DEVELOPMENTALBIOLOGY

148,590~601

Localization

(1991)

of Epidermal Growth Factor Receptors of the Enamel Organ of the Rat Incisor

B. MARTINEAU-DOIZI$’ Department

K. DICKSON,

H. WARSHAWSKY,’

of Anatomy,

McGill

University,

University

3610

Accepted

August

Street,

in Ceils

W. H. LAI, AND J. J. M. BERGERON Montreal,

Quebec,

Canada

H3A

2B2

29, 1991

Epidermal growth factor (EGF) is a peptide shown to effect precocious incisor tooth eruption in rat pups. Binding sites for EGF were visualized in the continuously erupting adult rat incisor by light and electron microscope radioautography after in viva injection of iZI-EGF. These binding sites represented EGF receptors because of (i) competition between ‘%I-EGF binding at 2 min after injection and a coinjected excess of unlabeled EGF; (ii) the receptor-mediated endocytosis of ia?-EGF at 15 and 30 min after injection; and (iii) the demonstration of EGF receptor kinase activation in viva The stem and the mitotic cells in the epithelial odontogenic organ at the growing end of the tooth develop into two nondividing layers of the enamel organ: (i) ameloblasts which secrete enamel and are suhsequently involved in the enamel maturation process, and (ii) papillary layer cells situated between the blood supply and the ameloblasts. Although few EGF receptors were present at the mitotic end, receptor density was highest at the mature end of the enamel organ. High levels of ‘%I-EGF binding were found on papillary layer cells and ruffle-ended, but not smooth-ended, ameloblasts. This implies a cyclical exteriorization and internalization of receptors during modulations between the two cell types. These data suggest that the EGF receptor mediates a major function of the enamel organ in the formation of enamel.

0 1991 Academic

Press. Inc.

INTRODUCTION

Mouse epidermal growth factor (EGF) was first discovered because of its action in precocious eyelid opening in the newborn mouse and its accelerating effect on incisor eruption (Cohen, 1962). Since then it has been demonstrated that EGF elicits mitogenic responses in a variety of cell types (reviewed by Carpenter and Cohen, 1979). The EGF receptor has been identified as a tyrosine kinase (Cohen et ah, 1982; Hunter and Cooper, 1985; Chen et aL, 1987; Ullrich and Schlessinger, 1990). In addition, the avian erythroblastosis v-erb B oncogene has been shown to be related to, and most probably derived from, the gene for the EGF receptor (Gamett et ah, 1986; Riedel et ab, 1987). These observations have established the EGF receptor as a proto-oncogene product triggering an intracellular cascade of events leading to enhanced mitogenesis (Hunter, 1985; Carpenter and Cohen, 1990). We have undertaken a study of the distribution of EGF receptors in the well-defined differentiating system of the rat incisor (Warshawsky and Smith, 1974; Smith and Warshawsky, 1975a,b, 1976; Warshawsky, 1988a,b) to investigate the tooth-related components

i Present Veterinaire,

address: Universite de Montreal, C.P. 5000, St. Hyacinthe, Montreal,

Faculti! de Medecine Quebec, Canada J2S

7C6.

*To whom dressed.

correspondence

and

0012-1606/91$3.00 Copyright All rights

Q 1991 by Academic Press, Inc. of reproduction in any form reserved.

reprint

requests

should

be ad-

590

that might contribute to one of the first described functions of EGF, namely, precocious tooth eruption (Cohen, 1962; Topham et al., 1987; Rhodes et al, 1987). In order to identify histologically the cell types in tooth harboring EGF receptors, we utilized the radioautographic method previously used to identify EGF target cells in bone (Martineau-Doize et al, 1988). MATERIALS

AND

METHODS

Experimental and control gmups. Eight male Sherman rats (n = 8) weighing 100 + 10 g, designated the experimental group, were anesthetized with sodium pentobarbital and injected via the external jugular vein with 5 x 10’ dpm of ‘%I-EGF in 0.2 ml of phosphate-buffered saline (PBS, pH 7.4). The EGF (200 Fg/ml, Receptor Grade, Collaborative Research Laboratories, Waltham, MA) was freshly iodinated using the chloramine-T method (sp act, 196 f 8.6 &i/pug; Lai et ah, 1986). The validity of the method had been extensively documented previously (Martineau-Doize et ab, 1988) and the intactness of lz51-EGF demonstrated in hone and liver. Eight rats of similar weight constituted the control group and received identical amounts of 1251-EGF plus 100 pg of unlabeled EGF. At 2 min (n = 2) and at 15 and 30 min (VX= 3) after injection, both groups of rats were perfused with lactate-containing Ringer’s solution through the left ventricle until blanching of the liver occurred (20-30 see). This was followed by perfusion fixation for 10 min with

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LOWER

RIGHT

ET AL.

