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Immunocytochemical Demonstration of Simultaneous Synthesis of Types I, III and V Collagen and Fibronectin in Mouse Embryonic Palatal Mesenchymal Cells In Vitro
Collagen ReI. Res. Vol. 7/1987, pp. 333-340
Immunocytochemical Demonstration of Simultaneous Synthesis of Types I, III and V Collagen and Fibronectin in Mouse Embryonic Palatal Mesenchymal Cells In Vitro K. KURISU, Y.OHSAKI, K. NAGATA, T. KUKIT A, H. YOSHIKAWA and T.INAI Second Department of Anatomy, Faculty of Dentistry, Kyushu University, Fukuoka, Japan.
Abstract An immunocytochemical study was made on the palatal mesenchymal cells obtained from mouse embryos during palatal development with special reference to synthesis of types of collagen and fibronectin in vitro. Most cells showed positive staining with antibodies against the four proteins examined. The staining for type I collagen was most intense among the four proteins and was distributed perinuclearly. The staining for type III collagen was quite similar as that for type I collagen but less intense, whereas that for type V collagen was weak and its staining pattern was different from those for types I and III collagen in that the surface of the plasma membrane, in addition to the perinuclear cytoplasm, showed weak staining for type V collagen. Antibodies to fibronectin showed perinuclear and extracellular fibrous staining. These data suggest that palatal mesenchymal cells synthesize types I, III, and V collagen and fibronectin simultaneously. Key words: collagen, fibronectin, immunocytochemistry, mouse embryo, palate.
Introduction During secondary palate formation, the collagen content of the palate has been found to increase significantly (Pratt and King, 1971; Hassel and Orkin, 1976); and administration of lathyrogenic agents, such as [3-aminopropionitrile (BAPN), which interfere with collagen fiber formation by preventing cross-linkage between newly synthesized collagen molecules (Pratt and King, 1972). These data suggest that collagen plays an active role in palatal development. At least ten genetically distinct types of collagen have been identified in vertebrates. As for the types of collagen found in palatal mesenchyme certain discrepancies exist among results obtained by various researchers. Uitto and Thesleff (1979) and Silver et al. (1981) observed type I and type III collagen, but Hassell and Orkin (1976) detected only type I in the developing palatal tissue of rat. Type V collagen was first isolated from human placenta (Burgeson et aI., 1976). Thereafter, type V collagen has been identified in various tissues and cells, including blood vessel wall (Chung et aI., 1976), bone (Rhodes and Miller, 1978), cartilage (Rhodes and Miller, 1978), corneal stroma (Poschl and von der Mark, 1980),
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skin (Brown and Weiss, 1979) and lung (Sano et a!., 1981). Silver et al. (1984) demonstrated that type V collagen was present in mouse palatal shelves and that its synthesis was stimulated by epidermal growth factor (EGF). However, it is not known whether there are distinct subpopulations in the palatal mesenchyme cells which synthesize different types of collagen (e.g. type I vs. type III). Fibronectin is a large glycoprotein present in serum as well as in intercellular matrix (Stenman et a!., 1978; Pearlstein et a!., 1980; Bayne et a!., 1984) and has been shown to have high affinity for collagen. Codistribution of fibronectin with collagen has been reported in mesenchymal tissue of palatal shelves (Silver et a!', 1981; Ohsaki and Kurisu, 1984). The cellular origin of fibronectin produced in vivo, however, is still unknown. Therefore, it is interesting to know if mesenchymal cells from developing palate synthesize different types of collagen andlor fibronectin. In the present study, mouse embryonic palatal mesenchymal cells were examined immunocytochemically for types I, III, and V collagen and fibronectin. Immunofluorescence double-staining technique demonstrated that these cells simultaneously synthesize all four macromolecules examined. Materials and Methods
Cell culture Isolation and culture procedures of mouse embryonic palatal mesenchymal (MEPM) cells used in the present study were previously described (Sasaki and Kurisu, 1983). Briefly, cells were isolated from palatal shelves of CF1 mouse embryos immediately before palatal fusion (day 14). Cells were plated on Lab-Tek plates and cultured at 37°C in an atmosphere of 95% air and 5% CO 2 for 3-7 days in alpha-modified Eagle's medium (a-MEM) containing 10% fetal calf serum (FCS) and the antibiotics penicillin (100 IU/ml) and streptomycin (100 f-lg/ml). The MEPM cells were plated at a cell density of 104 in each well of the plate. After 24 h, when most cells were attached, the medium was exchanged for fresh medium of the same type and the cultures were incubated for up to 6 days. The cells used in the present study were in less than the third passage level in order to retain the in vivo characteristics as closely as possible.
