Changes in the matrix proteins, fibronectin and collagen, during differentiation of mouse tooth germ

Changes in the matrix proteins, fibronectin and collagen, during differentiation of mouse tooth germ

DEVELOPMENTAL BIOLOGY 70, 116-126 (1979) Changes in the Matrix Proteins, Fibronectin and Collagen, Differentiation of Mouse Tooth Germ during I...

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DEVELOPMENTAL

BIOLOGY

70, 116-126

(1979)

Changes in the Matrix Proteins, Fibronectin and Collagen, Differentiation of Mouse Tooth Germ

during

I. THESLEFF, S. STENMAN, A. VAHERI, AND R. TIMPL* Departments of Pathology and Virology, University of Helsinki, Finland, and *Max Planck-Institut Biochemie, Martinsried, Federal Republic of Germany

fir

Received October 2, 1978; accepted November 29, 1978

The distribution of the matrix protein fibronectin was studied by indirect immunofluorescence in differentiating mouse molars from bud stage to the stage of dentin and enamel secretion, and compared to that of collagenous proteins procollagen type III and collagen type I. Fibronectin was seen in mesenchymal tissue, basement membranes, and predentin. The dental mesenchyme lost fibronectin staining when differentiating into odontoblasts. Fibronectin was not detected in mineralized dentin. Epithelial tissues were negative except for the stellate reticulum within the enamel organ. Particularly intense staining was seen at the epithelio-mesenchymal interface between the dental epithelium and mesenchyme. Fibronectin may here be involved in anchorage of the mesenchymal cells during their differentiation into odontoblasts. Procollagen type III was lost from the dental mesenchyme during odontoblast differentiation but reappeared with advancing vascularization of the dental papilla. Similarly, procollagen type III present in the dental basement membrane during the bud and cap stages disappeared from the cuspal area along with odontoblast differentiation. Weak staining was seen in predentin but not in mineralized dentin. The staining with anti-collagen type I antibodies was weak in dental mesenchyme but intense in predentin as well as in mineralized dentin. INTRODUCTION

glycans, and glycoproteins. This material has been thought to induce differentiation of the epithelial cells into ameloblasts (Ruth et al., 1974), although cell-tocell contacts between the epithe1ia.l and mesenchymal cells have also been proposed as mediators of the inductive signal (Slavkin and Bringas, 1976). The secretory product of the ameloblasts is the enamel matrix, which contains unique types of proteins, i.e., enamel proteins, not found in other tissues (Slavkin et al., 1977). In the developing tooth the distribution of collagens of types I, II, III, and IV were studied recently (Lesot et al., 1978). The specific fluorescence of collagen type III, which is characteristically expressed in embryonic newly formed tissues (Timpl et al., 1977), was shown to be weaker in dental than in nondental mesenchyme, suggesting a reduction of collagen type III during advancing mesenchymal cell differentiation.

The developing tooth offers a good model for studying the role of extracellular matrix materials during differentiation. In tooth development a sequence of epithelio-mesenchymal interactions takes place, and these are accompanied by changes in the composition of the extracellular matrix. During the early stages of morphogenesis when the mesenchymal tissue invades the epithelial bud and the bell-shaped tooth germ is formed, a basement membrane separates the epithelial and mesenchymal tissues. Transfilter studies have indicated that the basement membrane material is directly involved in the differentiation of the cells into odontoblasts mesenchymal (Thesleff et al., 1978). The differentiated odontoblasts then secrete predentin which is the organic matrix of dentin into the epithelio-mesenchymal interface. Predentin is composed of collagen, glycosamino116 OO12-16O6/79/050116-11$02.OO/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

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We now describe the distribution of fibronectin in the developing tooth from bud stage to advanced bell stage and compare it with the distributions of procollagen type III and collagen type I. Fibronectin, also known as LETS protein or cell surface protein (CSP), is a major component of connective tissue matrix and basement membranes (Linder et al., 1975; Stenman and Vaheri, 1978). It is produced by cultured fibroblastic cells in which it plays a major role in cell adhesion (Klebe, 1974; Yamada et al., 1975; Pearlstein, 1976; Grinnell, 1976; Hook et al., 1977; Hedman et al., in press). The role of fibronectin in the anchorage and positioning of cells is also suggested by its characteristic distribution in basement membranes (Stenman and Vaheri, 1978). The results show that major changes in the distribution of these proteins are associated with morphogenetic events and cell differentiation in the developing tooth. MATERIALS

