Cell Tissue Res (1992) 270:37%382
Cell&Tissue Research @ Springer-Verlag 1992
Embryonic chicken gizzard: immunolocalization of collagen and smooth muscle myosin Elke R. Paul, Truc Linh Vo, Andreas Meyer, and Ute Gr6schel-Stewart Institut ffir Zoologie, Technische Hochschule Darmstadt, Schnittspahnstrasse 10, W-6100 Darmstadt, Federal Republic of Germany Received October 23, 1991 / Accepted July 1, 1992
Summary. Antibodies to chicken gizzard myosin and to chicken skin collagen type I allow the myofibrillar and connective tissue development in the embryonic chicken gizzard to be followed. Fibroblasts are assumed to synthesize collagen prior to the onset of smooth muscle cell development in the muscle primordium (day 5); they are presumably also responsible for collagen synthesis close to the presumptive lamina propria and in the developing tubular glands (day 14 to 17). From day 6 to 8, myosin and collagen are colocalized intracellularly, and from day 9 onward collagen fibers start to appear extracellularly, eventually forming the trellis-like connective tissue septa that give the rhomboid profile found in the adult muscle. The close association of collagen and myosin in early development suggests that the muscle cells themselves produce and export collagen.
ment. Bennet and Cobb (1969) have reported that connective tissue fibers begin to delineate the myoblasts into bundles on day 9; their light-microscopic studies have been supported by electron microscopy (Gabella 1989), where small bundles of collagen fibers have been observed in the connective tissue septa around day 11. These studies have not solved the problem regarding the site of the synthesis of collagen; possible sites are the septal fibroblasts or the smooth muscle cells. In this paper, we describe the immunolocalization of collagen in the developing chicken gizzard, and the temporal and spatial relationship between the appearance of the intracellular contractile protein myosin and the extracellular matrix protein collagen, using a double immunofluorescent technique. Preliminary observations have been reported (Paul et al. 1991).
Key words: Gizzard - Embryo - Collagen - Myosin Antibodies - Chicken
Materials and methods Tissue processing
Chicken gizzard is a frequently used source of smooth muscle proteins for biochemical studies, and, in addition, an important organ for the study of the biochemical differentiation of smooth muscle (Saborio et al. 1979; Hirai and Hirabayashi 1983, 1986; Stuewer and Gr6schel-Stewart 1985; Kawamoto and Adelstein 1987; Yanagisawa etal. 1987). Using antibodies to adult smooth muscle myosin, we have observed small foci of immunoreactive myosin in the mesenchyme of the presumptive chicken gizzard as early as day 5/6 in ovo (Stuewer and Gr6schel-Stewart 1985), several days before thick myosin filaments become visible (Bennett and Cobb 1969) or spontaneous contraction can be recorded (Donahue and Bowen 1972). Since the stroma of smooth muscle presumably plays a mechanical role in the transmission of force, it is of great interest to follow the appearance o f collagen and collagen fibers during develop-
Correspondence to . U. Gr6schel-Stewart
Fertilized eggs of White Leghorn chickens were incubated at 3738° C, and embryos were harvested daily from day 5 to day 15 of incubation. Their gizzards were excised and immediately frozen in 2-methylbutane pre-cooled in liquid nitrogen. They were sectioned transversely and longitudinally at a thickness of 6 ~tm in a cryostat, the sections were then collected on gelatine-coated slides and air-dried. Small cubes of adult chicken gizzard muscle were treated correspondingIy.
