- Email: [email protected]
Changes in the distribution of type IV collagen, laminin, proteoglycan, and fibronectin during mouse tooth development
DEVELOPMENTAL
BIOLOGY
81, 182-192 (1981)
Changes in the Distribution of Type IV Collagen, Laminin,. Proteoglycan, and Fibronectin during Mouse Tooth Development I. THESLEFF,
H. J. BARRACH,
J. M. FOIDART,’
A. VAHERI,*
R. M. PRATT, AND G. R. MARTIN
Laboratory of Developmental Biology and Anomalies, National Institu.te oj’Denfa1 Research, National Institutes of Health, Bethesda., Maryla.nd 20.20~5~ and *Department qf Virology, University oj’ Helsinki, SF-00250 Helsinki 29, Finland Received March 10, 1980; accepted in revised
form
May
19, 1980.
The distribution of certain basement membrane (BM) components including type IV collagen, laminin, BM proteoglycan, and fibronectin was studied in developing mouse molar teeth, using antibodies or antisera specific for these substances in indirect immunofluorescence. At the onset of cuspal morphogenesis, type IV collagen, laminin, and BM proteoglycan were found to be present throughout the basement membranes of the tooth. Fibronectin was abundant under the inner enamel epithelium at the region of differentiating odontoblasts and also in the mesenchymal tissues. After the first layer of predentin had been secreted by the odontoblasts at the epithelial-mesenchymal interface, laminin remained in close association with the epithelial cells whereas type IV collagen, BM proteoglycan, and fibronectin were distributed uniformly throughout this area. Later when dentin had been produced and the epithelial cells had differentiated into ameloblasts, basement membrane components disappeared from the cuspal area. These matrix components were not detected in dentin while BM proteoglycan and fibronectin were present in predentin. The observed changes in the collagenous and noncollagenous glycoproteins and the proteoglycan appear to be closely associated with cell differentiation and matrix secretion in the developing tooth. INTRODUCTION
lar and Baird, 1969). After the deposition of a layer of predentin, the epithelial cells of the enamel organ differentiate into ameloblasts at the same time that the intervening basement membrane disintegrates and disappears. It has been suggested that the disappearance of the basement membrane is necessary for the differentiation of the epithelial cells into ameloblasts (Kallenbach, 1971; Slavkin and Bringas, 1976; Meyer et al., 1977; Kallenbach and Piesco, 1978). Several components have been isolated from basement membranes and include type IV collagen, laminin, a noncollagenous glycoprotein, and a heparan sulfateproteoglycan (BM proteoglycan). Laminin and BM proteoglycan were isolated from a murine tumor that produces basement membrane (Timpl et al., 1979; Hassell et a.Z.,1980). However, antibodies prepared against type IV collagen, laminin, and BM proteoglycan react with all authentic basement membranes indicating that they contain similar materials (Yaoita et al., 1978; Foidart et al., 1980; Hassell et al., 1980). Antibody to type IV collagen has been used to localize this protein in developing mouse and rat teeth (Lesot et al., 1978; Cournil et a.Z.,1979). The distribution of fibronectin, a glycoprotein component of many connective tissues, was studied in developing teeth (Thesleff et al., 1979). In the present study, we have compared the distribution of fibronectin to type IV collagen, laminin, and
Basement membranes are extracellular matrices that separate epithelial cells from underlying connective tissues. Basement membranes have varying functions including serving as a structural support for epithelial cells, as a barrier restricting the passage of macromolecules, and as a scaffold in tissue formation and repair (Kefalides, 1978). They have also been implied in epithelial mesenchymal interactions and cell differentiation during embryonic development (Bernfield and Banerjee, 1978; Hay, 1978; Thesleff, 1978). The formation of basement membranes during embryonic development may require epithelial mesenchymal interactions, as has been demonstrated during kidney tubule differentiation (Ekblom et al., 1980). The development of various tissues in the tooth appears to depend on epithelial mesenchymal interactions in which the basement membrane has important functions. For example, mesenchymal cells of the dental papilla align under the enamel epithelium, differentiate into odontoblasts, and secrete the collagenous predentin matrix. Separation of the epithelial cells and basement membrane from the mesenchymal cells interferes with odontoblast and predentin formation (Koch, 1967; Kol’ J.-M. Foidart is a “Charge de Recherche” of the Fonds National Belge de la Recherche Scientifique in Belgium. 182 0012-1606/81/010182-11$02.00/O Copyright All rights
1981 by Academic Press. Inc. reproduction in any form reserved.
183
BRIEF NOTES
FIG. 1. Light photomicrographs of mandibular first molars of Day 16 and 18 mouse embryos and Day 1 and 2 neonates. These sections were poststained with periodic acid-schiff (PAS) after immunofluorescent staining and the sections correspond to sections from the same region as those in Figs. 3-6. (A) In the Day 16 embryonic molar the epithelial enamel organ (E) surrounds a condensation of dental papilla mesenchyme (M). Dental lamina (DL) connects the tooth germ to oral epithelium (OE). (B) In the cuspal area of a Day 18 tooth germ the mesenchymal cells under the enamel epithelium (E) have differentiated into odontoblasts (0) and initiate secretion of predentin to the epitheliomesenchymal interface. (C) In the Day 1 neonatal molar more predentin (arrow) has been deposited and the epithelial cells (E) are polarizing. The area of cell differentiation extends further cervically. (D) In the Day 2 neonatal molar the cuspal ameloblasts (A) are fully polarized, more predentin (PD) is seen, and the dentin starts to mineralize.
BM proteoglycan using specific antibodies to these proteins in the developing mouse molar from the stage of mesenchymal cell differentiation until mineralization of dentin and enamel occurs. These studies show that major changes occur in the tissue distribution of these substances which appear to be related to the development of the tooth.
MATERIALS
AND METHODS
Prepa.ra.tion and jkation of tooth germs. First mandibular molars from Day 16 and 18 (day of birth) mouse fetuses and l-, 2-, and S-day-old neonates were used. Swiss Webster (NIH) females were mated overnight to fertile males and the detection of a vaginal plug the
184
DEVELOPMENTAL BIOLOGY
VOLUME 81, 1981
FIG. 2. Light photomicrographs of mandibular first molar of a Day 3 neonatal mouse. The sections of the cuspal area (A) and the intercuspal area (B) were stained with the von Kossa stain for mineral deposits and the areas correspond to those presented in immunofluorescent staining in Figs. ‘7 and 8. (A) In the cuspal area the mineralization has advanced from that in the Day 2 neonatal molar (Fig. 1D). The ameloblasts (A) have secreted enamel matrix (EM) which is partly mineralized. (B) In the intercuspal area dentin is also mineralized and the ameloblasts have polarized but enamel matrix has not been secreted.
following morning was considered Day 0 of gestation. 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 95% alcohol for 2 hr, dehydrated in absolute alcohol at 4°C for 1 hr, and transferred to xylene at 4°C for 1 hr. Tissues were then embedded in paraplast at 60°C and serially sectioned at 6 pm. Since autofluorescence was associated with the mineralized enamel in sections from Day 2 and 3 teeth, these sections were demineralized with 0.3 M EDTA (ethylenediaminotetraacetic acid) for 30 min prior to reaction with antibody. Demineralization of the section was verified by the von Kossa staining in some studies. Some of the sections were poststained with PAS (periodic acid-Schiff) stain.