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METHODS

Preparation and fixation of tooth germs, First mandibular molars from 14 to 19-dayold hybrid mouse embryos or 2-day-old newborns (corresponding to 21 days after conception) (CBA X BALB/C) were used (vaginal plug = day 0). The tooth germs were removed from the jaws under a dissecting microscope. The dental lamina connecting the tooth to oral epithelium was left intact, and a piece of oral epithelium and connective tissue overlying the tooth germ were left in place. The teeth were fixed in cold 94% alcohol for 4 hr and processed as recommended by Sainte-Marie (1962). They were embedded in Tissue Prep (Fisher Scientific Co., Fairlawn, New Jersey) at 64°C and serially sectioned at 6 pm. Some of the deparaffinized sections were demineralized with 0.3 M EDTA [(ethylenedinitrilo)tetraacetic acid] (20 min), and the demineralization was verified in the van Kossa staining. Anti-fibronectin serum. Anti-human fibronectin raised in rabbits was used throughout the study. The specificity of the

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antiserum (Stenman et al., 1977) and the characteristic interspecies cross-reaction (Kuusela et al., 1976) have been documented elsewhere. In control experiments the anti-fibronectin rabbit serum was substituted either with normal rabbit serum, with anti-fibronectin serum pretreated with purified plasma fibronectin (50 pg/ml), or with PBS (phosphate-buffered saline). Anti-collagen antibodies. Rabbit antibodies against bovine type III procollagen and type I collagen were prepared and rendered specific by immunoabsorption (Nowack et al., 1976). The purified antibodies showed distinct reaction with human, bovine, and mouse tissues when studied in immunofluorescence (Timpl et al., 1977). Antibodies against type III collagen and type III procollagen have been shown to have similar staining patterns (Nowack et al., 1976). The theoretical possibility that fibronectin and the collagenous proteins would cross-react immunologically has been excluded by passive hemagglutination and radioimmunoassay tests (Vaheri et al., 1978). Immunofluorescent staining. Deparaffinized and hydrated sections were treated with rabbit anti-fibronectin, anti-procollagen III or anti-collagen I or control sera for 30 min at room temperature. The stained sections were washed in PBS (three changes, 20 min) and stained with commercially obtained sheep fluorescein isothiocyanate-labeled anti-rabbit immunoglobulins. After three PBS washes (20 min) the preparation was mounted under a coverslip in isotonic Veronal-buffered (50 n-&f, pH 8.5) NaCl. Fluorescence microscopy. A Zeiss fluorescence microscope equipped with a HBO 200-W mercury high-pressure lamp, epi-illuminator III RS was used. Fluorescence filters for blue excitation light were: excitation filters BP 455-490, dichroic mirror FT 510, and emission filters LP 520 and KP 560. Autofluorescence was controlled by using uv emission light (filters UG 1, FT 420, and LP 418) or green emission light (filters

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BP 456, FT 580, and LP 590). With these filters autofluorescence appeared bluish or red, respectively. The fluorescence of fibronectin as well as that of the collagen was specific, since control stained sections were negative, and autofluorescence was absent or very weak. Light microscopy. For photography Agfapan 400 professional film was used. Fields were first photographed in fluorescence microscopy, then the same field was photographed in Nomarski differential interference contrast microscopy. Some of the sections were poststained for mineral deposits with the von Kossa stain. RESULTS