Antigens and antibodies Type I and type III collagens and fibronectin were extracted from skin and blood plasma of CF1 mice, respectively, and purified as described by Nowack et a!. (1976). Type V collagen was purified from kidney of CF1 mice by the method of Kresina and Miller (1979). Their purity was verified by sodium dodecyl sulfate gel electrophoresis. The purified antigens were used to immunize rabbits or guinea pigs; and the antibodies to type I and type 1II collagen and fibronectin were IgG, which were purified by afffinity chromatography using Sepharose-bound antigens. Antibody to type V collagen was obtained in the IgG-fraction of DEAE column chromatography. All antibody preparations were adsorbed by the other three antigens. For example, antibody to type I collagen was adsorbed by type III and type V collagen and fibronectin to make it monospecific for type I collagen.
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Immunofluorescence microscopy The cultures were fixed overnight at 4°C in a solution of absolute ethanol and glacial acetic acid (99:1). This was followed by two changes of ethanol, and two changes of xylene, all at 4 0c. Double-staining technique was performed as described by Engel et al. (1980). For example, in the case of double-staining for type I and type III collagens, the culture was incubated consecutively with rabbit antibodies to type I collagen, guinea pig antibodies to type III collagen, FITC-conjugated goat anti-guinea pig IgG (1:100 dilution) and finally with rhodamine-conjugated goat anti-rabbit IgG (1:200 dilution) . The culture was incubated in a moist chamber with primary and secondary antibodies for 1 hand 30 min, respectively, and rinsed with PBS between each step. All procedures were performed at room temperature. Finally the cultures were washed exhaustively with PBS and sealed with a PBS-buffered glycerol to which p-phenylenediamine had been added to decrease fluorescence fading as described by Johnson and Araujo (1981). The samples were observed and photographed with a Zeiss fluorescence microscope.
Fig. 1. Phase-contast micrograph of MEPM cells cultured for 3 days in a-MEM supplemented with fetal calf serum. Culture is subconfluent and most cells are polygonal in shape. Original magnification : x 430. Bar = 30 !J.m. Fig. 2. Immunofluorescence micrograph of ME PM cells incubated firstly with preimmune serum of rabbit and secondly with rhodamine-conjugated goat anti-rabbit IgG. No staining is observed. Original magnification: x 430. Bar = 30 !J.m.
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Fig. 3. Immunofluorescence microcraphs of MEPM cells cultured for 3 days and examined by the double-staining technique. The culture was incubated firstly with rabbit antibody to type I collagen and guinea pig antibody to type III collagen, and secondly with rhodamineconjugated goat anti-rabbit IgG and FITC-conjugated sheep anti-guinea pig IgG. (a) Rhodamine-fluorescence micrograph showing type I collagen. (b) FITC-fluorescence micrograph showing type III collagen. Most cells show positive staining for type I and type III collagen simultaneously except a few cells (arrows) which show weak staining for type III collagen. Original magnification: x 430. Bar = 30 ~m.
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Fig. 5. Immunofluorescence micrographs of MEPM cells examined by the double-staining technique. (a) Rhodamine-fluorescence micrograph showing type I collagen. (b) FITC-fluorescence micrograph showing fibronectin. Original magnification: X 430. Bar = 30 !tm.