INCISOR

Apic

A&B) Presecretory

c) Secretory

D)

Maturation

FIG. 1. (a) Skeletal preparation of the rat hcmimandible viewed from the lateral side. The in&al end is separated from the molars by a wide diastema. (b) Schematic representation of the lateral side of the continuously erupting rat incisor showing the zones of the enamel organ which were sampled including the odontogenic organ at the apical tip and the presccretory (A, B), secretory (C), and maturat.ion (D) stages of amelogenesis. The cross-sectional segments (A-D) show the relationship of the various epithelial cells of the enamel organ to the pulp and the odontoblasts in each of the stages of amelogenesis. Indicated are the enamel organ (EO), enamel (E), dentin (D) odontoblasts (Of, and pulp (P) in the various zones of amelogenesis. Also indicated are blood vessels (bv), odontogenic organ (oo), apical foramen (af), root sheath (rs), lateral cementoenamel junction (LCEJ), and mesial cementoenamel junction (MCEJ). (Segments A-D are from Smith and Warshawskg, 19’76.)

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FIG. 2. Sections of the enamel organ from the presecretory (a, c) and secretory (b, d) zones of an experimental (a, b) and a control rat (c, d) plus excess (50 pg) unlabeled EGF (control rat). Light microscope perfused 2 min after the injection of ‘%I-EGF (experimental rat) and ‘=I-EGF radioautographs of l-pm-thick Epon sections were exposed for 2 weeks (x600). In the presecretory zone of the experimental rat (a), silver grains overlie the outer dental epithelium (ODE) and stellate reticulum (SR). F ew silver grains are present over the stratum intermedium (SI). In the control rat (c), the number of silver grains over ODE and SR is markedly reduced. The inner dental epithelium (IDE) is very weakly labeled in both the experimental and the control rats. In the secretory zone (b) of the experimental rat, numerous silver grains are present over the cells of the papillary layer and the stratum intermedium (PL, SI). In the control rat (d), the labeling is reduced. Capillaries (C) are unlabeled. Also indicated are odontoblasts (0), predentin (Pd), and secretory ameloblasts (See).

2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) containing 0.05% CaCl,. The mandibles (Fig. la) were additionally fixed by immersion for 2 hr at 4°C and decalcified in 4.13% disodium EDTA (pH 7.3) for 16 days (Warshawsky and Moore, 1967). The decalcified mandibular incisors were sliced longitudinally and the mesial halves were cut into l-mm

cross-sectional segments (Fig. lb) and processed as described previously (McKee et ab, 1987). One-micrometer-thick longitudinal and cross sections of the incisor segments were prestained with iron hematoxylin, dipped in Kodak NTB2 liquid emulsion for light microscope radioautography (Kopriwa and Leblond, 1962), and developed after 1, 2, and 6 weeks of exposure.

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ET AL.

EGF

Receptors

FIG. 3. Quantitative light microscope radioautography showing the concentration of silver grains (mean f 1 variation, n = 2) over cells of the outer dental epithelium and papillary layer from the presecretory and secretory zones of the enamel organ. Higher concentration of labeling is present over sections from the experimental rats (EXP) with negligible labeling over sections from the controls. Conditions were as described for Fig. 2.

For electron microscope radioautography, 90-nm sections were cut with a diamond knife and prepared for radioautography (Kopriwa, 1973). After exposure for 3 months, fine grain development was carried out using the Agfa-Gevaert “Solution-Physical” technique (Kopriwa, 1975). The grids were then treated in glacial acetic acid for 2 to 30 see, stained with uranyl acetate and lead citrate, 5 and 2 min, respectively, and examined with a Philips 400 T electron microscope at 80 kv. Quantitative radioautography. Grains were counted in both light and electron microscope radioautographs from experimental and control rats perfused 2, 15, and 30 min after the injection of 1251-EGF. At the light microscope level silver grains were counted within the boundary of a Whipple micrometer ocular grid (Wild, Heerbrugg, Switzerland) at a magnification of 1000X, giving a dimension of 8.3 pm2 to each grid square. Grains were counted in lo-16 such areas over the cellular layers of the enamel organ in the presecretory, secretory, and maturation zones (see A to D in Fig. lb for orientation, histograms in Fig. 3 for presecretory and secretory zones, and Fig. 5 for the maturation zone). For electron microscopy, the silver grains were counted from electron micrographs that were photo-