Bud stage, 14 days. At this stage of development the tooth bud has invaginated from the oral epithelium to the underlying mesenchyme. The cells of the dental mesenchyme cannot yet be distinguished from other mesenchymal cells. Fibronectin was seen in the mesenchymal tissue and in the basement membrane, but not in the epithelium (Fig. 1). Procollagen type III was distributed similarly to fibronectin, but the mesenchyme immediately underlining the epithelial bud had relatively weak staining (Figs. 1 and 2). Cap stage, 16 days. Dental mesenchyme has now invaded the epithelial bud from its under surface. In the epithelial component of the tooth, the enamel organ, inner and outer enamel epithelia as well as the central loose stellate reticulum can be distinguished. The intensity of fibronectin-specific fluorescence was similar in the dental and nondental mesenchyme. Fibronectin was not seen in inner or outer enamel epithelia, or in oral epithelium, but the stellate reticulum was moderately stained (Fig. 1). Less procollagen type III was detected in the dental than in nondental mesenchyme. The basement membrane under the oral epithelium was more intensely stained than that between the dental epithelium and mesenthyme. No procollagen type III was seen in

the epithelial tissue (Figs. 1 and 2). Early bell stage, 17 days. Molar morphogenesis has now advanced to the cusp formation. The enamel epithelium extends further cervically. The distribution of fibronectin was essentially similar to that in the 16-day-old tooth germ (Fig. 1). The distribution of procollagen type III had changed considerably from that at 16 days. No fluorescence was seen in the mesenchymal tissue in the cusps, but only in the undifferentiated intercuspal and cervical areas. The basement membrane under the enamel epithelium was not stained except in the intercuspal and cervical areas. The mesenchyme and basement membrane under the oral epithelium were intensely stained (Fig. 1). Collagen type I was seen in dental mesenchyme and at the epithelio-mesenchymal interface. The fluorescence was, however, more intense in the mesenchymal tissue surrounding the tooth bud (Fig. 3). Odontoblast differentiation, 18 days. One layer of mesenchymal cells, differentiating odontoblasts, have aligned under the enamel epithelium at the cuspal tips, where the differentiation begins. Fibronectin staining was very intense in the basement membrane between enamel epithelium and dental mesenchyme, when compared to that under the oral epithelium. In dental mesenchyme fibronectin was evenly distributed except in the layer of odontoblasts which was only weakly stained. The distribution of fibronectin in the epithelia was similar to that seen earlier; i.e., moderate fluorescence was seen in stellate reticulum. The intensity of procollagen type III staining in dental mesenthyme had decreased further especially in the cuspal area (Figs. 1 and 2). Secretion ofpredentin, 19 days. The area of differentiated odontoblasts now extends further cervically, and the odontoblasts at the cuspal tips have secreted a thin layer of predentin. The vascularization in the mesenchyme has increased. Very intense fibronectin staining was

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FIG. 1. The mouse lower fit molar from bud (14d) to bell stage (18d) of development stained with antifibronectin (top) and anti-procollagen type III antibodies (bottom). x 70. Fibronectin is present in nondental and dental mesenchyme as well as in basement membranes. The oral epithelium (oe), the dental lamina (dl), and the enamel epithelia (ee) are negative. In the stellate reticulum (sr) within the enamel organ fibronectin can be detected. Intense staining with procollagen type III antibodies is seen in the mesenchyme and basement membrane under the oral epithelium. In dental mesenchyme (dm) the intensity of staining weakens with advancing differentiation. No procollagen type III is seen in epithelial tissue. For a description of the epitheliomesenchymal interface see Fig. 2.

seen throughout the epithelio-mesenchymal interface of the tooth. The areas that contained predentin, as judged by light microscopy, stained similarly to the basement membrane (Fig. 5). In the odontoblast layer little or no fibronectin was detected, whereas the rest of the dental mesenchyme, as well as the oral mesenchyme, was intensely stained. Capillary walls in the dental mesenchyme contained fibronectin (Figs. 4 and 5). Weak procollagen type III specific fluorescence was seen in predentin, and the

intensity in the mesenchyme had increased from that at 18 days (Figs. 4 and 6).