Materials Alpha-MEM and FCS was purchased from Flow Laboratories (North Ryde, Australia). Penicillin-streptomycin solution was obtained from Grand Island Biochemical (Grand Island, NY). Eight-chamber Lab-Tek plate were from Lab-Tek Products (Naperville, IL). FITC-conjugated goat anti-guinea pig IgG and rhodamine-conjugated goat anti-rabbit IgG were from Kirkegard and Perry Lab. (Gaithersburg, MD). Results When plated at a cell density of 104 per well, MEPM cells were distributed sparsely on day 3 (Figure 1). Most of the cells were polygonal in shape and had an oval nucleus. Cultures became confluent by 5 days after plating (data not shown). Control cultures stained with preimmune rabbit serum showed no positive staining (Figure 2). When the cells were exposed to antibody to type I collagen (Figure 3a) and that to type III collagen (Figure 3b) simultaneously, although the staining intensity was stronger for type I collagen than that for type III collagen, the perinuclear cytoplasm of most cells
..... Fig. 4. Immunofluorescence micrographs of MEPM cells examined by the double-staining technique. (a) Rhodamine-fluorescence micrograph showing type I collagen. (b) FITCfluorescence micrograph showing type V collagen. Although the staining is more intense for type I collagen than for type V collagen, most cells show positive staining for both types of collagens simultaneously. Not the presence of weak but positive staining for type V collagen on the plasma membrane. Original magnification: X 430. Bar = 30 !tm.
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showed positive staining for both antibodies, indicating the simultaneous presence of both types of collagen in MEPM cells. But, in a few cells very weak staining was observed for type III collagen. No staining for type I or III was observed in the extracellular matrix, indicating no deposition of either type of. collagen in the matrix under the culture conditions employed. When the cells were stained for type I (Figure 4a) and type V collagen (Figure 4b) simultaneously, the staining was much weaker for type V collagen than that for type I collagen, and the perinuclear cytoplasm in most cells again showed positive staining for both types of collagen. This suggests that MEPM cells may simultaneously synthesize both types of collagen. In the case for type V collagen, weak but positive staining was found also on the plasma membrane of the cells, suggesting deposition of type V collagen on the surface of the plasma membrane. Perinuclear cytoplasm of the cells showed positive staining for type I collagen (Figure Sa) and for fibronectin (Figure 5b) simultaneously, when the culture was doublestained for both macromolecules, suggesting simultaneous synthesis of them in MEPM cells. In the case of fibronectin, staining of fibers in the matrix was observed, indicating fibrous deposition of this macromolecule in the extracellular matrix. Discussion The present study suggests that, although there was some heterogeneity in morphology of the palatal mesenchymal cells from the mouse fetus, these cells simultaneously synthesized type I, III and V collagen and fibronectin in vitro. These results suggest that these types of collagen and fibronectin found in the extracellular matrix of the palatal shelf in vivo are secreted from the mesenchymal cells and that there were no distinct subpopulations of cells which synthesize different types of collagen or fibronectin. The simultaneous sythesis of different types of collagen by MEPM cells is not surprising, because this fact has been already reported for other cells in vitro, i.e., human fibroblasts (Gay et al., 1976), monkey aortic smooth muscle cells (Burke et al., 1977), rat liver-derived epithelial cells (Foidart et al., 1980) and rat glomerular epithelial and mesangial cells (Foidart et al., 1983). There is inconsistency among reports about the presence of type III collagen in the palatal shelves. Hassell and Orkin (1976) could not detect type III collagen in the palatal shelf of mouse fetuses by biochemical means. However, Uitto and Thesleff (1979), Silver et al. (1981), and Ohsaki and Kurisu (1984) detected type III collagen in the same tissue of mouse fetuses in vivo by means of immunohistochemical methods. The results obtained in the present study strongly support the observations shown in the latter studies. The amount of type III collagen synthesized in the palatal shelf is thought to be too small to be detected by the biochemical method employed by Hassell and Orkin (1976). In MEPM cells, anti-type V collagen antibodies showed positive staining not only the perinuclear cytoplasm but also on the cell surface. This observation is consistent with those of chick corneal fibroblasts (Poschl and von der Mark, 1980), human gingival fibroblast (Narayanan et al., 1985) and rat smooth muscle cells (Gay et al., 1981aj Gay et al., 1981b). This suggests that type V collagen contributes to the construction of the exocytoskeleton, as suggested by Gay et al. (1981a). Silver et al. (1984) found in their organ culture system that synthesis of type V collagen in mouse palatal shelves was stimulated by EGF. EGF have been shown to increase dramatically in the period of palatogenesis (Pratt et al., 1980), suggesting that type V collagen plays an important role in palate formation. There was very little matrix staining for any types
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of collagen examined; this was because incubations were done without ascorbic acid (Gay et aI., 1976). Although the presence of fibronectin in the palatal shelves has been demonstrated by Silver et al. (1981) and Ohsaki and Kurisu (1984) in vivo, the cellular origin of fibronectin is not clear. The present data suggest that MEPM cells synthesize fibronectin in vitro, indicating the possibility that cellular origin of fibronectin found in the extracellular matrix of palatal shelves is the mesenchymal cells of the tissues. Our immunoelectron microscope study on the distribution of fibronectin in palatal shelves revealed that positive staining for fibronectin was found in rER of mouse embryo palatal mesenchymal cells in vivo, indicating the synthesis of fibronectin in the cells in vivo also (Nagata et aI., unpublished). There are two types of fibronectin molecules, i.e. the cellular and plasma fibronectin, which are similar in structure (Hynes and Yamada, 1982; Pearlstein et aI., 1980). Since polyclonal antibodies that cross-react with both types of fibronectin were used in the present study, it is not to determine which type of fibronectin was observed, the intracellular or the extracellular.
