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DEVELOPMENTAL DYNAMICS 213:500–511 (1998)
PDGF-A and PDGFR-␣ Regulate Tooth Formation
Via Autocrine Mechanism During Mandibular
Morphogenesis In Vitro
YANG CHAI,1* PABLO BRINGAS, JR.,1 ALI MOGHAREI,1 CHARLES F. SHULER,1
AND HAROLD C. SLAVKIN2
1Center For Craniofacial Molecular Biology, School of Dentistry, University of Southern California, Los Angeles, California
2Craniofacial Development Section, National Institute of Arthritis and Musculoskeletal and Skin Disease, National Institute of
Health, Bethesda, Maryland
ABSTRACT
Platelet-derived growth factor
A (PDGF-A) binding to the PDGF receptor alpha
(PDGFR-␣) mediates signal transduction processes related to DNA synthesis, cell migrations,
cytodifferentiation, and wound healing. Recent
studies indicate that PDGFR-␣ functions during
cranial neural crest cell migrations and first
branchial arch morphogenesis (Stephenson et al.
[1991] Proc. Natl. Acad. Sci. USA 88:6–10; MorrisonGraham et al. [1992] Development 115:133–142;
Hu et al. [1995] Int. J. Dev. Biol. 39:939–945; Soriano [1997] Development 124:2691–2700). The present studies were designed to test the hypothesis
that PDGF-A, interacts with its cognate receptor
PDGFR-␣ via an autocrine mechanism that regulates the timing, rates, and size of embryonic
mouse tooth morphogenesis. Both PDGF-A and
PDGFR-␣ transcripts were coordinately expressed in mandibular prominences prior to and
during tooth formation using reverse transcriptase-polymerase chain reaction (RT-PCR). During the dental lamina stage, ligand and receptor
were present in both enamel organ epithelium
and adjacent mesenchymal cells. During the bud
stage, ligand and receptor were localized mainly
to the enamel organ epithelium. Exogenous
PDGF-A at 20 ng/ml enhanced tooth development to
reach the cap stage with increased tooth size (P F
0.05) using embryonic day (E)10 mandibular explants cultured in serumless, chemically defined
medium. A significant increase in DNA synthesis
was observed within enamel organ epithelium at
E10⫹4 when the mandibular explants were treated
with PDGF-A at 20 ng/ml. These data suggest that
PDGF-A and its cognate receptor (PDGFR-␣) regulate the size and stage of tooth development via an
autocrine mechanism during odontogenesis in vitro.
Dev. Dyn. 1998;213:500–511. r 1998 Wiley-Liss, Inc.
Key words: PDGF-A; PDGFR-␣; mouse tooth development; in vitro organ culture
INTRODUCTION
Platelet-derived growth factor (PDGF) is a disulfidebonded dimer of two subunit proteins; A-chain and
r 1998 WILEY-LISS, INC.
B-chain (Heldin and Westermark, 1990). Whereas all
dimeric combinations (AA, AB, BB) have been identified in various biological tissues, unique dimers have
been identified in specific tissues during amphibian and
mammalian embryonic development and wound healing (Stroobant and Waterfield, 1984; Heldin et al., 1986;
Hammacher et al., 1988a,b; Bowen-Pope et al., 1989;
Nilsen-Hamilton, 1989; Rizzino, 1989; Hart et al., 1990;
Orr-Urtreger et al., 1992, Schatteman, et al., 1996; Betsholtz and Raines, 1997). PDGF-A transcripts are expressed
by mouse teratocarcinoma stem cell lines and a host of
embryonic mouse tissues during development (Mercola
et al., 1990). The presence of PDGF-A is required for
normal murine cardiovascular and lung development
(Schatteman et al., 1996; Bostrom et al., 1996; Lindahl
et al., 1997; Betsholtz and Raines, 1997). PDGF receptor alpha (PDGFR-␣) is required for neural crest cell
development and for normal patterning of the somites
(Soriano, 1997). Multiple binding sites exist for the
different isoforms of PDGF (Hart et al., 1988; Heldin et
al., 1988). The biologically active high-affinity PDGF
binding site is a dimer of two receptor subunit proteins
designated PDGFR-␣ and PDGFR-␤ (Bishayee et al.,
1989; Hammacher et al., 1989; Heldin et al., 1989;
Seifert et al., 1989). The binding of PDGF isoforms to
different cell types appears to be determined by the
differential ability of the two PDGF subunit chains to
bind to the two receptor subunits (Seifert et al., 1989);
PDGF-A only binds to PDGFR-␣, whereas PDGF-B can
bind to PDGFR-␣ and PDGFR-␤.
PDGF isoforms and PDGFR-␣, ␤ are expressed at
critical phases of mouse embryogenesis, and have been
implicated in pre- and postimplantation embryogenesis
including the regulation of first branchial arch morphogenesis and many mesodermal derivatives during embryonic and fetal development (Rappolee et al., 1988;
Nilsen-Hamilton, 1989; Mercola et al., 1990; Schatteman et al., 1992; Orr-Urtreger et al., 1992; Morrison-
Grant sponsor: NIDR Center; Grant number: DE-09165.