Preparation of antibody to type IV collagen, laminin, BM proteogbycan, andjibronecti,n.Antibodies to type IV collagen and laminin were prepared in rabbits as described earlier (Yaoita et al., 1978; Foidart et a.l., 1980; and Timpl et ad., 1978). The antibodies were purified by cross immunoabsorption. Their specificity was verified by radioimmunoassay, ELISA assay, Ouchterlony immunodiffusion, immunoelectrophoresis, immunofluorescence blocking studies, and binding to agarose beads coated with various antigens (Yaoita et al., 1978; Liotta et a,l.,1979b; Foidart and Reddi, 1980; Foidart et al., 1980). Basement membrane proteoglycans were extracted
and purified from a transplantable mouse tumor, EHS sarcoma (Hassell et al., 1980). Antibodies to this proteoglycan were raised in rabbits and the antibody was purified by absorption to BM-proteoglycan Sepharose 4B columns. ELISA assays and immunofluorescence blocking assays were used to verify the specificity of the antibodies. These studies showed that the purified antibodies reacted with the antigen to which they were prepared but not to the other materials. Antiserum to human fibronectin was raised in rabbits. The specificity of the antiserum (Stenman et al., 19’77) and the characteristic interspecies cross-reaction have been documented (Kuusela et al., 1976). hmu.nojhorescen t sta.in,ing a:nd microscopy. The sections were quickly deparaffinized and hydrated, after which they were treated for 30 min at room temperature with the antibodies. The anti-type collagen antibody and anti-laminin antibody were used at 80 pg/ml and the antifibronectin antiserum was used at a dilution of 1 to 40. In control experiments, normal rabbit serum (1:40) or phosphate-buffered saline (PBS) was used. The sections were washed three times in PBS, pH 7.4, and then incubated for 30 min with fluorescein isothiocyanate-conjugated goat antibody to rabbit immunoglobulins (Cappel, Cochranville, Pa.). After three PBS washes (20 minj the preparation was mounted under a coverslip in glycerin-PBS (9:l) and viewed with a Leitz-Ortholux II epiilluminated fluorescence microscope.
BRIEF NOTES
185
FIG. 3. Sections of Day 16 embryonic molars stained with antibody to type IV collagen (A), antibody to laminin (B), antibody to BM proteoglycan (C), and with antiserum to libronectin CD). See Fig. 1A for PAS-stained section. Linear deposits are seen in oral and dental basement membranes and in the walls of blood vessels. The distributions of type IV collagen, laminin, and BM proteoglycan appear identical. The fluorescence of fibronectin is particularly intense in the dental basement membrane (arrow) and fibronectin is also present in mesenchymal tissue. No stain is observed in the epithelial cells.
RESULTS
Photomicrographs of histological sections showing the morphology of the mandibular first molar at the various ages studied are presented in Figs. 1 and 2. In the Day 16 fetus (Fig. lA), the epithelial enamel organ surrounds the mesenchyme cells of the dental papilla. The tissue has already begun to assume the shape of the mature tooth in that definite cusps have formed. At
this stage the cells of the dental papilla are not differentiated and a continuous basement membrane surrounds the entire enamel organ. Two days later (Fig. 1B) the development of the cusps is even more pronounced and mesenchymal cells in the cuspal regions of the tooth have differentiated into odontoblasts and secreted a predentin matrix. In the l-day-old neonate (Fig. lC), the areas with differentiated odontoblasts have extended in a cervical direction, and the epithelial
186
DEVELOPMENTAL BIOLOGY
VOLUME 81, 1981
FIG. 4. Sections of cuspal areas of Day 18 embryonic molars stained with antibody to type IV collagen (A), antibody to laminin (B), antibody to BM proteoglycan (C), and with antiserum to fibronectin (D). Corresponding areas stained with PAS are shown in Fig. 1B. The basement membrane region has been extended because of initiated predentin secretion. Type IV collagen, BM proteoglycan, and fibronectin are present in all of the widened basement membranes, but laminin is located in closer association with the epithelial cells (E).
cells in the cuspal areas are becoming polarized. One day later (Fig. lD), ameloblasts are fully polarized and initial mineralization of dentin in the cuspal areas is observed at the same time. In the 3-day-old neonate enamel matrix has been secreted by the ameloblasts and has mineralized (Figs. 2A, B). On Day 16, all the basement membranes in the fetal molar stained when exposed to antibodies to type IV collagen, laminin, BM proteoglycan, and fibronectin (Figs. 3A-D). The various basement membranes showed similar reactions with the antibodies to type IV colla-
gen, laminin, and BM proteoglycan. As reported previously (Thesleff et al., 1979), the fluorescence observed with antibody to fibronectin was more intense in those portions of the basement membranes beneath the inner enamel epithelium than in the basement membrane associated with the oral epithelium or the outer enamel epithelium. Fibronectin was also present in the mesenchymal tissue in contrast to the staining elicited by antibodies to the other substances which was limited to basement membranes. A similar pattern was noted on Day 18 (Figs. 411-D). Basement membranes reacted
BRIEF NOTES
FIG. 5. Sections of cuspal areas of Day 1 neonates stained with antibody to type IV collagen (A), antibody to laminin (BJ, antibody to BM proteoglycan (Cl, and with antiserum to fibronectin (D). The same area is shown stained with PAS in Fig. 1C. The basement membrane is stained with all antibodies in the cervical area, but no reaction is seen with any of the antibodies under the polarizing epithelial cells (El in the cuspal area. Predentin (PD) shows staining with anti-BM-proteoglycan antibodies and antiserum to fibronectin. Some staining is seen with antibody to type IV collagen antibodies but none with antilaminin antibodies.
