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The effect of epidermal growth factor on neonatal incisor differentiation in the mouse
DEVELOPMENTAL
BIOLOGY
124,532-543 (1987)
The Effect of Epidermal Growth Factor on Neonatal Incisor Differentiation in the Mouse R. TODD TOPHAM, DANIEL
J. CHIEGO, JR.,’ VINCENT H. GATTONE II, DUANE A. HINTON, AND ROBERT M. KLEIN
Department of Anatomy/Division
of Cell Biology, University of Kansas Medical Center, Kansas City, Kansas 6610.3
Received February 10, 1987; accepted in revised f&-m August 10, 1987' The effect of epidermal growth factor (EGF) on cellular differentiation of the neonatal mouse mandibular incisor was examined autoradiographically using tritiated thymidine (rH]TDR) and tritiated proline (rH]PRO). On Days 0 (day of birth), 1, and 2, EGF was administered (3 pg/g body wt) SCto neonates. Mice were killed on Days 1,4, ‘7,10, and 13 after birth and were injected with either [aHJl?DR or [3H]PR0 1 hr before death. [3H]TDR was used to analyze cell proliferation in eight cell types in the developing mouse incisor including upper (lingual) and lower (buccal) pulpal fibroblasts, preodontoblasts, inner and outer enamel epithelial cells (IEE and OEE), stratum intermedium (SI), stellate reticulum (SR), and periodontal ligament (PDL) t’ibroblasts. [3H]PR0 was used to analyze protein synthesis in ameloblasts, and their secretion products (enamel and dentin), as well as PDL fibroblasts. The selected EGF injection scheme elicited acceleration of incisor eruption with minimal growth retardation. At Day 1, the upper and lower pulp, preodontoblasts, SI, and SR showed a significant decrease in labeling index (LI) 24 hr after a single EGF injection. After multiple injections (Days 0, 1, 2), two LI patterns were observed. In lower pulp, preodontoblasts, IEE, SI, SR, and OEE, a posteruptive change in LI was observed. In contrast, the upper pulp and PDL regions demonstrated a direct temporal relationship with eruption. Autoradiographic analysis with [aH]PRO indicated that EGF treatment caused significant increases in grain counts per unit area in ameloblast, odontoblast, and PDL regions studied. Significant differences were found in all four regions studied (ameloblasts, enamel, odontoblasts, dentin) at the 45-pm-tall ameloblast level as well as ameloblasts and odontoblasts at the 30-pm level at 13 days of age. The PDL demonstrated significant differences at all locations studied (base, 30 pm, 45 pm,) in 4-, 7-, and 13-day-old mice. Morphologically, EGF-treated groups demonstrated premature differentiation of ameloblasts and odontoblasts at the light microscopic level. The data indicate that EGF alters DNA and protein synthesis as well as differentiation patterns during the eruption process. While EGF affects both DNA and protein synthesis, the alteration of differentiation may be secondary to mitogenic effects on proliferative compartments. In order to determine the cellular target for EGF within the newborn mouse incisor, in viva ‘*‘I-EGF binding was analyzed autoradiographically. EGF binding sites were demonstrated in the dental follicle, an area which contains cells believed to be the progenitors for the PDL, alveolar bone, and cementum. The results of the [3H]TDR, [3H]PR0, and ‘a51-EGF studies argue strongly that the PDL is the target for EGF within the 0 198’7 Academic Press, Inc. incisor.
INTRODUCTION
Previous studies using epidermal growth factor (EGF)’ have demonstrated that this low-molecularweight hormone has a wide range of biological activities in vivo and in vitro. EGF stimulates the growth of ectodermally (Carpenter and Cohen, 1979; Cohen and Elliott, 1963; Cohen 1965; Gospodarowicz, 1981) and endodermally (Konturek et aZ.,1981) derived tissues in vivo and promotes ectodermal and mesodermal growth in vitro (Adamson and Rees, 1981). Extensive analysis of i Present address: Department of Oral Biology, University of Michigan, School of Dentistry, Ann Arbor, MI 48104. a Abbreviations used: EGF, epidermal growth factor; EGF-R, epidermal growth factor receptor; Ep-Mes, epithelial-mesenchymal; [3H]PR0, tritiated proline; [3H]TDR, tritiated thymidine; IEE, inner enamel epithelium; LI, labeling index; OEE, outer enamel epithelium; PDL, periodontal ligament; SI, stratum intermedium; SR, stellate reticulum.
0012-1606187$3.00 Copyright All rights
0 1987 by Academic Press, Inc. of reproduction in any form reserved.
532
EGF effects on the growth and development of skin reveals that EGF stimulates the proliferation and keratinization of the epidermis of cultured embryonic skin (Cohen, 1965) and accelerates epidermal development in newborn mice (Cohen and Elliott, 1963), including the premature opening of the eyelids and accelerated eruption of the incisor teeth (Cohen, 1962). In organ systems such as the developing lung, EGF has been shown to have a trophic function (Goldin and Opperman, 1980). There have been few studies on developing teeth; however, it is known that EGF stimulates DNA synthesis in the enamel organ epithelium (Brownell and Rovero, 1980) and odontogenic cells (Steidler and Reade, 1981) in vitro. In addition, EGF receptors have been localized in both epithelial and mesenchymal compartments of fetal mouse molars (Partanen and Thesleff, 1987). Although the premature eruption of the mouse inci-
TOPHAM
ET AL.
EGF Effects on Neonatal Mouse hcisor
sor was used as the original bioassay for EGF, the mechanism by which EGF exerts its effect on incisor eruption and differentiation has not been investigated. The present study was designed to determine the cellular response of the continuously erupting incisor of the neonatal mouse to EGF administration in viva by analysis of tritiated thymidine ([3H]TDR) and tritiated proline (rH]PRO) incorporation into the various developing cellular compartments. In addition, the presence of EGF binding sites in the cellular compartments of the newborn mouse incisor was analyzed by in vivo injection of lz51-EGF followed by autoradiography. The hypothesis of this study is that EGF-accelerated eruption occurs by modification of normal differential rates of DNA and protein synthesis in cells which are sensitive to EGF (possess EGF receptors) during incisor development and eruption. MATERIALS
AND METHODS
Animals Breeding stock of Balb/c mice was obtained from Harlan/Sprague-Dawley (Indianapolis, IN). These animals were maintained in the KUMC Animal Care Unit on a 1%hr light/dark cycle (lights on 06:00-18:00) and given Purina Mouse Chow and water ad lib&m. Pups were weighed each day prior to the injections. Day zero was defined as the date of birth. Neonates were checked each day for evidence of tooth eruption or eyelid opening. Mice were killed by ether overdose at one of five time points (1,4,7,10, and 13 days) for both control and EGF groups. Mandibles for examination were immediately excised using a magnifying glass lamp and iris scissors and forceps. Tissues were then placed into Peter’s neutral buffered formalin (pH 7.4) for fixation. Preparation and Administration
of EGF Solution
EGF was obtained from Collaborative Research, Inc. (Lexington, MA), where it was produced according to the method of Savage and Cohen (1972). The EGF was received in lyophilized form, 100 pg per vial, and was solubilized by adding 1 cc of 0.15 M NaCl solution. The EGF was administered by SCinjection under the dorsal skin. A dosage of 3 /*g/g body wt of EGF per day was chosen. This dose was found to be effective in induction of tooth eruption in previous studies (Steidler and Reade, 1980). Controls were injected with an equal volume of 0.15 M NaCl solution. Our preliminary studies indicated that a 0, 1,2 day only series of injections had optimum results (Day 0 = day of birth), because early tooth eruption was elicited with minimal growth retardation. The preliminary studies included the following injection schemes: Day 0 only, Day 1 only, Day 2 only, Days 1,2,3, only, Days 2,3,4, only, and Days 3,4,5 only.
533
Preparation and Administration of Tritiated Thymidine and Tritiated Proline Tritiated proline (sp act 5.0 Ci/mmole; New England Nuclear, Boston, MA) and tritiated thymidine (sp act 6.7 Ci/mmole; ICN, Irvine, CA) were injected for a 1-hr pulse at dosages of 1 and 2 pCi/g body wt, respectively. Both isotopes were injected iv via the orbital branch of the anterior facial vein in animals 7 days old or less. Older animals (Days 10 and 13) were given intracardiac injections of the isotopes. No differences were found between intracardiac and intravenous injections in preliminary studies. Preparation of Tissue for Light Microscopy Whole mandibles were fixed for 20 hr in Peter’s neutral buffered formalin. Following fixation, mandibles were decalcified in EDTA sodium citrate (pH 7.4). Decalcification was verified by X-ray examination. Decalcified tissues were washed for 24 hr, then dehydrated, cleared with xylene, and embedded in paraplast. Mandibles were divided into right and left halves and then embedded in paraffin blocks with the buccal surface of the mandible facing out toward the cutting surface of the paraffin block. Sections were cut in 5-pm thicknesses and placed onto acid-cleaned, gelatin ChromAlum subbed glass slides. Following completion of sectioning, deparaffinization, exposure of autoradiograms, and staining with hematoxylin and eosin, the tissues were covered with acrytol and coverslipped. Autoradiography Slides were dipped in nuclear track emulsion, NTB-3 (Eastman-Kodak Co., Rochester, NY), and exposed in black boxes at 4°C. rH]TDR slides were exposed for 16 days; rH]PRO slides were exposed for 13 days. Exposure time was determined by use of control slides. All slides were developed as follows: 3.5 min in developer D-19 (Kodak) 10 set in distilled water (with a drop of D-19 added), 6 min in Kodak fixer, and 30 min in a water bath. Slides were air-dried overnight, followed by the completion of a standard H and E staining process. Analysis of Autoradiograms Analysis was done manually at 450X magnification. For rH]TDR autoradiograms, the percentage of labeled cells (LI) was determined by counting cells in eight specific areas (Fig. 3) of the enamel organ region: upper (lingual) pulp fibroblasts, lower (buccal) pulp fibroblasts, preodontoblasts, inner enamel epithelium (IEE), stratum intermedium, stellate reticulum, outer enamel epithelium (OEE), and periodontal ligament (PDL) fibroblasts. In the upper pulp area, a full grid was
534
DEVELOPMENTAL BIOLOGY
counted for each section (each grid box 20 pm on a side). Half a grid was counted over the lower pulp area, taking care not to include preodontoblast cells. At the apex of the loop, an arbitrary dividing line was placed between the IEE and OEE regions (Chiego et ab, 1981). Moving i&sally, in areas 3-8, counts were terminated at a point equal to the beginning of the dentino-enamel matrix. PDL cells (area 8) were counted from that point to the apex of the loop. Cells were considered labeled if they contained four or more grains. With [3H]PR0 autoradiograms, grains were counted per unit area, using ameloblast heights as landmarks for measurements (30, 45 pm). The 30-pm level was chosen as the approximate location of the dentinoenamel junction and the 45-pm level was selected as the location of the first mature ameloblasts (polarized nuclei and attainment of maximum cell height). Counts were made at those landmarks over odontoblast, dentin, enamel, ameloblast, and PDL layers (Fig. 5). Grid boxes used measured 10 pm on a side. Data were analyzed with an unpaired t test.
