Radioautographic demonstration of receptors for epidermal growth factor in various cells of the oral cavity.код для вставкиСкачать
THE ANATOMICAL RECORD 222:191-200 (1988) Radioautographic Demonstration of Receptors for Epidermal Growth Factor in Various Cells of the Oral Cavity MOON-IL CHO, YU LIN LEE, AND PHILIAS R. GARANT Department of Oral Biology and Pathology, School of Dental Medicine, State University of New York at Stony Brook, New York 11794-8700 ABSTRACT Mouse iodinated epidermal growth factor (EGF)was localizedby light and electron microscopic radioautography in basal cells of oral epithelium, papillary cells of the enamel organ, periodontal ligament fibroblasts, preodontoblast precursor cells, and preosteoblasts of the alveolar bone of 13-day-old Sprague-Dawleyrats. The specificity of binding in these cells was suggested by an observed reduction of about 90% in the labeling when excess unlabeled EGF was injected along with the 125I-EGF. In contrast, fully differentiated cells, such as ameloblasts. odontoblasts, and osteoblasts, were only poorly labeled. Quantitative analysis of the light microscopic radioautographs revealed that the papillary cells had the highest level of labeling (5.5grains per 100 pm2 of cell area). The significance of the rather high labeling of the preosteoblasts of the alveolar bone and the fibroblasts of the periodontal ligament is unknown. However, the well-known effect of EGF in producing precocious eruption of teeth may be a consequence of an effect on these two cell types. Epidermal growth factor (EGF) was originally isolated from male mouse submandibular glands (Cohen, 1962). It is a single-chain polypeptide of 53 amino acids with a molecular weight of 6,045 Da (Carpenter and Cohen, 1979). EGF is a potent mitogen for various cell types, both in vitro and in vivo (Carpenter and Cohen, 1979; Das, 1982). It is well documented that EGF binds to specific receptors on the cell membrane of its target cells and is internalized into an endosomal compartment (Carpenter and Cohen, 1976; Haigler, et al., 1979). One of the earliest findings with respect to the in vivo effects of EGF was the precocious eruption of incisors when it was administered to newborn mice (Cohen, 1962). EGF administered in vivo binds to cells in the enamel organ of developing teeth (Martineau-Doize,et al., 1987). The in vitro binding of EGF to mouse and human odontogenic tissue a t various stages of development has also been studied (Thesleff, 1987; Thesleff, et al., 1987; Partaneu and Thesleff, 1987). These recent demonstrations of the binding of EGF to various odontogenic tissues suggests that this growth factor may have a significant role in the differentiation of the enamel organ and dental follicle mesenchyme. The recent observation of the binding of EGF in the apical tissues of a developing human premolar root (Thesleff, et al., 1987) is in good agreement with the observed association of precocious tooth eruption and EGF (Cohen, 1962; Frindik, et al., 1985). However, the mechanism responsible for precocious eruption remains unknown. In this study, we have identified the cell types that bind EGF in vivo during the development of teeth. With lZ5I-EGFradioautography, a high number of binding sites for EGF were detected on the cell membranes of basal cells of oral epithelium, papillary cells of the enamel organ, periodontal ligament fibroblasts, and @ 1988 ALAN R. LISS, INC preosteoblasts. On the basis of these observations, we speculate that accelerated eruption of teeth resulting from treatment with EGF may be due t o a direct and/ or indirect effect on periodontal ligament fibroblasts and preosteoblasts. MATERIALS AND METHODS lodination of Epidermal Growth Factor Mouse EGF (receptor grade) was purchased from Collaborative Research, Inc. (Waltham, MA) and radioiodinated by the chloramine T technique (Hunter and Greenwood, 1962; Carpenter and Cohen, 1976)to a specific activity of approximately 100 pci/pg immediately before use. Injection of l2’I-EGF A total of three 13-day-old Sprague-Dawley rats weighing 23.5 +- 0.1 g were used. lZ5I-EGFwas injected through a jugular vein under ether anesthesia. In order to study the in vivo localization of specific binding sites for EGF by radioautography, two rats were injected with 100 pci of lZ5I-EGF(S.A. = approximately 100 pci/ p.g) in 0.1 ml of 0.05 M potassium phosphate buffer, pH 7.5, containing 0.075 M NaCl. To assess the specificity of binding, a control rat was injected with 0.1 ml of 0.05 M potassium phosphate buffer, pH 7.5, containing 100 pci of KI-EGF and an excess (50 pg) of unlabeled EGF, and 0.075 M NaC1. Tissue Preparation for Radioautography At 5 min after administration of the iodinated growth factor, the animals were anesthetized by intraperitoReceived November 18, 1987; accepted March 2,1988. 