in Rat Incisor

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graphed wherever silver grains were seen and printed at a final magnification of 37,260X. The silver grains were either single deposits or clusters of several deposits. A cluster was considered a single grain when it fit within the space of a silver bromide crystal (Kopriwa, 1975). To assign a cluster to one silver grain and to determine its center, a transparency with a 5.2-mm circle (corresponding to the mean 140-pm diameter of a silver bromide crystal at 37,260~ magnification) was placed over the cluster. Silver grains were then localized by attributing the silver grain directly to an underlying structure (direct scoring; Nadler, 1979). For grains related to the cell membrane and other membrane-bound cytoplasmic organelles, a Bausch and Lomb measuring magnifier was used to measure the distance between the center of each grain and the closest membrane and assigned to the nearest structure. EGF receptor autophosphorylation activity. Homogenates of the enamel organ were prepared from the longer, nonmitotic part of the enamel organ after removing the short, stem-cell-containing, and mitotic portion of the odontogenic organ. The homogenates were prepared from enamel organs freshly dissected off the surface of the enamel and immediately placed in a Dounce manual-type tissue grinder (Fisher Scientific) containing 0.5 ml of ice-cold 0.25 M sucrose in 50 mM Tris-HCl (pH 7.4), aprotinin (1000 KIU/ml), benzamidine (1 mM), phenylmethylsulfonyl fluoride (PMSF, 1 mM), and pepstatin (50 PUM). Homogenization was effected with 10 complete turns of the pestle. Enamel organ homogenates were prepared from uninjected rats (controls) and from rats injected with 1 pg EGF/lOO g body wt 2 min prior to sacrifice. In vitro autophosphorylation assays on 2 mg of homogenate protein were carried out in the presence of 0.05% Triton X-100 & EGF (1 pg/ml) and 5 yM[y-32P]ATP (3000 Ci/ mmol, DuPont, Canada, Mississauga, Ontario) for 1 min on ice followed by immunoprecipitation with monoclonal antibody to the EGF receptor (IgG-151, AE-4; Fig. 9), all procedures having been described previously (Lai et ah, 1989a). RESULTS

Distribution of lz51-EGF binding sites in the ennmel organ. Mitotic stem cells in the bulbous part of the odontogenic organ (Fig. lb, apical end; Smith and Warshawsky, 1975b, 1976; Smith, 1980; Abbott and Pratt, 1988), as well as the mitotic inner dental epithelial cells (presecretory ameloblasts) in the presecretory zone (Fig. lb, segments A and B; Smith and Warshawsky, 197513, 1976), showed a low level of radiolabeling with ‘251-EGF which was competitively inhibited by excess unlabeled EGF (not shown). However, major sites of ‘251-EGF binding were found with increasing intensity

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FIG. 4. Enamel organ in the maturation zone from a region of ruffle-ended amelohlasts (a, c) and from a region of smooth-ended ameloblasts (b, d) of experimental (a, b) and control rats (c, d) perfused 2 min after the injection of lZ51-EGF (experimental rat) and ‘=I-EGF plus excess unlabeled EGF (control rat). Light microscope radioautographs of l-pm-thick Epon sections were exposed for 2 weeks (X600). Numerous silver grains are present over the cells of the papillary layer (PL) facing both ruffle-ended (RA) and smooth-ended ameloblasts (SA) of the experimental rats (a, b), while they are nearly absent over similar regions of the control rats (c, d). Capillaries (C) in the papillary layer are unlabeled in the experimental and in the control rats. Weaker labeling is found over ruffle-ended ameloblasts (RA) with negligible labeling over smoothended ameloblasts (SA) in sections from the experimental rats (a, b). Comparable regions from control rats show negligible labeling (c, d).

in the presecretory, secretory, and maturation zones of the enamel organ (Fig. lb, segments C and D) and these were nearly completely inhibited by excess unlabeled EGF at 2 min after injection (Figs. 2-5). In the presecretory zone, radioautography at the light microscopic level demonstrated that the majority of ‘=I-EGF binding sites were over the outer dental epithe-

lium (Fig. 2a). This labeling was markedly reduced in sections from control animals (Fig. 2c), as verified by quantitation (Fig. 3). In the secretory zone the cells of the outer dental epithelium and the stellate reticulum, collectively known as the cells of the papillary layer, were highly and specifically labeled (Fig. 2b compared to 2d; Fig. 3). Further-

MARTINEAU-DOIZI?