*Correspondence to: Dr. Yang Chai, Center For Craniofacial Molecular Biology, School of Dentistry, University of Southern California,
Health Sciences Campus, 2250 Alcazar Street, CSA 103, Los Angeles,
CA 90033. E-mail: ychai@zygote.hsc.usc.edu
Received 11 November 1997; Accepted 14 September 1998
PDGF-A, PDGFR-␣ AND TOOTH FORMATION
Graham et al., 1992, Hu et al., 1995; Bostrom et al.,
1996; Soriano, 1997). The Patch mutation was identified as a deletion of the PDGFR-␣ gene that maps to
chromosome 5 (Stephenson et al., 1991; Smith et al.,
1991; Soriano, 1997). This mutation in homozygous
Ph/Ph demonstrates pleiotropic effects associated with
cranial and trunk neural crest cells and produces
significant first branchial arch deformities including
facial clefting, hemifacial microsomia, micrognathia,
adontia, open neural tube, heart deformities, and large
interstitial ‘‘blebs,’’ and is lethal by embryonic day (E)12
(Gruneburg and Truslove, 1960). One interpretation of
these studies is that a depletion of cranial neural
crest-derived ectomesenchymal cells, possibly resulting
from a defect in PDGF-A ligand-mediated signal transduction using the PDGFR-␣, produces first arch deformities including hypodontia and adontia. However, the
functions of PDGF-A or its cognate receptor during
tooth morphogenesis are unknown.
To investigate that PDGF-A signal transduction mediates instructive epithelial-mesenchymal interactions
which determine regional and temporal patterns of
early stages of tooth morphogenesis, (DNA synthesis,
tooth size, and tooth shape), we designed experiments
to test the hypothesis that both PDGF-A and PDGFR-␣
are coordinately expressed prior to and during initial
tooth formation, and that gain of function of PDGF-A
ligand up-regulates DNA synthesis in enamel organ
epithelium and thereby controls the size and shape
patterns for tooth morphogenesis. Tooth morphogenesis
and cytodifferentiation are dependent upon instructive
epithelial-mesenchymal interactions (Slavkin, 1974;
Lumsden, 1988; Jowett et al., 1993; Vainio et al., 1993;
Kratochwil et al., 1996). Tissue recombination experiments have clearly shown that specific regions within
ectodermally derived oral epithelium initiate tooth
formation (Lumsden, 1988; Kollar and Mina, 1991;
Vainio et al., 1993; Thesleff et al., 1996), whereas the
inductive role appears to shift from the oral epithelium
to adjacent mesenchymal during the bud stage of
mouse tooth morphogenesis (Kollar and Baird, 1969,
1970a,b; Ruch, 1984, Kratochwil et al., 1996). PDGF-A
and PDGFR-␣ may play a critical role in the signaling
required for tooth formation.
The present study reports that both PDGF-A and the
PDGFR-␣ transcripts are expressed during early mandibular morphogenesis. Both ligand and receptor are
colocalized to the enamel oral epithelium and crestderived ectomesenchymal cells adjacent to the dental
lamina at the initiation of tooth formation. As development advances into bud stage, however, both PDGF-A
and its cognate receptor (PDGFR-␣) are associated with
enamel organ epithelium, indicating an autocrine mechanism. Using a simple embryonic E10 mandibular
explant model in serumless, chemically defined medium, we found that PDGF-A gain of function at 20
ng/ml significantly enhanced the stage of development
and size of tooth organs (P ⬍ 0.05) in the mandibular
explants. Three-dimensional reconstruction showed a
501
Fig. 1. Platelet-derived growth factor A (PDGF-A) and PDGF receptor
alpha (PDGFR-␣) expression in mandibular prominences during different
culture stages using reverse transcriptase-polymerase chain reaction
(RT-PCR). A: The expression of PDGF-A transcripts. (M) marker X
174/Hae III. Lane 1: Late embryonic day (E)10 (42–44 somite pairs)
mandibular processes. Lane 2: Late E10 mandibular explants plus 2 days
in vitro. Lane 3: E10⫹4 days in vitro. Lane 4: E10⫹6 days in vitro. Lane
5: E10⫹9 days in vitro. Lane 6: E13 (Theiler stage 21) mandibular
prominences. Lane 7: Reverse-transcription negative control. Lane 8:
Polymerase chain reaction negative control. B: The expression of
PDGFR-␣ transcripts. The marker and the RNA samples for RT-PCR are
the same as in A. C: The expression of ␤-actin transcripts using the same
RNA samples for both A and B, indicating that there is sufficient amount of
mRMA from each sampling point. PCR products size: PDGF-A ⫽ 225
base pairs; PDGFR-␣ ⫽ 189 base pairs; ␤-actin ⫽ 209 base pairs.
significant overall increase in tooth size. An increased
DNA synthesis activity in the tooth organ was first seen
within cranial neural crest-derived mesenchymal cells
at E10⫹2 and then within the enamel organ epithelium
during the bud stage of tooth formation. Collectively,
this study demonstrated that PDGF-A and its cognate
receptor (PDGFR-␣) regulated the size and stage of
tooth development via an autocrine mechanism during
early odontogenesis.
RESULTS
PDGF-A and PDGFR-␣ Transcripts Identified
Before and During Tooth Formation
PDGF-A and PDGFR-␣ transcripts were both detected within microdissected first branchial arch segments at E9 (data not shown) and at E10 (Fig. 1A,B).