192 M.-I. CHO, Y.L. LEE, AND P.R. GARANT TABLE 1. Quantitative analysis of light microscopic radioautographic silver grains over various cell types of the developing first maxillary molars of 13-day-oldrats Experimental group Control group # Grains Mean # # Cells # Grains Mean # counted counted per cell counted counted per cell # Cells Cell types Oral epithelium basal cells Enamel organ: Presecretory ameloblasts Stratum intermedium Postsecretory ameloblasts Papillary cells pulp: Odontoblasts Bone: Osteoblasts Preosteoblasts Periodontal ligament fibroblasts 117 1,734 14.8 103 401 3.9 90 63 88 95 48 31 196 4,505 0.5 0.5 2.2 47.4 91 409 4.5 111 135 1.2 89 80 116 63 864 1,496 0.7 10.8 12.9 73 134 131 228 1.8 1.7 neal injection of Nembutal (1 mg/l00 g body weight) prior to an intracardiac perfusion with lactated Ringer's solution for 20 sec. This procedure was immediately followed by perfusion with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, for 10 min. The maxillae were dissected free of the surrounding tissues and further fxed in Karnovsky's fixative (Karnovsky, 1965) for an additional 3 hr. After rinsing in 0.1 M cacodylate buffer for 10 min, the maxillae were decalcified for: 4 days at 4°C in 0.1 M EDTA containing 3% glutaraldehyde. The areas containing the first maxillary molars were then mesiodistally sectioned into 1mm-thick slices. These slices, along with tissue containing the developing third molars, was postfixed for 1%hours in 1%OsO, in s-collidine buffer, pH 7.4. Tissues were then dehydrated in a graduated series of cold ethanols and propylene oxide prior to infiltration with Poly Bed mixture a t room temperature for 4 hr and were placed in flat embedding molds containing fresh embedding mixture. Polymerization was accomplished at 60°C for 48 hr. Electron Microscopic Radioautography To prepare electron microscopic radioautographs, the loop method of Caro and Van Tubergen (1962) was employed as described previously (Cho and Garant, (1981b). Thin sections approximately 100 nm thick were cut from four blocks of each animal and were coated with a crystalline monolayer of Ilford L4 emulsion. After exposure at 4"C, the sections were developed with Microdol-X. QuantitativeAnalysis of Radioautographs Identification of cell types with specific binding sites for epidermal growth factor A total of 12 blocks (two blocks from each maxillar; i.e., eight blocks from the experimental rats and four blocks from the control rat) were selected and prepared for light microscopic radioautographs under conditions identical to those described previously. Photographs were taken at x 1,000 in a Zeiss photomicroscope (Carl Zeiss, Inc., NY)and printed at a final magnification of x 5,000. The number of cells and silver grains over these cells were counted on the photographic prints. The cell types Tissue Processing for Morphology subjected to quantitative analysis of radioautographs Three 13-day-oldrats weighing 23.0 f 0.1 g were an- are listed in Table 1. esthetized by intraperitoneal injection of Nembutal (1 Binding sites for epidermal growth factor per 100 pm2 of mgI100 g body weight) before intracardiac perfusion cytoplasmic surface area with 2.5% glutaraldehyde in 0.1 M sodium cacodylate In an attempt to study the relative number of binding buffer, pH 7.4, for 10 min. All subsequent procedures were identical to those described in tissue preparation sites for EGF present on different cell types, only those for radioautography. However, for morphological study, cell types with high mean values of silver grains were en bloc staining in 1%uranyl acetate in 0.1 M maleate subjected to quantitative analysis. The areas were buffer, pH 6.2, was added between postfixation in 1% measured on the photographic prints with a Kontron OsO, and dehydration in alcohol (Cho and Garant, MOP analyzer