Antigens and antibodies The isolation of myosin from adult chicken gizzard smooth muscle, and the production and the specificity of the respective antibodies have been described (Gr6schel-Stewart et al. 1985, 1989). Collagen type I was extracted from adult chicken skin following the method of Chung and Miller (1974). Interrupted polyacrylamide electrophoresis (Sykes et al. 1976), in which in situ reduction converts e(III) polymers into monomers, has shown that skin collagen consists of cq(I) and e2(I) chains only (Fig. 1a). Antibodies were obtained after multiple injections of 0.2 mg of this type I collagen into rabbits. In immunoblots, they reacted with the ~1(I) and the
378
Fig. 1. Polyacrylamide gel electrophoresis on 6% gels (a, c, d) and immunoblotting with anti-collagen type I (b, e) of collagen extracted from adult chicken skin (a, b) and embryonic chicken gizzard muscle (c, d, e), using non-interrupted (c) and interrupted (a, d) electrophoresis with in situ reduction to allow separation of cq(III) chains, a Electrophoresis pattern of adult skin with cq(I) and c~2(I) chains; b the immunoblot thereof, c Electrophoresis pattern of non-reduced embryonic gizzard collagen with el(I) and ez(I) chains; d the same after reduction, showing the el(III) chain; e the immunoblot thereof
c~2(I) chains (Fig. 1b). Collagen isolated from embryonic chicken gizzard (day 17) showed the presence of el(I), e2(I) and cq(III) chains after interrupted electrophoresis (Fig. 1c, d), only the el(I) and ~2(I) chain, but not the el(III) chain, reacted with the above antiserum (Fig. 1e). The IgG fractions of immune and control sera were isolated according to Harboe and Ingild (1973). Anti-smooth muscle myosin was labeled with the fluorochrome tetramethylrhodamine isothiocyanate (TRITC) following the method of Hudson and Hay (1989).
aggregates that eventually become separated from each other by the increased formation of imbricated collagen nets (Fig. 2d, e). As the collagen secretions into the extracellular space increase, the intracellular collagen immunoreactivity decreases. The muscle cells in the bundles become more tightly packed, myosin immunoreactivity increases and the surrounding connective tissue septa thicken. By day 15, the typical rhomboid profile seen in adult muscle is reached (Fig. 2f). There are occasional collagen fibers in the spaces between individual muscle cells. In the adult muscle, the intercellular gaps are filled with collagen fibers, forming a fine network that enmeshes individual cells and groups of cells, and eventually merges with the collagen of the intramuscular septa (Fig. 2g): In addition to the myosin/collagen-reactive areas, a narrow continuous band of collagen-reactive (and myosin-negative) cells can be seen in longitudinal sections of day-8 gizzards close to the luminal epithelium (Fig. 3). This band persists throughout development. On days 13/14, when the smooth muscle cells have expanded throughout the presumptive gizzard mesenchyme (as seen in sections at day 17; Fig. 4d), the intramuscular connective tissue septa appear to merge with this band (Fig. 4a-c). The micrographs also show that, on day 14, the collagen immunoreactivity appears to expand into the glandular epithelial layer, reaching the apical cells on day 17 (Fig. 4 a-d).
Discussion
Immunohistochemistry For double-labelingimmunofluorescence,the unfixed sections were treated as follows: incubation with anti-collagen IgG (0.1 mg/ml) for 30 min, and visualization with fiuorescein isothiocyanate (FITC)-labeled goat-anti-rabbit IgG (1:80; Sigma, Deisenhofen, FRG). Photographs of the anti-collagen staining were taken using Kodak Tri-X Pan. After removal of the coverslip, the free binding sites on the goat-anti-rabbit IgG were saturated by incubation with rabbit non-immune IgG (2 mg/ml) for 60 rain. Myosin was then visualized with TRITC-labeled anti-smooth muscle myosin (0.15 mg/ml) for 30 rain; anti-myosin reactivity was photographed in identical areas that were documented before.