References Bayne, E. K., Anderson, M. J. and Fambrough, D. M.: Extracellular matrix organization in developing muscle: Correlation with acetylcholine receptor aggregates. J. Cell BioI. 99: 1486-1501, 1984. Brown, R. A. and Weiss, J. B.: Type V collagen: Possible shared identity of aA, aB and aC chains. FEBS Lett. 106: 71-75, 1979. Burgeson, R. E. and Adli, F. A. E., Kaitila, I. I. and Hollister, D. W.: Fetal membrane collagens: Identification of two new collagen alpha chains. Proc. Natl. Acad. Sci. USA 73: 2579-2583, 1976. Burke, J. M., Balain, G., Ross, R. and Bornstein, P.: Synthesis of type I and III pro collagen and collagen by monkey aortic smooth muscle cells in vitro. Biochemistry 16: 3243-3249, 1977. Chung, E., Rhodes, R. K. and Miller, E. J.: Isolation of three collagenous components of probable basement membrane origin from several tissues. Biochem. Biophys. Res. Comm. 71: 1167-1174,1976. Engel, D., Schroeder, H. E., Gay, R. and Clagett, J.: Fine structure of cultured human gingival fibroblasts and demonstration of simultaneous synthesis of type I and III collagen. Arch. Oral Bioi. 25: 283-296, 1980. Foidart, J. M., Berman, ].'J., Paglia, L., Rennard, S., Abe, S., Perantoni, A. and Martin, G. R.: Synthesis of fibronectin, laminin, and several collagens by a liver-derived epithelial line. Lab. Invest. 42: 525-532, 1980. Foidart, J. B., Foidart, J. M., Hassel, J. and Mahieu, P.: Localization by immunofluorescent microscopy of several collagen types and of a basement membrane proteoglycan in rat glomerular epithelial and mesangial cell cultures. Renal Physiol. Basel 6: 163-170,1983. Gay, S., Martin, G. R. Miiller, P. K., Timpl, R. and Kiihn, K.: Simultaneous synthesis of types I and III collagen by fibroblasts in culture. Proc. Acad. Natl. Sci. USA 73: 4037-4040, 1976. Gay, S., Rhodes, R. K., Gay, R. E. and Miller, E. J.: Collagen molecules comprised ofa1(V)chains (B-chains): An apparent localization in the exocytoskeleton. Collagen Rei. Res. 1: 53-58, 1981. Gay, S., Martinez-Hernandez, A., Rhodes, R. K. and Miller, E. J.: The collagenous exocytoskeleton of smooth muscle cells. Collagen ReI. Res. 1: 377-384, 1981. Hassell, J. R. and Orkin, B. W.: Synthesis and distribution of collagen in the rat palate during shelf elevation. Devel. Bioi. 49: 80-88, 1976.
