/. Embryol. exp. Morph. Vol. 58, pp. 195-208, 1980
Printed in Great Britain © Company of Biologists Limited 1980
195
Tunicamycin inhibits mouse tooth morphogenesis
and odontoblast differentiation in vitro
By IRMA THESLEFF 1 AND ROBERT M. PRATT 2
From the Laboratory of Developmental Biology and Anomalies,
National Institute of Dental Research, Bethesda
SUMMARY
Tunicamycin (TM), an antibiotic that selectively inhibits dolichol-mediated protein
glycosylation, inhibited morphogenesis and differentiation of odontoblasts in the molar
tooth germ in vitro. These effects of TM are reversible and dose-dependent, and in advanced
teeth the effect of TM was not complete unless the basement membrane was removed prior
to culture. TM did not prevent secretion of predentin or enamel when added to the cultures
after initiation of predentin secretion. TM dramatically inhibited protein glycosylation and
the accumulation of labeled proteoglycans and glycoproteins in the basement membrane.
Our previous studies indicated that odontoblast differentiation is triggered by an interaction
between the basement membrane and mesenchymal cells. We suggest that TM inhibits
odontoblast differentiation by causing alterations in the basement membrane which prevent
the necessary cell-matrix interaction required for odontoblast differentiation.
INTRODUCTION
The tooth bud is initiated when the oral epithelium grows into the underlying
mesenchyme. Tooth development reaches the bell stage when the epithelial
bud invaginates at its mesenchymal aspect and grows to surround a condensation
of mesenchymal cells, known as the dental papilla. At this time, cuspal morphogenesis begins and epithelial-mesenchymal cell differentiation and matrix
secretion take place. The mesenchymal cells first differentiate into odontoblasts
and secrete predentin. Subsequently, the epithelial cells apposing the predentin
polarize into ameloblasts and deposit the organic matrix of enamel. Interaction
between the epithelial and mesenchymal components of the tooth germ appears
to be a prerequisite for morphogenesis and differentiation since isolated tooth
epithelium or mesenchyme is not able to form dental structures (Koch, 1967;
Kollar & Baird, 1969).
The events that occur during these tissue interactions are not known, but
some information has been obtained from various experimental studies. Culture
of heterochronal associations of dental epithelium and mesenchyme has shown
1
Author's address: Department of Pathology, University of Helsinki, SF-00290 Helsinki
29, Finland.
2
Author's address: Laboratory of Developmental Biology and Anomalies, National
Institute of Dental Research, National Institutes of Health, Bethesda, MD 20205, U.S.A.
196
I. THESLEFF AND R. M. PRATT
that these tissues influence the mitotic activity of each other (Ruch, KarcherDjuricic & Thiebold, 1976). Transfilter studies have indicated that the mesenchymal cells need to come into close contact with the basement membrane
before they differentiate into odontoblasts (Thesleff, Lehtonen & Saxen, 1978).
The importance of the extracellular matrix has also been demonstrated in
studies in which inhibition of collagen synthesis or processing inhibited tooth
morphogenesis (Ruch, Fabre, Karcher-Djuricic & Staubli, 1974; Galbraith &
Kollar, 1974; Hetem, Kollar, Cutler & Yager, 1975). Diazo-oxo-norleucine
(DON) is a glutamine analogue which interferes with glycoconjugate synthesis
and glycosylation and inhibits rodent tooth morphogenesis and odontoblast
differentiation in vitro (Hurmerinta, Thesleff & Saxen, 1979). It was suggested
that DON exerted its effect on the developing tooth by preventing the interaction
between the mesenchymal cells and the basement membrane. However, in
addition to its inhibitory effect on glycoconjugate synthesis, DON also affects
purine biosynthesis (Buchanan, 1973).
Tunicamycin is an antibiotic that selectively interferes with glycoprotein
biosynthesis by inhibiting the formation of dolichol-bound N-acetylglucosamine
derivatives (Takatsuki, Kohno & Tamura, 1975). Consequently, the glycosylation of proteins at asparagine residues is prevented (Tkacz & Lampen, 1975;
Olden, Pratt & Yamada, 1978). In the present study we have examined the
effect of tunicamycin on tooth morphogenesis. Our results show that tunicamycin
inhibits the morphogenesis of cultured mouse tooth germs, and it selectively
inhibits the differentiation of odontoblasts if present at the early stages of
development. At later stages, differentiation is inhibited only if the basement
membrane is removed prior to cultivation.