E10 mandibular explants cultured for 2, 4, 6, and 9
days in a serumless, chemically defined medium continued to express PDGF-A and PDGFR-␣ transcripts as
detected by reverse transcriptase- polymerase chain
reaction (RT-PCR; Fig. 1A,B). Meanwhile, both PDGF-A
and PDGFR-␣ are present in vivo at E13 (Fig. 1 A,B,
lane 6). As demonstrated in our previous study, tooth
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CHAI ET AL.
Fig. 2. PDGF-A and PDGFR-␣ immunolocalization on adjacent serial
sections through the same tooth during early tooth morphogenesis in
vitro. A: At E10⫹2, PDGF-A was present in the dental lamina, basement
membrane, and adjacent mesenchymal cells. B: PDGFR-␣ was localized
to identical locations comparing to its ligand at E10⫹2. After 4 days,
PDGF-A (C) and PDGFR-␣ (D) were present in enamel organ epithelium
and its basement membrane of the tooth (tb). By 6 days, PDGF-A (E) and
PDGFR-␣ (F) remained in the enamel organ epithelium and basement
membrane, while adjacent dental mesenchyme presented with faint
positive staining. By the end of 9 days, PDGF-A (G) and PDGFR-␣ (H)
were present in enamel organ epithelium, basement membrane, and
adjacent dental mesenchyme as the tooth development getting ready to
advance into cap stage. Scale bar ⫽ 100 µm.
PDGF-A, PDGFR-␣ AND TOOTH FORMATION
503
Fig. 3. Dose response experiment showed that PDGF-A enhanced
tooth morphogenesis and enlarged the size or volume of the tooth.
A: E10⫹9 control group showed oral epithelium (oe) and bud stage tooth
development (tb). B: PDGF-A at 20 ng/ml enhanced development to the
cap stage and enlarged tooth size. Three-dimensional reconstruction
from serially sectioned mandibular explants were analyzed to define the
size of the tooth organ in E10 mandibles cultured for 9 days. C: Control.
D: PDGF-A at 20 ng/ml treated mandibular explant. Tooth organs are
indicated in red. Meckel’s cartilage is indicated in blue. The outline of
mandibular explants are shown in green. Explants are made to be
transparent and presented with increased intervals to show the tooth and
Meckel’s cartilage positions and size. Scale bar ⫽ 100 µm.
formation within E10 mandibular organ culture begins
at 1–2 days after the initiation of tissue culture and is
marked by the formation of dental lamina (Chai et al.,
1994). However, the expression of PDGF-A and
PDGFR-␣ are present before the initiation of tooth
formation and continues throughout the culture process. This is further demonstrated by the results of
immunolocalization of PDGF-A and PDGFR-␣ during
mandibular morphogenesis in vitro.
detected in the oral epithelium. Meanwhile, PDGFR-␣
was also localized to these mesenchymal cells and blood
vessels (data not shown). At E10⫹2, PDGF-A was
detected in enamel organ epithelium, basement membrane and adjacent mesenchymal cells (Fig. 2A) while
PDGFR-␣ had a similar distribution associated with
the dental lamina (Fig. 2B). Four days after the culture,
PDGF-A was present in the enamel organ epithelium
and the basement membrane (Fig. 2C). PDGFR-␣ was
also found within the same tissue type (Fig. 2D). As
tooth development continues to E10⫹6, PDGF-A and
PDGFR-␣ were localized to the enamel organ epithelium and basement membrane (Fig. 2C,D). At the
termination time of mandibular organ culture, PDGF-A
was located within enamel organ epithelium and surrounding dental mesenchyme (Fig. 2G), while PDGFR-␣
was localized within the enamel organ epithelium with
PDGF-A Colocalization With PDGFR-␣ During
Tooth Formation In Vitro
Immunohistochemistry on adjacent serial sections
from the same tooth germ demonstrated that PDGF-A
and PDGFR-␣ were associated with tooth formation
(Fig. 2). At E10, PDGF-A was present in the cranial
neural crest-derived mesenchymal cells, but it was not
504
CHAI ET AL.
detectable expression in adjacent dental mesenchyme
(Fig. 2H).
PDGF-A Induces Precocious
Tooth Morphogenesis
A number of growth factors have been found to be
pleiotropic, being potent mitogens at a particular concentration and target cell, or at a specific stage in
development, or as regulators of cell differentiation by
controlling gene expression and protein synthesis. Exogenous PDGF-A induced a dose-response effect on tooth
morphogenesis added as a supplement to the serumless, chemically defined medium used to culture E10
mandibular explants for 9 days in vitro (Fig. 3A–D). In
control nontreated explants as well as explants cultured with 2 ng/ml or 10 ng/ml PDGF-A (data not
shown), tooth formation reached the bud stage within 4
days in vitro and remained at the bud stage up to 9 days
in vitro (Fig. 3A). In contrast, PDGF-A at 20 ng/ml not
only advanced the tooth development into the early cap
stage but also increased the size of the tooth form (Fig.
3B). Statistical analyses indicated that tooth sizes were
50% larger in PDGF-A-treated cultures than the nontreated controls (P ⬍ 0.05). Three-dimensional reconstruction of serially sectioned mandibular explant further demonstrated the substantial increase of tooth size
when mandibular explants were treated with PDGF-A
at 20 ng/ml (Fig. 3D). This size increase was not limited
to a particular section, but was consistent throughout
the entire tooth organ when compared to controls (Fig.