with all four antibodies while mesenchyme was only stained by the antibody to fibronectin. In the cuspal regions in the area in which initial predentin had been deposited, laminin appeared to be limited in location and to be confined to the subepithelial portion of the basement membrane. Differences were noted in the staining of the structures of the molar tooth obtained from l-day-old neonatal mice (Figs. 5A-D). While the basement membrane in the cervical area and the area of recently deposited predentin stained with all four antibodies, the antigens
were absent from the basal surface of the polarizing epithelial cells in cuspal areas. The dentin itself did not react with any of the antibodies while the predentin stained intensely with antibody to BM proteoglycan and fibronectin, lightly with antibody to type IV collagen, and not at all with antibody to laminin. A weak reaction was seen in the ameloblasts with antibodies to laminin. Similar patterns were observed in teeth taken from 2and 3-day-old animals except that the reaction with type IV antibody was no longer observed in predentin and less basement membrane material was present
188
DEVELOPMENTAL BIOLOGY
(Figs. 6-8). In the 3-day-old teeth enamel matrix had not yet been secreted by the ameloblasts in the intercuspal region and the adjacent basement membrane reacted with antibodies to type IV collagen, laminin, and BM proteoglycan but did not react with antibody to fibronectin (Fig. 8). DISCUSSION
Basement membranes are among the first extracellular matrices to appear in the developing embryo. They
VOLUME 81, 1981
subsequently undergo constant remodeling to allow necessary changes in the size and shape of tissues (Hay, 1978). Further, the basement membranes are not of uniform size; for example, they are discontinuous at the lobular tips, and thickened in the clefts between lobules of the embryonic submaxillary gland (Bernfield and Banerjee, 1978). These variations in basement membrane thickness are due to differences in the synthesis or degradation of individual components and are probably related to the function of the basement membrane (Bernfield and Banerjee, 1978). In the case of the em-
FIG. 6. Sections of cuspal areas of Day 2. neonatal molars stained with antibody to type IV collagen (A), antibody to laminin (B), antibody to BM proteoglycan (C), and with antiserum to fibronectin (D). The same area stained with PAS is shown in Fig. 1D. Basement membrane has disappeared under the am&blasts (A) but still exists in the cervical regions. BM proteoglycan and fibronectin are seen in predentin (PD) but type IV collagen and laminin are not. No reaction is seen in dentin (D) with any of the antibodies. In the ameloblasts (A) some reaction is observed with antibody to laminin.
BRIEF NOTES
FIG. 7. Sections of cuspal areas of Day 3 neonatal molars stained with antibody to type IV collagen (A), antibody to laminin (B), antibody to BM proteoglycan (C), and with antiserum to fibronectin (D). The same area stained with the van Kossa stain for mineral (Fig. 2A). The sections were demineralized before reactions with antibody to remove the autofluorescent mineralized enamel matrix. The distributions of the basement membrane components resemble those in the Day 2 molars (Fig. 6). BM proteoglycan and fibronectin, but not type IV collagen and laminin, are present in predentin and in the mesenchymal tissue. The epithelial tissues are negative except the ameloblasts (A) which stain with antibody to laminin.
bryonic tooth, the basement membrane separating the epithelial enamel organ from the mesenchymal dental papilla may function as a trigger for the differentiation of dental papilla cells into odontoblasts (Thesleff, 1978). After the initiation of predentin secretion by the odontoblasts into the epithelial-mesenchymal interface the basement membrane disappears as part of the program in which the epithelial cells differentiate into ameloblasts (Nylen and Scott, 1958; Kallenbach, 1971: Slavkin
and Bringas, 19’76; Meyer et al., 1977; Kallenbach and Piesco, 1978). Several proteins have been isolated from basement membranes. These include basement membrane-specific macromolecules such as type IV collagen, laminin, and BM proteoglycan. Fibronectin, which occurs in many connective tissues and in blood, also occurs in many basement membranes. Type IV collagen has been localized to the lamina densa of the epidermal-dermal
DEVELOPMENTALBIOLOGY
VOLUME 81, 1981
FIG. 8. Sections of the intercuspal areas of Day 3 neonatal molars stained with antibody to type antibody to BM proteoglycan (C), and with antiserum to tibronectin (D). Same area stained with von between the polarized ameloblasts (A) and the mineralized dentin (D) type IV collagen, laminin, fibronectin. In predentin BM proteoglycan shows intense staining. The walls of blood vessels show
basement membrane while laminin appears to be in higher concentration in the lamina lucida (Yaoita et al., 1978; Foidart et al., 1980). BM proteoglycan and fibronectin have not been localized within the zones of basement membrane (Mayer et al., 1979). Previous studies on the developing tooth have shown that its basement membranes react with antibody to type IV collagen and to fibronectin (Lesot et al., 1978; Cournil et al., 1979; Thesleff et al., 1979). Here we studied the distribution of type IV collagen, laminin, fibronectin, and BM proteoglycan at various stages of tooth development. These studies showed that type IV col-
IV collagen (A), antibody to laminin (B), Kossa staining (Fig. 2B). In the interface and BM proteoglycan are seen, but not intense staining with all antibodies.
lagen, laminin, and BM proteoglycan were evenly distributed along the basement membranes of the tooth on Day 16. Fibronectin was not evenly distributed as reported previously (Thesleff et al., 1979), but was present in higher amounts in the basement membrane under the inner enamel epithelium. The difference in distribution may be related to the differentiation of the mesenchymal cells. Further, the fibronectin in the basement membranes may have been derived in part from the mesenchymal cells while the other components may have been produced by epithelial cells. As shown previously (Thesleff et al., 19’78; Meyer et al., 1978), both
BRIEF NOTES
191