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Hynes, R. o. and Yamada, K. M.: Fibronectin: Multifunctional modular glycoproteins. J. Cell. Bioi. 95: 369-377, 1982. Johnson, G. D. and Araujo, G. M. C. N.: A simple method of reducing the fading of immunofluorescence during microsopy. J. Immunol. Methods 42: 349-350, 1981. Konomi, H., Hayashi, T., Nakayasu, K. and Arima, M.: Localization of type V collagen and type IV collagen in human cornea, lung, and skin. Immunohistochemical evidence by anti-collagen antibodies characterized by immunoelectroblotting. Am. J. Pathol. 116: 417-426, 1984. Kresina, T. F. and Miller, E. ].: Isolation and characterization of basement membrane collagen from human placental tissue. Evidence for the presence of two genetically distinct collagen chains. Biochemistry 18: 3089-3097, 1979. Nagata, K., Ohsaki, Y. and Kurisu, K.: Immunoelectron microscope study on the distribution of type I and type III collagen, and fibronectin in palatal shelves of mouse fetuses in vivo. (in preparation). Narayanan, A. S., Clagett,]. A. and Page, R. c.: Effect of inflammation on the distribution of collagen types, I, III, IV, and V and type I trimer and fibronectin in human gingivae. J. Dent. Res. 64: 1111-1116, 1985. Nowack, H., Gay, S., Wick, G., Becker, U. and Timpl, R.: Preparation and use in immunohistology of antibodies specific for type I and type III collagen and pro collagen. J. Immunol. Methods. 12: 117-124, 1976. Ohsaki, Y and Kurisu, K.: Immunohistochemical localization of collagen types in palate of glucocorticoid-treated mouse fetuses. J. Dent. Res. 63: 564, 1984. Pearlstein, E., Gold, L. I. and Garcia-Pardo, A.: Fibronectin: A review of its structure and biological activity. Molec. Cell Biochem. 29: 103-128, 1980. Poschl, A. and von der Mark, K.: Synthesis of type V collagen by chick corneal fibroblasts in vivo and in vitro. FEBS Lett. 115: 100-104, 1980. Pratt, R. M. and King, C. T. G.: Collagen synthesis in the secondary palate of the developing rat. Archs. Oral Bioi. 16: 1181-1185, 1971. Pratt, R. M. and King, C. T. G.: Inhibition of collagen cross-linking associated with ~ aminopronionitrile-induced cleft palate in the rat. Devel. Bioi. 27: 322-328, 1972. Pratt, R. M., Yoneda, T., Silver, M. H. and Salamon, D. S.: Involvement of glucocorticoids and epidermal growth factor in secondary palate development. In: Current research trends in prenatal craniofacial development, ed. by Pratt, R. M and Christiansen, R. L., ElsevierlNorth-Holland, New York, pp. 235-252, 1980. Rhodes, R. K. and Miller, E. J.: Physiochemical characterization and molecular organization of the collagen A and B chains. Biochemistry, 17: 3442-3448, 1978. Sano, ]., Fujiwara, S., Sato, S., Ishizaki, M., Sugisaki, Y., Yajima, G. and Nagai, Y.: AB (type V) and basement membrane (type IV) collagens in the bovine lung parenchyma: Electron microscopic localization by the peroxidase-labeled antibody method. Biomed. Res. 2: 20-29, 1981. Sasaki, S. and Kurisu, K.: Effect of triamcinolone acetonide on proliferation and collagen and glycosaminoglycan syntheses in palatal mesenchymal cells from the mouse fetus. J. Craniofac. Genet. Devel. Bioi. 3: 351-369, 1983. Silver, M. H., Foidart, ]. M and Pratt, R. M.: Distribution of fibronectin and collagen during mouse limb and palate development. Differentiation 18: 141-149, 1981. Silver, M. H., Murray, ]. C. and Pratt, R. M.: Epidermal growth factor stimulates type-V collagen synthesis in cultured murine palatal shelves. Differentiation 27: 205-208, 1984. Stenman, S. and Vaheri, A.: Distribution of a major connective tissue protein, fibronectin, in normal human tissues. J. Exp. Med. 147: 1054-1064, 1978. Vitto, V.-]. and Thesleff, I.: Effect of hydrocortisone on collagen synthesis in cultured mouse palatal explants. Arch. Oral Bioi. 24: 575-583, 1979. Dr. K. Kurisu, Second Department of Anatomy, Faculty of Dentistry 61, Kyushu University, Maidashi, Higashi-Ku, Fukuoka 812, Japan.
Immunocytochemical Demonstration of Simultaneous Synthesis of Types I, III and V Collagen and Fibronectin in Mouse Embryonic Palatal Mesenchymal Cells In Vitro K. KURISU, Y.OHSAKI, K. NAGATA, T. KUKIT A, H. YOSHIKAWA and T.INAI Second Department of Anatomy, Faculty of Dentistry, Kyushu University, Fukuoka, Japan.