3C).
PDGF-A Stimulates DNA Synthesis of Enamel
Oral Epithelium During Odontogenesis In Vitro
DNA synthesis was monitored in E10 explants cultured for 9 days in chemically defined medium. Immunodetection of 5’-bromo-2’-deoxyuridine (BrdU) labeling of DNA was used to identify epithelial or
mesenchymal cells engaged in DNA synthesis. Whereas
a few epithelial cells were labeled with BrdU at the
initial incisor and molar dental lamina stage of tooth
morphogenesis (Fig. 4A,B), the majority of dividing
cells in both incisor and molar tooth buds were identified within the condensed dental mesenchymal cells at
4 and 9 days of culture (Fig. 4C–F).
Exogenous PDGF-A ligand at 20 ng/ml induced precocious cap stage tooth morphogenesis with increased
number of cells showing positive BrdU labeling in
incisor and molar tooth organs (Fig. 5A–F). At E10⫹2,
PDGF-A increased DNA synthesis activity mainly in
the cranial neural crest-derived mesenchyme (Fig.
5A,B). Interestingly, PDGF-A (20 ng/ml) significantly
increased cell proliferation within molar enamel organ
epithelium at E10⫹4 (Fig. 5D), which might have
contributed to the advancement of tooth development
from bud stage to cap stage at E10⫹9 (Fig. 5F).
Furthermore, quantitation of percentage of BrdUlabeled cells showed that PDGF-A at 20 ng/ml significantly increased the DNA synthesis (fourfold) within
enamel organ epithelium (Fig. 6 and Table 1). Meanwhile, the proliferation of cranial neural crest-derived
dental mesenchymal cells remained relatively comparable to the control group during bud stage tooth
morphogenesis.
PDGF-A at 20 ng/ml also significantly increased the
size (P ⬍ 0.05) of the tooth organ as compared to
nontreated bud stage controls. However, the proliferation of enamel organ epithelium in PDGF-A-treated
mandibular explants did not advance these epithelial
cells to become ameloblasts which are marked by the
expression of amelogenin (data not shown). In previous
studies, dose responses of exogenous epidermal growth
factor (EGF) and transforming growth factor-␤2 (TGF␤2)
in this E10 mandibular explant in vitro model resulted
in significantly smaller and less-developed tooth primordium (Shum et al., 1993; Chai et al., 1994). The
presence of PDGF-A and other possible factors presumably act with EGF and TGF ␤ subtypes to regulate the
size and shape of tooth form during morphogenesis of
the mandibular prominence.
DISCUSSION
This study presents detailed analyses of the biological activity of PDGF-A during tooth morphogenesis in a
serumless, chemically defined medium. It is also the
first report describing the localization of both PDGF-A
and PDGFR-␣ during initiation and subsequent morphogenesis of developing embryonic mouse tooth organs.
An autocrine functional role for PDGF-A and its cognate receptor was demonstrated during the early inductive stage of tooth morphogenesis. Both ligand and its
cognate receptor transcripts are expressed in the mandibular prominence prior to and during the initiation of
embryonic mouse tooth formation, and both gene products were immunolocalized mainly to the enamel organ
epithelium. Gain of function of PDGF-A in a serumless,
chemically defined in vitro explant culture model resulted in precocious tooth development reaching the
cap stage, whereas endogenous PDGF-A appears sufficient for the initiation and formation of small bud stage
tooth organs. In light of the earlier identification of the
Patch mutant mouse showing severe first branchial
arch dysmorphogenesis, open neural tube, hypodontia,
and adontia (Gruneberg and Truslove, 1960; MorrisonGraham et al., 1992), and the discovery that the Ph/Ph
results from a deletion of the PDGFR-␣ (Stephenson et
al., 1991), the present investigation provides additional
evidence to support the hypothesis that PDGF-mediated signal transduction functions in early inductive
epithelial-mesenchymal interactions that are required
for the initiation and progression of tooth morphogenesis.
PDGF-A Ligand Binds to PDGFR-␣ to Regulate
Tooth Morphogenesis In Vitro
The hypothesis that PDGF-A ligand-mediated signal
transduction is involved in early developmental processes is based on the proposition that the timing and
PDGF-A, PDGFR-␣ AND TOOTH FORMATION
position of expression implies function, and that the
major function of PDGF ligands is to regulate DNA
synthesis of odontogenic epithelium and surrounding
mesenchymal cells (Rizzino, 1989; Nilsen-Hamilton,
1989; Mercola et al., 1990; Orr-Urtreger and Lonai,
1992; Orr-Urtreger et al., 1992; Betsholtz and Raines,
1997; Soriano, 1997). The present report provides new
evidence which indicates PDGF-A and PDGFR-␣ act
mainly through an autocrine mechanism in regulating
early tooth morphogenesis in vitro. In particular,
PDGF-A and its cognate receptor are present in both
odontogenic epithelium and condensed mesenchyme at
E10⫹2 during the formation of the dental lamina. The
cell/tissue distribution of the growth factor and its
cognate receptor suggest a role in regulating epithelialmesenchymal interaction during the initiation stage of
tooth morphogenesis (Fig. 2A,B). Four days after the
culture, however, both PDGF-A and its cognate receptor
PDGFR-␣ are mainly present within the ectodermally
derived odontogenic epithelium (Fig. 2C,D) and continued to be present until E10⫹9. Collectively, our results
indicate that PDGF-A and its cognate receptor are
mainly responsible for regulating the proliferation of
odontogenic epithelial cells during early tooth morphogenesis in vitro.