FOIDART,J. M., and REDDI, A. H. (1980). Immunofluorescent localization of type IV collagen and laminin during endochondral bone differentiation and regulation by pituitary growth hormone. Develop. Biol. 74, 130-136. HASSELL, J., ROBEY.P. G., BARRACH, H. J., WILCZEK, J., RENNARD, S. I., and MARTIN, G. R. (1980). Isolation of a heparin sulfate-containing proteoglycan from basement membrane. Proc. Nut. Acad. Sci. USA 77, 4494-4498. HAY, E. D. (1978). Role of basement membranes in development and differentiation. In “Biology and Chemistry of Basement Membranes” (N. A. Kefalides, ed.), pp. 119-136. Academic Press, New York/London. KALLENBACH. E. (1971). Electron microscopy of the differentiating rat incisor ameloblast. J. Ultrastruct. Res. 35, 508-531. KALLENBACH, E. J., and PIESCO,N. P. (1978). The changing morpholog?; of the epithelium-mesenchyme interface in the differentiation zone of growing teeth of selected vertebrates and its relationship to possible mechanism of differentiation. J. Biol. Buccale 6, 229-240. KEFALIDES, N. A. (ed.) (1978). “Biology and Chemistry of Basement Membranes.” Academic Press, New York/London. KOCH, W. E. (1967). In vitro differentiation of tooth rudiments of embryonic mice. I. Transfilter interaction of embryonic incisor tissues. J. Erp. Zoo/. 165, 155-170. KOLLAR, E. J., and BAIRD, G. R. (1969). The influence of the dental papilla on the development of tooth shape in embryonic mouse tooth germs. J. Embryol. Exp. Morphol. 21, 131-148. KUUSELA, P., RUOSLAHTI,E., ENGVALL, E., and VAHERI, A. (1976). Immunological interspecies cross-reactions of fibroblast surface antigen (fibronectin). Immunochemistry 43,639-642. LESOT, H., VON DER MARK, K., and RUCH, J. V. (1978). Localization par immunofluorescence des types de collagene synthethises par l’ebauche dentaire chez l’embryon de Souris. C. R. Acad. Sci. Paris 286, 765-768. LIOTTA, L. A., ABE, S., GEHRONROBEY,P., and MARTIN, G. R. (1979a). Preferential digestion of basement membrane collagen by an enzyme derived from a metastatic tumor. Proc. Nut. Acad. Sci. USA 76, 2268-2272. LIOTTA, L. A., WICHA, M. S., FOIDART,J. M., RENNARD,S. I., GARBISA, S., and KIDWELL, W. R. (1979b). Hormonal requirements for basement membrane collagen deposition by cultured rat mammary epithelium. Lab. Invest. 41, 511-518. MAYER, B. W., JR., HAY, E. D., and HYNES, R. 0. (1979). Electron microscopic localization of fibronectin in embryonic chick trunk and area vasculosa, utilizing ferritin-conjugated antibodies. Amt. Rec. REFERENCES 193, 616. BERNFIELD,M. R., and BANERJEE,S. D. (1978). The basal lamina in MEYER, J. M., FABRE, M., STAUBLI, A., and RUCH, J. V. (1977). Relations cellulaires au tours de l’odontogenese. J. Biol. Buccale 5, epithelial-mesenchymal morphogenetic interactions. In “Biology 107-109. and Chemistry of Basement Membranes” (N. A. Kefalides, ed.), pp. MEYER, J. M., KARCHER-DJURICIC,V., OSMAN, M., and RUCH, J. V. 137-148. Academic Press, New York/London. (1978). Aspects ultrastructuraux de la reconstitution de la memCOURNIL,I., LEBLOND,C. P., POMPONIO,J., HAND, A. R., SEDERLOF, brane basale dans des associations de constituants dentaires culL., and MARTIN, G. R. (1979). Immunohistochemical localization of tivees in vitro. C. R. Acad. Sci. Paris 287, 329-331. procollagens. I. Light microscopic distribution of procollagen I, III NYLEN, U., and SCOTT.D. B. (1958). An electron microscopic study of and IV antigenicity in the rat tooth by the indirect peroxidase-antithe early stages of dentinogenesis. Pub. Health Serv. Pub. 613,1-55. peroxidase method. J. Histochem. Cytochem. 27, 1059-1069. EKBLOM,P., ALITALO, K., VAHERI, A., TIMPL, R., and SAXEN,L. (1980). SLAVKIN, H. C., and BRINGAS,P. (1976). Epithelial-mesenchyme interactions during odontogensis, IV. Morphological evidence for diInduction of a basement membrane glycoprotein in embryonic kidrect heterotypic cell-cell contacts. Develop. Biol. 50, 428-442. ney: Possible role of laminin in morphogenesis. Proc. N&l. Acad. Sci. USA 77, 485-489. STENMAN,S., WARTIOVAARA,J.. and VAHERI, A. (1977). Changes in FOIDART,J. M., BERE, E. W., YAAR, M., RENNARD,S., GULLINO, M., the distribution of a major fibroblast protein, fibronectin, during mitosis and interphase. J. Cell Biol. 74, 453-467. MARTIN, G. R., and KATZ, S. I. (1980). Distribution and immunoelectron microscopic localization of laminin, a non-collagenous THESLEFF,I. (1978). Role of the basement membrane in odontoblast basement membrane glycoprotein. Lab. Iwest. 42, 336-342. differentiation. J. Biol. Buccale 6. 241-249.
epithelium and mesenchyme appear to be required for the production of the dental basement membrane. When predentin secretion was initiated in the region of the cusps, laminin remained associated with the basement membrane next to the epithelial cells while the other components were distributed throughout the expanded basement membrane. This is in accordance with the ultrastructural observations that laminin is present in the lamina lucida next to the epithelial cell surface (Foidart et al., 1980). Later, as more predentin was deposited, the basement membrane was degraded. Fibronectin appeared to be lost first while the other components were removed en bloc. It is well known that the degradation of collagen is carried out by highly specific proteases, the collagenases. Recent studies indicate that the collagenases that cleave type I, II, and III collagens do not cleave type IV collagen (Woolley et al., 1978; Timpl et al., 1978). Rather type IV collagen is cleaved by a different enzyme (Liotta et al., 1979a). Presumably, the degradation of the basement membrane of the developing tooth requires such a type IV collagenase and other enzymes for the other basement membrane components. These studies indicate that type IV collagen, BM proteoglycan, and laminin are constant components of the basement membranes in the developing tooth and are probably deposited in specific proportions. The alignment of mesenchymal cells along the basement membrane and their differentiation into odontoblasts may be associated with an increased production of fibronectin. The removal of the basement membrane apparently &curs at the same time that epithelial cells polarize into ameloblasts.! This suggests that specific receptors on the epithelial cells react with components of the basement membrane. While these interactions endure the cells divide; when they are lost, the cells differentiate.
192
DEVELOPMENTALBIOLOGY
THESLEFF, I., LEHTONEN, E., and SAXEN, L. (1978). Basement membrane formation in transfilter tooth culture and its relation to odontoblast differentiation. myerentiation 10, 71-79. THESLEFF,I., STENMAN,S., VAHERI, A., and TIMPL, R. (1979). Changes in the matrix proteins, fibronectin and collagen, during differentiation of mouse tooth germ. Develop. Biol. 70, 116-126. TIMPL, R., RHODE, H., ROBEY,P. G., RENNARD,S. I., FOIDART,J. M., and MARTIN, G. R. (1979). Laminin - a glycoprotein from basement membranes. J. Biol. Chem. 254.9933-9937.
VOLUME81, 1981
TIMPI., R., MARTIN, G. R., and BRUCKNER,P. (1978). Structure of basement membrane collagen obtained from a mouse tumor. In “Frontiers in Matrix Biology,” Vol. 7, pp. 130-141. Karger, Basel. WOOLLEY,D. E., GLANVILLE, R. w., ROBERTS,R. R., and EVANSON, J. M. (1978). Purification characterization and inhibition of human skin collagenases. Biochem. J. 169, 265-276. YAOITA, H., FOIDART,J. M., and KATZ, S. I. (1978). Localization of the collagenous component in skin basement membrane. J. Invest. Dermatol. 70, 191-193.