VOLUME 124, 1987
1 ’
“CON
t t t 0
1 2 3
*,,+,,*,,+I 4 5 6
7
El 9 10111213
AGE FIG. 2. Graph of body weight changes caused by EGF administration. (t) indicate injection days; (k) indicates sacrifice days; E, eruption day; 0, opening of eyelids. (p i 0.05*, p < O.Ol**, p < O.OOl***.)
EGF was obtained from New England Nuclear Co., diluted in PBS, and injected into newborn mice at a concentration of 0.25 &i/g body wt. Mice were anesthetized with nembutal and injections were made by the intracardiac route using a dual stopcock syringe. Two minutes after the injection mice were perfused with PBS to remove unbound radiolabeled EGF and subsequently perfused with 2.0% glutaraldehyde in 0.1 M phosphate buffer. Controls received the same dosage of 1251-EGF Binding Studies lz51-EGF plus a 500-fold excess of cold (unlabeled) EGF. The method used for analyzing the distribution of Mandibles were then removed and immersion-fixed EGF-binding sites in the newborn mouse incisor was overnight before preparation for methacrylate embedsections were cut at 2 pm and adapted from the method of Chabot et al. (1986). lz51- ding. Methacrylate mounted on acid-cleaned, gelatin-subbed slides for autoradiography using NTB-2 nuclear track emulsion. Slides were exposed for 12 weeks and subsequently Eyelid Eruption Body weight trend (mean) stained lightly using a stain containing basic Fuchsin opening Injection days observed E or C days days Day 0 Day 7 Day 10 and Methylene blue. Slides were viewed and photographed on a Nikon Labophot microscope using both 9 0, 1, 2-E 1.70 6.38 8.26 bright- and dark-field optics. 1, 2, 3-E 10 1.55 4.53 6.08 A 3, 4, 5-E 1 2, 3, 4-E
9 9
2.04 1.55
4.53 3.12
6.50 4.78
O-E 1-E B 2-E I
9 10 10
11 11 12
1.77 2.72 1.80
5.08 4.07 4.77
7.17 6.82 6.92
None-C None-C
9 9
12 12
1.63 2.22
4.80 5.30
6.82 7.20
10 9 9 9
1.68 1.97 1.89 1.82
4.17 5.30 4.90 4.20
7.13 6.90 8.08 6.52
12 11 12 12 13 12
1.75 2.03 1.82 1.78 1.90 1.68
4.82 5.53 6.35 4.91 5.47 5.53
7.53 7.24 7.45 6.38 7.93 6.87
0, 0, 0, 0,
1, Z-E 1, Z-E 1, 2-E 1, Z-E
0, 0, 0, 0, 0, 0,
1, 2-c ic 1, 2-c 1, 2-c 1, 2-c 1, 2-c 1, 2-c,
10 9 10 10 10 10
FIG. 1. Optimization of injection protocol. (A) Three-day series injection schemes, (B) single day only injection schemes, (C) data from 0, 1, 2, day injection scheme used. “None-C” indicates data from control mice.
RESULTS
Preliminary
Studies
Preliminary experiments were performed to determine the optimal EGF dosage required for induction of precocious eruption of the incisors, in conjunction with the least weight loss. Earlier research with EGF demonstrated that continued daily injections caused steady body weight reduction in the experimental animals, which resulted in a high rate of mortality. It was anticipated that a specific period of EGF sensitivity (during the first postnatal week) could be identified and used to optimize the accelerated rate of eruption. Several single day only and 3-day injection schemes were tested (Fig. 1). It was determined that injections on Days 0, 1, and 2 provided the best results in terms of precocious incisor eruption coupled with the least growth retardation. Experimental mice treated by this method displayed a temporary weight loss followed by recovery (Fig. 2), as well as the desired effect of premature eruption of inci-
TOPHAM ET AL.
EGF Eflects on Neonatal
sor teeth as obtained earlier by Cohen (1962; Cohen and Elliott, 1963). Tritiated
Thymidine
Autoradiographic
Data
Eight specific areas within the mouse incisor were examined autoradiographically with rH]TDR as previously described (Fig. 3). Autoradiographs of the loop region displayed increased labeling in experimental animals compared to controls. In the upper pulp cell region associated with the epithelial (Hertwig’s root sheath) (Fig. 4a) significant differences in labeling indices were found at all time points except Day 10. The lower pulp region associated with the cervical loop (Fig. 4b) displayed significant LI differences at the l- and 13-day time points, while in the preodontoblast (Fig. 4c) and IEE (Fig. 4d) regions, highly significant differences were observed following eruption at the lo- and 13-day time points. The SI layer of cells (Fig. 4e) displayed a
similar pattern, as EGF administration appeared to maintain DNA synthesis after eruption at a higher level than control values. In the areas with the fewest labeled cells, SR (Fig. 4f) and OEE (Fig. 4g), significant differences were again seen at the lo- and 13-day intervals. Finally, in the PDL region, significant differences between control and EGF-treated groups were observed at all time points except Day 1 (Fig. 4h). Tritiated
Proline
@ inner enamel epithaliw @ stratum intemmdiun
(IEE)
FIG. 3. Diagram of sagittal (longitudinal) section through the incisor showing cellular areas analyzed with [‘Hj’I’DR. The upper (lingual) pulp region was defined as the pulpal area associated with Hertwig’s root sheath and the lower (buccal) pulp was delineated as pulpal cells associated with the cervical loop.
Autoradiographic
Data
Specific regions near the base of the developing mouse incisor were examined autoradiographically using [3H]PR0, as previously described (Fig. 5). Autoradiographs of incisor sections from 13-day-old mice displayed increased labeling in EGF-treated mice compared to controls. Ameloblast cell heights of 30 and 45 pm were chosen for reference points. Data from this study indicated that significant differences between control and EGF-treated groups were present at the 30-pm level in both ameloblasts and odontoblasts at the 13-day time point (Fig. 6). At the 45-pm level, significant differences were found in all four layers studied: ameloblasts, enamel, odontoblasts, and dentin (Fig. 7). Significant differences between groups at this level were also found in ameloblast and dentin layers at the 7-daytime point. Interestingly, in all PDL regions studied (base, 30+m, and 45-pm levels), highly significant differences between groups were seen at the 4- and i’-day time points (Fig. 8). This highly significant difference (p < 0.001) was especially obvious at all positions studied at the 4-day time point. Significant differences between EGF-treated and control groups were also found to be present at all three PDL levels studied at the 13-day interval (Fig. 8). Morphological
@ upper pulp fibroblasts @ lower pulp fibroblasts
535
Mouse Incisor
Observations
After EGF treatment, light microscopic morphological differences were observed in the maturation of odontoblasts and ameloblasts at the base of the loop region. As seen on photomicrographs from lo-day-old EGF-treated mice (Fig. 9a), ameloblasts and odontoblasts appeared more ordered and linear than cells from control mice (Fig. 9b). The secretory cells in the EGF-treated group appeared to organize earlier, at a point nearer to the loop, along the epithelial-mesenchyma1 (Ep-Mes) interface. In addition, polarization of the nuclei toward the basal aspect of the cell, typical of mature ameloblasts and odontoblasts, occurred prematurely, followed by earlier deposition of the enamel layer in EGF-treated animals. The precocious polarization of odontoblasts and ameloblasts was also seen at
536
VOLUME 124,1987
DEVELOPMENTAL BIOLOGY
UPPER
LOVER
PULP
PULP
t: 25. % 20: a
i?lS
0 CON 10
1
4
?
13
10
AGE
PRE0DOrvrOBlAsls
INNER
ENAMBL
EPlTEEUUY
0
0 CON
CON
e
0
1
4
7
13
10
AGE
16 OUTER
R
BNAMEL
EPrniBuUY
h
c1 .\
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FIG. 4. Labeling
ECF
Q
CON
3
I
1
4
7 AGE
10
13
0 I
index data in eight [3HjTDR areas analyzed
earlier time points with termination of DNA synthesis occurring at a point closer to the loop (Fig. 10). [*251jEGF Binding
l
Studies
Autoradiographs from newborn mouse incisors demonstrated specific labeling in the dental follicle area. Figure lla demonstrates a bright-field photomicrograph illustrating the loop region of the incisor. Figure llb demonstrates the autoradiographic binding of lz51EGF in the PDL and dental follicle region using dark-
4
GE
10
13
(a-h) (p i 0.05*, p c. O.Ol**, p < O.OOl***).
field photomicrography. Controls did not demonstrate localization of grains in the dental follicle region. DISCUSSION
The experiments in this study analyzed [3H]TDR and [3H]PR0 incorporation into compartments of the developing mouse incisor during the first two postnatal weeks and the localization of EGF binding sites in the newborn incisor. These data will be discussed in two sections: (1) cellular and organ sensitivity to EGF and
TOPHAMETAL.
537
EGF Efects on Neonatal Mouse Incisor 50 g
40
-5 30 ag
d
20
4
10 A
au 5
7
10
13
7
10
13
40
do m 30
5 s
0
20 10 0
1
4
DAY
Regions
Counted
FIG. 7. Graphs showing results of the [‘HIPRO study at the 45-pm (cell height) ameloblast level. Grain counts were taken over ameloblasts (a), enamel (b), dentin (c), and odontoblasts (d), as shown in Fig. 5.
-1 FIG. 5. Diagram of sagittal (longitudinal) section through the incisor showing cellular areas analyzed with [3H]PR0. Two landmarks were selected for reference points, the ameloblasts cell heights of 30 and 45 Nrn.
a v, kl 4 E =
25 20
q
CONTROL EGF
70
15 9 bg
5
"4:
5
30 5:
0
b
60
10
20 10 01
257
70 60
DAY
FIG. 6. Graphs showing results of the [3H]PR0 study at the 30-pm (cell height) ameloblast level. Grain counts were taken over ameloblast (a) and odontoblast (b) regions, as shown in Fig. 5; p values: p < 0.05*, p i O.Ol**, and p < O.OOl***.
FIG. 8. Graphs showing results of the [3H]PR0 study of the PDL compartment. Grain counts were taken at three sites in the PDL: 45-pm (cell height) ameloblast level (a), 30-pm (cell height) ameloblast level (b), and a basal loop position (c), as shown in Fig. 5.
FIG. 9. r3H]TDR autoradiographs from EGF and control (lo-day-old mice). EGF-treated odontoblasts and ameloblasts appear more ordered and linear (9a) than those of controls (9b). Secretory cells treated with EGF appear to organize at a point nearer to the loop than controls (c) (arrowheads). Nuclei of maturing cells migrate basally prematurely (b) ( small arrows), and subsequently begin deposition of the enamel layer earlier than in controls (a) (dark arrow). 538
TOPHAM ET AL.
EGF Effects on Neonatal
Mouse Incisor
FIG. 10. [$H]TDR autoradiographs from EGF and control (I-day-old mice). In EGF mice (b), the odontoblasts and ameloblasts appearedmore organized with a more linear arrangement. After EGF treatment, nuclei migrated toward functional secretory positions at an earlier point (closer to the loop), and no longer displayed [aH]TDR labeling (left of bracket). p, pulp, o, odontoblasts, a, ameloblasts.