after cell boundaries were drawn with 1981a). One-micrometer sections cut in a mesiodistal a red color marker. The mean value of silver grains per 100 pm2 of cell was obtained for each cell type (Table 2). plane were stained with 1%toluidine blue. tight Microscopic Radioautography Three t o four 1-km thick sections from each block were mounted on glass slides, coated with Kodak NTB2 liquid emulsion, exposed at 4"C, and developed as previously described (Cho and Garant, 1981b). The radioautographs were subsequently stained with 1%toluidine blue in sodium acetate buffer. RESULTS Light Microscopic Morphology In 13-day-oldrats used in this study, the mesial cusp of the first maxillary molar was located directly under the oral epithelium. Root development was well advanced. The collagenous fibers of the periodontal ligament were arranged obliquely from the root surface EPIDERMAL GROWTH FACTOR RECEPTORS IN THE ORAL CAVITY 193 TABLE 2. Quantitative analysis of light microscopic radioautomaphic silver grains over unit area (100 pn2) of cell volume Cell types # Grains/100 fim2 Basal cells of oral epithelium Papillary cells in association with postsecretory ameloblasts Preosteoblasts Periodontal ligament fibroblasts 3.4 5.5 1.9 2.1 Light microscopic radioautographs from the experimental animals contained numerous silver grains, mostly restricted to the basal cells (Fig. 2a). Within the basal cells, the majority of silver grains appeared to be located at both the basal and lateral portions of the cells (Fig. 2c). The apical portion of the cell was relatively free of silver grains (Fig. 2c). Electron microscopic radioautographs confirmed these observations (Fig. 2d). The number of silver grains on the cells of the basal layer was reduced dramatically from 14.8/cell in the experimental tissues t o 3.9kell in the control tissues (Fig. 2b, Table 1). Enamel organ Light microscopic radioautography of the enamel organ in the experimental animals clearly showed that neither the presecretory, secretory, and postsecretory ameloblasts nor the cells of the stratum intermedium were labeled (Fig. 3a, b). Heavy labeling was observed along the peripheral region of all papillary cells (Fig. 3b). Ultrastructural morphology of this region showed a large number of microvillilike structures projecting into the intercellular spaces. Electron microscopic radioautography revealed specific localization of silver grains along the cell membrane proper, but not on the microvilli (Fig. 3d, el. The number of silver grains was greatly reduced over papillary cells of the control rat, although the pattern of labeling remained the same as in the experimental group (Fig. 3c, Table 1). Bone Fig. 1. Light micrograph showing the mesial developing root of the first maxillary molar of a 13-day-old rat x 100. Low-magnification micrograph (inset)illustrates the mesial half of the developing tooth with its enamel organ under the oral epithelium (ep). x 23. The rectangular area of inset shows an area similar to that shown in Fig. 1. A = ameloblasts; AB = alveolar bone; D = dentin; E = enamel; ES = enamel space; HR = Hertwig's root sheath Od = odontoblasts; P = pulp; PD = predentin; PDL = periodontal ligament. In areas of active bone formation, light microscopic radioautography from both the experimental and control animals demonstrated that clearly identifiable osteoblasts (including osteoclasts; result not shown) remained unlabeled (Fig. 4a, b). However, prominent labeling was observed over fibroblastic cells near the osteoblasts (Fig. 4a). A small number of grains remained in association with these cells in the control group (Fig. 4b, Table 1). Electron microscopic radioautography clearly confirmed that the labeled fibroblastic cells were relatively immature cells, perhaps preosteoblasts in the process of migrating toward the prebone surface between adjacent osteoblasts (Fig. 4c). coronally toward the alveolar bone. Odontoblasts were Periodontal ligament The developing periodontal ligament of the first maxlocated along the predentin surface in the pulp (Fig. 1). illary molars of 13-day-old rats was characterized by Radioautographic Localization of Binding Sites for '251-EGF fewer collagen fibrils and more fibroblasts, compared Oral epithelium with a fully developed and functional periodontal ligThe oral epithelium covering the first developing ament (Fig. 5a). Periodontal ligament fibroblasts exmaxillary molars was a typical stratified squamous epi- hibiting a high degree of polarization and orientation thelium (Figs. 2a,c). were heavily labeled along their cell boundaries in the 194 M.