freeze-substitution, the present study revealed intercellular junctional structures that had not been noted clearly before by the usual chemical fixation. Since it is generally presumed that the cryofixed cells are closer to the native state than those prepared by chemical fixation (Heuser et al., 1979; Hirokawa and Kirino, 1980; Reese and Reese, 1981),the present features possibly represent a more natural morphology of junctional complexes in the embryonic chick heart. Some of the intercellular structures prepared after freeze-subFig. 11. Bundles of intermediate filaments approached and asso- stitution appeared to provide more information than ciated with the desmosome (open arrows).Asterisks indicate a mesh- those obtained with replicas of chemically fixedlglycerwork of fine fibrils. x 93,000. Bar = 0.2 km. inated or physically fixedldeep-etched materials. Further immunocytochemical studies using tissues prepared in this manner (Ichikawa et al., 1989) will be thin filaments and interwoven fibrils reinforcing the required to clarify the nature of the structures revealed by the present method. plasmalemma at the fasciae should also be clarified. In embryonic guinea pig myocardium, desmosomes We observed physically fixed desmosomes, which were cut perpendicularly and tilted so as t o expose the associated with a pair of facsimile lines, which are in trilaminar unit membranes of the two apposing halves. turn apposed to the sarcoplasmic reticulum, have been The fine structures can also be more clearly demon- described (Forbes and Sperelakis, 1975). Such strucstrated by this method than by the conventional chem- tures were not found in embryonic hearts in the ical fixation with or without the lanthanum treatment present and other studies (Hirakow and Sugi, 1990; method (Forbes and Sperelakis, 1975; Kelly, 1966; Manasek, 1968, 1970; Muir, 1957; Rash et al., 1970; Rayns et al., 1969);they include the central lamina and Spira, 1971). They appear to be formed in the embryos
Fig. 12. Bundles of intermediate filaments bridged desmosomes formed in a series at the lateral cell boundary (open arrows). x 65,000. Bar = 0.2 pm.
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Fig. 13. Structural features of intercellular junctions, based on freeze-substitution observations of the embryonic chick myocardium. In the intercellular space of the fascia adherens (left), bridging structures (small black arrowheads) with central thickenings (small white arrowheads) are present. Thin filaments (larger black arrowheads) from myofibrils of the terminal sarcomere attach to the fibrous mat (*), which reinforces the junctional plasmalemma. Fine fibrils (small arrows) crosslink terminal thin filaments. In the intercellular space of
the desmosomes (right), central laminae (small white arrowheads) and traversing filaments (small black arrowheads) are seen. Intermediate filaments (open arrows) associate laterally with the fibrous mat (*). 1, external leaflet of the desmosomal plasmalemma; 2, internal leaflet; 3, electron-lucent layer; 4, desmosomal plaque with uniform electron density; 5, desmosomal protuberances with less electron density.
of certain species or may be too transient during development to be detected.
Fawcett, D.W.and McNutt, N.S. (1969) The ultrastructure of the cat myocardium. I. Ventricular papillary muscle. J . Cell Biol., 42:l-45. Forbes, M.S. and Sperelakis, N. (1975) The “imaged-desmosome”:A component of intercalated discs in embryonic guinea pig myocardium. Anat. Rec., 133:243-257. Hamburger, V. and Hamilton, H.L. (1951) A series of normal stages in development of the chick embryo. J . Morphol., 8849-92. Heuser, J.E., Reese, T.S., Dennis, M.J., Jan, Y., Jan, L., and Evans, L. (1979) Synaptic vesicle exocytosis captured by quick freezing and correlated with quanta1 transmitter release. J . Cell Biol., 81:275300. Hirakow, R. and Sugi, Y. (1990) Intercellular junction and cytoskeleton organization in embryonic chick myocardial cells. In: Developmental Cardiology: Morphogenesis and Function. E.B. Clark and A. Takao, eds. Futura, Mount K i m , NY, pp. 95-113. Hirokawa, N. and Kirino, T. (1980) An ultrastructural study of nerve and glial cells by freeze-substitution. J. Neurocytol., 5395-406. Hirokawa, N. and Heuser, J.E. (1981) Quick-freeze, deep-etch visualization of the cytoskeleton beneath surface differentiations of intestinal epithelial cells. J . Cell Biol., 91:399-409. Ho, E. and Shimada, Y.(1978) Formation of the epicardium studied with the scanning electron microscope. Dev. Biol., 66:579-585.