Results
On day 5 in ovo, small fibrillar structures reactive with antibodies to chicken collagen type I are scattered throughout the gizzard primordium (Fig. 2 a); anti-myosin-reactive cells are not visible. From day 6 to day 8, first islets, then narrow bands of cells in the central part of the mesenchyme are immunoreactive for both collagen and myosin. The paired micrographs in Fig, 2b show that the two antigens are colocalized in a narrow cytoplasmic rim, with the unstained nucleus still occupying most of the cell. Structural reorganizations become evident from day 9 onward in transverse sections of the myosin/collagen-reactive areas. Small collagen fibers start to appear in the extracellular space (Fig. 2c), and the smooth muscle cells begin to assemble into tuft-like
According to Bennet and Cobb (1969), the most active period of cell division in the embryonic chicken gizzard is around day 6, when the cells of the primordium are still undifferentiated and indistinguishable from fibroblasts. At this stage (day 5), randomly dispersed collagen fibers can be visualized with antibodies to chicken collagen I. Since cells immunoreactive to smooth muscle myosin are not yet seen at this stage, one must assume that the collagen is synthesized by fibroblasts or undifferentiated myoblast precursors. The same must apply to the luminal collagen-immunoreactive band; these cells seem to be responsible for the formation of the prominent lamina propria of the adult muscle. According to Cornselius (1925), the pseudo-stratified columnar cells of the luminal epithelium start to invaginate into the subjacent connective tissue layer (the later lamina propria) on day 14. Longitudinal clefts appear from day 15 onward; they initiate the formation of tubular glands. By days 17/18, these glands have a single layered epithelium, and are deeply immersed in the connective tissue. The narrow spaces between the individual glands are filled with a network of collagen fibers. Thus, the inward movement of the tubular glands, rather than an expansion of collagen-immunoreactivity toward the apical cells, must be responsible for the staining patterns seen in Fig. 4. The sudden onset of the synthesis of all major contractile proteins (Saborio et al. 1979; Hirai and Hirabayashi 1983, 1986; Stuewer and Gr6schel-Stewart
379
Fig. 2a-g. Paired micrographs of transverse sections of fresh frozen gizzards sequentially stained with a-collagen type I (a-col; indirect method, FITC-label) and a-smooth muscle myosin (a-myo; direct method, TRITC-label). Identical areas are indicated by arrows, a Day-5 embryo shows disperse collagen staining only (a-col); b day-7 embryo exhibit intracellular codistribution of collagen (aco[) and myosin (a-myo); c in day-9 embryos, small extracellnlar collagen fibers appear; d-f between clay 11 to day 15, trellis-like collagen networks form; g adult muscle shows the intramuscular connective tissue septa and fine collagen network around each ceil. × 270
380
1985), presumably following the peak of cell division, is in accordance with the observation of Gabella (1989) that all smooth muscle cells develop uniformly and that all have the same degree of differentiation, with no intermediate forms being visible. In a subsequent paper (Paul et al., in preparation) we will show however that the smooth muscle cells appear to synthesize both muscular and cytoplasmic myosin at an early stage. Extracellular collagen fibers were first seen by Bennett and Cobb (1969) on day 9, and on day 11 in electron micrographs (Gabella 1989). Using collagen antibodies, we can follow the assembly of muscle bundles by trellis-like connective tissue septa from day 9 to the adult stage. By day 15, the adult rhomboid pattern is reached (Blessing and Miiller 1974) and the only further change seen is an additional fine collagen network surrounding all individ-
ual muscle cells in the adult. Likewise, Gabella (1989) describes a slightly larger number of collagen fibers in the post-hatching period. Extensive studies have been performed on collagen types of embryonic skeletal muscle. Chicken myoblasts have been shown by biochemical and immunological methods to synthesize collagen types I and III in vitro (Bailey et al. 1979) and to participate in the deposition of extracellular connective tissue fibers in skeletal muscle (Sasse et al. 1981). With specific anti-type I and III collagen sera, Duance et al. (1977) have localized type I mainly in the epimysium, and type [II preferentially in the perimysium of bullock muscle. Cultured smooth muscle cells (Chamley-Campbell et al. 1979) and developing rat aorta (Ross and Klebanoff 1971) produce collagen types I and III in varying proportions. In the adult intestinal
381
Fig. 3. Paired micrographs of longitudinal sections of day-8 embryonic gizzard stained with a-collagen (a-col) and a-myosin (a-myo). Note the additional collagen-positive band close to the lumen (L). Composite picture, x 55
Fig. 4a-d. Micrographs of longitudinal sections of embryonic chicken gizzard (luminal part), stained with a-collagen type I (a-col) and a-myosin (a-rnyo). a Da2-14 embryo shows collagen-immunoreactivity in the intramuscular septa (is), the future lamina propria (lp) and the basal part of the glandular epithelium (ge); b in day-16 embryos, progression of the epithelial staining is seen; c a day-17 embryo exhibits collagen reactivity throughout ge; d a corresponding section staining with a-myosin (a-rnk,o). × 270
382 muscle, type I a n d I I I are also the m o s t a b u n d a n t species (Epstein a n d M u n d e r l o h 1975). Since fibroblasts in culture also simultaneously synthesize type I and I I I collagen ( G a y et al. 1976), it is n o t surprising that the embryonic chicken gizzard exhibits b o t h collagen types, as d e m o n s t r a t e d in this paper. The e m b r y o n i c cq(I) a n d c~2(I) chains, b u t n o t the cq(III) chains, have been shown by i m m u n o b l o t t i n g to react with the antibodies to adult chicken skin collagen type I; this justifies the use o f these antibodies for the localization o f collagen in the developing gizzard. A l t h o u g h the nature o f the link between the collagen fibers and the muscle cells is n o t k n o w n , it is generally assumed t h a t their intimate association is essential for binding the s m o o t h muscle cells together and for the transmission o f tension generated by the myofilaments (Mullins and G u n t h e r o t h 1965).