Abstract An immunocytochemical study was made on the palatal mesenchymal cells obtained from mouse embryos during palatal development with special reference to synthesis of types of collagen and fibronectin in vitro. Most cells showed positive staining with antibodies against the four proteins examined. The staining for type I collagen was most intense among the four proteins and was distributed perinuclearly. The staining for type III collagen was quite similar as that for type I collagen but less intense, whereas that for type V collagen was weak and its staining pattern was different from those for types I and III collagen in that the surface of the plasma membrane, in addition to the perinuclear cytoplasm, showed weak staining for type V collagen. Antibodies to fibronectin showed perinuclear and extracellular fibrous staining. These data suggest that palatal mesenchymal cells synthesize types I, III, and V collagen and fibronectin simultaneously. Key words: collagen, fibronectin, immunocytochemistry, mouse embryo, palate.
Introduction During secondary palate formation, the collagen content of the palate has been found to increase significantly (Pratt and King, 1971; Hassel and Orkin, 1976); and administration of lathyrogenic agents, such as [3-aminopropionitrile (BAPN), which interfere with collagen fiber formation by preventing cross-linkage between newly synthesized collagen molecules (Pratt and King, 1972). These data suggest that collagen plays an active role in palatal development. At least ten genetically distinct types of collagen have been identified in vertebrates. As for the types of collagen found in palatal mesenchyme certain discrepancies exist among results obtained by various researchers. Uitto and Thesleff (1979) and Silver et al. (1981) observed type I and type III collagen, but Hassell and Orkin (1976) detected only type I in the developing palatal tissue of rat. Type V collagen was first isolated from human placenta (Burgeson et aI., 1976). Thereafter, type V collagen has been identified in various tissues and cells, including blood vessel wall (Chung et aI., 1976), bone (Rhodes and Miller, 1978), cartilage (Rhodes and Miller, 1978), corneal stroma (Poschl and von der Mark, 1980),
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skin (Brown and Weiss, 1979) and lung (Sano et a!., 1981). Silver et al. (1984) demonstrated that type V collagen was present in mouse palatal shelves and that its synthesis was stimulated by epidermal growth factor (EGF). However, it is not known whether there are distinct subpopulations in the palatal mesenchyme cells which synthesize different types of collagen (e.g. type I vs. type III). Fibronectin is a large glycoprotein present in serum as well as in intercellular matrix (Stenman et a!., 1978; Pearlstein et a!., 1980; Bayne et a!., 1984) and has been shown to have high affinity for collagen. Codistribution of fibronectin with collagen has been reported in mesenchymal tissue of palatal shelves (Silver et a!', 1981; Ohsaki and Kurisu, 1984). The cellular origin of fibronectin produced in vivo, however, is still unknown. Therefore, it is interesting to know if mesenchymal cells from developing palate synthesize different types of collagen andlor fibronectin. In the present study, mouse embryonic palatal mesenchymal cells were examined immunocytochemically for types I, III, and V collagen and fibronectin. Immunofluorescence double-staining technique demonstrated that these cells simultaneously synthesize all four macromolecules examined. Materials and Methods
Cell culture Isolation and culture procedures of mouse embryonic palatal mesenchymal (MEPM) cells used in the present study were previously described (Sasaki and Kurisu, 1983). Briefly, cells were isolated from palatal shelves of CF1 mouse embryos immediately before palatal fusion (day 14). Cells were plated on Lab-Tek plates and cultured at 37°C in an atmosphere of 95% air and 5% CO 2 for 3-7 days in alpha-modified Eagle's medium (a-MEM) containing 10% fetal calf serum (FCS) and the antibiotics penicillin (100 IU/ml) and streptomycin (100 f-lg/ml). The MEPM cells were plated at a cell density of 104 in each well of the plate. After 24 h, when most cells were attached, the medium was exchanged for fresh medium of the same type and the cultures were incubated for up to 6 days. The cells used in the present study were in less than the third passage level in order to retain the in vivo characteristics as closely as possible.
Antigens and antibodies Type I and type III collagens and fibronectin were extracted from skin and blood plasma of CF1 mice, respectively, and purified as described by Nowack et a!. (1976). Type V collagen was purified from kidney of CF1 mice by the method of Kresina and Miller (1979). Their purity was verified by sodium dodecyl sulfate gel electrophoresis. The purified antigens were used to immunize rabbits or guinea pigs; and the antibodies to type I and type 1II collagen and fibronectin were IgG, which were purified by afffinity chromatography using Sepharose-bound antigens. Antibody to type V collagen was obtained in the IgG-fraction of DEAE column chromatography. All antibody preparations were adsorbed by the other three antigens. For example, antibody to type I collagen was adsorbed by type III and type V collagen and fibronectin to make it monospecific for type I collagen.