BIOLOGY
81, 182-192 (1981)
Changes in the Distribution of Type IV Collagen, Laminin,. Proteoglycan, and Fibronectin during Mouse Tooth Development I. THESLEFF,
H. J. BARRACH,
J. M. FOIDART,’
A. VAHERI,*
R. M. PRATT, AND G. R. MARTIN
Laboratory of Developmental Biology and Anomalies, National Institu.te oj’Denfa1 Research, National Institutes of Health, Bethesda., Maryla.nd 20.20~5~ and *Department qf Virology, University oj’ Helsinki, SF-00250 Helsinki 29, Finland Received March 10, 1980; accepted in revised
form
May
19, 1980.
The distribution of certain basement membrane (BM) components including type IV collagen, laminin, BM proteoglycan, and fibronectin was studied in developing mouse molar teeth, using antibodies or antisera specific for these substances in indirect immunofluorescence. At the onset of cuspal morphogenesis, type IV collagen, laminin, and BM proteoglycan were found to be present throughout the basement membranes of the tooth. Fibronectin was abundant under the inner enamel epithelium at the region of differentiating odontoblasts and also in the mesenchymal tissues. After the first layer of predentin had been secreted by the odontoblasts at the epithelial-mesenchymal interface, laminin remained in close association with the epithelial cells whereas type IV collagen, BM proteoglycan, and fibronectin were distributed uniformly throughout this area. Later when dentin had been produced and the epithelial cells had differentiated into ameloblasts, basement membrane components disappeared from the cuspal area. These matrix components were not detected in dentin while BM proteoglycan and fibronectin were present in predentin. The observed changes in the collagenous and noncollagenous glycoproteins and the proteoglycan appear to be closely associated with cell differentiation and matrix secretion in the developing tooth. INTRODUCTION
lar and Baird, 1969). After the deposition of a layer of predentin, the epithelial cells of the enamel organ differentiate into ameloblasts at the same time that the intervening basement membrane disintegrates and disappears. It has been suggested that the disappearance of the basement membrane is necessary for the differentiation of the epithelial cells into ameloblasts (Kallenbach, 1971; Slavkin and Bringas, 1976; Meyer et al., 1977; Kallenbach and Piesco, 1978). Several components have been isolated from basement membranes and include type IV collagen, laminin, a noncollagenous glycoprotein, and a heparan sulfateproteoglycan (BM proteoglycan). Laminin and BM proteoglycan were isolated from a murine tumor that produces basement membrane (Timpl et al., 1979; Hassell et a.Z.,1980). However, antibodies prepared against type IV collagen, laminin, and BM proteoglycan react with all authentic basement membranes indicating that they contain similar materials (Yaoita et al., 1978; Foidart et al., 1980; Hassell et al., 1980). Antibody to type IV collagen has been used to localize this protein in developing mouse and rat teeth (Lesot et al., 1978; Cournil et a.Z.,1979). The distribution of fibronectin, a glycoprotein component of many connective tissues, was studied in developing teeth (Thesleff et al., 1979). In the present study, we have compared the distribution of fibronectin to type IV collagen, laminin, and
Basement membranes are extracellular matrices that separate epithelial cells from underlying connective tissues. Basement membranes have varying functions including serving as a structural support for epithelial cells, as a barrier restricting the passage of macromolecules, and as a scaffold in tissue formation and repair (Kefalides, 1978). They have also been implied in epithelial mesenchymal interactions and cell differentiation during embryonic development (Bernfield and Banerjee, 1978; Hay, 1978; Thesleff, 1978). The formation of basement membranes during embryonic development may require epithelial mesenchymal interactions, as has been demonstrated during kidney tubule differentiation (Ekblom et al., 1980). The development of various tissues in the tooth appears to depend on epithelial mesenchymal interactions in which the basement membrane has important functions. For example, mesenchymal cells of the dental papilla align under the enamel epithelium, differentiate into odontoblasts, and secrete the collagenous predentin matrix. Separation of the epithelial cells and basement membrane from the mesenchymal cells interferes with odontoblast and predentin formation (Koch, 1967; Kol’ J.-M. Foidart is a “Charge de Recherche” of the Fonds National Belge de la Recherche Scientifique in Belgium. 182 0012-1606/81/010182-11$02.00/O Copyright All rights
1981 by Academic Press. Inc. reproduction in any form reserved.
183
BRIEF NOTES
FIG. 1. Light photomicrographs of mandibular first molars of Day 16 and 18 mouse embryos and Day 1 and 2 neonates. These sections were poststained with periodic acid-schiff (PAS) after immunofluorescent staining and the sections correspond to sections from the same region as those in Figs. 3-6. (A) In the Day 16 embryonic molar the epithelial enamel organ (E) surrounds a condensation of dental papilla mesenchyme (M). Dental lamina (DL) connects the tooth germ to oral epithelium (OE). (B) In the cuspal area of a Day 18 tooth germ the mesenchymal cells under the enamel epithelium (E) have differentiated into odontoblasts (0) and initiate secretion of predentin to the epitheliomesenchymal interface. (C) In the Day 1 neonatal molar more predentin (arrow) has been deposited and the epithelial cells (E) are polarizing. The area of cell differentiation extends further cervically. (D) In the Day 2 neonatal molar the cuspal ameloblasts (A) are fully polarized, more predentin (PD) is seen, and the dentin starts to mineralize.
BM proteoglycan using specific antibodies to these proteins in the developing mouse molar from the stage of mesenchymal cell differentiation until mineralization of dentin and enamel occurs. These studies show that major changes occur in the tissue distribution of these substances which appear to be related to the development of the tooth.
MATERIALS
AND METHODS
Prepa.ra.tion and jkation of tooth germs. First mandibular molars from Day 16 and 18 (day of birth) mouse fetuses and l-, 2-, and S-day-old neonates were used. Swiss Webster (NIH) females were mated overnight to fertile males and the detection of a vaginal plug the
184
DEVELOPMENTAL BIOLOGY
VOLUME 81, 1981
FIG. 2. Light photomicrographs of mandibular first molar of a Day 3 neonatal mouse. The sections of the cuspal area (A) and the intercuspal area (B) were stained with the von Kossa stain for mineral deposits and the areas correspond to those presented in immunofluorescent staining in Figs. ‘7 and 8. (A) In the cuspal area the mineralization has advanced from that in the Day 2 neonatal molar (Fig. 1D). The ameloblasts (A) have secreted enamel matrix (EM) which is partly mineralized. (B) In the intercuspal area dentin is also mineralized and the ameloblasts have polarized but enamel matrix has not been secreted.
following morning was considered Day 0 of gestation. 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 95% alcohol for 2 hr, dehydrated in absolute alcohol at 4°C for 1 hr, and transferred to xylene at 4°C for 1 hr. Tissues were then embedded in paraplast at 60°C and serially sectioned at 6 pm. Since autofluorescence was associated with the mineralized enamel in sections from Day 2 and 3 teeth, these sections were demineralized with 0.3 M EDTA (ethylenediaminotetraacetic acid) for 30 min prior to reaction with antibody. Demineralization of the section was verified by the von Kossa staining in some studies. Some of the sections were poststained with PAS (periodic acid-Schiff) stain.