Acknowledgements. This work was supported by the Deutsche Forschungsgemeinschaft (Ste 105/25-2). We wish to thank Ms. Anna-Luise Christian, Ms. Renate Franke, and Ms. Ruth Rohr for their able assistance.
References Bailey AJ, Shellswell GB, Duance CV (1979) Identification and change of collagen types in differentiating myoblasts and developing chick muscle. Nature 278 : 67-68 Bennett T, Cobb JLS (1969) Studies on the avian gizzard: morphology and innervation of the smooth muscle. Z Zellforsch 96:173-185 Blessing MH, Mfiller G (1974) Myoglobin concentration in the chicken, especially in the gizzard (a biochemical, light and electron microscopic study). Comp Biochem Physiol [A] 47:535540 Chamley-Campbell J, Campbell GR, Ross R (1979) The smooth muscle cell in culture. Physiol Rev 59 : 1-61 Chung E, Miller EJ (1974) Collagen polymorphism: characterization of molecules with the chain composition [~l(III)]s in human tissuesl Science 183 : 1200-1201 Cornselius C (1925) Morphologie, Histologie und Embryologie des Muskelmagens der V6gel. Gegenbaurs Morphol Jahrb 54: 507559 Donahoe JR, Bowen JM (1972) Analysis of the spontaneous motility of the avian embryonic gizzard. Am J Vet Res 33:1835-1848 Duance VC, Restall DJ, Beard H, Bourne FJ, Bailey AJ (1977) The location of three collagen types in skeletal muscle. FEBS Lett 79 : 248-252
Epstein EH, Munderloh NH (1975) Isolation and characterization of CNBr peptides of human [~l(III)] 3 collagen and tissue distribution of [~l(III)]3 collagens. J Biol Chem 250:9304-9312 Gabella G (1989) Development of smooth muscle: ultrastructural study of the chick embryo gizzard. Anat Embryol 180:213-226 Gay S, Martin GR, Miiller PK, Timpl R, Kiihn K (1976) Simultaneous synthesis of types I and III collagen by fibroblasts in culture. Proc Natl Acad Sci USA 73:4037-4040 Gr6schel-Stewart U, Rakousky C, Franke R, Peleg I, Kahane I, Eldor A, Muhlrad A (1985) Immunohistochemical studies with antibodies to myosins from the cytoplasm and membrane fraction of human blood platelets. Cell Tissue Res 241 : 399-404 Gr6schel-Stewart U, Magel E, Paul E, Neidlinger AC (1989) Pig brain homogenates contain smooth muscle myosin and cytoplasmatic myosin isoforms. Cell Tissue Res 257:137-139 Harboe N, Ingild A (1973) Immunisation, isolation of immunoglobulins, estimation of antibody titre. Scand J Immunol 2:3541 Hirai S, Hirabayashi T (1983) Developmental changes of protein constituents in chicken gizzards. Dev Biol 97:483-493 Hirai S, Hirabayashi T (1986) Development of myofibrils in the gizzard of chicken embryos. Intracellular distribution of structural proteins and development of contractility. Cell Tissue Res 243 : 487-493 Hudson L, Hay FC (1989) Practical immunology, 3rd edn. Blackwell, Oxford London Edinburgh Kawamoto S, Adelstein RS (1987) Characterization of myosin heavy chains in cultured aorta smooth muscle cells. J Biol Chem 262:7282-7288 Mullins GL, Guntheroth WG (1965) A collagen net hypothesis for force transference of smooth muscle. Nature 206 : 592-594 Paul E, Lin Voh T, Gr6schel-Stewart U (1991) Immunolocalization of collagen in the developing