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Immunofluorescence microscopy The cultures were fixed overnight at 4°C in a solution of absolute ethanol and glacial acetic acid (99:1). This was followed by two changes of ethanol, and two changes of xylene, all at 4 0c. Double-staining technique was performed as described by Engel et al. (1980). For example, in the case of double-staining for type I and type III collagens, the culture was incubated consecutively with rabbit antibodies to type I collagen, guinea pig antibodies to type III collagen, FITC-conjugated goat anti-guinea pig IgG (1:100 dilution) and finally with rhodamine-conjugated goat anti-rabbit IgG (1:200 dilution) . The culture was incubated in a moist chamber with primary and secondary antibodies for 1 hand 30 min, respectively, and rinsed with PBS between each step. All procedures were performed at room temperature. Finally the cultures were washed exhaustively with PBS and sealed with a PBS-buffered glycerol to which p-phenylenediamine had been added to decrease fluorescence fading as described by Johnson and Araujo (1981). The samples were observed and photographed with a Zeiss fluorescence microscope.
Fig. 1. Phase-contast micrograph of MEPM cells cultured for 3 days in a-MEM supplemented with fetal calf serum. Culture is subconfluent and most cells are polygonal in shape. Original magnification : x 430. Bar = 30 !J.m. Fig. 2. Immunofluorescence micrograph of ME PM cells incubated firstly with preimmune serum of rabbit and secondly with rhodamine-conjugated goat anti-rabbit IgG. No staining is observed. Original magnification: x 430. Bar = 30 !J.m.
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Fig. 3. Immunofluorescence microcraphs of MEPM cells cultured for 3 days and examined by the double-staining technique. The culture was incubated firstly with rabbit antibody to type I collagen and guinea pig antibody to type III collagen, and secondly with rhodamineconjugated goat anti-rabbit IgG and FITC-conjugated sheep anti-guinea pig IgG. (a) Rhodamine-fluorescence micrograph showing type I collagen. (b) FITC-fluorescence micrograph showing type III collagen. Most cells show positive staining for type I and type III collagen simultaneously except a few cells (arrows) which show weak staining for type III collagen. Original magnification: x 430. Bar = 30 ~m.
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Fig. 5. Immunofluorescence micrographs of MEPM cells examined by the double-staining technique. (a) Rhodamine-fluorescence micrograph showing type I collagen. (b) FITC-fluorescence micrograph showing fibronectin. Original magnification: X 430. Bar = 30 !tm.
Materials Alpha-MEM and FCS was purchased from Flow Laboratories (North Ryde, Australia). Penicillin-streptomycin solution was obtained from Grand Island Biochemical (Grand Island, NY). Eight-chamber Lab-Tek plate were from Lab-Tek Products (Naperville, IL). FITC-conjugated goat anti-guinea pig IgG and rhodamine-conjugated goat anti-rabbit IgG were from Kirkegard and Perry Lab. (Gaithersburg, MD). Results When plated at a cell density of 104 per well, MEPM cells were distributed sparsely on day 3 (Figure 1). Most of the cells were polygonal in shape and had an oval nucleus. Cultures became confluent by 5 days after plating (data not shown). Control cultures stained with preimmune rabbit serum showed no positive staining (Figure 2). When the cells were exposed to antibody to type I collagen (Figure 3a) and that to type III collagen (Figure 3b) simultaneously, although the staining intensity was stronger for type I collagen than that for type III collagen, the perinuclear cytoplasm of most cells
..... Fig. 4. Immunofluorescence micrographs of MEPM cells examined by the double-staining technique. (a) Rhodamine-fluorescence micrograph showing type I collagen. (b) FITCfluorescence micrograph showing type V collagen. Although the staining is more intense for type I collagen than for type V collagen, most cells show positive staining for both types of collagens simultaneously. Not the presence of weak but positive staining for type V collagen on the plasma membrane. Original magnification: X 430. Bar = 30 !tm.