Preparation of antibody to type IV collagen, laminin, BM proteogbycan, andjibronecti,n.Antibodies to type IV collagen and laminin were prepared in rabbits as described earlier (Yaoita et al., 1978; Foidart et a.l., 1980; and Timpl et ad., 1978). The antibodies were purified by cross immunoabsorption. Their specificity was verified by radioimmunoassay, ELISA assay, Ouchterlony immunodiffusion, immunoelectrophoresis, immunofluorescence blocking studies, and binding to agarose beads coated with various antigens (Yaoita et al., 1978; Liotta et a,l.,1979b; Foidart and Reddi, 1980; Foidart et al., 1980). Basement membrane proteoglycans were extracted
and purified from a transplantable mouse tumor, EHS sarcoma (Hassell et al., 1980). Antibodies to this proteoglycan were raised in rabbits and the antibody was purified by absorption to BM-proteoglycan Sepharose 4B columns. ELISA assays and immunofluorescence blocking assays were used to verify the specificity of the antibodies. These studies showed that the purified antibodies reacted with the antigen to which they were prepared but not to the other materials. Antiserum to human fibronectin was raised in rabbits. The specificity of the antiserum (Stenman et al., 19’77) and the characteristic interspecies cross-reaction have been documented (Kuusela et al., 1976). hmu.nojhorescen t sta.in,ing a:nd microscopy. The sections were quickly deparaffinized and hydrated, after which they were treated for 30 min at room temperature with the antibodies. The anti-type collagen antibody and anti-laminin antibody were used at 80 pg/ml and the antifibronectin antiserum was used at a dilution of 1 to 40. In control experiments, normal rabbit serum (1:40) or phosphate-buffered saline (PBS) was used. The sections were washed three times in PBS, pH 7.4, and then incubated for 30 min with fluorescein isothiocyanate-conjugated goat antibody to rabbit immunoglobulins (Cappel, Cochranville, Pa.). After three PBS washes (20 minj the preparation was mounted under a coverslip in glycerin-PBS (9:l) and viewed with a Leitz-Ortholux II epiilluminated fluorescence microscope.
BRIEF NOTES
185
FIG. 3. Sections of Day 16 embryonic molars stained with antibody to type IV collagen (A), antibody to laminin (B), antibody to BM proteoglycan (C), and with antiserum to libronectin CD). See Fig. 1A for PAS-stained section. Linear deposits are seen in oral and dental basement membranes and in the walls of blood vessels. The distributions of type IV collagen, laminin, and BM proteoglycan appear identical. The fluorescence of fibronectin is particularly intense in the dental basement membrane (arrow) and fibronectin is also present in mesenchymal tissue. No stain is observed in the epithelial cells.
RESULTS
Photomicrographs of histological sections showing the morphology of the mandibular first molar at the various ages studied are presented in Figs. 1 and 2. In the Day 16 fetus (Fig. lA), the epithelial enamel organ surrounds the mesenchyme cells of the dental papilla. The tissue has already begun to assume the shape of the mature tooth in that definite cusps have formed. At
this stage the cells of the dental papilla are not differentiated and a continuous basement membrane surrounds the entire enamel organ. Two days later (Fig. 1B) the development of the cusps is even more pronounced and mesenchymal cells in the cuspal regions of the tooth have differentiated into odontoblasts and secreted a predentin matrix. In the l-day-old neonate (Fig. lC), the areas with differentiated odontoblasts have extended in a cervical direction, and the epithelial
186
DEVELOPMENTAL BIOLOGY
VOLUME 81, 1981
FIG. 4. Sections of cuspal areas of Day 18 embryonic molars stained with antibody to type IV collagen (A), antibody to laminin (B), antibody to BM proteoglycan (C), and with antiserum to fibronectin (D). Corresponding areas stained with PAS are shown in Fig. 1B. The basement membrane region has been extended because of initiated predentin secretion. Type IV collagen, BM proteoglycan, and fibronectin are present in all of the widened basement membranes, but laminin is located in closer association with the epithelial cells (E).
cells in the cuspal areas are becoming polarized. One day later (Fig. lD), ameloblasts are fully polarized and initial mineralization of dentin in the cuspal areas is observed at the same time. In the 3-day-old neonate enamel matrix has been secreted by the ameloblasts and has mineralized (Figs. 2A, B). On Day 16, all the basement membranes in the fetal molar stained when exposed to antibodies to type IV collagen, laminin, BM proteoglycan, and fibronectin (Figs. 3A-D). The various basement membranes showed similar reactions with the antibodies to type IV colla-
gen, laminin, and BM proteoglycan. As reported previously (Thesleff et al., 1979), the fluorescence observed with antibody to fibronectin was more intense in those portions of the basement membranes beneath the inner enamel epithelium than in the basement membrane associated with the oral epithelium or the outer enamel epithelium. Fibronectin was also present in the mesenchymal tissue in contrast to the staining elicited by antibodies to the other substances which was limited to basement membranes. A similar pattern was noted on Day 18 (Figs. 411-D). Basement membranes reacted
BRIEF NOTES
FIG. 5. Sections of cuspal areas of Day 1 neonates stained with antibody to type IV collagen (A), antibody to laminin (BJ, antibody to BM proteoglycan (Cl, and with antiserum to fibronectin (D). The same area is shown stained with PAS in Fig. 1C. The basement membrane is stained with all antibodies in the cervical area, but no reaction is seen with any of the antibodies under the polarizing epithelial cells (El in the cuspal area. Predentin (PD) shows staining with anti-BM-proteoglycan antibodies and antiserum to fibronectin. Some staining is seen with antibody to type IV collagen antibodies but none with antilaminin antibodies.