chicken gizzard (abstract). Eur J Cell Biol 54:49 Ross R, Klebanoff SJ (1971) The smooth muscle cell. I. In vivo synthesis of connective tissue proteins. J Cell Biol 50:159-171 Saborio JL, Segure M, Flores M, Garca R, Palmer E (1979) Differential expression of gizzard actin genes during chick embryogenesis. J Biol Chem 254:11119-11125 Sasse J, Mark H, Ktihl U, Dessau W, Mark K (1981) Origin of collagen types I, III and V in cultures of avian skeletal muscle. Dev Biol 83:79-89 Stuewer D, Gr6schel-Stewart U (1985) Expression of immunoreactive myosin and myoglobin in the developing chicken gizzard. Roux's Arch Dev Biol 194:417-424 Sykes B, Puddle B, Francis M, Smith R (1976) The estimation of two collagens from human dermis by interrupted gel electrophoresis. Biochem Biophys Res Commun 72:1472-1479 Yanigasawa M, Hamada Y, Kasuragawa, Y, Imamura M, Mikawa T, Maskai T (1987) Complete primary structure of vertebrate smooth muscle myosin heavy chain deduced from its complementary DNA sequence. J Mol Biol 198:143-157
Cell&Tissue Research @ Springer-Verlag 1992
Embryonic chicken gizzard: immunolocalization of collagen and smooth muscle myosin Elke R. Paul, Truc Linh Vo, Andreas Meyer, and Ute Gr6schel-Stewart Institut ffir Zoologie, Technische Hochschule Darmstadt, Schnittspahnstrasse 10, W-6100 Darmstadt, Federal Republic of Germany Received October 23, 1991 / Accepted July 1, 1992
Summary. Antibodies to chicken gizzard myosin and to chicken skin collagen type I allow the myofibrillar and connective tissue development in the embryonic chicken gizzard to be followed. Fibroblasts are assumed to synthesize collagen prior to the onset of smooth muscle cell development in the muscle primordium (day 5); they are presumably also responsible for collagen synthesis close to the presumptive lamina propria and in the developing tubular glands (day 14 to 17). From day 6 to 8, myosin and collagen are colocalized intracellularly, and from day 9 onward collagen fibers start to appear extracellularly, eventually forming the trellis-like connective tissue septa that give the rhomboid profile found in the adult muscle. The close association of collagen and myosin in early development suggests that the muscle cells themselves produce and export collagen.
ment. Bennet and Cobb (1969) have reported that connective tissue fibers begin to delineate the myoblasts into bundles on day 9; their light-microscopic studies have been supported by electron microscopy (Gabella 1989), where small bundles of collagen fibers have been observed in the connective tissue septa around day 11. These studies have not solved the problem regarding the site of the synthesis of collagen; possible sites are the septal fibroblasts or the smooth muscle cells. In this paper, we describe the immunolocalization of collagen in the developing chicken gizzard, and the temporal and spatial relationship between the appearance of the intracellular contractile protein myosin and the extracellular matrix protein collagen, using a double immunofluorescent technique. Preliminary observations have been reported (Paul et al. 1991).