Several lines of evidence are considered which support our hypothesis: (1) a number of endogenous growth
factors including TGF-␣, EGF, TGF-␤1, TGF-␤2, or
TGF-␤3 are expressed in odontogenic epithelium and/or
mesenchyme prior to and during early mouse tooth
formation which do not increase the size nor advance
the stages of tooth morphogenesis. (Kronmiller et al.,
1990; Heikinheimo et al., 1993; Shum et al., 1993; Chai
et al., 1994); (2) underexpression of TGF-␤2 results in
an increased size and precocious advancement in the
stage of tooth morphogenesis, supporting the suggestion that endogenous growth factor(s) play critical roles
in regulating tooth development, and that some of these
function to inhibit DNA synthesis, growth and morphogenesis (Chai et al., 1994); (3) PDGF-A (this report),
fibroblast growth factor (FGF), as well as insulin-like
growth factor (IGF-II) all increase DNA synthesis,
growth, and induce precocious tooth morphogenesis
(Neubuser et al., 1997; Kettunen and Thesleff, 1998;
unpublished results), resulting in late cap stage tooth
organs with attendant epithelial and mesenchymal cell
differentiation. Furthermore, the expression pattern of
PDGF-A and PDGFR-␣ strongly suggests that PDGF-A
is an important mediator for enamel organ epithelial
cell proliferation during the process of odontogenesis. It
is also noted, however, that PDGF-A or PDGFR-␣
immunolocalization need not correspond to the site of
PDGF-A or PDGFR-␣ synthesis. This can only be
determined by in situ hybridization. Although the
possibility is remote, PDGF-A could conceivably diffuse
and collect on cell surfaces of an adjacent PDGFR-␣
expressing tissue. In addition to an autocrine mechanism, a paracrine mechanism is not completely ruled
out.
505
PDGF-A Induces DNA Synthesis in Odontogenic
Epithelium During Early Tooth Formation
In Vitro
Overexpression of PDGF-A, achieved by supplementation of PDGF-A (20 ng/ml) to a serumless, chemically
defined medium devoid of exogenous growth factors,
produced a significant increase in DNA synthesis first
seen within the mesenchyme at E10⫹2 (Fig. 5A,B) and
then within the ectodermally derived enamel organ
epithelium at E10⫹4 (Fig. 5D). This increase in proliferation of enamel organ epithelium advanced the tooth
development into cap stage at E10⫹9 (Fig. 5F). Coexpression of PDGF-A and PDGFR-␣ in the enamel organ
epithelium during the progression of early tooth development (Fig. 2) indicates an autocrine mechanism that
PDGF-A is able to induce the proliferation of these
epithelial cells. The number of BrdU-labeled mesenchymal cells remains high throughout the culture period in
both control and exogenous PDGF-A-treated explants.
It is, however, the significant increase in DNA synthesis
within enamel organ epithelium which might be responsible for the advancement of both size and stage of tooth
development following the addition of exogenous
PDGF-A. These observations support the suggested
functions for PDGF-A and PDGFR-␣ in postimplantation mouse embryogenesis (Mercola et al., 1990; Morrison-Graham et al., 1992; Orr-Urtreger and Lonai, 1992;
Orr-Urtreger et al., 1992). Furthermore, our study
indicates that PDGF-A and its cognate receptor
PDGFR-␣ transcripts are expressed by cranial neural
crest-derived mesenchymal cells within the forming
first arch and early tooth primordium, subsequently
expressed in the enamel organ epithelium, and are
responsible for mediating the critical epithelial-mesenchymal interaction and subsequent proliferation of
enamel organ epithelial cells in odontogenesis. These
observations also support the interpretation that cranial neural crest-derived tooth mesenchymal cells are
deficient in the Patch mutant, resulting in hypodontia
and adontia due to the deletion of the PDGFR-␣.
PDGF-A may control several downstream regulatory
genes such as bone morphogenetic protein 4 (BMP-4)
and several transcription factors (Lef1, Msx-1, and
Msx-2). Several recent studies have elegantly demonstrated that BMP-4 and the transcription factors are
initially expressed in oral epithelium prior to the
initiation of tooth formation; moreover, these regulatory molecules also shift from initial expression within
oral epithelium prior to tooth formation to expression
within adjacent mesenchyme during the bud stage of
tooth morphogenesis (Mackenzie et al., 1991, 1992;
Vainio et al., 1993; Kratochwil et al., 1996; Jernvall et
al., 1998). Homologous recombinations in transgenic
mice have produced a null mutation for Msx-1 which
results in tooth development arrest at the terminal bud
stage in the homozygous animals (Satokata and Maas,
1994). Certain exogenous growth factor signals can
substitute for the odontogenic placode as the initiation
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CHAI ET AL.