ACKNOWLEDGMENTS This work was supported by grants from the following: General Scientific Research (B02454109)and Special Project Research (59116003) of the Japanese Ministry of Education, Science, and Culture; the National Center of Neurology and Psychiatry (NCNP) of the Japanese Ministry of Health and Welfare; the Uehara Memorial Foundation; the Japan Cardiovascular Research Foundation; and the Kazato Foundation. The authors wish to thank Mrs. K. Shimizu and Mr. N. Nakamura for their technical assistance. REFERENCES Benshalon G. and Reese T.S. (1985) Ultrastructural observations on the cytoarchitecture of axons processed by rapid-freezing and freeze-substitution. J. Neurocytol., 14:943-960.
CELL JUNCTIONS IN EMBRYONIC CARDIAC MUSCLE Ichikawa. M., Sasaki, K., and Ichikawa, A. (1989) Immunocytochemical localization of amylase in gerbil salivary gland acinar cells processed by rapid freezing and freeze-substitution fixation. J . Histochem. Cytochem., 37:185-194. Kelly, D.E. (1966) Fine structure of desmosomes, hemidesmosomes and an adepiderdemal globular layer in developing newt epidermis. J . Cell Biol., 2851-72. Kelly, D.E., and Kuda, A.M. (1981) Traversing filaments in desmosoma1 and hemidesmosomal attachments: Freeze-fracture approaches toward their characterization. Anat. Rec., 199:l-14. Kurkiewicz, T. (1909) Zur Kenntnis der Histogenese des Herzmuskels der Wirbeltiere. Bull. Acad. Sci. Cracovie, 1909:148-191. Manasek, F.J. (1968) Embryonic development of the heart. I. A light and electron microscopic study of myocardial development in the early chick embryo. J . Morphol., 125:329-366. Manasek, F.J. (1969) Embryonic development of the heart. 11. Formation of the epicardium. J . Embryol. Exp. Morphol., 22:333-348. Manasek, F.J. (1970) Histogenesis of the embryonic myocardium. Am. J . Cardiol., 25149-168. McNutt, N.S. (1970) Ultrastructure of intercellular junctions in adult and developing cardiac muscle. Am. J . Cardiol., 25169-183. McNutt, N.S. and Fawcett, D.W. (1969) The ultrastructure of the cat myocardium. 11. Atrial muscle. J . Cell Biol., 42:46-67. Muir, A.R. (1957) An electron microscope study of the embryology of the intercalated disc in the heart of the rabbit. J . Biochem. Biophys. Cytol., 3:193-202. Rash, J.E., Biesel, J.J., and Gey, G.O. (1970) Three classes of filaments in cardiac differentiation. J. Ultrastruct. Res., 33:408-435. Raviola, E.R., Goodenough, D.A., and Raviola, G. (1980) Structure of rapid frozen gap junctions. J. Cell Biol., 87:273-279.
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Rayns, D.G., Simpson, F.O., and Ledingham, J.M. (1969) Ultrastructure of desmosomes in mammalian intercalated disc: Appearance after lanthanum treatment. J. Cell Biol., 42:322-326. Reese, R.P. and Reese, T.S. (1981) New structural features of freezesubstituted neuritic growth cones. Neuroscience, 6:247-254. Shimada, Y. and Ho, E. (1980) Scanning electron microscopy of the embryonic heart: Formation of the epicardium and surface structure of the four heterotype cells that constitute the embryonic heart. In: Etiology and Morphogenesis of Congenital Heart Disease. R. Van Praagh and A. Takao, eds. Futura, Mount Kisco, NY, pp. 63-80. Shimada, Y., Ho, E., and Toyota, N. (1981) Epicardial covering over myocardial wall in the chicken embryo as seen with the scanning electron microscope. Scan. Electron Microsc. 1981/11:275-280. Shimoyama, Y ., Hirohashi, S., Hirano, S., Noguchi, M., Shimosato, Y., and Takeichi, M. (1989) Cadherin cell-adhesion molecules in human epithelial tissue and carcinomas. Cancer Res., 49:21282133. Spira, A.W. (1971) Cell junctions and their role in transmural diffusion in the embryonic chick heart. Z. Zellforsh., 120:463-487. Takeichi, M. (1988) The cardherins: Cell-cell adhesion molecules controlling animal morphogenesis. Development, 102:639-655. Tatsuoka, H. and Reese, T.S. (1989) New structural features of synapses in the anteroventral cochlear nucleus prepared by direct freezing and freeze-substitution. J . Comp. Neurol., 290:343-357. Volk, T. and Geiger, B. (1986) A-CAM: A 135 kD receptor of intercellular adherens junctions. I. Immunoelectron microscopic localization and biochemical studies. J . Cell Biol., 103:1441-1450.