with all four antibodies while mesenchyme was only stained by the antibody to fibronectin. In the cuspal regions in the area in which initial predentin had been deposited, laminin appeared to be limited in location and to be confined to the subepithelial portion of the basement membrane. Differences were noted in the staining of the structures of the molar tooth obtained from l-day-old neonatal mice (Figs. 5A-D). While the basement membrane in the cervical area and the area of recently deposited predentin stained with all four antibodies, the antigens
were absent from the basal surface of the polarizing epithelial cells in cuspal areas. The dentin itself did not react with any of the antibodies while the predentin stained intensely with antibody to BM proteoglycan and fibronectin, lightly with antibody to type IV collagen, and not at all with antibody to laminin. A weak reaction was seen in the ameloblasts with antibodies to laminin. Similar patterns were observed in teeth taken from 2and 3-day-old animals except that the reaction with type IV antibody was no longer observed in predentin and less basement membrane material was present
188
DEVELOPMENTAL BIOLOGY
(Figs. 6-8). In the 3-day-old teeth enamel matrix had not yet been secreted by the ameloblasts in the intercuspal region and the adjacent basement membrane reacted with antibodies to type IV collagen, laminin, and BM proteoglycan but did not react with antibody to fibronectin (Fig. 8). DISCUSSION
Basement membranes are among the first extracellular matrices to appear in the developing embryo. They
VOLUME 81, 1981
subsequently undergo constant remodeling to allow necessary changes in the size and shape of tissues (Hay, 1978). Further, the basement membranes are not of uniform size; for example, they are discontinuous at the lobular tips, and thickened in the clefts between lobules of the embryonic submaxillary gland (Bernfield and Banerjee, 1978). These variations in basement membrane thickness are due to differences in the synthesis or degradation of individual components and are probably related to the function of the basement membrane (Bernfield and Banerjee, 1978). In the case of the em-
FIG. 6. Sections of cuspal areas of Day 2. neonatal molars stained with antibody to type IV collagen (A), antibody to laminin (B), antibody to BM proteoglycan (C), and with antiserum to fibronectin (D). The same area stained with PAS is shown in Fig. 1D. Basement membrane has disappeared under the am&blasts (A) but still exists in the cervical regions. BM proteoglycan and fibronectin are seen in predentin (PD) but type IV collagen and laminin are not. No reaction is seen in dentin (D) with any of the antibodies. In the ameloblasts (A) some reaction is observed with antibody to laminin.
BRIEF NOTES
FIG. 7. Sections of cuspal areas of Day 3 neonatal molars stained with antibody to type IV collagen (A), antibody to laminin (B), antibody to BM proteoglycan (C), and with antiserum to fibronectin (D). The same area stained with the van Kossa stain for mineral (Fig. 2A). The sections were demineralized before reactions with antibody to remove the autofluorescent mineralized enamel matrix. The distributions of the basement membrane components resemble those in the Day 2 molars (Fig. 6). BM proteoglycan and fibronectin, but not type IV collagen and laminin, are present in predentin and in the mesenchymal tissue. The epithelial tissues are negative except the ameloblasts (A) which stain with antibody to laminin.
bryonic tooth, the basement membrane separating the epithelial enamel organ from the mesenchymal dental papilla may function as a trigger for the differentiation of dental papilla cells into odontoblasts (Thesleff, 1978). After the initiation of predentin secretion by the odontoblasts into the epithelial-mesenchymal interface the basement membrane disappears as part of the program in which the epithelial cells differentiate into ameloblasts (Nylen and Scott, 1958; Kallenbach, 1971: Slavkin
and Bringas, 19’76; Meyer et al., 1977; Kallenbach and Piesco, 1978). Several proteins have been isolated from basement membranes. These include basement membrane-specific macromolecules such as type IV collagen, laminin, and BM proteoglycan. Fibronectin, which occurs in many connective tissues and in blood, also occurs in many basement membranes. Type IV collagen has been localized to the lamina densa of the epidermal-dermal
DEVELOPMENTALBIOLOGY
VOLUME 81, 1981
FIG. 8. Sections of the intercuspal areas of Day 3 neonatal molars stained with antibody to type antibody to BM proteoglycan (C), and with antiserum to tibronectin (D). Same area stained with von between the polarized ameloblasts (A) and the mineralized dentin (D) type IV collagen, laminin, fibronectin. In predentin BM proteoglycan shows intense staining. The walls of blood vessels show
basement membrane while laminin appears to be in higher concentration in the lamina lucida (Yaoita et al., 1978; Foidart et al., 1980). BM proteoglycan and fibronectin have not been localized within the zones of basement membrane (Mayer et al., 1979). Previous studies on the developing tooth have shown that its basement membranes react with antibody to type IV collagen and to fibronectin (Lesot et al., 1978; Cournil et al., 1979; Thesleff et al., 1979). Here we studied the distribution of type IV collagen, laminin, fibronectin, and BM proteoglycan at various stages of tooth development. These studies showed that type IV col-
IV collagen (A), antibody to laminin (B), Kossa staining (Fig. 2B). In the interface and BM proteoglycan are seen, but not intense staining with all antibodies.
lagen, laminin, and BM proteoglycan were evenly distributed along the basement membranes of the tooth on Day 16. Fibronectin was not evenly distributed as reported previously (Thesleff et al., 1979), but was present in higher amounts in the basement membrane under the inner enamel epithelium. The difference in distribution may be related to the differentiation of the mesenchymal cells. Further, the fibronectin in the basement membranes may have been derived in part from the mesenchymal cells while the other components may have been produced by epithelial cells. As shown previously (Thesleff et al., 19’78; Meyer et al., 1978), both
BRIEF NOTES
191