Fig. 4. Using 5-bromo-2’-deoxyuridine (BrdU) as cell proliferation
marker to follow the DNA synthesizing activity in both enamel oral
epithelium and condensed mesenchymal cells in the control group of
E10⫹9 cultured mandibles. A, B: At 2 days, both incisor (i) and molar (m)
enamel organ epithelium showed cell proliferation activity. The condensed mesenchymal cells surrounding both the incisor and molars also
revealed DNA synthesizing activity. C, D: By 4 days, cell proliferation
activity had increased in the mesenchymal cells surrounding both the
incisor and molar enamel organ epithelium. There were few cells stained
positive with Brdu within enamel organ epithelium of both incisor and
molar tooth organs. E, F: At 9 days, there were still large numbers of
condensed mesenchymal cells with positive BrdU labeling, while only a
few cells were positive in both the incisor and molar enamel organ
epithelium. Scale bar ⫽ 100 µm.
center for tooth morphogenesis and induce transcription factors such as Lef1, Msx1, and Msx2 within the
primitive odontogenic mesenchyme (Vainio et al., 1993;
Kratochwil et al., 1996; Thesleff and Nieminen, 1996;
Jernvall et al., 1998). Further study on the interaction
of different growth factors/receptors, such as PDGF-A/
PDGFR-␣, and transcription factors, such as Lef1,
Msx1, 2, can help us to understand the molecular
mechanism that regulates the morphogenesis and cytodifferentiation of different phenotypes during embryogenesis.
In summary, the present investigation provides evidence that PDGF-A and its cognate receptor PDGFR-␣
transcripts are expressed in the first branchial arch
PDGF-A, PDGFR-␣ AND TOOTH FORMATION
507
Fig. 5. PDGF-A at 20 ng/ml increased the proliferation enamel organ
epithelial cells and resulted in the advancement of tooth development. A,
B: At 2 days, there was increased cell proliferation activity in both enamel
organ epithelium and condensed mesenchymal cells in incisors (i) as well
as molars (m) compared to the controls (Fig. 4). C, D: By 4 days, enamel
organ epithelial cells showed increased cell proliferation activity, especially in the molar (m) as indicated by the intense Brdu staining. The
condensed mesenchymal cells surrounding the enamel organ epithelium
also revealed intense DNA synthesizing activity, but without a difference
compared to the control groups. E, F: By 9 days, cell proliferation activity
continued to be active in both incisor (i) and molars (m), especially in the
enamel organ epithelium compared to the controls. PDGF-A at 20 ng/ml
enhanced tooth development to reach the cap stage. Scale bar ⫽ 100 µm.
prior to the initiation of tooth development. Immunostaining evidence for both ligand and its cognate receptor indicates localization within the cranial neural
crest-derived tooth mesenchymal cells during the initial dental lamina stages of morphogenesis. During
later bud stage, PDGF-A and its receptor (PDGFR-␣)
are predominantly localized to the enamel organ epithelial cells which are highly responsive to PDGF-A-
induced DNA synthesis. These observations support
the hypothesis that PDGF-A and its cognate receptor
PDGFR-␣ act through an autocrine mechanism to
regulate tooth morphogenesis. As seen in the control
group, a constantly active DNA synthesis activity and
the expression of PDGFR-␣ are mainly associated with
the cranial neural crest-derived mesenchymal cells
which may in part reflect the well-documented influ-
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CHAI ET AL.
(Millipore, Bedford, MA), and supported on a stainless
steel raft. All explants were positioned with the oral
epithelium face upward, opposite to the filter surface.
Approximately 6–8 explants per culture dish were
cultured in Grobstein Falcon dishes under optimal
humidity conditions in an atmosphere containing 5%
CO2 and 95% air. The culture dishes were filled with
BGJb medium (Gibco, Grand Island, New York) supplemented with 0.1 mg/ml ascorbic acid and 50 units
penicillin/streptomycin (Flow Laboratories, McLean,
VA). The medium was changed every other day and
kept within a pH range of 7.2 to 7.4. All mandibular
explants were cultured for 9 days in vitro.
Reverse Transcription Polymerase
Chain Reaction
Fig. 6. PDGF-A at 20 ng/ml increased the percentage of BrdU-labeled
odontogenic epithelial cells by almost fourfold. In the control group, only
8.6% of total counted cells within enamel organ epithelium were labeled
with BrdU, while PDGF-A at 20 ng/ml increased BrdU-labeled odontogenic epithelial cells to 33.4%.
ence of dental papilla mesenchyme on tooth shape and
form. Addition of exogenous PDGF-A initially induced
DNA synthesis activity within the cranial neural crestderived mesenchymal cells adjacent to the enamel oral
epithelium and then dramatically increased cell proliferative activity within the enamel organ epithelium at
E10⫹4, which may contribute to the advancement of
tooth morphogenesis from bud to cap stage in this in
vitro system.
EXPERIMENTAL PROCEDURES
Mouse Embryos
Virgin female Swiss-Webster mice, obtained from
Simonsen Labs (Gilroy, CA), were housed for at least 2
weeks in the breeding colony with other animals of the
same age in a controlled light and temperature environment with ad libitum food and water. By mating these
mice with adult male mice (11–14 weeks old) for 3
hours, timed pregnancies were obtained. The presence
of a vaginal plug was used as an indication of pregnancy
day 0. At selected developmental stages, the pregnant
mice were killed by cervical dislocation. Mouse embryos
were dissected from uterine decidum and staged by
their external features according to Theiler’s method
(1989). Mandibular processes including the tuberculum
impar were removed from embryos by microdissection
and used as explants for organ culture (Slavkin et al.,
1989; Shum et al., 1993; Chai et al., 1994).