Intercellular Junctions in Embryonic Chick Cardiac Muscle Revealed by Rapid Freezing and Freeze-Substitution MASAYUKI SHIOZAKI AND YUTAKA SHIMADA Department of Anatomy and Cell Biology, School of Medicine, Chiba University, Chiba 280, Japan
KEY WORDS
Cryofixation, Development, Fasciae adherentes, Desmosomes, Myocardium
ABSTRACT Using the method of rapid freezing and freeze-substitution, the embryonic chick cardiac muscle was investigated by transmission electron microscopy. Initially, the intercellular junctional complexes (fasciae adherentes and desmosomes) were formed in close proximity to each other along a nearly straight line. Subsequently, the separation of fasciae from desmosomes took place to form intercalated discs. The cell membranes of fasciae adherentes were reinforced with highly interwoven fine fibrils at which myofibrils terminated. The intercellular space of fasciae was bridged with fine fibrillar structures seemingly connected by a thin line at their middle portions. In the intercellular space of desmosomes, central lamina and traversing filaments were clearly observed. The outer and inner leaflets of the desmosomal plasmalemma were asymmetrically differentiated; the outer leaflet was thinner than the inner leaflet. On the inner side of the cell membrane, an electron-lucent layer and a dense desmosomal plaque were observed. The latter structure had protrusions with less electron density towards the cytoplasmic side. Further inside, a meshwork of fine fibrils was seen along and toward which bundles of intermediate filaments ran. The results obtained with freeze-substitution appeared to provide more information than those with thin sections after conventional fixation or with replicas of chemically fixedlglycerinated or physically fixedldeep-etched materials. INTRODUCTION and positioned on a specimen holder such that the The formation and elaboration of the cell iunctions outer surface of the heart faced a copper block cooled by characteristic of cardiac muscle have been studied by liquid helium (about 4°K).The specimens quick-frozen several investigators with the usual chemical fixation with a Polaron Slammer E7200 were placed into 2% methods (Forbes and Sperelakis, 1975; Manasek, 1968, OsO, in acetone at -79°C and substituted for 1.5-2.5 1970; McNutt, 1970; Muir, 1957; Rash et al., 1970; days. They were then warmed in steps; at -20°C for 2 Spira, 1971). Recently, rapid freezing followed by hr, at 4°C for 2 hr, and finally at room temperature for freeze-substitution has proved useful for observing the 2 hr. They were then soaked in 100% methanol. Some biological ultrastructure, since cryofixed materials are preparations were stained en bloc with 2-5% uranyl closer to the native state than those prepared by chem- acetate in 100% methanol (Raviola et al., 1980)for 2 hr ical fixation (Heuser et al., 1979; Hirokawa and Kirino, and then 0.2-1.0% hafnium chloride (Benshalon and Reese, 1985; Tatsuoka and Reese, 1989) in 100%meth1980; Reese and Reese, 1981). The outer myocardial surface of the mature heart is anol for 0.5-3.0 hr. All specimens were passed through covered with epicardial cells and connective tissue, but propylene oxide and embedded in Epon 812. Thin secthat of early embryos is uncovered or covered only with tions were cut parallel to the contact surface. The seca single layer of epicardial cells (Ho and Shimada, tions were double-stained with uranyl acetate and lead 1978; Kurkiewicz, 1909; Manasek, 1969; Shimada and citrate and examined with a JEOL JEM 1200 EX11 Ho, 1980; Shimada et al., 19811, making procedures electron microscope operating at 80 kV. Stereopairs of such as slicing the tissue with a razor blade or a tissue electron micrographs were taken by tilting the grid chopper to expose the concerned areas unnecessary. 27". This material is therefore ideally suited for examinaRESULTS tion with rapid freezing by the metal contact method. Good freezing was obtained to a depth of approxiWe have reinvestigated these structures based on the apparent contribution of the newly developed method mately 15 pm from the metal contact surface of the heart in this study. Since the epicardial cells were eiof physical fixation followed by freeze-substitution. ther absent at most of the surface areas on the myoMATERIALS AND METHODS Fertile white Leghorn eggs were incubated a t 37°C for 2-5 days to yield embryos ranging from stages 12 to 26 of Hamburger and Hamilton (1951). The entire emReceived June 6, 1991; accepted in revised form July 12, 1991. bryos were excised and placed in phosphate-buffered Address reprint requests to Yutaka Shimada, Department of Anatomy, School saline (PBS). Their hearts were quickly dissected out of Medicine, Chiba University, Chiba 280, Japan.