MATERIAL AND METHODS
Tooth buds were dissected from mouse fetuses of days 14-17 of gestation.
Male and female Swiss Webster (NIH) mice were allowed to mate overnight
and the detection of a vaginal plug was considered day 0 of gestation. The
lower jaw of each embryo was removed, submerged in phosphate-buffered
saline (PBS, pH 7-4), and the first mandibular molars, containing a piece of
overlying oral epithelium, were dissected out. In order to remove the basement
membrane, the molars of day-16 fetuses were treated with 2-25 % trypsin-0-75 %
pancreatin for 15 min at 4 °C. The tissues were either placed directly in culture
after enzyme treatment or were first incubated for 30 min in culture medium
at 22 °C. In the latter case, the epithelial and mesenchymal components of the
tooth bud were then mechanically separated, and cleaned of adherent tissues.
The mesenchyme was first placed on the Millipore filter followed by the inner
enamel epithelium which was placed in direct contact with the mesenchyme.
A Trowell-type organ culture system was used, with the explants supported
by a Millipore filter (25 [im thick, 0-45 /«n porosity) on a metal grid. The
Inhibition of tooth morphogenesis by tunicamycin
197
medium consisted of BGJb medium (GIBCO) supplemented with 100 i.u./ml
penicillin, 100/*g/ml streptomycin, 0-25/*g/ml Fungizone, 20% horse serum
(GIBCO), 10 % chick embryo extract, and 150 /*g/ml ascorbic acid. The culture
dishes were kept in a humidified incubator at 37 °C in an atmosphere of 5 %
CO2 in air. The explants were cultured for 2-14 days, and the medium was
changed every other day. The tissues were fixed in Carnoy's or Zenker's
solutions, embedded in Paraplast and serially sectioned at 6 fim. Deparaffinized
sections were stained with periodic acid-Schiff reagent (PAS) or Mallory's
phosphotungstic acid-hematoxylin.
The incorporation of labelled precursors into glycoproteins and proteoglycans
was examined by autoradiography. During the third day of culture, the' explants
were exposed for 6 h to media containing 50/tCi/ml of either D - [ 2 - 3 H ( N ) ] mannose (specific activity 18/*Ci/mmol), L-[6-3H]-fucose (specific activity
13-1 Ci/mmol), or [35S] sodium sulfate, all from New England Nuclear.
Afterwards the explants were washed three times in PBS containing 5 mM
unlabelled precursor. Explants were fixed in Carnoy's solution, embedded in
Paraplast and sectioned at 6 fim. The deparaffinized and hydrated sections were
dipped in nuclear track emulsion NTB-2 (Kodak). The slides were exposed at
4 °C for 1-4 weeks, developed, and stained with PAS.
Tunicamycin was a gift from Dr Gakuzo Tamura via the Drug Evaluation
Branch of the National Cancer Institute. It was dissolved in DMSO (dimethylsurf oxide) and stored at - 7 0 °C and used in concentrations from 0-01 /^g/ml
to 2/tg/ml. In most experiments, TM at a concentration of 0-15/^g/ml was
used, and an equivalent volume of DMSO was included in control medium.
RESULTS
Cultures of whole tooth rudiments
At day 14 the molar tooth bud is in the cap stage of development, and the
epithelial enamel organ partially surrounds the dental papilla mesenchyme.
When cultured in control medium, the tooth germs reached the bell stage by
4 days of culture, and by 10 days the differentiated odontoblasts had secreted
predentin and the ameloblasts had polarized (Fig. 1A). In the presence of TM
(0-15 /6g/ml), the developmental stage of the tooth germs was the same after
4 days of culture as at the onset of cultivation (Fig. IB); after 10 days the
tooth rudiment had regressed and the epithelia were heavily keratinized.