Mandibular Organ Culture
A modified Trowell method was used to culture
mandibular processes as previously described (Slavkin
et al., 1989). Dissected mandibular processes (E10,
42–44 somite pairs) were placed on type AA, 0.8 µm
pore size Millipore filter paper 6 mm in diameter
RT-PCR was performed according to Rappolee et al.
(1988). E10 and E13 mandibular processes as well as
E10 mandibular explants cultured for 2, 4, 6, and 9
days were isolated and placed on dry ice to ensure the
stability of RNA within explants. Five mandibular
explants or tissue isolates were pooled for each RNA
extraction. The 5’ and 3’ amplimers used in the present
studies were designed based on the cDNA sequences of
PDGFR-␣ and PDGF-A (Orr-Urtreger and Lonai, 1992;
Mercola et al, 1990). The primer set specific for PDGFR-␣
was for a 189-base pair product using primers 5’-AGG AAG
CCA TTC CTG CA (17 mer), and 3’-CTT GAC ACT GCG
GTG GTG (18 mer; Orr-Urteger and Lonai, 1992). The
PDGF-A was amplified with a product size 435 using a pair
of primers of 5’-CTC CAG CGA CTC TTG GAG ATA G (22
mer) and 3’-TTCAGG TTG GAG GTC GCACAT G (22 mer;
Mercola et al., 1990). The internal positive control was
␤-actin, a 209-base-pair product using primers 5’-GAC ATC
CGCAAAGAC C (16 mer) and 3’-CCACAT CTG CTG GAA
G (16 mer). The amplification of ␤-actin confirmed that the
amount of RNA in each sample was not degraded and
differences in signal were not due to differences in the
amount of RNA amplified. The PCR reaction mixture
contained PCR buffer with 1.5–2.5 mM magnesium
chloride, 1.0 mM dNTP, 0.25 unit Taq polymerase
(Amplitaq, Perkin-Elmer Cetus, Norwalk, CT), 2 µl of
each of the 5’ and 3’ amplimers, and 4 µl of the reverse
transcription product. PCR was done in thermal cycles
(ERICOMP, San Diego, CA). Subsequently, the PCR
products were visualized on a 4% agarose gel (NuSeive
[FMC, Rockland, ME]: BRL Agarose [BRL, Gaithersburg, MD]; 3:1; w/w) by staining with 1 µg/ml ethidium
bromide. The size of the amplified product was evaluated using (⫻174 RF DNA/Hae III marker (BRL). The
amplified products were verified by comparison with
PDGFR-␣ and PDGF-A plasmid controls and product
sequence analysis. In PCR control, diethylpolycarbonate (DEPC)-treated water was used to replace the
reverse transcription product as a negative control.
␤-actin was used as an internal positive control.
509
PDGF-A, PDGFR-␣ AND TOOTH FORMATION
TABLE 1. Number of BrdU-Labeled Cells in Odontogenic Epitheliuma
Section
1
2
3
4
5
Mean ratio
S.D.
BK 1b
9 (102)
11 (91)
13 (109)
6 (81)
10 (96)
Control
BK 2
5 (79)
9 (98)
8 (97)
8 (93)
8 (95)
0.086
0.011
BK 3
7 (85)
8 (91)
5 (78)
5 (76)
6 (68)
BK 1
36 (103)
43 (142)
38 (131)
41 (121)
34 (119)
PDGF-A (20 ng/ml)
BK 2
48 (136)
40 (125)
26 (72)
46 (129)
40 (116)
0.334
0.017
BK 3
43 (124)
42 (128)
44 (134)
47 (131)
41 (121)
a58-bromo-28-deoxyuridine
(BrdU) labeled cells and the total number of cells counted (in parentheses) within enamel organ epithelium. Three E10⫹9 cultured mandibular explants were randomly
chosen from each group. All sections contained tooth organs. Two adjacent sections were chosen
with at least 40 µm distance between them to avoid the same cells being counted twice.
Platelet-derived growth factor-A (PDGF-A) at 20 ng/ml substantially (P ⬍ 0.05) increased the
percentage of BrdU-labeled cells within enamel organ epithelium.
bBK 1, 2, and 3 ⫽ E10⫹9 cultured mandibular explants 1, 2, and 3.
PDGF-A and PDGFR-␣ PCR Products
Sequence Analysis
The PCR products were confirmed using PCR product
nucleic acid sequence analysis (Tracy and Mulcahy,
1991). Ten microliters of PCR product observed on an
agarose gel was eluted through a GLASSMAX (BRL)
spin column. The resulting elucidate (40 µl) was found
to be free of the primers, unincorporated dNTPs, enzymes, etc. The eluted PCR product (7.5 µl) along with
3.2 pMoles PDGF-A or PDGFR-␣ primers and water up
to a total volume of 10.5 µl was provided to the
Microchemical Core Facility of the USC Norris Cancer
Center at this Institution. A fluorescent cycle sequencing reaction was performed and analyzed on an Applied
Biosystems (ABI) automated sequencer (Model 373A).