EXP

CONTROL

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EXP.

CONTROL

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ET AL.

EXP.

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Receptors

CONTROL

PAPILLARY LAYER

FIG. 5. Quantitative light microscope radioautography showing the concentration of silver grains over cells of the maturation zone of the enamel organ (mean ? i variation, n = 2). The highest concentrations of silver grains occur over the papillary layer with ruffle-ended ameloblasts (RA) also showing considerable labeling. Controls in both cases were very weakly labeled. Smooth-ended ameloblasts (SA) were similar to controls in labeling intensity. Conditions were as described for Fig. 4.

more, cells of the stratum intermedium of the secretory zone also showed prominent specific labeling (Figs. 2b and 2d). In both the presecretory and the secretory zones low but specific labeling was observed over ameloblasts (Figs. 2a and 2b). In the maturation zone of the enamel organ, ruffleended ameloblasts showed high specific labeling (Fig. 4a compared to 4c; Fig. 5). Little specific labeling was observed over smooth-ended ameloblasts (Fig. 4b compared to 4d; Fig. 5). Papillary layer cells showed the highest specific labeling (Figs. 4a and 4b compared to 4c and 4d; Fig. 5) and no difference was observed whether such cells were located adjacent to ruffle-ended (Fig. 4a) or smooth-ended ameloblasts (Fig. 4b). Electron microscope radioautography and internalixation. In the papillary layer, at early times after injection (2 min), the majority of silver grains (58%) was found in association with the plasma membrane (Figs. 6a and 7a). This proportion fell to 37% by 30 min with a temporal accumulation in endosomal components (Fig. 6b, 15 min; Fig. 6c, 30 min) from 20% at 2 min to 35% by 30 min (Fig. ?‘a). Electron microscope analysis of grain distribution over ruffle-ended ameloblasts (Figs. 6d, 6e, and 7b)

in Rat Incise

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showed that initial labeling at the cell surface (42%) was followed by a peak of endosomal labeling (65%) at 15 min with progressive temporal accumulation of 1251EGF label in lysosome-like structures. The radioautographic data demonstrated that in the enamel organ, the highest number of specific binding sites for 1251-EGF occurred in papillary layer cells and ruffle-ended ameloblasts in the maturation zone. In both cell types the labeled ligand was internalized into endosomes (defined as electron lucent tubulovesicular structures of 123 -t 45 nm in diameter) and multivesicular bodies. Little labeling was found in lysosome-like structures (dense staining vesicles, 230 f 65 nm in diameter often with a clear halo separating the membrane and the dense content; Fig. 6b) in papillary layer cells even at 30 min after injection (Fig. 7a, -5%). In contrast, labeling accumulated markedly in lysosome-like structures of ameloblasts between 15 and 30 min after the injection of 1251-EGF (Figs. 6d and 7b) coincident with the drop in endosomal labeling over the same time interval. Since internalization of EGF has been temporally associated with its degradation within lysosomes (Dunn and Hubbard, 1984; Dunn et al., 1986; Cooper et al., 1988), we evaluated the contribution of papillary layer cells and ruWe-ended ameloblasts to EGF degradation. The data in Fig. 8 show a loss of radiolabel from papillary layer cells with time but a significant increase over the same period of time in the content of radiolabel in ruffle-ended ameloblasts. Thus, despite the negligible lysosomal labeling in papillary layer cells (Fig. 7a), these cells lost total radiolabeling with time as seen in Fig. 8. Paradoxically, the adjacent ruffle-ended ameloblasts, although showing significant lysosomal labeling (Fig. 7b), increased their total content of labeling over the same time period (Fig. 8). EGF receptor kinase activation. The final criterion used to conclude that the ‘251-EGF binding sites in the enamel organ represented bona&de receptors was the demonstration of EGF receptor kinase activation. When enamel organ homogenates were prepared from control rats and incubated with [T-~~P]ATP in the presence or absence of EGF, ligand-dependent phosphorylation of a band at 170 kDa was observed after immunoprecipitation with a well-characterized monoclonal antibody to the rat EGF receptor (Lai et ab, 1989a; Fig. 9 compare lanes 1 and 2). Since the gel was previously treated with hot alkali, the radiolabel represented phosphorylated tyrosine residues (Cooper et al., 1983). When identical experiments were carried out using enamel organ homogenates from rats injected with EGF (1 pg) 2 min prior to sacrifice an enhanced signal was observed (Fig. 9, lanes 3 and 4) as expected when there has been a prior in viva activation of the receptor (Lai et al., 198910).Control immunoprecipitations using nonspecific antisera revealed