0 1992 WILEY-LISS, INC.
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Figs. 1-3. Intercellular junctions of embryonic chick cardiac muscle at early stages. Initially, fasciae adherentes (black arrows and double arrows) and desmosomes (white arrows) were formed near each other along a nearly straight line. Early fasciae (double arrows
in Figs. 1 and 2) had no relation to myofibrils (Mf), but they later (black arrows in Figs. 2 and 3) became associated with developing myofibrils (arrowheads in Figs. 2 and 3). Fig. 1, ~ 7 3 , 0 0 0 ;Fig. 2, x 78,000; Fig. 3, x 63,000. Bar = 0.2 wm.
CELL JUNCTIONS IN EMBRYONIC CARDIAC MUSCLE
Figs. 4 and 5. Stereopair micrographs of fasciae adherentes (black arrows) and desmosomes (white arrows). Figure 4 is a higher magnification of the rectangle in Figure 3. Small bundles of filaments (arrowheads) separated from myofibrils (Mf) terminated at fasciae at acute angles. Bundles of intermediate filaments (open arrows) were associated with desmosomes. Fig. 4, x 95,000; Fig. 5 , x 50,000. Bar = 0.2 bm.
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Fig. 6. Somewhat matured muscle possessing step-type intercalated discs. Fasciae adherentes (black arrows) situated at the traversing segment and desmosomes (white arrows) on the longitudinal segment of the cells. Myofibrils (Mf) were connected with fasciae almost at right angles (arrowheads). X 37,000. Bar = 0.5 &m.
cardium (at and before stage 17) or still very thin (less than 10 bm at stage 26; Manasek, 1969), both the muscle directly contacted by the metal and that frozen through the epicardium were well preserved morphologically. Wide areas of well-frozen myocytes could be observed by sectioning the tissues tangentially to the surface. During this period, although the development of the myocardium generally proceeded steadily, the myocardial cells at varying stages of differentiation were always encountered in any given heart and, even within a single cell, myofibrils a t various stages of development were also found (see also Manasek, 1968, 1970). In the early myocardium, the cardiac myocytes were joined to each other by fasciae adherentes and desmosomes at points where they came into contact. Initially, these junctional structures were small in size and were formed in close proximity to each other in a nearly straight line along the lateral cell borders (Figs. 1-5). Subsequently, each junctional structure was enlarged and the separation of fasciae from desmosomes took place. Although desmosomes remained a t the lateral cell surface, fasciae became oriented at right angles to the longitudinal axis of the cells and, thus, step-type junctional specializations (intercalated discs) characteristic of the myocardium were formed (Fig. 6). Early fasciae adherentes, which could be recognized by dense material along the inner aspects of the plasma membranes, had no structural relation with nascent myofibrils (Figs. 1, 2). They soon became associated with small bundles of thin filaments, which were separated from developing myofibrils. They approached and terminated a t the fasciae a t an acute angle (Figs. 2-5). As the fasciae changed their position relative to desmosomes, myofibrils came to be connected with the fasciae at oblique or nearly right angles (Fig. 6).
The sarcoplasmic surface of the membranes confronted a t the fasciae adherentes was reinforced with a layer (thickness: 20-60 nm) of interwoven fine fibrils (diameter: 7-15 nm). The thin filaments of the terminal sarcomeres were seen to end in this fibrous mat of apposing cells (Figs. 3 - 5 , 7 , 8 ) . Between thin filaments near their insertion, crosslinking fibrillar structures (diameter: 7-18 nm) were observed. The intercellular space of the junctions was 30-40 nm in width and was bridged by fine fibrils (diameter: less than 4 nm) (Figs. 7, 8). These fine fibrillar bridges were thickened in their middle portion, thus appearing to form a thin line that connected them at their centers (Fig. 8). The opposing cell surfaces a t the desmosomal areas were about 30 nm apart (Figs. 2-5, 9-12). In their intercellular space, the central lamina (thickness: less than 3 nm) and fine traversing filaments (diameter: less than 2 nm) were clearly seen (Figs. 4, 9-11). Intersections of these two structures were thickened (Figs. 4, 9, 10). The external and internal leaflets of the desmosomal plasmalemma were asymmetrically differentiated. The outer leaflet, 2-4 nm thick, was a thin line possessing thickenings to which the traversing filaments appeared to attach. The inner leaflet, 3-5 nm thick, was a continuous dense line. On the inner surface of the desmosomal membrane, an electron-lucent layer 4-8 nm thick was present. On the cytoplasmic side of this layer, an electron-dense, desmosomal plaque 20-30 nm thick was seen. Its outer portion was a uniformly dark layer (thickness: 4-6 nm), while the inner part consisted of small protuberances (height: 15-25 nm) extending from the former and possessing less electron density. Some traversing filaments appeared to reach the desmosomal plaque through the plasmalemma and the electron-lucent layer (Fig. 9).