-I. CHO, Y.L. LEE, AND P.R. GARANT Fig. 2. Ramoautographs showing localization of '25I-EGF on oral epithelium. a: Light microscopic radioautograph of oral epithelium from an experimental animal reveals a heavy labeling with silver grains on the basal cells (arrowheads).x 120. b The number of grains is dramatically decreased on the basal cells from the control animal x 120. c: Cells of the cornified (C),granulosum (G),and spinosum (S)layers are free of labeling, in contrast to heavy labeling of basal layer (B).X 900. d Eledon microscopic radioautography demonstrates localization of silver grains (arrowheads) along the basal and lateral protions of the cell membrane of the basal cells (B) from an experimental animal. x 2,700. S = spinosum layer; F = fibroblasts. Fig. 3. Radioautographs showing the localization of ‘“I-EGF on the enamel organ. a: Light microscopic radioautograph of developing third molar shows that both presecretory amelohlasts (A) and cells of the stratum intermedium (SI) of an experimental animal remain unlabeled. x 350.D = dentin. b Papillary cells (P)of an experimental animal are, however, heavily labeled, whereas only a small number of grains are present on the papillary cells from the control animal ( c ) x 400. A = amelohlasts; B = blood vessel. d Electron microscopic radioautograph of papillary cells (P) from an experimental animal localizes the silver grains on the cell membrane. x 1,200.A = ameloblasts; M = microvilli. e: The silver grains (arrowheads) are present on the cell membrane of papillary cells, hut not on the microvilli (MI.X 2,700. 196 M.-I. CHO, Y.L. LEE, AND P.R. GARANT Fig. 4. Radiographs showing localization of lZ5I-EGFon bone tissue. a: Preosteoblasts (arrowheads)from an experimental animal demonstrate heavy labeling. x400. The number of grains on those from the control animal (b) are reduced dramatically. x 400. Osteoblasts (Ob) and osteocytes (OC) remain unlabeled. B = bone; pb = prebone. c: Electron micrsocopic radioautography of an experimental animal exhibiting a preosteoblast (POB) migrating (arrow) toward prebone (PB) between two osteoblasts (OBI. x 2,000. Fig. 5. Radioautographs showing the localization of 1251-EGFin the periodontal ligament. a: Light microscopic radioautographs from an experimental animal demonstrating numerous silver grains over periodontal ligament, x 120. AB = alveolar bone; D = dentin. b: Only a small number of grains are present on the periodontal ligament from the control animal. X 120. AB = alveolar bone; D = dentin. c: Most silver grains are located over cell membrane of periodontal ligament fibroblasts (F). x 500. d Electron microscopic radioautograph illustrating the localization of silver grains (arrowheads)near the cell membrane of the fibroblasts (F). X 2,000. 198 M.-I. CHO, Y.L. LEE, AND P.R. GARANT experimental animals (Fig. 5a, c). A marked reduction in number of silver grains was observed over these cells from the control group (Fig. 5b, Table 1).Electron microscopic radioautography clearly revealed the localization of silver grains along the cell membrane of periodontal ligament fibroblasts (Fig. 5d). Odontoblasts Odontoblasts at different stages of cytodifferentiation were identified along the forming root surface (Fig. 1). The precursor cells, aligned next to the root sheath, did not show any special orientation or evidence of alignment to the epithelial cells (Fig. 6a). As differentiation progresses, odontoblasts become closely packed, elongated, and polarized and show evidence of secretion of the dentinal matrix (Fig. 6b). Mature odontoblasts, characterized by the presence of odontoblastic processes, form predentin and dentin (Fig. 6c). Light microscopic radioautography of 1251-EGFdemonstrated that neither newly differentiated odontoblasts nor fully mature odontoblasts bind EGF (Fig. 6b, c). However, a small number of grains were found over the cells in the subodontoblastic layer, as well as over odontoblast precursor cells close t o the root sheath (Fig. 6a, c). Quantitative Analysis of Radioautographs