596

FIG. 6. Electron microscope radioautographs (a), 15 min (b), and 30 min (c) after the injection

DEVELOPMENTAL

of papillary of ‘=I-EGF

BIOLOGY

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layer cells in the maturation (experimental rats). Silver

148, 1991

zone of the enamel organ from rats sacrificed ai 2 min grains at 2 min (a) are found over the highly convoluted

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Receptors

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as evaluated with a well-characterized monoclonal antibody to the EGF receptor. Most noteworthy was the observation that the in vitro autophosphorylation activity of the receptor was greater in enamel homogenates derived from EGF-injected animals. This has been previously interpreted in liver total membrane fractions (Lai ef al., 198913) as evidence for a prior in viva activation of the receptor leading to enhanced in vitro activity. Hence, EGF injection led within 2 min to an activation of the EGF receptor tyrosine kinase. Since the only cells showing binding of EGF at 2 min were papillary layer cells and ruffle-ended ameloblasts then it must be these cells that harbored EGF receptors which were responsive to the injected EGF. Lack of correlation between mitosis and EGF receptor localization. Although the odontogenic organ is a renewing cell system, mitosis occurs in only two sites, (1) stem cell division in the bulbous part and (2) differentiated cell division related to all four layers of the enamel organ in the presecretory zone. Beyond the region of ameloblasts facing dentin, no cell division is seen in the enamel organ (Smith and Warshawsky, 1975b, 1976). Thus, in terms of differentiation, the apical end of the tooth is “embryonic,” while the incisal end is “mature.” This is opposite to the density of EGF receptors, where with the exception of the mitotic cell population in the odontogenic organ, which showed only low levels of EGF receptors, the target cells of the enamel organ with the highest densities of EGF receptors are mature and not mitotic. Indeed, many cells with comparably high densities of EGF receptors do not belong to highly mitotic populations (e.g., liver, Cohen et al., 1982; brain, McKanna and Cohen, 1989; bone, Martineau-Doize et al., 1988; and placental syncytiotrophoblasts, Lai and Guyda, 1984; Goustin et al., 1985). Thus, this study attributed a nonmitogenic function to the EGF receptors on most of the enamel organ. EGF is known to cause precocious incisor eruption, but in association with decreased tooth size, an effect that cannot be attributed to a mitogenic function on the odontogenic cells (Rhodes et al., 1987). Therefore, it is unlikely that the cells described here are related directly to tooth eruption, and it seems more likely that precocious eruption would be mediated by EGF target cells in the periodontal ligament (Topham et al., 1987; Cho et al., 1988a,b). Predicted significance ?f EGF receptors in the enamel organ. The major collective function of the enamel or-

no immunoprecipitable y-32P-labeled EGF receptors (Fig. 9, lanes 5 and 6). Thus, in enamel organ homogenates derived from control (uninjected) rats the EGF receptor was identified after in vitro phosphorylation based on (1) its recognition by the monoclonal antibody IgG-151, AE-4 as evaluated in immunoprecipitates; (2) its molecular weight of 170 kDa (Lai et al., 1989a,b; Chandler et ah, 1985; Carpenter, 1987); (3) the ligand dependency of its in vitro phosphorylation; and (4) its autophosphorylation on tyrosine residues as deduced from gels treated with hot alkali (Cooper et ah, 1983). DISCIJSSION