Mineralization and enamel secretion, 21 days. The odontoblasts have secreted more predentin which has started to mineralize at the cuspal tips. The cells of the enamel epithelium have differentiated into ameloblasts and started to secrete enamel matrix on the surface of the mineralized dentin. The enamel matrix mineralizes shortly after it has been secreted. Fibronectin was not seen in mineralized dentin although the fluorescence in unmin-

FIG. 2. The epithelio-mesenchymal interface of bud (14d), cap (16d), and bell stage (lad) tooth germs. The same sections stained with anti-procollagen type III antibodies are shown unstained in Nomarski optics. x 400. In the bud stage intense staining is seen in the basement membrane (bm) surrounding the invaginating epithelial bud. The layer of mesenchymal cells (m) closely underlying the bud is less intensely stained than the rest of the mesenchymal tissue. In the cap stage procollagen type III is seen in the basement membrane (bm) under the enamel epithelium (ee). In the bell stage, where differentiation of the mesenchymal cells into odontoblasts has started, the dental mesenchyme (dm) and the basement membrane of the differentiated cuspal area are negative. Procollagen type III is seen in dental mesenchyme only in the intercuspal area (ica) and in the epitheliomesenchymal interface in the undifferentiated cervical area (see Fig. 1). 120

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FIG. 3. The lower first molar at the early bell stage (17d) and at the time of mineralization and enamel secretion (Zld) stained with anti-type I collagen antibodies. The section of the 2Id tooth germ was demineralized with EDTA prior to immunofluorescent staining. x 70. Collagen type I is seen in mesenchymal tissue and in the basement membranes. The fluorescence is more intense in the mesenchyme surrounding the tooth germ than in the dental mesenchyme (dm). Intense staining is seen in predentin (pd) and in mineralized dentin (md).

eralized predentin was intense. The mineral deposits as visualized by von Kossa staining were in close codistribution with areas negative for fibronectin. The mineralized areas were also fibronectin-negative in sections demineralized with EDTA. The thin layer of enamel matrix was weakly autofluorescent but did not stain for fibronectin (Figs. 4 and 5). The dental mesenchyme showed intense procollagen type III staining. Weak staining was seen in predentin but not in mineralized dentin (Figs. 4 and 6), even after demineralization with EDTA. Collagen type I was seen in dental mesenchyme, predentin, and dentin. The fluorescence was more intense in the dentin of demineralized sections (Fig. 3). The collagenous proteins were not detected in enamel matrix. DISCUSSION

The present results show that libronectin, procollagen type III, and collagen type I are present in the dental tissues throughout the differentiation of the tooth germ. Significant changes, however, take place in their distribution with advancing morphogenesis and differentiation. The observed changes in the distributions were not due to differences in the processing of the tooth

germs of different developmental stages, as proven by the observation that the staining in the oral mesenchyme and basement membrane remained fairly constant throughout the study. The distribution of various collagenous proteins (types I, II, III, and IV) was recently reported by Lesot et al. (1978) in cap- and bell-staged mouse molars and incisors. Type IV collagen was detected in the basement membranes; type II collagen was not seen. The findings on distributions of types I and III collagens were similar to those presented here. In addition to the cap and bell stage of tooth development, we also studied the distribution of these collagens in the bud stage and in the stage of dentin mineralization and enamel matrix secretion. Loss of procollagen type III, which was evident in the cap- and bellstaged tooth germs (Lesot et al., 1978), was already observed in the bud stage of development, where the layer of mesenchymal cells directly underlying the epithelial bud was less intensely stained than the rest of the mesenchyme. The reduction of procollagen type III from the dental mesenchyme might be one of the features associated with the specificity of this tissue. Experiments in which heterotypic epithelial and mesenchy-

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mal tissues have been grown in different combinations have indicated that the dental papilla mesenchyme is the only tissue from which odontoblasts differentiate (Kollar and Baird, 1970). Furthermore, the absence of procollagen type III in the cuspal areas and its presence in the intercuspal area may be associated with cusp formation in tooth morphogenesis. An analogous loss of type III collagen has been suggested to be an important feature also during morphogenesis of skin and cornea (Reddi, 1976;

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von der Mark et al., 1977). Loss of fibronectin from mesenchymal cells differentiating into odontoblasts was a further characteristic change with advancing development. The preodontoblast layer contained only little fibronectin, and in fully polarized odontoblasts the fibronectin was confined to the basement membrane to which they were attached. This redistribution of fibronectin in mesenchymal cells during differentiation is analogous to that observed during differentiation of