Silver grain counts in experimental tissue indicated that a small number of grains (less than 2.2 grains per cell) were observed over ameloblasts, stratum intermedium cells, osteoblasts, osteocytes, osteoclasts (result not shown), and odontoblasts (Table 1).However, a high number of grains per cell was observed over papillary cells (47.4) in association with the postsecretory ameloblast, basal cell (14.8) of oral epithelium, periodontal ligament fibroblast (12.9), and preosteoblast (10.8) (Table 1). In contrast, the number of silver grains localized over these cells types was signficantly reduced in control tissue to approximately one-tenth the number recorded in the experimental groups (Table 1). This difference may indicate that the silver grains observed in the experimental animals represent specific binding sites for EGF. The relative number of specific binding sites for EGF present on each cell type was obtained by counting the silver grains per unit area (100 pm2)of cell volume for each cell type. It was found that papillary cells showed the highest number (5.5),followed by basal cells of oral epithelium (3.4), periodontal ligament fibroblasts (2.1), and preosteoblasts (1.9) (Table 2). DISCUSSION It has been established that 1251-labeledhormones and EGF maintain biologic activity and an ability to bind to their specific receptors on the cell membrane Fig. 6. Radioautographs depicting localization of '"1-EGF durign den(Carpenter and cohen, 1976; Bergeron et al., 1977, 1978; tinogenesis. a: The epithelial root sheath (E)and the subadjacent odonBarazzone et al., 1980; Warshawsky et al., 1980; Fehl- toblast precursor cells (arrow) slightly labeled. x 400. P = pulp cells. mann et al., 1982; Silver et al., 1982; Nanney et al., b Newly differentiated odontoblasts (Od) are unlabeled, while some 1986). Radioautography has been used for the in vivo silver grains are loeated on subodontoblasticfibroblasts, shown at higher in 6e (arrow). x 400. c: Mature odontoblasts (Od) show and in vitro demonstration of receptor sites for calci- magnification no labeling. A small number of silver grains are associated with fibroblast tonin (Warshawsky et al., 1980), insulin (Bergeron et (arrow) adjacent to the odontoblast layer. ~ 5 0 0D. = dentin; OP = al., 1977), growth hormone (Barazzone et al., 1980),and odontoblastic process; PD = predentin. EPIDERMAL GROWTH FACTOR RECEPTORS IN THE ORAL CAVITY 199 EGF (Nanney et al., 1984, 1986; Martineau-Doize et EGF on root formation and vascular changes during al., 1987; Thesleff, 1987; Thesleff et al., 1987; Partinen tooth eruption. In addition to root formation and vasand Thesleff, 1287).Binding sites for lZsI-EGFin normal cular changes, the development of the periodontal lighuman skin were located primarily on the mitotically ament and the formation and resorption of bone have active basal keratinocytes and appeared in diminished been regarded as major factors responsible for tooth numbers as the degree of differentiation of these cells eruption (Ten Cate, 1980a, b; Avery, 1987).Among these progressed (Nanney et al., 1984,1986). Our results sup- factors, the periodontal ligament has been regarded as port these findings; in addition, our electron micro- the most likely source for generating the force required scopic radioautographic results demonstrate that EGF for tooth eruption. Although the generation of traction receptors are located primarily along the basal and lat- within the periodontal ligament remains unclear (Ten eral cell membranes of the basal cells and only rarely Cate, 1980a, b), it has been suggested that collagen molecules and/or the fibroblasts themselves may be the on the apical surface of these cells. Recently, McKee et al. (1986) investigated the entry major sources of contractile force generation (Thomas, and penetration of various I251-labeledproteins with dif- 1964; Bellows et al., 1982a, b; Ten Cate, 1980a, b). Considering that collagen fibrils are a product of fiferent molecular weights into the enamel organ and enamel of the rat incisor. They reported heavy labeling broblasts and that their organization and turnover are of papillary cells by lZ5I-EGFbut were unable to deter- manipulated by fibroblasts, it may be concluded that mine whether this labeling was specific or nonspecific. EGF may influence the rate of