FIG. 11. Autoradiograph of newborn mouse incisor demonstrating photomicrograph (a) illustrates the loop region. Dark-field enlargement follicle region. Controls did not display labeling in these regions.
specific labeling in the dental follicle area and PDL. Bright-field autoradiograph (b) shows binding of ‘a?-EGF in the PDL and dental
540
DEVELOPMENTALBIOLOGY
(2) EGF-induced alterations of cell proliferation, tein synthesis, and morphology.
pro-
Cellular and Organ Sensitivity to EGF Studies of neonatal rat tissues have demonstrated that postnatal Days 0 to 3 are critical time points for elicitation of EGF effects on incisor eruption, ear canal opening, and eyelid unfusion (Hoath, 1986). Our experimental results concerning premature eruption and eyelid opening were in agreement with earlier findings by Cohen (1962; Cohen and Elliott, 1963), and indicate that these tissues are subject to a period of EGF sensitivity during the first neonatal week. In the present experiments EGF was found to alter [3H]TDR and [3H]PR0 incorporation in all cell compartments studied during the first two postnatal weeks. The pattern of EGF-induced alteration of cell proliferation and protein synthesis differed temporally and from compartment to compartment. Generally, EGF appeared to prolong or maintain a higher level of DNA synthesis in all regions studied, following eruption. The effect of EGF on incisor eruption and associated EGFinduced changes in rH]TDR and [3H]PR0 may be due to either direct or indirect effects on the incisor. It is possible that the large, pharmacological dose of EGF used in these studies (3 pg/g body wt) led to indirect effects, including alteration of differentiation-related hormones such as the glucocorticoid or thyroid hormones. EGF concentration in neonatal mouse skin is altered by hypothyroid and hyperthyroid conditions (Hoath et al., 1983); however, EGF administration, even in large doses, does not appear to alter serum thyroxine levels (Lakshmanan et aL, 1985). In addition, relatively low doses of EGF (500 rig/g body wt) have been shown to accelerate tooth eruption in newborn rats (Hoath, 1986). The presence of EGF and EGF-Rs in the developing tooth has been investigated only to a limited extent, although EGF-R and EGF concentrations have been studied in other organs. EGF-Rs are present in fetal organs (Adamson et ah, 1981) and EGF may also be present in tissue and fluids to influence rapid metabolic sequences such as growth, differentiation, and tissue repair (King, 1985). For example, in the salivary glands EGF and mRNA for EGF are detectable in granular convoluted tubule cells of male mice at 20 days of age and EGF concentration increases during postnatal life (Gresik and Barka, 1978; Gresik and Azmitia, 1980; Gresik et al., 1985). EGF-Rs have been localized in the basal proliferative areas of neonatal skin, with receptor number decreasing as growth rates decline with age (Green et al, 1983). There is some evidence for involvement of EGF in tooth development. Mouse prepro-EGF mRNA has been localized in the mouth region in an area normally asso-
VOLUME124,1987
ciated with the incisor, suggesting that EGF may be synthesized by cells within the tooth (Rall et ah, 1985). Partanen and Thesleff (1987) indicated that in mouse embryonic molars in vitro, EGF-Rs are present on the dental epithelium during the bud stage with an absence of EGF-Rs on the dental mesenchymal cells. EGF-Rs were localized within the dental mesenchyme in the cap stage with an absence of binding in the dental epithelium, and in later stages of development, lz51-EGF binding was demonstrated in the dental follicle area. The dental follicle is believed to contain progenitor cells for the PDL, alveolar bone, and cementum. The results of the present study indicate that the dental follicle and PDL of the newborn mouse incisor also possess EGF-Rs and may be the target for EGF during postnatal development. EGF-Induced Alteration of Cell Proliferation, Protein Synthesis, and Morphology All cellular compartments of the neonatal mouse incisor demonstrated an alteration of cell proliferation during both normal eruption and EGF-accelerated eruption. At early time points, several zones (upper and lower pulp, preodontoblasts, IEE, SR, and SI) demonstrated an initial decrease in LI 24 hr after a single injection of EGF. The decrease in LI in these zones may be due to initial down-regulation of EGF-R within the incisor. EGF has been demonstrated to regulate expression of its own receptor in vitro (Clark et aZ.,1985). Down-regulation of receptors in vivo has been demonstrated in fetal tissues following intraamniotic injection of EGF (Adamson and Warshaw, 1982). The mitogenie effect exhibited by long-term administration of EGF in the present study may be due to EGF stimulation of EGF-R synthesis. Stimulation of EGF-R synthesis by EGF treatment has been demonstrated with WB cells in vitro. In that system EGF stimulates DNA synthesis and EGF-R degradation; however, there is a counterbalancing by an enhancement of EGF-R synthesis (Earp et ah, 1986). During the remainder of the experimental period in the present study, stimulation of cell proliferation occurred in all cellular compartments. This effect of EGF is similar to the mitogenic effect exhibited in numerous other developing organs (Steidler and Reade, 1980; Goldin and Opperman, 1980). EGF has also been shown to have a stimulatory effect on the enamel organ epithelium from fetal mice (Brownell and Rovero, 1980) and odontogenic cells from bovine fetuses (Steidler and Reade, 1981). A selective effect of EGF has been demonstrated in the case of the dental mesenchyme, which plays an essential role in the response of the tooth toward EGF. While dental mesenchymal cell proliferation is inhibited during the in vitro growth of the mouse molars, dissociated dental mesenchymal cells are stimulated by EGF (Partanen et al., 1985). The inhibition of
TOPHAMETAL.
EGF Effects on Neonatal Mouse Itiw
cell proliferation in the dental mesenchyme of organcultured embryonic molars may be mediated by EGFRs (Partanen and Thesleff, 1987). This alteration of the dental mesenchyme may cause the dramatic inhibition of the terminal differentiation of odontoblasts and ameloblasts observed in this system following EGF administration (Partanen et al, 1985). Two general patterns of cell proliferation were observed in the present studies. A pattern of late stimulation of cell proliferation in the SI, IEE, OEE, SR, lower pulp, and preodontoblast regions may indicate a secondary effect of EGF on these compartments. The earlier alterations of cellular proliferation in the PDL and upper pulp highlight the potential involvement of these zones in the EGF-accelerated eruption process. In the upper pulp region there is an increase in cell proliferation seen before normal eruption (during Days 7-10) and prior to EGF-accelerated eruption (during Days 4-7). Most previous studies of altered eruption rates have used the continuously erupting rodent incisor as a model system. A direct relationship between accelerated eruption and altered cell production in the IEE and pulp has been demonstrated following shortening of the incisor at the occlusal surface (Ness and Smale, 1959; Michaeli and Weinreb, 1968; Michaeli et al., 1972; Zajicek et aL, 1972). Although increased IEE cell proliferation occurred in the present study, it did not appear to be directly related to the eruptive response on a temporal basis. Recent studies indicate that the number of PDL fibroblasts per cell layer, the total number of PDL fibroblasts, and the daily production rate of PDL fibroblasts are increased dramatically following altered eruption (Weinreb et al, 1985). Those data corroborate our present findings to a certain extent, although they probably do not represent a causal relationship between PDL cell proliferation and eruption. A direct relationship between PDL cell proliferation and eruption has been refuted by several previous studies (Berkovitz, 1971; Berkovitz, 1972; Moxham and Berkovitz, 1974; Pitaru et ak, 1976); however, it is difficult to compare the results of those studies to those of the present work because of the differences between the impeded, continuously erupting adult rodent incisor and the initial postnatal development and eruption of the growing incisor. In the present experiments, EGF induced a distinct alteration of the patterns of [3H]PR0 incorporation into odontoblasts and ameloblasts at several locations near the apical loop region. Studies in other organ systems have also demonstrated increased protein synthesis and a stimulation of differentiation processes following EGF treatment (Cohen and Elliott, 1963; Franklin and Lynch, 1979). Protein synthetic processes have not been directly studied in the impeded rat incisor model, although histomorphometric data indicate that long-
541
term shortening of the incisor causes a reduction in dentin apposition (Weinreb et al, 1985). The stimulation of C3H]PR0 incorporation into ameloblasts and odontoblasts observed in the present study (particularly at 7 and 13 days) appear to contradict those findings. Other points to consider, however, are that (1) effects on rH]PRO incorporation into ameloblasts and odontoblasts may be secondary to cell proliferative alterations; and (2) EGF may alter degradation as well as synthesis of protein, so that overall turnover of protein differs in the EGF-accelerated incisor. EGF altered [3H]PR0 incorporation in the PDL, particularly between Days 4-7, immediately before eruption, but as discussed above, EGF may alter protein turnover rate. Using the unimpeded continuously erupting incisor Slootweg (1976) found no difference in the amount or metabolism of collagen, but an increase in the amount and a slight decrease in the turnover rate of noncollagenous protein. Others have demonstrated a slightly faster turnover rate in the PDL of unimpeded incisor teeth (Beertsen and Everts, 1977). In another study, altered eruption rate did not affect the turnover of insoluble collagen, structural noncollagenous proteins, or sulfated glycosaminoglycans (Van den Bos and Tonino, 1984). These authors suggested that remodeling due to accelerated eruption may not affect the activity of PDL components since their metabolism is probably already maximal. Morphologically, observations during the present study have revealed a more linear and organized appearance of preodontoblasts and preameloblasts in the group compared to EGF-treated controls. In EGFtreated mice, cells appeared to polarize earlier (at a point nearer to the apex of the loop), while secretion of enamel and predentin matrices is initiated in the EpMes interface at a point nearer to the apex of the loop than was seen in control tissues. EGF-induced acceleration of differentiation has previously been reported in a wound healing study in which EGF was applied topically to rabbit ear skin, having an organizing and stimulatory effect on fibroblasts in the epidermal layers of the wound (Franklin and Lynch, 1979). The alteration of odontoblast and ameloblast differentiation observed in the present study may result from disturbances of the complex cellular interactions along the basal lamina (Kallenbach and Piesco, 1978; Slavkin, 1979; Hurmerinta and Thesleff, 1981; Ruth et aL, 1983). Alterations in the synthesis of collagen (types I and IV), laminin, and fibronectin associated with the basal lamina occur during the embryonic development of the teeth (Trelstad and Slavkin, 1974; Lesot et al, 1981; Thesleff et ah, 1979; Thesleff et ah, 1981), and are involved in differentiation of the continuously erupting incisor throughout life. The morphological changes observed in the current study, combined with the findings of increased [3H]PR0
542
DEVELOPMENTALBIOLOGY
incorporation and secretion nearer to the apical loop region. indicate that a more ranid differentiation of odintdblasts and ameloblasts may occur after EGF treatment. These data appear to contradict those of Thesleff and Partanen (1984), and Partanen et al. (1985), who have demonstrated that EGF inhibits the terminal differentiation of odontoblasts and ameloblasts during the bell stage of molar development in vitro. The three main differences between our study and that of Partanen et al. include (1) molar model vs incisor model, (2) fetal animals vs neonates, and (3) in vitro vs in vivo methods. making comnarisons difficult. The key to understanding these-conflicting data may lie in determining the temporal sequence of the selective effects of EGF during development, since EGF does not inhibit morphogenesis in tooth germs that have reached the late cap stage or early bell stage (15 to 16 days gestation) of development (Partanen et cd., 1985). A recent study by Rhodes et al. (1987) indicates that EGF induces a reduction in mandibular and maxillary incisal area, as measured from radiographs, during accelerated eruption. These authors suggest that the reduction in tooth size they observed at 7 days after daily EGF treatment (6.5 /*g/g body wt) indicates that EGF does not have a mitogenic effect on cellular compartments of the mouse incisor in vivo. Our cell proliferation results appear to directly contradict this suggestion. Rhodes et al. also suggest that the PDL may be the target for EGF. Our data from [3H]TDR, [3H]PR0, and ‘251-EGF studies argue strongly that EGF may induce precocious eruption of the incisor through its action on the PDL. The accelerated eruption induced by exogenous EGF administration appears to affect both cell proliferation and protein synthesis by PDL cells. The relationship between eruption rate and cell proliferation and protein synthesis in the PDL requires further investigation during EGF-accelerated eruption. This work was completed as partial fulfillment for the Master of Arts degree awarded to R. Todd Topham from the Graduate School, University of Kansas Medical Center. The authors thank Susan G. Kramer for her technical assistance and Wanda Hinton for typing the manuscript. This project was supported by Public Health Service Grant DE07148 from the National Institute of Dental Research and by BRSG SO7 RR05373 awarded by the Biomedical Research Support Grant Program, Division of Research Resources, National Institutes of Health. REFERENCES ADAMSON,E. D., DELLER, M. H., and WARSHAW, J. B. (1981). Functional EGF receptors are present on mouse embryo tissues. Nature (London) 291.656-659. ADAMSON,E. D., and REES, A. R. (1981). Epidermal growth factor receptors. Mol. CeU B&hem. 34,129-152. ADAMSON, E. D., and WARSHAW, J. B. (1982). Down-regulation of epidermal growth factor receptors in mouse embryos. Dew. Biol 99, 430-434. BEERTSEN,W., and EVERTS,V. (1977). The site of remodelling of collagen in the periodontal ligament of the mouse incisor. Anat. Rec. 189,479-498.