Key words: Gizzard - Embryo - Collagen - Myosin Antibodies - Chicken
Materials and methods Tissue processing
Chicken gizzard is a frequently used source of smooth muscle proteins for biochemical studies, and, in addition, an important organ for the study of the biochemical differentiation of smooth muscle (Saborio et al. 1979; Hirai and Hirabayashi 1983, 1986; Stuewer and Gr6schel-Stewart 1985; Kawamoto and Adelstein 1987; Yanagisawa etal. 1987). Using antibodies to adult smooth muscle myosin, we have observed small foci of immunoreactive myosin in the mesenchyme of the presumptive chicken gizzard as early as day 5/6 in ovo (Stuewer and Gr6schel-Stewart 1985), several days before thick myosin filaments become visible (Bennett and Cobb 1969) or spontaneous contraction can be recorded (Donahue and Bowen 1972). Since the stroma of smooth muscle presumably plays a mechanical role in the transmission of force, it is of great interest to follow the appearance o f collagen and collagen fibers during develop-
Correspondence to . U. Gr6schel-Stewart
Fertilized eggs of White Leghorn chickens were incubated at 3738° C, and embryos were harvested daily from day 5 to day 15 of incubation. Their gizzards were excised and immediately frozen in 2-methylbutane pre-cooled in liquid nitrogen. They were sectioned transversely and longitudinally at a thickness of 6 ~tm in a cryostat, the sections were then collected on gelatine-coated slides and air-dried. Small cubes of adult chicken gizzard muscle were treated correspondingIy.
Antigens and antibodies The isolation of myosin from adult chicken gizzard smooth muscle, and the production and the specificity of the respective antibodies have been described (Gr6schel-Stewart et al. 1985, 1989). Collagen type I was extracted from adult chicken skin following the method of Chung and Miller (1974). Interrupted polyacrylamide electrophoresis (Sykes et al. 1976), in which in situ reduction converts e(III) polymers into monomers, has shown that skin collagen consists of cq(I) and e2(I) chains only (Fig. 1a). Antibodies were obtained after multiple injections of 0.2 mg of this type I collagen into rabbits. In immunoblots, they reacted with the ~1(I) and the
378
Fig. 1. Polyacrylamide gel electrophoresis on 6% gels (a, c, d) and immunoblotting with anti-collagen type I (b, e) of collagen extracted from adult chicken skin (a, b) and embryonic chicken gizzard muscle (c, d, e), using non-interrupted (c) and interrupted (a, d) electrophoresis with in situ reduction to allow separation of cq(III) chains, a Electrophoresis pattern of adult skin with cq(I) and c~2(I) chains; b the immunoblot thereof, c Electrophoresis pattern of non-reduced embryonic gizzard collagen with el(I) and ez(I) chains; d the same after reduction, showing the el(III) chain; e the immunoblot thereof
c~2(I) chains (Fig. 1b). Collagen isolated from embryonic chicken gizzard (day 17) showed the presence of el(I), e2(I) and cq(III) chains after interrupted electrophoresis (Fig. 1c, d), only the el(I) and ~2(I) chain, but not the el(III) chain, reacted with the above antiserum (Fig. 1e). The IgG fractions of immune and control sera were isolated according to Harboe and Ingild (1973). Anti-smooth muscle myosin was labeled with the fluorochrome tetramethylrhodamine isothiocyanate (TRITC) following the method of Hudson and Hay (1989).
aggregates that eventually become separated from each other by the increased formation of imbricated collagen nets (Fig. 2d, e). As the collagen secretions into the extracellular space increase, the intracellular collagen immunoreactivity decreases. The muscle cells in the bundles become more tightly packed, myosin immunoreactivity increases and the surrounding connective tissue septa thicken. By day 15, the typical rhomboid profile seen in adult muscle is reached (Fig. 2f). There are occasional collagen fibers in the spaces between individual muscle cells. In the adult muscle, the intercellular gaps are filled with collagen fibers, forming a fine network that enmeshes individual cells and groups of cells, and eventually merges with the collagen of the intramuscular septa (Fig. 2g): In addition to the myosin/collagen-reactive areas, a narrow continuous band of collagen-reactive (and myosin-negative) cells can be seen in longitudinal sections of day-8 gizzards close to the luminal epithelium (Fig. 3). This band persists throughout development. On days 13/14, when the smooth muscle cells have expanded throughout the presumptive gizzard mesenchyme (as seen in sections at day 17; Fig. 4d), the intramuscular connective tissue septa appear to merge with this band (Fig. 4a-c). The micrographs also show that, on day 14, the collagen immunoreactivity appears to expand into the glandular epithelial layer, reaching the apical cells on day 17 (Fig. 4 a-d).