FIG. 4. Bell-staged lower first molars at the time of initial predentin secretion (19d) and mineralization and enamel secretion (21d) stained with anti-fibronectin (top) and anti-type III procollagen antibodies (bottom). X 70. Fibronectin is evenly distributed in the dental (dm) and oral (om) mesenchyme inside and outside the tooth bud. Predentin is intensely stained, but the mineralized dentin at the cuspal tips shows little or no tibronectin fluorescence. Fibronectin is not seen in the oral and enamel epithelia, but appears in the stellate reticulum (sr) within the enamel organ. The enamel matrix shows weak autofluroescence. Weak staining with anti-procollagen type III antibodies is seen in predentin but not in mineralized dentin (for details see Fig. 5). Intense fluorescence is seen in the mesenchyme (om) and basement membrane under the oral epithelium.

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FIG. 5. The epithelio-mesenchymal interface of tooth germs at the time of predentin secretion (19d) and mineralization (21d). The same sections stained with anti-fibronectin antibodies are shown unstained in Nomarski optics. The 21d tooth was also stained for mineral deposits according to the von Kossa method (right). x 180. Intense fluorescence is seen in predentin (pd). The odontoblasts (ob) are only weakly stained when compared to the undifferentiated mesenchymal cells (dm). Fibronectin is not seen in the enamel epithelium (ee) and the stratum intermedium (si), whereas the epithelial stellate reticulum (sr) shows moderate fluorescence. Fibronectin is absent from mineralized dentin (md), which is visualized in the von Kossa staining.

kidney tubules in the metanephric mesenthyme both in chick and mouse embryos (Linder et al., 1975; Wartiovaara et al., 1976). The composition of predentin, the organic matrix of dentin, has not been thoroughly characterized. Collagen appears to

be the major component; in addition glycosaminoglycans and unidentified glycoproteins are present (Thomas and Leaver, 1977). Our results show that fibronectin is one of the glycoproteins in predentin. However, after mineralization of dentin fibronectin was not detected even in deminer-

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alized sections. A similar loss of fibronectin has been observed during terminal maturation of extracellular matrices such as bone, cartilage, and tendon (Linder et al., 1975; Dessau et al., 1978; Hassell et al., 1978; Lehto et al., in preparation). The major collagen type in dentin is type

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I (Scott and Veis, 1976; Lesot and Ruth, 1979), and its presence in predentin was demonstrated also in this study. Concerning the presence of type III collagen in dentin, contradictory results have been obtained. No collagen type III was detected in dentin and predentin by biochemical

FIG. 6. The epithelio-mesenchymal interface of tooth germs at the time of predentin secretion (19d) and mineralization (21d). The same sections stained with anti-procollagen type III antibodies are shown unstained in Nomarski optics. Weak fluorescence is seen in predentin (pd) but not in mineralized dentin (md). The staining in the dental mesenchyme (dm) is more intense than at earlier stages. No procollagen type III is seen in epithelial tissue.

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methods (Scott and Veis, 1976; Muncksgaard et al., 1978), whereas in immunofluorescent studies (reported in abstract form, Cournil and Pomponio, 1977) this type of collagen was seen in dentin. We observed weak fluorescence with anti-procollagen type III antibodies in predentin but not in mineralized dentin. Neither procollagen type III nor collagen type I was seen in any of the epithelial tissues. Fibronectin was also absent from the oral epithelium, from the inner and outer enamel epithelia, and from the stratum intermedium cells associated with inner enamel epithelium. However, in the stellate reticulum inside the enamel organ moderate fibronectin staining was seen. The stellate reticulum cells rise from the cells of the dental bud when matrix material accumulates between the central cells of the bud. Our results show that also libronectin accumulates in the stellate reticulum. Previously fibronectin has been detected in epithelial cells of mesenchymal and endodermal origin (Linder et al., 1975; Chen et al., 1977). In ectoderm-derived epithelium fibronectin has not been detected even in cell culture conditions (cf. Vaheri and Mosher, 1978). This raises the possibility that the fibronectin in the stellate reticulum may be derived from the circulation, as fibronectin is known to be a major plasma protein (cf. Vaheri and Mosher, 1978. Fibronectin is a major component of the basement membranes. It conceivably plays some role in the positioning and anchorage of cells, a function assigned for basement membranes (Vracko, 1974). In the case of tooth development an additional function has been assigned to the basement membrane. A close contact between the basement membrane and the differentiating mesenchymal cells is required for the differentiation of odontoblasts in transfilter cultivation of dental epithelium and mesenchyme (Thesleff et al., 1978). These contacts were suggested to be crucial for the