VOLUME124, 1987
BERKOVITZ,B. K. B. (1971). The effect of root transection on the unimpeded eruption rate of the rat incisor. Arch. Oral Biol. 16, 1033-1043. BERKOVITZ,B. K. B. (1972). The effect of preventing eruption on the proliferative basal tissues of the rat lower incisor. Arch. Oral Biol. 17.12’79-1288. BROWNELL,A. G., and ROVERO,L. J. (1980). DNA synthesis of enamel organ epithelium in vitro is enhanced by co-cultivation with non-viable mesenchymal cells. J. Dent. Res. 59,1075-1080. CARPENTER, G., and COHEN, S. (1979). Epidermal growth factor. Annu. Rev. Biochem. 48,193-216. CHABOT,J. G., WALKER, P., and PELLETIER,G. (1986). Distribution of epidermal growth factor binding sites in the adult rat liver. Amer. J. Physiol. 250, 760-764. CHIEGO, D. J., JR., KLEIN, R. M., and AVERY, J. K. (1981). Tritiated thymidine autoradiographic study of the effects of inferior alveolar nerve resection on the proliferative compartments of the mouse incisor formative tissues. Arch. Oral Biol. 26. 83-89. CLARK, A. J. L., ISHII, S., RICHERT,N., MERLIN;, G. T., and PASTAN,I. (1985). Epidermal growth factor regulates the expression of its own receptor. Proc. Natl. Acad Sci USA 82,8374-8378. COHEN, S. (1962). Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal. J. Biol. Chem. 237,1555-1562. COHEN, S. (1965). The stimulation of epidermal proliferation by a specific protein (EGF). Dev. Biol 12,394-407. COHEN,S., and ELLIOTT, G. A. (1963). The stimulation of epidermal keratinization by a protein isolated from the submaxillary gland of the mouse. J. Invest. Derm. 40,1-5. EARP, H. E., AUSTIN, K. S., BLAISDELL,J., RUBIN, R. A., NELSON,K. G., LEE, L. W., and GRISHAM, J. W. (1986). Epidermal growth factor (EGF) stimulates EGF receptor synthesis. J. Biol. Chem. 261, 4777-4780. FRANKLIN, J. D., and LYNCH, J. B. (1979). Effects of topical applications of epidermal growth factor on wound healing. Plust. Reconstr. Surg. 64,766-770. GOLDIN,G. V., and OPPERMAN,L. A. (1980). Induction of supernumerary tracheal buds and the stimulation of DNA synthesis in the embryonic chick lung and trachea by epidermal growth factor. J. Embrgol. Exp. Morphol. 60,235~243. GOSPODAROWICZ, D. (1981). Epidermal and nerve growth factors in mammalian development. Annu. Rev. Physiol. 43,251-263. GREEN, M. R., BASKETTER,D. A., COUCHMAN,J. R., and REES, D. A. (1983). Distribution and number of epidermal growth factor receptors in skin is related to epithelial cell growth. Dev. Biol. 100, 506-512. GRESIK,E., and AZMITIA, E. (1980). Age related changes in NGF, EGF and protease in the granular convoluted tubules of the mouse submandibular gland: A morphological and immunocytochemical study. J. Gerontol 35,520-525. GRESIK, E., and BARKA, T. (1978). Immunocytochemical localization of epidermal growth factor during the postnatal development of the submandibular gland of the mouse. Amer. J. Anat. 151, l-10. GRESIK,E., GUBITS,R. M., and BARKA, T. (1985). In situ localization of mRNA for epidermal growth factor in the submandibular gland of the mouse. J. Histochem. Cytochem. 33,1235-1240. HOATH, S. B. (1986). Treatment of the neonatal rat with epidermal growth factor: Differences in time and organ response. Pediutr. Res. 20,468-472. HOATH, S. B., LAKSHMANAN,J., SCOTT,S. M., and FISHER,D. A. (1983). Effect of thyroid hormones on epidermal growth factor concentration in neonatal mouse skin. Endocrinology 112,308-314. HURMERINTA,K., and THESLEFF,I. (1981). Ultrastructure of the epithelial-mesenchymal interface in the mouse tooth germ. J. Cruniofacial Genet. Dev. Biol 1,191-202. KALLENBACH,E., and PIESCO,N. P. (1978). The changing morphology
TOPHAM ET AL.
EGF Efects on Neonatal Mouse Incisor
of the epithelium-mesenchyme interface in the differentiation zone of growing teeth of selected vertebrates and its relationship to possible mechanisms of differentiation. J. Biol. Buccule 6,229-240. KING, L. E., JR. (1985). What does epidermal growth factor do and how does it do it? J. Invest. De+rmatoL84,165-167. KONTUREK, S. J., RADECKI, T., BRZOZOWISKI, T., PIASTUCKI, I., DEM-
BINSKI, A. DEMBINSKI-KIEC, A., ZMUDA, A., GRYGLEWSKI,R., and GREGORY, H. (1981). Gastric cytoprotection by epidermal growth factor: Role of endogenous prostaglandins and DNA synthesis. Gastroenterology 81,438-443. LAKSHMANAN,J., PERHEENTUPA,J., HOATH, S. B., KIM, H., GRUTERS, A., ODELL,C., and FISHER, D. A. (1985). Epidermal growth factor in mouse ocular tissue: Effects of thyroxine and exogenous epidermal growth factor. Pediatr. Res. 19,315-319. LESOT, H., OSMAN, M., and RUCH, J. V. (1981). Immunofluorescent localization of collagens, fibronectin, and laminin during terminal differentiation of odontoblasts. Dev. BioL 82,371-381. MICHAELI, Y., and WEINREB, M. M. (1968). Role of attritional and occlusal contact in the physiology of the rat incisor. III. Prevention of attrition and occlusal contact in the nonarticulating incisor. J. Dent. Res. 47, 633-640. MICHAELI, Y., WEINREB, M. M., and ZAJIECK, G. (1972). Role of attrition and occlusal contact in the physiology of rat incisors. V. Life cycle of inner enamel epithelial cells at various rates of eruption. J. Dent. Res. 51,960-963. MOXHAM, B. J., and BERKOVITZ,B. K. B. (1974). The effects of root transection on the unimpeded eruption rate of the rabbit mandibular incisor. Arch. oral Biol 19,903-909. NESS,A. R., and SMALE, D. E. (1959). The distribution of mitoses and cells in the tissues bounded by the socket wall of the rabbit mandibular incisor. Proc. R. Sot. London 151,106-128. PARTANEN, A., EKBLOM, P., and THESLEFF, I. (1985). Epidermal growth factor inhibits morphogenesis and cell differentiation in cultured mouse embryonic teeth. Dew. BioL 111,84-94. PARTANEN,A., and THESLEFF,I. (1987). Localization and quantitation of ‘%I-epidermal growth factor binding in mouse embryonic tooth and other embryonic tissues at different developmental stages. Deu. BioL 120,186-197. PITARU, S., MICHAELI, Y., ZAJICEK, G., and WEINREB, M. M. (1976). Role of attrition and occlusal contact in the physiology of the rat incisor. X. The part played by the periodontal ligament in the eruptive process. J. Dent. Res. 55,819-824. RALL, L. B., SCOTT,J., BELL, G. I., CRAWFORD,R. J., PENSCHOW,J. D., NIALL, H. D., and COGHLAN,J. P. (1985). Mouse prepro-epidermal growth factor synthesis by the kidney and other tissues. Nature (London) 313,228-231.
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RHODES,J. A., FITZGIBBON,D. H., MACCHIARULO,P. A., and MURPHY, R. A. (1987). Epidermal growth factor-induced precocious incisor eruption is associated with decreased tooth size. Dev. BioL 121, 247-252. RUCH, J. V., LESOT, H., KARCHER-DJURICIC,V., MEYER, J. M., and MARK, M. (1983). Epithelial-mesenchymal interactions in tooth germs: Mechanisms of differentiation. J. BioL Buccale 11,173-193. SAVAGE,C. R., JR., and COHEN,S. (1972). Epidermal growth factor and a new derivative: Rapid isolation procedures and biological and chemical characterization. J. BioL Chem 247,7609-7611. SLAVKIN, H. C. (1979). The nature and nurture of epithelial-mesenchymal interactions during tooth morphogenesis. J. BioL Buccale 6, 189-203.
SLOOTWEG,R. N. (1976). Changes of collagen and non-collagenous proteins in the peridontal ligament during acceleration of the eruption. A biochemical and histological investigation in different sectors of the periodontal ligament in the guinea pig incisor. Thesis, University of Utrecbt, The Netherlands. STEIDLER,N. E., and READE, R. C. (1980). Histomorphological effects of epidermal growth factor on skin and oral mucosa in neonatal mice. Arch. Oral BioL 25, 37-43. STEIDLER,N. E., and READE, P. C. (1981). Epidermal growth factor on proliferation of odontogenic cells in culture. J. Dent. Res. 60, 1977-1982.