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Figs. 7 and 8. Higher magnification of fascia adherens. On the cytoplasmic side of the plasmalemma, a fibrous mat (*I was seen, at which thin filaments (large arrowheads) of myofibrils (Mf) terminated. Between these thin filaments, crosslinking fibrils (black arrows) were visible. In the intercellular space of the fascia, bridgingstructures (small arrowheads) were seen. When properly oriented and cut, thickenings of bridges at their middle portions were visible (small white arrowheads in Fig. 8). Fig. 7, x 98,000. Bar = 0.2 km; Fig. 8, x 140,000.Bar = 0.1 km.
Further into the cytoplasmic side of the plaque, a meshwork of fine fibrils (diameter: about 5 nm) was present. This meshwork was 70-90 nm thick and became more evident in en bloc preparations stained with uranyl acetate and hafnium chloride (Figs. 4, 10-12). Intermediate filaments in the cytoplasm converged near the desmosomes to form bundles. These bundles ran parallel to the desmosomal plaque and associated laterally with the fine fibrillar lattice. Occasionally, intermediate filaments bridging neighboring desmosomes were seen where a series of desmosomes were formed (Fig. 12). Figure 13 shows a structural model of a fascia adherens and desmosomes based on the present observations by rapid freezing and freeze-substitution of embryonic cardiac muscle. DISCUSSION In support of the assumptions of previous studies with chemical fixation (Forbes and Sperelakis, 1975; Manasek, 1968,1970; McNutt, 1970; Muir, 1957; Rash et al., 1970;Spira, 1971), the present study (by physical fixation followed by freeze-substitution) has demonstrated that membranes of myocytes are initially closely apposed where minute amounts of dense materials are associated. In these areas, fasciae adherentes and desmosomes can be distinguished and are seen to be formed side by side along the lateral cell boundaries. Cells with more extensive contacts demonstrate that
Fig. 8.
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Figs. 9 and 10. Higher magnification of desmosomes without (Fig. 9) and with (Fig. 10)en bloc staining. In the intercellular space, central laminae (small white arrowheads) and traversing filaments (small black arrowheads) were discernible. The external leaflet (1) of the desmosomal plasmalemma was thinner than the internal leaflet (2). Traversing filaments reached the desmosomal plaque through the plasmalemma and the electron-lucent layer (3). From the uniformly
dark, outer part (4) of the desmosomal plaque, processes with less electron density (5) extended. On the cytoplasmic side of the desmosoma1 plaque, a meshwork of fine fibrils (*) was seen. Bundles of intermediate filaments (open arrows) converged on the cytoplasmic side of the meshwork. Fig. 9, x 290,000; Fig. 10, x 120,000. Bar = 0.1 Fm.