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transmission of the inductive signal leading to odontoblast differentiation. The present results show that fibronectin staining in this particular basement membrane was prominent at the time of odontoblast differentiation. A role of fibronectin as a mediator of the contact between the basement membrane and the mesenchymal cells is an interesting possibility. This study was supported by grants from the Emil Aaltonen foundation and the Sigrid Juselius Foundation, by Grant No. CA 17373 of the National Cancer Institute, and by Deutsche Forschungsgemeinschaft. REFERENCES CHEN, L. B., MAITLAND, N., GALLIMORE, P. H., and MCDOUGALL, J. K. (1977). Detection of the large external transformation-sensitive protein on some epithelial cells. Exp. Cell Res. 106,39-46. COURNIL, I., and POMPONIO, J. (1977). Localization of procollagen I and III antigenicity in sections of rat incisor tooth, using the peroxidase-antiperoxidase technique. Anat. Rec. 187, 557-558. DESSAU, W., SASSE, J., TIMPL, R., and VON DER MARK, K. (1978). Role of fibronectin and collagen types I and II in chondrocytic differentiation in vitro. Ann. N. Y. Acad. Sci. 312.404-405. GRINNELL, F. (1976). Cell spreading factor-occurrence and specificity of action. Exp. Cell Res. 102,

51-62. HASSELL, J. R., PENNYPACKER, J. P., YAMADA, K. M., and PRATT, P. M. (1978). Changes in cell surface proteins during normal and vitamin A-inhibited chondrogenesis in vitro. Ann. N.Y. Acad. Sci. 312, 406-409. HEDMAN, K., KURKINEN, M., ALITALO, K., VAHERI, A., JOHANSSON, S., and H~BK, M. Isolation of the pericellular matrix of human fibroblast cultures. J. Cell Biol., in press. Hook, M., RUBIN, K., OLDBERG, A., OBRINK, B., and VAHERI, A. (1977). Cold insoluble globulin (fibronectin) mediates the adhesion of rat liver cells to plastic petri dishes. Biochem. Biophys. Res. Commun. 79, 726-733. KLEBE, R. J. (1974). Isolation of a collagen-dependent cell attachment factor. Nature (London) 250, 24% 251. KOLLAR, E. J., and BAIRD, G. R. (1970). Tissue interactions in developing mouse tooth germs. II. The inductive role of the dental papillae. J. Embryol. Ejcp. Morphol. 24, 173-186. KUUSELA, P., RUOSLAHTI, E., ENGVALL, E., and VAHERI, A. (1976). Immunological interspecies cross-reactions of fibroblast surface antigen (fibronectin). 13,639~642. Immunochemistry