This is the first report to demonstrate that 1251-EGF binding sites in cells of the enamel organ are bona fide receptors for EGF. The receptor concentration is low at the growing end of the tooth and increases toward the mature end. Based on the cell types in the enamel organ harboring high concentrations of EGF receptors, we propose a direct role for the EGF receptor in enamel formation. Correlation of binding sites with receptors. Radioautography was used to locate ‘251-EGF binding sites in the epithelial enamel organ of the rat incisor (see reviews in Warshawsky, 1988a,b). Three criteria were used to verify that they were EGF receptors: (i) cell surface localization of binding sites at 2 min after injection into experimental rats which was competitively inhibited by excess unlabeled EGF in control rats; (ii) the internalization of ‘%I-EGF at 15 and 30 min after injection which was again competed for by unlabeled EGF indicating receptor-mediated endocytosis; and (iii) EGF receptor kinase activation in vivo. Thus, by following the rationale of the radioautographic method used to detect binding of labeled biologically active peptides (Bergeron and Posner, 1979; Bergeron et al., 1985; Martineau-Doize et al., 1988), specific binding sites for ‘251-EGF were observed over the outer dental epithelium of the presecretory zone, the papillary layer cells of the secretory and maturation zones, and ruffle-ended ameloblasts of the maturation zone. All the cell types that showed specific binding sites also internalized the 1251-EGF into endocytic components in a time-dependent fashion. The competition with unlabeled EGF at each time interval demonstrated that the endocytosis was saturable and therefore receptor mediated. Finally, whole enamel organ homogenates revealed EGF receptor kinase activation -

plasma membrane (PM). By 15 min (b) grains are found clustered over multivesicular endosomes (MVB). By 30 min (c) grains are observed over the periphery of lysosome-like structures (ly) as well as occasional coated pits (cp) at the cell surface. Magnification bars are 2 Frn for (a) and 1 pm for (b) and (c). Electron microscope radioautographs of ruffle-ended ameloblasts from rats sacrificed at 15 min (d) and 30 min (e) after the injection of ‘=I-EGF showing silver grains over the periphery of endosomes (E) and lysosomes (1~). Also indicated on the micrographs are nucleus (N), mitochondria (M), rough endoplasmic reticulum (rER), plasma membrane (PM), pigment granules (PG), stacked saccules of the Golgi apparatus (G), endothelial cell (Endo), and coated vesicles (cv). Magnification bars are 1 Grn for (d) and 0.5 pm for (e). Exposures were 3 months.

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FIG. 7. Quantitative analysis of silver grain distribution over cell organelles in electron microscope radioautographs of the papillary layer (a) and the ruffle-ended ameloblasts (b) in the maturation zone at various times after the injection of lz51-EGF into experimental rats. Markedly less labeling over the plasma membrane is found in ruffle-ended ameloblasts at 15 and 30 min (b) in comparison to cells of the papillary layer (a). By contrast, uptake into endosomes at 15 min and lysosomes at 30 min is more prominent in the ruffle-ended ameloblasts (b) than in the papillary layer cells (a). The category designated as “others” includes cytosol, extracellular spaces, endothelial cells, capillary lamina, and perivascular spaces.

gan is the production of enamel by an essentially twostage process of secretion followed by maturation. During the presecretory stage, the renewing cells provided by mitosis of stem cells are organized into four distinct layers. These cells then undergo proliferation with subsequent differentiation into cells that no longer divide. The highest level of EGF receptors in the enamel organ was present on cells of the outer dental epithelium and the papillary layer, with the latter being the most highly labeled (Martineau-Doize et ab, 1987; Cho et al., 1988a,b). This outer layer of the enamel organ is in direct contact with a basement membrane and is very close to an extensive plexus of fenestrated capillaries. In order to reach the ameloblasts, the intravenously injected EGF had to pass from these capillaries across the papillary layer, We propose that the papillary layer cells extend the EGF influence to the ruffle-ended ame-

loblasts by a paracrine-like mechanism. This would account for a higher content of ‘%I-EGF at later time intervals in ruffle-ended ameloblasts than in papillary layer cells. Lai et al. (1989a), Murthy et al. (1986), and Felder et al. (1990) have demonstrated that the EGF receptor recycles. Furthermore, Lai et al. (198913) and Felder et al. (1990) demonstrated that the extent of recycling is regulated, and they have postulated that the tyrosine kinase activity and/or unique substrates found at the level of the endosomal membrane may regulate the sorting of endocytic material into lysosomes. Hence, the high level of lz51-EGF associated with the plasma membrane of papillary layer cells even at 30 min after injection, the internalization into endosomes, and the negligible uptake into lysosomes are consistent with a recycling mode of receptor traffic in these cells. This could serve