THESLEFF,I., BARRACH,H. J., FOIDART,J. M., VAHERI, A., R.M., and MARTIN, G. R. (1981). Changes in the distribution of type IV collagen, laminin, proteoglycan, and fibronectin during mouse tooth development. Dev. BioL 81, 182-192. THESLEFF, I., and PARTANEN, A. (1984). Growth factors and tooth morphogenesis. INSERM 125,73-88. THESLEFF,I., STENMAN,S., VAHERI, A., and TIMPL, R. (1979). Changes in the matrix proteins, fibronectin and collagen, during differentiation of mouse tooth germ. Den BioL 70,116-126. TRELSTAD,R. L., and SLAVKIN, H. C. (1974). Collagen synthesis by the epithelial enamel organ of the embryonic rabbit tooth. B&hem. Biophys. Res. Commun. 59,443-449. VAN DEN Bos, T., and TONINO,G. J. M. (1984). Composition and metabolism of the extracellular matrix in the periodontal ligament of impeded and unimpeded rat incisors. Arch. oral BioL 29,893-897. WEINREB,M., JR., STEIGMAN,S., ZAJICEK,G., and MICHAELI, Y. (1985). Odontoblast turnover in the impeded and unimpeded rat incisor derived from computerized histomorphology. Anat. Rec. 211, 218-225.
ZAJICEK,G., MICHAELI, Y., and WEINREB,M. M. (1972). Kinetics of the inner enamel epithelium in the adult rat incisor during accelerated eruption. Cell Tissue Kinet. 5, 35-39.
BIOLOGY
124,532-543 (1987)
The Effect of Epidermal Growth Factor on Neonatal Incisor Differentiation in the Mouse R. TODD TOPHAM, DANIEL
J. CHIEGO, JR.,’ VINCENT H. GATTONE II, DUANE A. HINTON, AND ROBERT M. KLEIN
Department of Anatomy/Division
of Cell Biology, University of Kansas Medical Center, Kansas City, Kansas 6610.3
Received February 10, 1987; accepted in revised f&-m August 10, 1987' The effect of epidermal growth factor (EGF) on cellular differentiation of the neonatal mouse mandibular incisor was examined autoradiographically using tritiated thymidine (rH]TDR) and tritiated proline (rH]PRO). On Days 0 (day of birth), 1, and 2, EGF was administered (3 pg/g body wt) SCto neonates. Mice were killed on Days 1,4, ‘7,10, and 13 after birth and were injected with either [aHJl?DR or [3H]PR0 1 hr before death. [3H]TDR was used to analyze cell proliferation in eight cell types in the developing mouse incisor including upper (lingual) and lower (buccal) pulpal fibroblasts, preodontoblasts, inner and outer enamel epithelial cells (IEE and OEE), stratum intermedium (SI), stellate reticulum (SR), and periodontal ligament (PDL) t’ibroblasts. [3H]PR0 was used to analyze protein synthesis in ameloblasts, and their secretion products (enamel and dentin), as well as PDL fibroblasts. The selected EGF injection scheme elicited acceleration of incisor eruption with minimal growth retardation. At Day 1, the upper and lower pulp, preodontoblasts, SI, and SR showed a significant decrease in labeling index (LI) 24 hr after a single EGF injection. After multiple injections (Days 0, 1, 2), two LI patterns were observed. In lower pulp, preodontoblasts, IEE, SI, SR, and OEE, a posteruptive change in LI was observed. In contrast, the upper pulp and PDL regions demonstrated a direct temporal relationship with eruption. Autoradiographic analysis with [aH]PRO indicated that EGF treatment caused significant increases in grain counts per unit area in ameloblast, odontoblast, and PDL regions studied. Significant differences were found in all four regions studied (ameloblasts, enamel, odontoblasts, dentin) at the 45-pm-tall ameloblast level as well as ameloblasts and odontoblasts at the 30-pm level at 13 days of age. The PDL demonstrated significant differences at all locations studied (base, 30 pm, 45 pm,) in 4-, 7-, and 13-day-old mice. Morphologically, EGF-treated groups demonstrated premature differentiation of ameloblasts and odontoblasts at the light microscopic level. The data indicate that EGF alters DNA and protein synthesis as well as differentiation patterns during the eruption process. While EGF affects both DNA and protein synthesis, the alteration of differentiation may be secondary to mitogenic effects on proliferative compartments. In order to determine the cellular target for EGF within the newborn mouse incisor, in viva ‘*‘I-EGF binding was analyzed autoradiographically. EGF binding sites were demonstrated in the dental follicle, an area which contains cells believed to be the progenitors for the PDL, alveolar bone, and cementum. The results of the [3H]TDR, [3H]PR0, and ‘a51-EGF studies argue strongly that the PDL is the target for EGF within the 0 198’7 Academic Press, Inc. incisor.
INTRODUCTION
Previous studies using epidermal growth factor (EGF)’ have demonstrated that this low-molecularweight hormone has a wide range of biological activities in vivo and in vitro. EGF stimulates the growth of ectodermally (Carpenter and Cohen, 1979; Cohen and Elliott, 1963; Cohen 1965; Gospodarowicz, 1981) and endodermally (Konturek et aZ.,1981) derived tissues in vivo and promotes ectodermal and mesodermal growth in vitro (Adamson and Rees, 1981). Extensive analysis of i Present address: Department of Oral Biology, University of Michigan, School of Dentistry, Ann Arbor, MI 48104. a Abbreviations used: EGF, epidermal growth factor; EGF-R, epidermal growth factor receptor; Ep-Mes, epithelial-mesenchymal; [3H]PR0, tritiated proline; [3H]TDR, tritiated thymidine; IEE, inner enamel epithelium; LI, labeling index; OEE, outer enamel epithelium; PDL, periodontal ligament; SI, stratum intermedium; SR, stellate reticulum.
0012-1606187$3.00 Copyright All rights
0 1987 by Academic Press, Inc. of reproduction in any form reserved.
532
EGF effects on the growth and development of skin reveals that EGF stimulates the proliferation and keratinization of the epidermis of cultured embryonic skin (Cohen, 1965) and accelerates epidermal development in newborn mice (Cohen and Elliott, 1963), including the premature opening of the eyelids and accelerated eruption of the incisor teeth (Cohen, 1962). In organ systems such as the developing lung, EGF has been shown to have a trophic function (Goldin and Opperman, 1980). There have been few studies on developing teeth; however, it is known that EGF stimulates DNA synthesis in the enamel organ epithelium (Brownell and Rovero, 1980) and odontogenic cells (Steidler and Reade, 1981) in vitro. In addition, EGF receptors have been localized in both epithelial and mesenchymal compartments of fetal mouse molars (Partanen and Thesleff, 1987). Although the premature eruption of the mouse inci-
TOPHAM
ET AL.
EGF Effects on Neonatal Mouse hcisor
sor was used as the original bioassay for EGF, the mechanism by which EGF exerts its effect on incisor eruption and differentiation has not been investigated. The present study was designed to determine the cellular response of the continuously erupting incisor of the neonatal mouse to EGF administration in viva by analysis of tritiated thymidine ([3H]TDR) and tritiated proline (rH]PRO) incorporation into the various developing cellular compartments. In addition, the presence of EGF binding sites in the cellular compartments of the newborn mouse incisor was analyzed by in vivo injection of lz51-EGF followed by autoradiography. The hypothesis of this study is that EGF-accelerated eruption occurs by modification of normal differential rates of DNA and protein synthesis in cells which are sensitive to EGF (possess EGF receptors) during incisor development and eruption. MATERIALS
AND METHODS
Animals Breeding stock of Balb/c mice was obtained from Harlan/Sprague-Dawley (Indianapolis, IN). These animals were maintained in the KUMC Animal Care Unit on a 1%hr light/dark cycle (lights on 06:00-18:00) and given Purina Mouse Chow and water ad lib&m. Pups were weighed each day prior to the injections. Day zero was defined as the date of birth. Neonates were checked each day for evidence of tooth eruption or eyelid opening. Mice were killed by ether overdose at one of five time points (1,4,7,10, and 13 days) for both control and EGF groups. Mandibles for examination were immediately excised using a magnifying glass lamp and iris scissors and forceps. Tissues were then placed into Peter’s neutral buffered formalin (pH 7.4) for fixation. Preparation and Administration
of EGF Solution
EGF was obtained from Collaborative Research, Inc. (Lexington, MA), where it was produced according to the method of Savage and Cohen (1972). The EGF was received in lyophilized form, 100 pg per vial, and was solubilized by adding 1 cc of 0.15 M NaCl solution. The EGF was administered by SCinjection under the dorsal skin. A dosage of 3 /*g/g body wt of EGF per day was chosen. This dose was found to be effective in induction of tooth eruption in previous studies (Steidler and Reade, 1980). Controls were injected with an equal volume of 0.15 M NaCl solution. Our preliminary studies indicated that a 0, 1,2 day only series of injections had optimum results (Day 0 = day of birth), because early tooth eruption was elicited with minimal growth retardation. The preliminary studies included the following injection schemes: Day 0 only, Day 1 only, Day 2 only, Days 1,2,3, only, Days 2,3,4, only, and Days 3,4,5 only.
533
Preparation and Administration of Tritiated Thymidine and Tritiated Proline Tritiated proline (sp act 5.0 Ci/mmole; New England Nuclear, Boston, MA) and tritiated thymidine (sp act 6.7 Ci/mmole; ICN, Irvine, CA) were injected for a 1-hr pulse at dosages of 1 and 2 pCi/g body wt, respectively. Both isotopes were injected iv via the orbital branch of the anterior facial vein in animals 7 days old or less. Older animals (Days 10 and 13) were given intracardiac injections of the isotopes. No differences were found between intracardiac and intravenous injections in preliminary studies. Preparation of Tissue for Light Microscopy Whole mandibles were fixed for 20 hr in Peter’s neutral buffered formalin. Following fixation, mandibles were decalcified in EDTA sodium citrate (pH 7.4). Decalcification was verified by X-ray examination. Decalcified tissues were washed for 24 hr, then dehydrated, cleared with xylene, and embedded in paraplast. Mandibles were divided into right and left halves and then embedded in paraffin blocks with the buccal surface of the mandible facing out toward the cutting surface of the paraffin block. Sections were cut in 5-pm thicknesses and placed onto acid-cleaned, gelatin ChromAlum subbed glass slides. Following completion of sectioning, deparaffinization, exposure of autoradiograms, and staining with hematoxylin and eosin, the tissues were covered with acrytol and coverslipped. Autoradiography Slides were dipped in nuclear track emulsion, NTB-3 (Eastman-Kodak Co., Rochester, NY), and exposed in black boxes at 4°C. rH]TDR slides were exposed for 16 days; rH]PRO slides were exposed for 13 days. Exposure time was determined by use of control slides. All slides were developed as follows: 3.5 min in developer D-19 (Kodak) 10 set in distilled water (with a drop of D-19 added), 6 min in Kodak fixer, and 30 min in a water bath. Slides were air-dried overnight, followed by the completion of a standard H and E staining process. Analysis of Autoradiograms Analysis was done manually at 450X magnification. For rH]TDR autoradiograms, the percentage of labeled cells (LI) was determined by counting cells in eight specific areas (Fig. 3) of the enamel organ region: upper (lingual) pulp fibroblasts, lower (buccal) pulp fibroblasts, preodontoblasts, inner enamel epithelium (IEE), stratum intermedium, stellate reticulum, outer enamel epithelium (OEE), and periodontal ligament (PDL) fibroblasts. In the upper pulp area, a full grid was
534
DEVELOPMENTAL BIOLOGY
counted for each section (each grid box 20 pm on a side). Half a grid was counted over the lower pulp area, taking care not to include preodontoblast cells. At the apex of the loop, an arbitrary dividing line was placed between the IEE and OEE regions (Chiego et ab, 1981). Moving i&sally, in areas 3-8, counts were terminated at a point equal to the beginning of the dentino-enamel matrix. PDL cells (area 8) were counted from that point to the apex of the loop. Cells were considered labeled if they contained four or more grains. With [3H]PR0 autoradiograms, grains were counted per unit area, using ameloblast heights as landmarks for measurements (30, 45 pm). The 30-pm level was chosen as the approximate location of the dentinoenamel junction and the 45-pm level was selected as the location of the first mature ameloblasts (polarized nuclei and attainment of maximum cell height). Counts were made at those landmarks over odontoblast, dentin, enamel, ameloblast, and PDL layers (Fig. 5). Grid boxes used measured 10 pm on a side. Data were analyzed with an unpaired t test.