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showed positive staining for both antibodies, indicating the simultaneous presence of both types of collagen in MEPM cells. But, in a few cells very weak staining was observed for type III collagen. No staining for type I or III was observed in the extracellular matrix, indicating no deposition of either type of. collagen in the matrix under the culture conditions employed. When the cells were stained for type I (Figure 4a) and type V collagen (Figure 4b) simultaneously, the staining was much weaker for type V collagen than that for type I collagen, and the perinuclear cytoplasm in most cells again showed positive staining for both types of collagen. This suggests that MEPM cells may simultaneously synthesize both types of collagen. In the case for type V collagen, weak but positive staining was found also on the plasma membrane of the cells, suggesting deposition of type V collagen on the surface of the plasma membrane. Perinuclear cytoplasm of the cells showed positive staining for type I collagen (Figure Sa) and for fibronectin (Figure 5b) simultaneously, when the culture was doublestained for both macromolecules, suggesting simultaneous synthesis of them in MEPM cells. In the case of fibronectin, staining of fibers in the matrix was observed, indicating fibrous deposition of this macromolecule in the extracellular matrix. Discussion The present study suggests that, although there was some heterogeneity in morphology of the palatal mesenchymal cells from the mouse fetus, these cells simultaneously synthesized type I, III and V collagen and fibronectin in vitro. These results suggest that these types of collagen and fibronectin found in the extracellular matrix of the palatal shelf in vivo are secreted from the mesenchymal cells and that there were no distinct subpopulations of cells which synthesize different types of collagen or fibronectin. The simultaneous sythesis of different types of collagen by MEPM cells is not surprising, because this fact has been already reported for other cells in vitro, i.e., human fibroblasts (Gay et al., 1976), monkey aortic smooth muscle cells (Burke et al., 1977), rat liver-derived epithelial cells (Foidart et al., 1980) and rat glomerular epithelial and mesangial cells (Foidart et al., 1983). There is inconsistency among reports about the presence of type III collagen in the palatal shelves. Hassell and Orkin (1976) could not detect type III collagen in the palatal shelf of mouse fetuses by biochemical means. However, Uitto and Thesleff (1979), Silver et al. (1981), and Ohsaki and Kurisu (1984) detected type III collagen in the same tissue of mouse fetuses in vivo by means of immunohistochemical methods. The results obtained in the present study strongly support the observations shown in the latter studies. The amount of type III collagen synthesized in the palatal shelf is thought to be too small to be detected by the biochemical method employed by Hassell and Orkin (1976). In MEPM cells, anti-type V collagen antibodies showed positive staining not only the perinuclear cytoplasm but also on the cell surface. This observation is consistent with those of chick corneal fibroblasts (Poschl and von der Mark, 1980), human gingival fibroblast (Narayanan et al., 1985) and rat smooth muscle cells (Gay et al., 1981aj Gay et al., 1981b). This suggests that type V collagen contributes to the construction of the exocytoskeleton, as suggested by Gay et al. (1981a). Silver et al. (1984) found in their organ culture system that synthesis of type V collagen in mouse palatal shelves was stimulated by EGF. EGF have been shown to increase dramatically in the period of palatogenesis (Pratt et al., 1980), suggesting that type V collagen plays an important role in palate formation. There was very little matrix staining for any types
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of collagen examined; this was because incubations were done without ascorbic acid (Gay et aI., 1976). Although the presence of fibronectin in the palatal shelves has been demonstrated by Silver et al. (1981) and Ohsaki and Kurisu (1984) in vivo, the cellular origin of fibronectin is not clear. The present data suggest that MEPM cells synthesize fibronectin in vitro, indicating the possibility that cellular origin of fibronectin found in the extracellular matrix of palatal shelves is the mesenchymal cells of the tissues. Our immunoelectron microscope study on the distribution of fibronectin in palatal shelves revealed that positive staining for fibronectin was found in rER of mouse embryo palatal mesenchymal cells in vivo, indicating the synthesis of fibronectin in the cells in vivo also (Nagata et aI., unpublished). There are two types of fibronectin molecules, i.e. the cellular and plasma fibronectin, which are similar in structure (Hynes and Yamada, 1982; Pearlstein et aI., 1980). Since polyclonal antibodies that cross-react with both types of fibronectin were used in the present study, it is not to determine which type of fibronectin was observed, the intracellular or the extracellular.
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