sarcomeric thin and intermediate filaments attach to the respective junctional structures. Later, fasciae adherentes are oriented at the transverse segments and desmosomes on the longitudinal segments of the intercalated discs. Initially, myofibrils insert into developing fasciae adherentes at acute angles, but during development reorientation occurs so that fibril insertion becomes approximately 90". It appears likely that contractile forces, which are transmitted from one cell to another via the fasciae adherentes while cells are attached to each other at their lateral sides with desmosomes, are responsible for this reorientation. A detail of the region a t the fasciae adherentes,
which does not seem to have been noticed before by the usual chemical fixation (Fawcett and McNutt, 1969; McNutt and Fawcett, 19691, was the occurrence of bridging structures between the membranes confronted, with a thin line connecting the center of these bridges. In quick-freeze, deep-etch replicas of an adherens junction of mouse intestinal epithelial cells, similar bridging structures have been observed (Hirokawa and Heuser, 19811, but the central lines have not been noted. It is necessary to clarify whether these structures are associated with cell-adhesion-relatedproteins such as A-CAM (Volk and Geiger, 19861 or cadherins (Shimoyama et al., 1989; Takeichi, 1988). Furthermore, the nature of the crosslinking fibrils between
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traversing filaments in the intercellular space, the electron-lucent layer between the sarcolemma and the electron-dense desmosomal plaque, and protrusions of desmosomal plaques with less electron density. The extension of traversing filaments in the intercellular space to the desmosomal plaque through the sarcolemma and the electron-lucent layer has not been reported before. Freeze-fracture or freeze-fractureldeepetch methods (Hirokawa and Heuser, 1981; Kelly and Kuda, 19811, although revealing the traversing filaments in desmosomes, failed to demonstrate the central lamella, which is one of the features that has been used to distinguish this type of junction in thin-sectioned materials. Thus, by using the method of rapid freezing and freeze-substitution, the present study revealed intercellular junctional structures that had not been noted clearly before by the usual chemical fixation. Since it is generally presumed that the cryofixed cells are closer to the native state than those prepared by chemical fixation (Heuser et al., 1979; Hirokawa and Kirino, 1980; Reese and Reese, 1981),the present features possibly represent a more natural morphology of junctional complexes in the embryonic chick heart. Some of the intercellular structures prepared after freeze-subFig. 11. Bundles of intermediate filaments approached and asso- stitution appeared to provide more information than ciated with the desmosome (open arrows).Asterisks indicate a mesh- those obtained with replicas of chemically fixedlglycerwork of fine fibrils. x 93,000. Bar = 0.2 km. inated or physically fixedldeep-etched materials. Further immunocytochemical studies using tissues prepared in this manner (Ichikawa et al., 1989) will be thin filaments and interwoven fibrils reinforcing the required to clarify the nature of the structures revealed by the present method. plasmalemma at the fasciae should also be clarified. In embryonic guinea pig myocardium, desmosomes We observed physically fixed desmosomes, which were cut perpendicularly and tilted so as t o expose the associated with a pair of facsimile lines, which are in trilaminar unit membranes of the two apposing halves. turn apposed to the sarcoplasmic reticulum, have been The fine structures can also be more clearly demon- described (Forbes and Sperelakis, 1975). Such strucstrated by this method than by the conventional chem- tures were not found in embryonic hearts in the ical fixation with or without the lanthanum treatment present and other studies (Hirakow and Sugi, 1990; method (Forbes and Sperelakis, 1975; Kelly, 1966; Manasek, 1968, 1970; Muir, 1957; Rash et al., 1970; Rayns et al., 1969);they include the central lamina and Spira, 1971). They appear to be formed in the embryos
Fig. 12. Bundles of intermediate filaments bridged desmosomes formed in a series at the lateral cell boundary (open arrows). x 65,000. Bar = 0.2 pm.
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Fig. 13. Structural features of intercellular junctions, based on freeze-substitution observations of the embryonic chick myocardium. In the intercellular space of the fascia adherens (left), bridging structures (small black arrowheads) with central thickenings (small white arrowheads) are present. Thin filaments (larger black arrowheads) from myofibrils of the terminal sarcomere attach to the fibrous mat (*), which reinforces the junctional plasmalemma. Fine fibrils (small arrows) crosslink terminal thin filaments. In the intercellular space of
the desmosomes (right), central laminae (small white arrowheads) and traversing filaments (small black arrowheads) are seen. Intermediate filaments (open arrows) associate laterally with the fibrous mat (*). 1, external leaflet of the desmosomal plasmalemma; 2, internal leaflet; 3, electron-lucent layer; 4, desmosomal plaque with uniform electron density; 5, desmosomal protuberances with less electron density.
of certain species or may be too transient during development to be detected.
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ACKNOWLEDGMENTS This work was supported by grants from the following: General Scientific Research (B02454109)and Special Project Research (59116003) of the Japanese Ministry of Education, Science, and Culture; the National Center of Neurology and Psychiatry (NCNP) of the Japanese Ministry of Health and Welfare; the Uehara Memorial Foundation; the Japan Cardiovascular Research Foundation; and the Kazato Foundation. The authors wish to thank Mrs. K. Shimizu and Mr. N. Nakamura for their technical assistance. REFERENCES Benshalon G. and Reese T.S. (1985) Ultrastructural observations on the cytoarchitecture of axons processed by rapid-freezing and freeze-substitution. J. Neurocytol., 14:943-960.
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