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LESOT, H., VON DER MARK, K., and RUCH, J. V. (1978). Localisation par immunofluorescence des types de collagene synthethisb par l’ebauche dentaire chez l’embryon de Souris. C.R. Acad. Sci. Paris 286, 765-768. LESOT, H., and RUCH, J. V. (1979). Analyse des types de collagene synthetisis par l’ebauche dentaire et ses constituants dissocies chez l’embryon de Souris. Biol. Cell. 34, in press. LINDER, E., VAHERI, A., RUOSLAHTI, E., and WARTIOVAARA, J. (1975). Distribution of fibroblast surface antigen in the developing chick embryo. J. Exp. Med. 142,41-49. MUNKSGAARD, E. C., RHODES, M., MAYNE, R., and BUTLER, W. T. (1978). Collagen synthesis and secretion by rat incisor odontoblasts in organ culture. Ear. J. Biochem. 82,609-617. NOWACK, H., GAY S., WICK, G., BECKER, U., and TIMPL, R. (1976). Preparation and use in immunohistology of antibodies specific for type I and type III collagen and procollagen. J. Zmmunol. Methods 12, 117-124. PEARLSTEIN, E. (1976). Plasma membrane glycoprotein which mediates adhesion of fibroblasts to collagen. Nature (London) 262,497-500. REDDI, A. H. (1976). Collagen and cell differentiation. In “Biochemistry of Collagen” (G. N. Ramachandran and A. H. Reddi, eds), pp. 449-479. Plenum Press, New York. RUCH, J. V., FABRE, M., KARCHER-DJURICIC, V., and ST~UBLI, A. (1974). The effects of L-azetidine-2carboxylic acid (analogue of proline) on dental cytodifferentiations in vitro. Differentiation 2, 211220. SAINTE-MARIE, G. (1962). A paraffin embedding technique for studies employing immunofluorescence. Histochem. Cytochem. 10,250-256. SCOTT, P. G., and VEIS, A. (1976). The cyanogen bromide peptides of bovine soluble and insoluble collagens. Connect. Tissue Res. 4, 107-129. SLAVKIN, H. C., and BRINGAS, P. (1976). Epithelialmesenchyme interactions during odontogenesis IV. Morphological evidence for direct heterotypic cellcell contacts. Develop. Biol. 50,428-442. SLAVKIN, H. C., TRUMP, G. N., SCHONFELD, S., BROWNELL, A., SORGENTE, N., and LEE-OWN, V. (1977). Epigenetic regulation of enamel protein synthesis

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during epithelial-mesenchymal interaction. In “Cell Interactions in Differentiation” (M. Karkinen-Jaaskelainen, L. Saxen, and L. Weiss, eds.), pp. 209-226. Academic Press, New York and London. STENMAN, S., WARTIOVAARA, J., and VAHERI, A. (1977). Changes in the distribution of a major fibroblast protein, libronectin, during mitosis and interphase. J. Cell Biol. 74, 453-467. STENMAN, S., and VAHERI, A. (1978). Distribution of a major connective tissue protein, fibronectin, in normal human tissues. J. Exp. Med. 147,1054-1064. THESLEFF, I., LEHTONEN, E., and SAXJ?N, L. (1978). Basement membrane formation in transfdter tooth culture and its relation to odontoblast differentiation. Differentiation 10, 71-79. THOMAS, M., and LEAVER, A. G. (1977). The less acidic glycoproteins of the organic matrix of human dentine. Arch. Oral Biol. 22, 545-549. TIMPL, R., WICK, G., and GAY, A. (1977). Antibodies to distinct types of collagens and procollagens and their application in immunohistology. J. Zmmunol. Methods 18, 165-182. VAHERI, A., KURKINEN, M., LEHTO, V-P., LINDER, E., and TJMPL, R. (1978). Pericellular matrix proteins, fibronectin and (pro)collagen: Codistribution in cultured fibroblasts and loss in transformation. Proc. Nat. Acad. Sci. USA 75,4944-4948. VAHERI, A., and MOSHER, D. (1978). High molecular weight, cell surface-associated glycoprotein (fibronectin) lost in malignant transformation. Biochirit. Biophys. Acta 516, l-25. VON DER MARK, K., VON DER MARK, H., TIMPL, R., and TRELSTAD, R. L. (1977). Immunofluorescent localization of collagen types I, II, and III in the embryonic chick eye. Develop. Biol. 59, 75-85. VRACKO, R. (1974). Basal lamina scaffold anatomy and significance for maintenance of orderly tissue structure. Amer. J. Pathol. 77, 314-338. WARTIOVAARA, J., STENMAN, S., and VAHERI, A. (1976). Changes in expression of fibroblast surface antigen (SFA) during cytodifferentiation and heterokaryon formation. Differentiation 5, 85-89. YAMADA, K. M., YAMADA, S. S., and PASTAN, I. (1975). The major cell surface glycoprotein of chick embryo fibroblasts is an agglutinin. Proc. Nat. Acad. Sci. USA 72, 3158-3162.