Discussion
Immunohistochemistry For double-labelingimmunofluorescence,the unfixed sections were treated as follows: incubation with anti-collagen IgG (0.1 mg/ml) for 30 min, and visualization with fiuorescein isothiocyanate (FITC)-labeled goat-anti-rabbit IgG (1:80; Sigma, Deisenhofen, FRG). Photographs of the anti-collagen staining were taken using Kodak Tri-X Pan. After removal of the coverslip, the free binding sites on the goat-anti-rabbit IgG were saturated by incubation with rabbit non-immune IgG (2 mg/ml) for 60 rain. Myosin was then visualized with TRITC-labeled anti-smooth muscle myosin (0.15 mg/ml) for 30 rain; anti-myosin reactivity was photographed in identical areas that were documented before.
Results
On day 5 in ovo, small fibrillar structures reactive with antibodies to chicken collagen type I are scattered throughout the gizzard primordium (Fig. 2 a); anti-myosin-reactive cells are not visible. From day 6 to day 8, first islets, then narrow bands of cells in the central part of the mesenchyme are immunoreactive for both collagen and myosin. The paired micrographs in Fig, 2b show that the two antigens are colocalized in a narrow cytoplasmic rim, with the unstained nucleus still occupying most of the cell. Structural reorganizations become evident from day 9 onward in transverse sections of the myosin/collagen-reactive areas. Small collagen fibers start to appear in the extracellular space (Fig. 2c), and the smooth muscle cells begin to assemble into tuft-like
According to Bennet and Cobb (1969), the most active period of cell division in the embryonic chicken gizzard is around day 6, when the cells of the primordium are still undifferentiated and indistinguishable from fibroblasts. At this stage (day 5), randomly dispersed collagen fibers can be visualized with antibodies to chicken collagen I. Since cells immunoreactive to smooth muscle myosin are not yet seen at this stage, one must assume that the collagen is synthesized by fibroblasts or undifferentiated myoblast precursors. The same must apply to the luminal collagen-immunoreactive band; these cells seem to be responsible for the formation of the prominent lamina propria of the adult muscle. According to Cornselius (1925), the pseudo-stratified columnar cells of the luminal epithelium start to invaginate into the subjacent connective tissue layer (the later lamina propria) on day 14. Longitudinal clefts appear from day 15 onward; they initiate the formation of tubular glands. By days 17/18, these glands have a single layered epithelium, and are deeply immersed in the connective tissue. The narrow spaces between the individual glands are filled with a network of collagen fibers. Thus, the inward movement of the tubular glands, rather than an expansion of collagen-immunoreactivity toward the apical cells, must be responsible for the staining patterns seen in Fig. 4. The sudden onset of the synthesis of all major contractile proteins (Saborio et al. 1979; Hirai and Hirabayashi 1983, 1986; Stuewer and Gr6schel-Stewart
379
Fig. 2a-g. Paired micrographs of transverse sections of fresh frozen gizzards sequentially stained with a-collagen type I (a-col; indirect method, FITC-label) and a-smooth muscle myosin (a-myo; direct method, TRITC-label). Identical areas are indicated by arrows, a Day-5 embryo shows disperse collagen staining only (a-col); b day-7 embryo exhibit intracellular codistribution of collagen (aco[) and myosin (a-myo); c in day-9 embryos, small extracellnlar collagen fibers appear; d-f between clay 11 to day 15, trellis-like collagen networks form; g adult muscle shows the intramuscular connective tissue septa and fine collagen network around each ceil. × 270
380
1985), presumably following the peak of cell division, is in accordance with the observation of Gabella (1989) that all smooth muscle cells develop uniformly and that all have the same degree of differentiation, with no intermediate forms being visible. In a subsequent paper (Paul et al., in preparation) we will show however that the smooth muscle cells appear to synthesize both muscular and cytoplasmic myosin at an early stage. Extracellular collagen fibers were first seen by Bennett and Cobb (1969) on day 9, and on day 11 in electron micrographs (Gabella 1989). Using collagen antibodies, we can follow the assembly of muscle bundles by trellis-like connective tissue septa from day 9 to the adult stage. By day 15, the adult rhomboid pattern is reached (Blessing and Miiller 1974) and the only further change seen is an additional fine collagen network surrounding all individ-