FOIDART,J. M., and REDDI, A. H. (1980). Immunofluorescent localization of type IV collagen and laminin during endochondral bone differentiation and regulation by pituitary growth hormone. Develop. Biol. 74, 130-136. HASSELL, J., ROBEY.P. G., BARRACH, H. J., WILCZEK, J., RENNARD, S. I., and MARTIN, G. R. (1980). Isolation of a heparin sulfate-containing proteoglycan from basement membrane. Proc. Nut. Acad. Sci. USA 77, 4494-4498. HAY, E. D. (1978). Role of basement membranes in development and differentiation. In “Biology and Chemistry of Basement Membranes” (N. A. Kefalides, ed.), pp. 119-136. Academic Press, New York/London. KALLENBACH. E. (1971). Electron microscopy of the differentiating rat incisor ameloblast. J. Ultrastruct. Res. 35, 508-531. KALLENBACH, E. J., and PIESCO,N. P. (1978). The changing morpholog?; of the epithelium-mesenchyme interface in the differentiation zone of growing teeth of selected vertebrates and its relationship to possible mechanism of differentiation. J. Biol. Buccale 6, 229-240. KEFALIDES, N. A. (ed.) (1978). “Biology and Chemistry of Basement Membranes.” Academic Press, New York/London. KOCH, W. E. (1967). In vitro differentiation of tooth rudiments of embryonic mice. I. Transfilter interaction of embryonic incisor tissues. J. Erp. Zoo/. 165, 155-170. KOLLAR, E. J., and BAIRD, G. R. (1969). The influence of the dental papilla on the development of tooth shape in embryonic mouse tooth germs. J. Embryol. Exp. Morphol. 21, 131-148. KUUSELA, P., RUOSLAHTI,E., ENGVALL, E., and VAHERI, A. (1976). Immunological interspecies cross-reactions of fibroblast surface antigen (fibronectin). Immunochemistry 43,639-642. LESOT, H., VON DER MARK, K., and RUCH, J. V. (1978). Localization par immunofluorescence des types de collagene synthethises par l’ebauche dentaire chez l’embryon de Souris. C. R. Acad. Sci. Paris 286, 765-768. LIOTTA, L. A., ABE, S., GEHRONROBEY,P., and MARTIN, G. R. (1979a). Preferential digestion of basement membrane collagen by an enzyme derived from a metastatic tumor. Proc. Nut. Acad. Sci. USA 76, 2268-2272. LIOTTA, L. A., WICHA, M. S., FOIDART,J. M., RENNARD,S. I., GARBISA, S., and KIDWELL, W. R. (1979b). Hormonal requirements for basement membrane collagen deposition by cultured rat mammary epithelium. Lab. Invest. 41, 511-518. MAYER, B. W., JR., HAY, E. D., and HYNES, R. 0. (1979). Electron microscopic localization of fibronectin in embryonic chick trunk and area vasculosa, utilizing ferritin-conjugated antibodies. Amt. Rec. REFERENCES 193, 616. BERNFIELD,M. R., and BANERJEE,S. D. (1978). The basal lamina in MEYER, J. M., FABRE, M., STAUBLI, A., and RUCH, J. V. (1977). Relations cellulaires au tours de l’odontogenese. J. Biol. Buccale 5, epithelial-mesenchymal morphogenetic interactions. In “Biology 107-109. and Chemistry of Basement Membranes” (N. A. Kefalides, ed.), pp. MEYER, J. M., KARCHER-DJURICIC,V., OSMAN, M., and RUCH, J. V. 137-148. Academic Press, New York/London. (1978). Aspects ultrastructuraux de la reconstitution de la memCOURNIL,I., LEBLOND,C. P., POMPONIO,J., HAND, A. R., SEDERLOF, brane basale dans des associations de constituants dentaires culL., and MARTIN, G. R. (1979). Immunohistochemical localization of tivees in vitro. C. R. Acad. Sci. Paris 287, 329-331. procollagens. I. Light microscopic distribution of procollagen I, III NYLEN, U., and SCOTT.D. B. (1958). An electron microscopic study of and IV antigenicity in the rat tooth by the indirect peroxidase-antithe early stages of dentinogenesis. Pub. Health Serv. Pub. 613,1-55. peroxidase method. J. Histochem. Cytochem. 27, 1059-1069. EKBLOM,P., ALITALO, K., VAHERI, A., TIMPL, R., and SAXEN,L. (1980). SLAVKIN, H. C., and BRINGAS,P. (1976). Epithelial-mesenchyme interactions during odontogensis, IV. Morphological evidence for diInduction of a basement membrane glycoprotein in embryonic kidrect heterotypic cell-cell contacts. Develop. Biol. 50, 428-442. ney: Possible role of laminin in morphogenesis. Proc. N&l. Acad. Sci. USA 77, 485-489. STENMAN,S., WARTIOVAARA,J.. and VAHERI, A. (1977). Changes in FOIDART,J. M., BERE, E. W., YAAR, M., RENNARD,S., GULLINO, M., the distribution of a major fibroblast protein, fibronectin, during mitosis and interphase. J. Cell Biol. 74, 453-467. MARTIN, G. R., and KATZ, S. I. (1980). Distribution and immunoelectron microscopic localization of laminin, a non-collagenous THESLEFF,I. (1978). Role of the basement membrane in odontoblast basement membrane glycoprotein. Lab. Iwest. 42, 336-342. differentiation. J. Biol. Buccale 6. 241-249.
epithelium and mesenchyme appear to be required for the production of the dental basement membrane. When predentin secretion was initiated in the region of the cusps, laminin remained associated with the basement membrane next to the epithelial cells while the other components were distributed throughout the expanded basement membrane. This is in accordance with the ultrastructural observations that laminin is present in the lamina lucida next to the epithelial cell surface (Foidart et al., 1980). Later, as more predentin was deposited, the basement membrane was degraded. Fibronectin appeared to be lost first while the other components were removed en bloc. It is well known that the degradation of collagen is carried out by highly specific proteases, the collagenases. Recent studies indicate that the collagenases that cleave type I, II, and III collagens do not cleave type IV collagen (Woolley et al., 1978; Timpl et al., 1978). Rather type IV collagen is cleaved by a different enzyme (Liotta et al., 1979a). Presumably, the degradation of the basement membrane of the developing tooth requires such a type IV collagenase and other enzymes for the other basement membrane components. These studies indicate that type IV collagen, BM proteoglycan, and laminin are constant components of the basement membranes in the developing tooth and are probably deposited in specific proportions. The alignment of mesenchymal cells along the basement membrane and their differentiation into odontoblasts may be associated with an increased production of fibronectin. The removal of the basement membrane apparently &curs at the same time that epithelial cells polarize into ameloblasts.! This suggests that specific receptors on the epithelial cells react with components of the basement membrane. While these interactions endure the cells divide; when they are lost, the cells differentiate.
192
DEVELOPMENTALBIOLOGY
THESLEFF, I., LEHTONEN, E., and SAXEN, L. (1978). Basement membrane formation in transfilter tooth culture and its relation to odontoblast differentiation. myerentiation 10, 71-79. THESLEFF,I., STENMAN,S., VAHERI, A., and TIMPL, R. (1979). Changes in the matrix proteins, fibronectin and collagen, during differentiation of mouse tooth germ. Develop. Biol. 70, 116-126. TIMPL, R., RHODE, H., ROBEY,P. G., RENNARD,S. I., FOIDART,J. M., and MARTIN, G. R. (1979). Laminin - a glycoprotein from basement membranes. J. Biol. Chem. 254.9933-9937.
VOLUME81, 1981
TIMPI., R., MARTIN, G. R., and BRUCKNER,P. (1978). Structure of basement membrane collagen obtained from a mouse tumor. In “Frontiers in Matrix Biology,” Vol. 7, pp. 130-141. Karger, Basel. WOOLLEY,D. E., GLANVILLE, R. w., ROBERTS,R. R., and EVANSON, J. M. (1978). Purification characterization and inhibition of human skin collagenases. Biochem. J. 169, 265-276. YAOITA, H., FOIDART,J. M., and KATZ, S. I. (1978). Localization of the collagenous component in skin basement membrane. J. Invest. Dermatol. 70, 191-193.