tooth eruption by its By comparing the labeling in animals injected with lZ5I- effect on periodontal ligament fibroblasts. FurtherEGF only and animals injected with both W-EGF and more, a mitogenic effect on preosteoblasts might inan excess of unlabeled epithelial growth factor, Mar- crease the rate of alveolar bone formation, which could tineau-Doize et al. (1987) obtained evidence suggesting also contribute to tooth eruption. a ligand-receptor interaction. Our results agree with their findings. ACKNOWLEDGMENTS Of interest is the fact that of all cells examined in This research was supported by grants #DE03745 our study, the papillary cells had the highest number and #DE06165 from the National Institute of Dental of binding sites for epithelial growth factor (5.5 per 100 Research, National Institutes of Health. We thank Mrs. pm2 unit area of cell volume), even exceeding that of Champa Codipilly for her excellent technical assisthe basal cells of the oral epithelium (3.4 per 200 pm2)>.tance. The help of Mrs. Kris Vandenberg in preparation The physiologic signficance of this abundance of EGF of the manuscript is greatly appreciated. binding by papillary cells remains to be established. Note added in proof: The possible roles of EGF on Although the mitogenic effect of EGF, its binding and tooth eruption was further studied by Rodes et al. (Deintracellular degradation have been intensively studied vel. Biol. 121:247-252,1987) and Topham et al. (Devel. in cultured fibroblasts (Carpenter and Cohen, 1974, Biol. 124532443, 1987). 1979), the in vivo localization of specific binding sites LITERATURE CITED for EGF on fibroblasts has received little attention. Nanney et al. (1984) were unsuccessful in localizing Barrazzone, P., M.A. Lesniak, P. Gorden, E. Van Obherghen, J.-L. Carpentier, and L. Orci 1980 Binding, internalization, and lysosomal binding sites for EGF on the fibroblasts of human skin, association of '"I-human growth hormone in cultured human l p using both radioautographic and immunocytochemical phocytes: A quantitative morphological and biochemical study. J . Cell techniques. They postulated that skin fibroblasts may Biol. 87:360-369. express only a small number of EGF receptors. We also Bellows, C.G., A.H. Melcher, U. Bhargava, and J.E. Aubin 1982a Fibroblast contrating three-dimensional collagen gels exhibit ultrastruchave observed only a small number of W-EGF binding ture consistent with either contraction or proteii synthesis. J. sites on fibroblasts located in the connective tissue adUltrastmct. Res 78:178-192. jacent to oral epithelium (manuscript in preparation). Bellows, C.G., A.H. Melcher, and J.E. Aubin 1982a Association between In view of the small amount of EGF that is bound by tension and orientation of periodontal ligament fibroblasts and exogenous collagen fibres in collagen gel in vitro. J . Cell Sci 58: skin and oral epithelial fibroblasts, the relatively high 125-138. amount observed to be associated with fibroblasts of Bergeron, J.J.M., G. Levine, R. Sikstrom, D. OShaughnessy, B. Kopriwa, the periodontal ligament (2.1 per 100 pm2) and with N.H. Nadler, and B.I. Posner 1977 Polypeptide hormone binding sites preosteoblasts (1.9 per 100 pmz)(Table 2) takes on spein uiuo: Initial localization of Y-labeled insulin to hepatocyte plasmalemma as visualized by electron mi-pe radioautography. Proc. cial significance. Our results indicate that fibroblasts Natl. Acad. Sci. U.S.A. 74.5051-5055. residing on different areas of body connective tissue Bergeron, J.J.M., B.I. Posner, Z. Josefsberg, and R. Sikstrom 1978 Inmay deploy different concentrations of receptors for EGF tracellular polypeptide hormone receptors. The demonstration of speand may be under different degrees of epithelial growth cific binding sites for insulin and human growth hormone in Golgi fractions isolated from the liver of female rats. J. Biol. Chem. factor control (manuscript in preparation). 253:4058-4066. One of the earliest findings of an in vivo effect for Cam, L.G., and R.P. Tubergen 1962 High resultion autoradioautography. EGF was precocious incisor eruption when this factor I. Methods. J . Cell Biol. 15t173-188. was injected into newborn mice (Cohen, 1962). Neither Camenter. G.. and S. Cohen 1976 '"I-labeled human eDidermd erowth ;ador: 'Binding, internalization, and degradation h human" fibrothe mechanisms for tooth eruption in general nor the blasts. J. Cell Biol. 71:159-171. mechanism responsible for precocious incisor eruption Carpenter, G., and S. Cohen 1979 Epidermal growth factor. Ann. Rev. resulting from injection of EGF are fully understood. Biochem. 