VOLUME 124, 1987
1 ’
“CON
t t t 0
1 2 3
*,,+,,*,,+I 4 5 6
7
El 9 10111213
AGE FIG. 2. Graph of body weight changes caused by EGF administration. (t) indicate injection days; (k) indicates sacrifice days; E, eruption day; 0, opening of eyelids. (p i 0.05*, p < O.Ol**, p < O.OOl***.)
EGF was obtained from New England Nuclear Co., diluted in PBS, and injected into newborn mice at a concentration of 0.25 &i/g body wt. Mice were anesthetized with nembutal and injections were made by the intracardiac route using a dual stopcock syringe. Two minutes after the injection mice were perfused with PBS to remove unbound radiolabeled EGF and subsequently perfused with 2.0% glutaraldehyde in 0.1 M phosphate buffer. Controls received the same dosage of 1251-EGF Binding Studies lz51-EGF plus a 500-fold excess of cold (unlabeled) EGF. The method used for analyzing the distribution of Mandibles were then removed and immersion-fixed EGF-binding sites in the newborn mouse incisor was overnight before preparation for methacrylate embedsections were cut at 2 pm and adapted from the method of Chabot et al. (1986). lz51- ding. Methacrylate mounted on acid-cleaned, gelatin-subbed slides for autoradiography using NTB-2 nuclear track emulsion. Slides were exposed for 12 weeks and subsequently Eyelid Eruption Body weight trend (mean) stained lightly using a stain containing basic Fuchsin opening Injection days observed E or C days days Day 0 Day 7 Day 10 and Methylene blue. Slides were viewed and photographed on a Nikon Labophot microscope using both 9 0, 1, 2-E 1.70 6.38 8.26 bright- and dark-field optics. 1, 2, 3-E 10 1.55 4.53 6.08 A 3, 4, 5-E 1 2, 3, 4-E
9 9
2.04 1.55
4.53 3.12
6.50 4.78
O-E 1-E B 2-E I
9 10 10
11 11 12
1.77 2.72 1.80
5.08 4.07 4.77
7.17 6.82 6.92
None-C None-C
9 9
12 12
1.63 2.22
4.80 5.30
6.82 7.20
10 9 9 9
1.68 1.97 1.89 1.82
4.17 5.30 4.90 4.20
7.13 6.90 8.08 6.52
12 11 12 12 13 12
1.75 2.03 1.82 1.78 1.90 1.68
4.82 5.53 6.35 4.91 5.47 5.53
7.53 7.24 7.45 6.38 7.93 6.87
0, 0, 0, 0,
1, Z-E 1, Z-E 1, 2-E 1, Z-E
0, 0, 0, 0, 0, 0,
1, 2-c ic 1, 2-c 1, 2-c 1, 2-c 1, 2-c 1, 2-c,
10 9 10 10 10 10
FIG. 1. Optimization of injection protocol. (A) Three-day series injection schemes, (B) single day only injection schemes, (C) data from 0, 1, 2, day injection scheme used. “None-C” indicates data from control mice.
RESULTS
Preliminary
Studies
Preliminary experiments were performed to determine the optimal EGF dosage required for induction of precocious eruption of the incisors, in conjunction with the least weight loss. Earlier research with EGF demonstrated that continued daily injections caused steady body weight reduction in the experimental animals, which resulted in a high rate of mortality. It was anticipated that a specific period of EGF sensitivity (during the first postnatal week) could be identified and used to optimize the accelerated rate of eruption. Several single day only and 3-day injection schemes were tested (Fig. 1). It was determined that injections on Days 0, 1, and 2 provided the best results in terms of precocious incisor eruption coupled with the least growth retardation. Experimental mice treated by this method displayed a temporary weight loss followed by recovery (Fig. 2), as well as the desired effect of premature eruption of inci-
TOPHAM ET AL.
EGF Eflects on Neonatal
sor teeth as obtained earlier by Cohen (1962; Cohen and Elliott, 1963). Tritiated
Thymidine
Autoradiographic
Data
Eight specific areas within the mouse incisor were examined autoradiographically with rH]TDR as previously described (Fig. 3). Autoradiographs of the loop region displayed increased labeling in experimental animals compared to controls. In the upper pulp cell region associated with the epithelial (Hertwig’s root sheath) (Fig. 4a) significant differences in labeling indices were found at all time points except Day 10. The lower pulp region associated with the cervical loop (Fig. 4b) displayed significant LI differences at the l- and 13-day time points, while in the preodontoblast (Fig. 4c) and IEE (Fig. 4d) regions, highly significant differences were observed following eruption at the lo- and 13-day time points. The SI layer of cells (Fig. 4e) displayed a
similar pattern, as EGF administration appeared to maintain DNA synthesis after eruption at a higher level than control values. In the areas with the fewest labeled cells, SR (Fig. 4f) and OEE (Fig. 4g), significant differences were again seen at the lo- and 13-day intervals. Finally, in the PDL region, significant differences between control and EGF-treated groups were observed at all time points except Day 1 (Fig. 4h). Tritiated
Proline
@ inner enamel epithaliw @ stratum intemmdiun
(IEE)
FIG. 3. Diagram of sagittal (longitudinal) section through the incisor showing cellular areas analyzed with [‘Hj’I’DR. The upper (lingual) pulp region was defined as the pulpal area associated with Hertwig’s root sheath and the lower (buccal) pulp was delineated as pulpal cells associated with the cervical loop.
Autoradiographic
Data
Specific regions near the base of the developing mouse incisor were examined autoradiographically using [3H]PR0, as previously described (Fig. 5). Autoradiographs of incisor sections from 13-day-old mice displayed increased labeling in EGF-treated mice compared to controls. Ameloblast cell heights of 30 and 45 pm were chosen for reference points. Data from this study indicated that significant differences between control and EGF-treated groups were present at the 30-pm level in both ameloblasts and odontoblasts at the 13-day time point (Fig. 6). At the 45-pm level, significant differences were found in all four layers studied: ameloblasts, enamel, odontoblasts, and dentin (Fig. 7). Significant differences between groups at this level were also found in ameloblast and dentin layers at the 7-daytime point. Interestingly, in all PDL regions studied (base, 30+m, and 45-pm levels), highly significant differences between groups were seen at the 4- and i’-day time points (Fig. 8). This highly significant difference (p < 0.001) was especially obvious at all positions studied at the 4-day time point. Significant differences between EGF-treated and control groups were also found to be present at all three PDL levels studied at the 13-day interval (Fig. 8). Morphological
@ upper pulp fibroblasts @ lower pulp fibroblasts
535
Mouse Incisor
Observations
After EGF treatment, light microscopic morphological differences were observed in the maturation of odontoblasts and ameloblasts at the base of the loop region. As seen on photomicrographs from lo-day-old EGF-treated mice (Fig. 9a), ameloblasts and odontoblasts appeared more ordered and linear than cells from control mice (Fig. 9b). The secretory cells in the EGF-treated group appeared to organize earlier, at a point nearer to the loop, along the epithelial-mesenchyma1 (Ep-Mes) interface. In addition, polarization of the nuclei toward the basal aspect of the cell, typical of mature ameloblasts and odontoblasts, occurred prematurely, followed by earlier deposition of the enamel layer in EGF-treated animals. The precocious polarization of odontoblasts and ameloblasts was also seen at
536
VOLUME 124,1987
DEVELOPMENTAL BIOLOGY
UPPER
LOVER
PULP
PULP
t: 25. % 20: a
i?lS
0 CON 10
1
4
?
13
10
AGE
PRE0DOrvrOBlAsls
INNER
ENAMBL
EPlTEEUUY
0
0 CON
CON
e
0
1
4
7
13
10
AGE
16 OUTER
R
BNAMEL
EPrniBuUY
h
c1 .\
0’
FIG. 4. Labeling
ECF
Q
CON
3
I
1
4
7 AGE
10
13
0 I
index data in eight [3HjTDR areas analyzed
earlier time points with termination of DNA synthesis occurring at a point closer to the loop (Fig. 10). [*251jEGF Binding
l
Studies
Autoradiographs from newborn mouse incisors demonstrated specific labeling in the dental follicle area. Figure lla demonstrates a bright-field photomicrograph illustrating the loop region of the incisor. Figure llb demonstrates the autoradiographic binding of lz51EGF in the PDL and dental follicle region using dark-
4
GE
10
13
(a-h) (p i 0.05*, p c. O.Ol**, p < O.OOl***).
field photomicrography. Controls did not demonstrate localization of grains in the dental follicle region. DISCUSSION
The experiments in this study analyzed [3H]TDR and [3H]PR0 incorporation into compartments of the developing mouse incisor during the first two postnatal weeks and the localization of EGF binding sites in the newborn incisor. These data will be discussed in two sections: (1) cellular and organ sensitivity to EGF and
TOPHAMETAL.
537
EGF Efects on Neonatal Mouse Incisor 50 g
40
-5 30 ag
d
20
4
10 A
au 5
7
10
13
7
10
13
40
do m 30
5 s
0
20 10 0
1
4
DAY
Regions
Counted
FIG. 7. Graphs showing results of the [‘HIPRO study at the 45-pm (cell height) ameloblast level. Grain counts were taken over ameloblasts (a), enamel (b), dentin (c), and odontoblasts (d), as shown in Fig. 5.