The resulting sequences were compared to the August,
1994, Genebank database and the results confirmed a
100% match of the observed PCR products with that of
PDGFR-␣ and PDGF-A.
Light Microscope Immunocytochemistry
Mandibular explants were fixed in Carnoy’s fixative
solution for 6 hr at 4°C, dehydrated in 100% ethanol
overnight, cleared in xylene, and embedded in paraffin.
Serial sections of 5 µm were cut and mounted onto
slides coated with Histostick (Accurate Chemical &
Scientific Co., Westbury, NY). The average cultured
mandibular explant represented approximately 160
five-micron sections inclusive of the anterior (rostral),
middle, and posterior extensions of Meckel’s cartilage
which were used for orientation. The slides were heated
in 60°C oven for 45 min and subsequently hydrated to
water through a series of decreasing concentration of
ethanol. Following the protocol of Zymed StreptavidinBiotin system (Zymed Laboratories Inc., San Francisco,
CA), anti-PDGFR-␣ and anti-PDGF-A antibodies (UBI,
Lake Placid, NY) developed in rabbits were used as the
primary antibody at a dilution of 1:100 for antiPDGFR-␣, and 1:150 for anti-PDGF-A. The immunostaining was done on adjacent serial sections from the
same tooth germ to localize both PDGF-A and its
cognate receptor. Normal rabbit serum at a 1:100
dilution was used as negative control. The primary
antibody was incubated overnight at room temperature
in a moisture chamber prior to washing with PBS and
reacting with goat-anti-rabbit secondary antibody. Adult
mouse lung was used as positive control. PDGFR-␣ and
PDGF-A antigens were indicated by orange-red coloration. The slides were counterstained with hematoxylin.
To verify the distribution patterns of PDGF-A and
PDGFR-␣, another set of primary antibodies were
obtained from Santa Cruz Biotechnology (Santa Cruz,
CA). Immunostainings using these antibodies confirmed our findings.
PDGF-A Stimulation of DNA Synthesis
The highly purified recombinant PDGF-A isoform
was commercially obtained (UBI, Lake Placid, NY), and
used in overexpression experiments at concentrations
ranging from 0.25 ng to 40 ng/ml diluted in serumless,
chemically defined medium. DNA synthesis activity
was monitored in E10 mandibular explants using BrdU
in chemically defined medium. Mandibular explants
were incubated with BrdU at 100 µM in serumless
chemically defined medium for 2 hours at 4°C. Then
these mandibles were placed in the tissue culture dish
with either control medium or medium plus PDGF-A at
20 ng/ml. The mandibular organ culture process was
identical to the procedure described above. The mandibular explants were harvested and fixed for immunostaining at E10⫹2, 4 and 9 days of culture. Mandibular
explants were fixed in Carnoy’s fixative solution for 6 hr
at 4°C, dehydrated in 100% ethanol overnight, cleared
in xylene, and embedded in paraffin. Serial sections of 5
µm were cut and mounted onto slides coated with
Histostick (Accurate Chemical & Scientific Co., Westbury, NY). The slides were heated in 60°C oven for 45
min and subsequently hydrated to water through a
series of decreasing concentration of ethanol. Then the
slides were treated with 2 N HCl for 60 min at 37°C to
510
CHAI ET AL.
denature DNA and followed with neutralization with
Borate buffer (pH 8.5) for three times (10 min each).
The slides were rinsed in PBS. Following the protocol of
Zymed Streptavidin-Biotin system (Zymed Laboratories Inc.), immunohistochemistry was performed.
Morphometric and Statistical Analysis
In order to evaluate the morphological effect induced
by PDGF-A treatment on tooth formation, the size of
tooth bud was measured. Three mandibles were randomly selected from each treatment group (control,
PDGF-A at 2 ng/ml, 10 ng/ml, and 20 ng/ml). Each
section of tooth bud was traced throughout the serial
sections of mandibular explant. The volume of the tooth
bud was calculated. Using Epistat Statistical Package
one-way, one-level analysis of variance (ANOVA) was
applied to test for significant changes in the size of
tooth bud among nontreated control, PDGF-A at 2
ng/ml, 10 ng/ml, and 20 ng/ml treated groups. A difference was considered statistically significant if the P
value was less than 0.05 (P ⬍ 0.05). The null hypothesis
for this test was that there was no difference in the size
of tooth bud for the various groups in the PDGF-A
treatment experiments.
Computer Analysis
With the same enlargement, the outlines on the
adjacent serial sections of mandible were traced using a
Jandel digitizing tablet into the PC3D software program (Jandel Scientific, Sausalito, CA). Three-dimensional reconstructions were completed on an IBM PC-AT
with a Cordata color monitor. The PC3D program
provides the option of rotating each reconstruction
around three axes (x, y, and z; Chai et al., 1993).
Evaluation of DNA Synthesis Activity During
Early Tooth Morphogenesis
BrdU-labeled cells and the total number of cells
within the enamel organ epithelium of a tooth bud were
counted from five randomly selected sections per mandibular explant. Three mandibular explants were evaluated from each experimental group. The results were
presented in Table 1.
ACKNOWLEDGMENTS
The authors thank Miss Marie Ly for her technical
assistance. This study was supported by NIDR, NIH
center grant DE-09165.
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