48:193-216. Of interest are the recent studies of Thesleff showing Cho, M.I., and P.R. Garant 1981a Sequential events in the formation of collagen secretion granules with special reference to the development signficant uptake of EGF by apical tissues of the deof segment-long-spacing-hke aggregates. Anat. Rec. 199: veloping root (Thesleff et al., 1987).They reported heavy 309-320. labeling of blood vessel walls and dental follicle mes- Cho, M.I., and P.R. Garant 1981b An electron microscopic radioautographic study of collagen secretion in periodontal ligament fibroblasts enchyme. They speculated on the possible influence of 200 M.-I. CHO, Y.L. LEE, AND P.R. GARANT of the mouse: I. Normal fibroblasts. h a t . Rec. 201.577-586. Cohen, S. 1962 Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal. J . Biol. Chem. 237r1555-1562. Das, M. 1982 Epidermal growth factor: Mechanisms of action. Int. Rev. Cyt~l.,78r233-256. Fehlmann, M., J.-L. Carpentier, L.C. Alphonse, P. Thamm, D. Saunders, D. Brandenburg, L. Orci, and P. Freychet 1982 Biochemical and morphological evidence that the insulin receptor is internalized with insulin in hepatocytes. J . Cell Biol. 93:82-87. Frindik, J.P., S.F. Kemp, R.H. Fiser, H. Schedewil, and M.J. Elders 1985 Phenotypic expression in Donohue syndrome (leprechaunism): A role for epidermal growth factor. J. Pediatr. 107t428-430. Haigler, H.T., J.A. McKanna, and S. Cohen 1979. Direct visualization of the binding and internalization of a fenitin conjugate of epidermal growth factor in human carcinoma cells A 431. J. Cell Biol. 81t382395. Hunter, W.M., and F.G. Greenwood 1962 Preparation of lSII-labelled human growth hormone of high specific activity. Nature (Lond.) 194:495-496. Karnovsky, M.J. 1965 A formaldehyde-glutaraldehyde futative of high osmolality for use in electron mircroscopy. J. Cell Biol. 27:137A. Martineau-Doize, B., W.H. Lai, H. Warshawsky, and J.J.M. Bergeron 1987 Specific binding sites for epidermal growth factor in bone and incisor enamel organ of the rat. In Development of Diseases of Cartilage and Bone Matrix. Alan R. Liss, Inc., New York, pp, 389-399. McKee, M.D., B. Martineau-Doize, and H. Warshawsky 1986 Penetration of various molecular weight proteins into the enamel organ and enamel of the rat incisor. Arch. oral Biol. 31: 287-296. Nanney, L.B.,M. Magid, C.M. Stoscheck, and L.E. King 1984 Epidermal growth factor binding and receptor distribution in normal human skin and appendages. J . Invest. Dermatol. 83:385-393. Nanney, L.B., C.M. Stoscheck, M. Magid, and L.E. King 1986 Altered lZ5I-epidermalgrowth factor binding and receptor distribution in psoriasis. J. Invest. Dermatol. 84t260-265. Portanen, AM., and I. Thesleff 1987 Localization and quantitation of 1251-epidermalgrowth factor binding in mouse embryonic tooth and other embryonic tissues at different developmental stages. Dev. Biol. 120:186-197. Silve, C.M., G.H. Hradek,A.L. Jones, andC.D.Arnaud 1982Parathyroid hormone receptor in intact embryonic chicken bone: Characterization and cellular localization. J. Cell Biol. 94:379-386. Ten Cate, A.R. 1980a Physiological tooth movement: Eruption and shedding. In: Oral Histology, Development, Structure and Function. The C.V. Mosby Company, St. Louis, pp. 270-283. Ten Cate, A.R. 1980b Tooth eruption. In: Organ's Oral Histology and Embryology. Bhaskar, S.N., ed. The C.V. Mosby Company, St. Louis, pp. 371-385. Thesleff, I. 1987 Epithelial cell rests of Malazziz bind epidermal growth factor intensely. J . Periodont. Res. 22:419-421. Thesleff, I., A.M. Portanen, and L. Rihtniemi 1987 Localization of epidermal growth factor receptors in mouse incisors and human premolars during emption. Eur. J. Orthop. 9:24-32. Thomas, N.R. 1964 The role of collagen maturation in alveolar bone growth and tooth eruption. J . Dent. Res. 43:947. Warshawsky, H., D. Goltzman, M.F. Rouleau, and J.J.B. Bergeron 1980 Direct in vivo demonstration by radioautography of specific binding sites for calcitonin in skeletal and renal tissues of the rat. J. Cell Biol. 85:682-694.