-1 FIG. 5. Diagram of sagittal (longitudinal) section through the incisor showing cellular areas analyzed with [3H]PR0. Two landmarks were selected for reference points, the ameloblasts cell heights of 30 and 45 Nrn.
a v, kl 4 E =
25 20
q
CONTROL EGF
70
15 9 bg
5
"4:
5
30 5:
0
b
60
10
20 10 01
257
70 60
DAY
FIG. 6. Graphs showing results of the [3H]PR0 study at the 30-pm (cell height) ameloblast level. Grain counts were taken over ameloblast (a) and odontoblast (b) regions, as shown in Fig. 5; p values: p < 0.05*, p i O.Ol**, and p < O.OOl***.
FIG. 8. Graphs showing results of the [3H]PR0 study of the PDL compartment. Grain counts were taken at three sites in the PDL: 45-pm (cell height) ameloblast level (a), 30-pm (cell height) ameloblast level (b), and a basal loop position (c), as shown in Fig. 5.
FIG. 9. r3H]TDR autoradiographs from EGF and control (lo-day-old mice). EGF-treated odontoblasts and ameloblasts appear more ordered and linear (9a) than those of controls (9b). Secretory cells treated with EGF appear to organize at a point nearer to the loop than controls (c) (arrowheads). Nuclei of maturing cells migrate basally prematurely (b) ( small arrows), and subsequently begin deposition of the enamel layer earlier than in controls (a) (dark arrow). 538
TOPHAM ET AL.
EGF Effects on Neonatal
Mouse Incisor
FIG. 10. [$H]TDR autoradiographs from EGF and control (I-day-old mice). In EGF mice (b), the odontoblasts and ameloblasts appearedmore organized with a more linear arrangement. After EGF treatment, nuclei migrated toward functional secretory positions at an earlier point (closer to the loop), and no longer displayed [aH]TDR labeling (left of bracket). p, pulp, o, odontoblasts, a, ameloblasts.
FIG. 11. Autoradiograph of newborn mouse incisor demonstrating photomicrograph (a) illustrates the loop region. Dark-field enlargement follicle region. Controls did not display labeling in these regions.
specific labeling in the dental follicle area and PDL. Bright-field autoradiograph (b) shows binding of ‘a?-EGF in the PDL and dental
540
DEVELOPMENTALBIOLOGY
(2) EGF-induced alterations of cell proliferation, tein synthesis, and morphology.
pro-
Cellular and Organ Sensitivity to EGF Studies of neonatal rat tissues have demonstrated that postnatal Days 0 to 3 are critical time points for elicitation of EGF effects on incisor eruption, ear canal opening, and eyelid unfusion (Hoath, 1986). Our experimental results concerning premature eruption and eyelid opening were in agreement with earlier findings by Cohen (1962; Cohen and Elliott, 1963), and indicate that these tissues are subject to a period of EGF sensitivity during the first neonatal week. In the present experiments EGF was found to alter [3H]TDR and [3H]PR0 incorporation in all cell compartments studied during the first two postnatal weeks. The pattern of EGF-induced alteration of cell proliferation and protein synthesis differed temporally and from compartment to compartment. Generally, EGF appeared to prolong or maintain a higher level of DNA synthesis in all regions studied, following eruption. The effect of EGF on incisor eruption and associated EGFinduced changes in rH]TDR and [3H]PR0 may be due to either direct or indirect effects on the incisor. It is possible that the large, pharmacological dose of EGF used in these studies (3 pg/g body wt) led to indirect effects, including alteration of differentiation-related hormones such as the glucocorticoid or thyroid hormones. EGF concentration in neonatal mouse skin is altered by hypothyroid and hyperthyroid conditions (Hoath et al., 1983); however, EGF administration, even in large doses, does not appear to alter serum thyroxine levels (Lakshmanan et aL, 1985). In addition, relatively low doses of EGF (500 rig/g body wt) have been shown to accelerate tooth eruption in newborn rats (Hoath, 1986). The presence of EGF and EGF-Rs in the developing tooth has been investigated only to a limited extent, although EGF-R and EGF concentrations have been studied in other organs. EGF-Rs are present in fetal organs (Adamson et ah, 1981) and EGF may also be present in tissue and fluids to influence rapid metabolic sequences such as growth, differentiation, and tissue repair (King, 1985). For example, in the salivary glands EGF and mRNA for EGF are detectable in granular convoluted tubule cells of male mice at 20 days of age and EGF concentration increases during postnatal life (Gresik and Barka, 1978; Gresik and Azmitia, 1980; Gresik et al., 1985). EGF-Rs have been localized in the basal proliferative areas of neonatal skin, with receptor number decreasing as growth rates decline with age (Green et al, 1983). There is some evidence for involvement of EGF in tooth development. Mouse prepro-EGF mRNA has been localized in the mouth region in an area normally asso-
VOLUME124,1987
ciated with the incisor, suggesting that EGF may be synthesized by cells within the tooth (Rall et ah, 1985). Partanen and Thesleff (1987) indicated that in mouse embryonic molars in vitro, EGF-Rs are present on the dental epithelium during the bud stage with an absence of EGF-Rs on the dental mesenchymal cells. EGF-Rs were localized within the dental mesenchyme in the cap stage with an absence of binding in the dental epithelium, and in later stages of development, lz51-EGF binding was demonstrated in the dental follicle area. The dental follicle is believed to contain progenitor cells for the PDL, alveolar bone, and cementum. The results of the present study indicate that the dental follicle and PDL of the newborn mouse incisor also possess EGF-Rs and may be the target for EGF during postnatal development. EGF-Induced Alteration of Cell Proliferation, Protein Synthesis, and Morphology All cellular compartments of the neonatal mouse incisor demonstrated an alteration of cell proliferation during both normal eruption and EGF-accelerated eruption. At early time points, several zones (upper and lower pulp, preodontoblasts, IEE, SR, and SI) demonstrated an initial decrease in LI 24 hr after a single injection of EGF. The decrease in LI in these zones may be due to initial down-regulation of EGF-R within the incisor. EGF has been demonstrated to regulate expression of its own receptor in vitro (Clark et aZ.,1985). Down-regulation of receptors in vivo has been demonstrated in fetal tissues following intraamniotic injection of EGF (Adamson and Warshaw, 1982). The mitogenie effect exhibited by long-term administration of EGF in the present study may be due to EGF stimulation of EGF-R synthesis. Stimulation of EGF-R synthesis by EGF treatment has been demonstrated with WB cells in vitro. In that system EGF stimulates DNA synthesis and EGF-R degradation; however, there is a counterbalancing by an enhancement of EGF-R synthesis (Earp et ah, 1986). During the remainder of the experimental period in the present study, stimulation of cell proliferation occurred in all cellular compartments. This effect of EGF is similar to the mitogenic effect exhibited in numerous other developing organs (Steidler and Reade, 1980; Goldin and Opperman, 1980). EGF has also been shown to have a stimulatory effect on the enamel organ epithelium from fetal mice (Brownell and Rovero, 1980) and odontogenic cells from bovine fetuses (Steidler and Reade, 1981). A selective effect of EGF has been demonstrated in the case of the dental mesenchyme, which plays an essential role in the response of the tooth toward EGF. While dental mesenchymal cell proliferation is inhibited during the in vitro growth of the mouse molars, dissociated dental mesenchymal cells are stimulated by EGF (Partanen et al., 1985). The inhibition of
TOPHAMETAL.
EGF Effects on Neonatal Mouse Itiw
cell proliferation in the dental mesenchyme of organcultured embryonic molars may be mediated by EGFRs (Partanen and Thesleff, 1987). This alteration of the dental mesenchyme may cause the dramatic inhibition of the terminal differentiation of odontoblasts and ameloblasts observed in this system following EGF administration (Partanen et al, 1985). Two general patterns of cell proliferation were observed in the present studies. A pattern of late stimulation of cell proliferation in the SI, IEE, OEE, SR, lower pulp, and preodontoblast regions may indicate a secondary effect of EGF on these compartments. The earlier alterations of cellular proliferation in the PDL and upper pulp highlight the potential involvement of these zones in the EGF-accelerated eruption process. In the upper pulp region there is an increase in cell proliferation seen before normal eruption (during Days 7-10) and prior to EGF-accelerated eruption (during Days 4-7). Most previous studies of altered eruption rates have used the continuously erupting rodent incisor as a model system. A direct relationship between accelerated eruption and altered cell production in the IEE and pulp has been demonstrated following shortening of the incisor at the occlusal surface (Ness and Smale, 1959; Michaeli and Weinreb, 1968; Michaeli et al., 1972; Zajicek et aL, 1972). Although increased IEE cell proliferation occurred in the present study, it did not appear to be directly related to the eruptive response on a temporal basis. Recent studies indicate that the number of PDL fibroblasts per cell layer, the total number of PDL fibroblasts, and the daily production rate of PDL fibroblasts are increased dramatically following altered eruption (Weinreb et al, 1985). Those data corroborate our present findings to a certain extent, although they probably do not represent a causal relationship between PDL cell proliferation and eruption. A direct relationship between PDL cell proliferation and eruption has been refuted by several previous studies (Berkovitz, 1971; Berkovitz, 1972; Moxham and Berkovitz, 1974; Pitaru et ak, 1976); however, it is difficult to compare the results of those studies to those of the present work because of the differences between the impeded, continuously erupting adult rodent incisor and the initial postnatal development and eruption of the growing incisor. In the present experiments, EGF induced a distinct alteration of the patterns of [3H]PR0 incorporation into odontoblasts and ameloblasts at several locations near the apical loop region. Studies in other organ systems have also demonstrated increased protein synthesis and a stimulation of differentiation processes following EGF treatment (Cohen and Elliott, 1963; Franklin and Lynch, 1979). Protein synthetic processes have not been directly studied in the impeded rat incisor model, although histomorphometric data indicate that long-
541