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to prolong the presentation of EGF to the ameloblasts for extended biological effects. Predicted signi$cance of EGF receptors on ameloblasts. Low levels of EGF receptors are present on proliferating and differentiating ameloblasts. Enamel secretory ameloblasts are also low in receptors for EGF. Only when the maturation ameloblasts are in the ruffleended state of their modulation do they reveal high concentrations of EGF receptors capable of recognizing exogenously administered EGF. Ruffle-ended ameloblasts have been implicated in calcium transport with subsequent deposition of Ca2+ into crystals (Bawden, 1989). Smooth-ended ameloblasts did not reveal receptors for EGF. The observation of Smith et al. (1987) that ameloblasts modulate their morphology from ruffle-ended to smooth-ended with a characteristic frequency of approximately 2 hr (T1,.. N 2 hr) indicates that modulation of ameloblast morphology is more rapid than the T,,, of EGF receptors (Tl,2 = 10 hr; Krupp et al., 1982; Stoscheck and Carpenter, 1984; Savoie et al., 1986). This suggests a sequestration of EGF receptors into smoothended ameloblasts and an exteriorization onto the sur-

EGF in vivo EGF in vitro IgG-151, AE-4

+

+ +

+ +

170 kDa-

+ + +

+ -

+ + -

^

-EGF-R

,. 123456 FIG. 9. Identification of EGF receptor kinase activity in enamel organ homogenates. Homogenates were isolated at 0 time (lanes 1 and 2) or 2 min (lanes 3 and 4) after the injection of 1 pg EGF/lOO g. Enamel organ homogenates were prepared and incubated in the absence (lanes 1 and 3) or presence of 1 rig/ml EGF followed by incubation with [y-3”P]ATP and immunoprecipitation with the monoclonal antibody IgG-151, AE-4 and then subjected to SDS-PAGE and radioautography. Lanes 5 and 6 refer to immunoprecipitations carried out with rabbit anti-mouse IgG instead of monoclonal antibody IgG151, AE-4. The position of the EGF receptor (170 kDa) is indicated. Exposure, 1 week.

faces of ruffle-ended ameloblasts during the cyclic modulations in ameloblast morphology. The functional significance of the unexpected high concentrations of receptors in papillary layer cells and ruffle-ended ameloblasts as either the cause or the consequence of modulation remains speculative. Only further functional studies can evaluate the molecular relationships between EGF binding, ameloblast modulation, and calciotropic events in enamel. It is noteworthy that EGF receptors in bone are limited to a small subset of morphologically distinct cells (Martineau-Doize et al., 1988). In bone the relevance of EGF to calcium release is well documented (Ibbotson et ah, 1985). The relationship, if any, between EGF binding in cells of bone (Martineau-Doizk et al., 1988) and tooth with a common function in the regulation of calcification and calcium homeostasis remains to be elucidated. This work was supported by grants from the Medical Research Council of Canada to Dr. H. Warshawsky and the National Cancer Institute of Canada to Dr. J. J. M. Bergeron. Dr. B. Martineau-Doizk was a recipient of a Medical Research Council Fellowship.

REFERENCES

PL

RA 2 MIN

I

PL 15 MIN

PL

RA I

RA

30 MIN

FIG. 8. Quantitative light microscope radioautography showing the concentration of silver grains over papillary and ruffle-ended ameloblasts in the maturation zone. Note the temporal loss of lZI-EGF from cells of the papillary layer (PL) and concomitant accumulation of -‘IEGF in cells of the ruffle-ended ameloblasts (RA). Conditions were as described for Fig. 4.

ABBOTT, B. D., and PRATT, R. M. (1988). EGF receptor expression in the developing tooth is altered by exogenous retinoic acid and EGF. Dev. Bid. 128, 300-304. BAWDEN, J. W. (1989). Calcium transport during mineralization. Arut. Rec. 224,226-233. BERGERON, J. J. M., CRUZ, J., KHAN, M. N., and POSNER, B. I. (1985). Uptake of insulin and other ligands into receptor rich endocytic components of target cells: The endosomal apparatus. Annu. Rev. PhJ.siol. 47, 383-403. BERGERON, J. J. M., and POSNER, 8. I. (1979). hl t:ivo studies on the initial localization and fate of polypeptide hormone receptors by the technique of quantitative radioautography. J Histochem. Cytochem. 27, 1512-1513.

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