ual muscle cells in the adult. Likewise, Gabella (1989) describes a slightly larger number of collagen fibers in the post-hatching period. Extensive studies have been performed on collagen types of embryonic skeletal muscle. Chicken myoblasts have been shown by biochemical and immunological methods to synthesize collagen types I and III in vitro (Bailey et al. 1979) and to participate in the deposition of extracellular connective tissue fibers in skeletal muscle (Sasse et al. 1981). With specific anti-type I and III collagen sera, Duance et al. (1977) have localized type I mainly in the epimysium, and type [II preferentially in the perimysium of bullock muscle. Cultured smooth muscle cells (Chamley-Campbell et al. 1979) and developing rat aorta (Ross and Klebanoff 1971) produce collagen types I and III in varying proportions. In the adult intestinal
381
Fig. 3. Paired micrographs of longitudinal sections of day-8 embryonic gizzard stained with a-collagen (a-col) and a-myosin (a-myo). Note the additional collagen-positive band close to the lumen (L). Composite picture, x 55
Fig. 4a-d. Micrographs of longitudinal sections of embryonic chicken gizzard (luminal part), stained with a-collagen type I (a-col) and a-myosin (a-rnyo). a Da2-14 embryo shows collagen-immunoreactivity in the intramuscular septa (is), the future lamina propria (lp) and the basal part of the glandular epithelium (ge); b in day-16 embryos, progression of the epithelial staining is seen; c a day-17 embryo exhibits collagen reactivity throughout ge; d a corresponding section staining with a-myosin (a-rnk,o). × 270
382 muscle, type I a n d I I I are also the m o s t a b u n d a n t species (Epstein a n d M u n d e r l o h 1975). Since fibroblasts in culture also simultaneously synthesize type I and I I I collagen ( G a y et al. 1976), it is n o t surprising that the embryonic chicken gizzard exhibits b o t h collagen types, as d e m o n s t r a t e d in this paper. The e m b r y o n i c cq(I) a n d c~2(I) chains, b u t n o t the cq(III) chains, have been shown by i m m u n o b l o t t i n g to react with the antibodies to adult chicken skin collagen type I; this justifies the use o f these antibodies for the localization o f collagen in the developing gizzard. A l t h o u g h the nature o f the link between the collagen fibers and the muscle cells is n o t k n o w n , it is generally assumed t h a t their intimate association is essential for binding the s m o o t h muscle cells together and for the transmission o f tension generated by the myofilaments (Mullins and G u n t h e r o t h 1965).
Acknowledgements. This work was supported by the Deutsche Forschungsgemeinschaft (Ste 105/25-2). We wish to thank Ms. Anna-Luise Christian, Ms. Renate Franke, and Ms. Ruth Rohr for their able assistance.
References Bailey AJ, Shellswell GB, Duance CV (1979) Identification and change of collagen types in differentiating myoblasts and developing chick muscle. Nature 278 : 67-68 Bennett T, Cobb JLS (1969) Studies on the avian gizzard: morphology and innervation of the smooth muscle. Z Zellforsch 96:173-185 Blessing MH, Mfiller G (1974) Myoglobin concentration in the chicken, especially in the gizzard (a biochemical, light and electron microscopic study). Comp Biochem Physiol [A] 47:535540 Chamley-Campbell J, Campbell GR, Ross R (1979) The smooth muscle cell in culture. Physiol Rev 59 : 1-61 Chung E, Miller EJ (1974) Collagen polymorphism: characterization of molecules with the chain composition [~l(III)]s in human tissuesl Science 183 : 1200-1201 Cornselius C (1925) Morphologie, Histologie und Embryologie des Muskelmagens der V6gel. Gegenbaurs Morphol Jahrb 54: 507559 Donahoe JR, Bowen JM (1972) Analysis of the spontaneous motility of the avian embryonic gizzard. Am J Vet Res 33:1835-1848 Duance VC, Restall DJ, Beard H, Bourne FJ, Bailey AJ (1977) The location of three collagen types in skeletal muscle. FEBS Lett 79 : 248-252
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