term shortening of the incisor causes a reduction in dentin apposition (Weinreb et al, 1985). The stimulation of C3H]PR0 incorporation into ameloblasts and odontoblasts observed in the present study (particularly at 7 and 13 days) appear to contradict those findings. Other points to consider, however, are that (1) effects on rH]PRO incorporation into ameloblasts and odontoblasts may be secondary to cell proliferative alterations; and (2) EGF may alter degradation as well as synthesis of protein, so that overall turnover of protein differs in the EGF-accelerated incisor. EGF altered [3H]PR0 incorporation in the PDL, particularly between Days 4-7, immediately before eruption, but as discussed above, EGF may alter protein turnover rate. Using the unimpeded continuously erupting incisor Slootweg (1976) found no difference in the amount or metabolism of collagen, but an increase in the amount and a slight decrease in the turnover rate of noncollagenous protein. Others have demonstrated a slightly faster turnover rate in the PDL of unimpeded incisor teeth (Beertsen and Everts, 1977). In another study, altered eruption rate did not affect the turnover of insoluble collagen, structural noncollagenous proteins, or sulfated glycosaminoglycans (Van den Bos and Tonino, 1984). These authors suggested that remodeling due to accelerated eruption may not affect the activity of PDL components since their metabolism is probably already maximal. Morphologically, observations during the present study have revealed a more linear and organized appearance of preodontoblasts and preameloblasts in the group compared to EGF-treated controls. In EGFtreated mice, cells appeared to polarize earlier (at a point nearer to the apex of the loop), while secretion of enamel and predentin matrices is initiated in the EpMes interface at a point nearer to the apex of the loop than was seen in control tissues. EGF-induced acceleration of differentiation has previously been reported in a wound healing study in which EGF was applied topically to rabbit ear skin, having an organizing and stimulatory effect on fibroblasts in the epidermal layers of the wound (Franklin and Lynch, 1979). The alteration of odontoblast and ameloblast differentiation observed in the present study may result from disturbances of the complex cellular interactions along the basal lamina (Kallenbach and Piesco, 1978; Slavkin, 1979; Hurmerinta and Thesleff, 1981; Ruth et aL, 1983). Alterations in the synthesis of collagen (types I and IV), laminin, and fibronectin associated with the basal lamina occur during the embryonic development of the teeth (Trelstad and Slavkin, 1974; Lesot et al, 1981; Thesleff et ah, 1979; Thesleff et ah, 1981), and are involved in differentiation of the continuously erupting incisor throughout life. The morphological changes observed in the current study, combined with the findings of increased [3H]PR0
542
DEVELOPMENTALBIOLOGY
incorporation and secretion nearer to the apical loop region. indicate that a more ranid differentiation of odintdblasts and ameloblasts may occur after EGF treatment. These data appear to contradict those of Thesleff and Partanen (1984), and Partanen et al. (1985), who have demonstrated that EGF inhibits the terminal differentiation of odontoblasts and ameloblasts during the bell stage of molar development in vitro. The three main differences between our study and that of Partanen et al. include (1) molar model vs incisor model, (2) fetal animals vs neonates, and (3) in vitro vs in vivo methods. making comnarisons difficult. The key to understanding these-conflicting data may lie in determining the temporal sequence of the selective effects of EGF during development, since EGF does not inhibit morphogenesis in tooth germs that have reached the late cap stage or early bell stage (15 to 16 days gestation) of development (Partanen et cd., 1985). A recent study by Rhodes et al. (1987) indicates that EGF induces a reduction in mandibular and maxillary incisal area, as measured from radiographs, during accelerated eruption. These authors suggest that the reduction in tooth size they observed at 7 days after daily EGF treatment (6.5 /*g/g body wt) indicates that EGF does not have a mitogenic effect on cellular compartments of the mouse incisor in vivo. Our cell proliferation results appear to directly contradict this suggestion. Rhodes et al. also suggest that the PDL may be the target for EGF. Our data from [3H]TDR, [3H]PR0, and ‘251-EGF studies argue strongly that EGF may induce precocious eruption of the incisor through its action on the PDL. The accelerated eruption induced by exogenous EGF administration appears to affect both cell proliferation and protein synthesis by PDL cells. The relationship between eruption rate and cell proliferation and protein synthesis in the PDL requires further investigation during EGF-accelerated eruption. This work was completed as partial fulfillment for the Master of Arts degree awarded to R. Todd Topham from the Graduate School, University of Kansas Medical Center. The authors thank Susan G. Kramer for her technical assistance and Wanda Hinton for typing the manuscript. This project was supported by Public Health Service Grant DE07148 from the National Institute of Dental Research and by BRSG SO7 RR05373 awarded by the Biomedical Research Support Grant Program, Division of Research Resources, National Institutes of Health. REFERENCES ADAMSON,E. D., DELLER, M. H., and WARSHAW, J. B. (1981). Functional EGF receptors are present on mouse embryo tissues. Nature (London) 291.656-659. ADAMSON,E. D., and REES, A. R. (1981). Epidermal growth factor receptors. Mol. CeU B&hem. 34,129-152. ADAMSON, E. D., and WARSHAW, J. B. (1982). Down-regulation of epidermal growth factor receptors in mouse embryos. Dew. Biol 99, 430-434. BEERTSEN,W., and EVERTS,V. (1977). The site of remodelling of collagen in the periodontal ligament of the mouse incisor. Anat. Rec. 189,479-498.
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BERKOVITZ,B. K. B. (1971). The effect of root transection on the unimpeded eruption rate of the rat incisor. Arch. Oral Biol. 16, 1033-1043. BERKOVITZ,B. K. B. (1972). The effect of preventing eruption on the proliferative basal tissues of the rat lower incisor. Arch. Oral Biol. 17.12’79-1288. BROWNELL,A. G., and ROVERO,L. J. (1980). DNA synthesis of enamel organ epithelium in vitro is enhanced by co-cultivation with non-viable mesenchymal cells. J. Dent. Res. 59,1075-1080. CARPENTER, G., and COHEN, S. (1979). Epidermal growth factor. Annu. Rev. Biochem. 48,193-216. CHABOT,J. G., WALKER, P., and PELLETIER,G. (1986). Distribution of epidermal growth factor binding sites in the adult rat liver. Amer. J. Physiol. 250, 760-764. CHIEGO, D. J., JR., KLEIN, R. M., and AVERY, J. K. (1981). Tritiated thymidine autoradiographic study of the effects of inferior alveolar nerve resection on the proliferative compartments of the mouse incisor formative tissues. Arch. Oral Biol. 26. 83-89. CLARK, A. J. L., ISHII, S., RICHERT,N., MERLIN;, G. T., and PASTAN,I. (1985). Epidermal growth factor regulates the expression of its own receptor. Proc. Natl. Acad Sci USA 82,8374-8378. COHEN, S. (1962). Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal. J. Biol. Chem. 237,1555-1562. COHEN, S. (1965). The stimulation of epidermal proliferation by a specific protein (EGF). Dev. Biol 12,394-407. COHEN,S., and ELLIOTT, G. A. (1963). The stimulation of epidermal keratinization by a protein isolated from the submaxillary gland of the mouse. J. Invest. Derm. 40,1-5. EARP, H. E., AUSTIN, K. S., BLAISDELL,J., RUBIN, R. A., NELSON,K. G., LEE, L. W., and GRISHAM, J. W. (1986). Epidermal growth factor (EGF) stimulates EGF receptor synthesis. J. Biol. Chem. 261, 4777-4780. FRANKLIN, J. D., and LYNCH, J. B. (1979). Effects of topical applications of epidermal growth factor on wound healing. Plust. Reconstr. Surg. 64,766-770. GOLDIN,G. V., and OPPERMAN,L. A. (1980). Induction of supernumerary tracheal buds and the stimulation of DNA synthesis in the embryonic chick lung and trachea by epidermal growth factor. J. Embrgol. Exp. Morphol. 60,235~243. GOSPODAROWICZ, D. (1981). Epidermal and nerve growth factors in mammalian development. Annu. Rev. Physiol. 43,251-263. GREEN, M. R., BASKETTER,D. A., COUCHMAN,J. R., and REES, D. A. (1983). Distribution and number of epidermal growth factor receptors in skin is related to epithelial cell growth. Dev. Biol. 100, 506-512. GRESIK,E., and AZMITIA, E. (1980). Age related changes in NGF, EGF and protease in the granular convoluted tubules of the mouse submandibular gland: A morphological and immunocytochemical study. J. Gerontol 35,520-525. GRESIK, E., and BARKA, T. (1978). Immunocytochemical localization of epidermal growth factor during the postnatal development of the submandibular gland of the mouse. Amer. J. Anat. 151, l-10. GRESIK,E., GUBITS,R. M., and BARKA, T. (1985). In situ localization of mRNA for epidermal growth factor in the submandibular gland of the mouse. J. Histochem. Cytochem. 33,1235-1240. HOATH, S. B. (1986). Treatment of the neonatal rat with epidermal growth factor: Differences in time and organ response. Pediutr. Res. 20,468-472. HOATH, S. B., LAKSHMANAN,J., SCOTT,S. M., and FISHER,D. A. (1983). Effect of thyroid hormones on epidermal growth factor concentration in neonatal mouse skin. Endocrinology 112,308-314. HURMERINTA,K., and THESLEFF,I. (1981). Ultrastructure of the epithelial-mesenchymal interface in the mouse tooth germ. J. Cruniofacial Genet. Dev. Biol 1,191-202. KALLENBACH,E., and PIESCO,N. P. (1978). The changing morphology
TOPHAM ET AL.
EGF Efects on Neonatal Mouse Incisor
of the epithelium-mesenchyme interface in the differentiation zone of growing teeth of selected vertebrates and its relationship to possible mechanisms of differentiation. J. Biol. Buccule 6,229-240. KING, L. E., JR. (1985). What does epidermal growth factor do and how does it do it? J. Invest. De+rmatoL84,165-167. KONTUREK, S. J., RADECKI, T., BRZOZOWISKI, T., PIASTUCKI, I., DEM-
BINSKI, A. DEMBINSKI-KIEC, A., ZMUDA, A., GRYGLEWSKI,R., and GREGORY, H. (1981). Gastric cytoprotection by epidermal growth factor: Role of endogenous prostaglandins and DNA synthesis. Gastroenterology 81,438-443. LAKSHMANAN,J., PERHEENTUPA,J., HOATH, S. B., KIM, H., GRUTERS, A., ODELL,C., and FISHER, D. A. (1985). Epidermal growth factor in mouse ocular tissue: Effects of thyroxine and exogenous epidermal growth factor. Pediatr. Res. 19,315-319. LESOT, H., OSMAN, M., and RUCH, J. V. (1981). Immunofluorescent localization of collagens, fibronectin, and laminin during terminal differentiation of odontoblasts. Dev. BioL 82,371-381. MICHAELI, Y., and WEINREB, M. M. (1968). Role of attritional and occlusal contact in the physiology of the rat incisor. III. Prevention of attrition and occlusal contact in the nonarticulating incisor. J. Dent. Res. 47, 633-640. MICHAELI, Y., WEINREB, M. M., and ZAJIECK, G. (1972). Role of attrition and occlusal contact in the physiology of rat incisors. V. Life cycle of inner enamel epithelial cells at various rates of eruption. J. Dent. Res. 51,960-963. MOXHAM, B. J., and BERKOVITZ,B. K. B. (1974). The effects of root transection on the unimpeded eruption rate of the rabbit mandibular incisor. Arch. oral Biol 19,903-909. NESS,A. R., and SMALE, D. E. (1959). The distribution of mitoses and cells in the tissues bounded by the socket wall of the rabbit mandibular incisor. Proc. R. Sot. London 151,106-128. PARTANEN, A., EKBLOM, P., and THESLEFF, I. (1985). Epidermal growth factor inhibits morphogenesis and cell differentiation in cultured mouse embryonic teeth. Dew. BioL 111,84-94. PARTANEN,A., and THESLEFF,I. (1987). Localization and quantitation of ‘%I-epidermal growth factor binding in mouse embryonic tooth and other embryonic tissues at different developmental stages. Deu. BioL 120,186-197. PITARU, S., MICHAELI, Y., ZAJICEK, G., and WEINREB, M. M. (1976). Role of attrition and occlusal contact in the physiology of the rat incisor. X. The part played by the periodontal ligament in the eruptive process. J. Dent. Res. 55,819-824. RALL, L. B., SCOTT,J., BELL, G. I., CRAWFORD,R. J., PENSCHOW,J. D., NIALL, H. D., and COGHLAN,J. P. (1985). Mouse prepro-epidermal growth factor synthesis by the kidney and other tissues. Nature (London) 313,228-231.
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