In the day-15 fetus, the molar has reached the bell stage of development and
the onset of cuspal morphogenesis is already evident. TM prevented further
cuspal morphogenesis when added to these cultured rudiments. Differentiation
of odontoblasts, however, was not completely prevented and, in three of nine
TM-treated tooth germs, some predentin was secreted by polarized odontoblasts
in the cuspal area (Fig. 1C, D).
Cuspal morphogenesis is more advanced in the day-16 and day-17 molars,
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I. THESLEFF AND R. M. P R A T T
B
Fig. 1. Photomicrographs illustrating the effect of tunicamycin (TM, 0-15/tg/ml)
on the development of mouse molar tooth germs in culture. Periodic acid-Schiff
stain.
(A) Tooth germ of a day-14 fetus cultured for 10 days in control medium. Cuspal
morphogenesis is advanced; the differentiated odontoblasts (O) have secreted
predentin (PD) and the ameloblasts (A) have polarized.
(B) A day-14 tooth germ cultured for 4 days in the presence of TM. Morphogenesis
has been inhibited, and the mesenchymal cells (M) have not differentiated. Dental
epithelium (E).
(C) A day-15 tooth germ cultured for 7 days in control medium. Well-developed
cusps are seen; differentiated odontoblasts (O) have secreted predentin (PD).
(D) A day-15 tooth germ cultured for 7 days in the presence of TM. Cuspal morphogenesis has been inhibited. This is one of the three explants where the odontoblasts
at the cusps differentiated and secreted predentin (PD). In the remaining six
explants no predentin was seen.
Inhibition of tooth morphogenesis by tunicamycin
199
but predentin secretion is not yet observed. In control cultures of day-16 and
-17 molars, well-developed cusps were seen after 7 days of cultivation. Differentiated odontoblasts which had secreted predentin were observed. Ameloblasts
had polarized (Fig. 2 A) and, in a few explants, secreted enamel. In the presence
of TM at 0-15 /*g/ml, cuspal development in the tooth germs remained at the
same stage as it was at the onset of culture; no predentin was secreted in four
out of six day-16 molars cultured for 7 days (Fig. 2B). In day-17 molars, TM
did not prevent predentin secretion, but cuspal morphogenesis was inhibited
(not shown).
The effect of TM was reversible. In experiments where the day-16 tooth
germs were transferred to control medium after 4 days of culture in the presence
of TM (0-15/tg/ml), predentin secretion and ameloblast polarization were
observed after 10 days of culture (Fig. 2C). Under these conditions, the explants
were smaller than the controls, but cuspal morphogenesis was well advanced.
TM did not prevent predentin secretion, ameloblast polarization or enamel
secretion if the day-16 tooth germs were cultured for 4 days in control medium
prior to addition of TM. After 4 days in control medium, the polarized odontoblasts had secreted the first layer of predentin, but ameloblasts were not
polarized. During the following 10 days in the presence of TM, additional
predentin was secreted, and by the end of this period the ameloblasts had
polarized and begun to secrete enamel (Fig. 2D).
Cultures of enzyme-treated tooth germs
Incubation of tooth germs in the 2-25% trypsin-0-75% pancreatin solution
results in the removal of the basement membrane (Thesleff et al. 1978). The
enzyme-treated molars of day-16 fetuses, when grown in control medium,
developed in the same manner as control molars. After 6 days in culture, welldeveloped cusps were observed, predentin had been secreted by the differentiated
odontoblasts, and the ameloblasts had polarized (Fig. 2E). TM prevented
cuspal morphogenesis, differentiation of the mesenchymal cells into odontoblasts and subsequent predentin secretion and ameloblast polarization in all
12 explants (Fig. 2F). No pycnotic nuclei indicative of cell death were observed
in these cultures, and the effect of TM was reversible. The explants transferred
to control medium after 4 days of culture in the presence of TM (0-15 /*g/ml)
recovered in the same manner as non-enzymically-treated tooth germs (see
above).
Cultures of recombined epithelium and mesenchyme
When dental epithelium and mesenchyme of day-16 molars were enzymically
separated and recombined in culture, odontoblasts and ameloblasts differentiated in a manner similar to that of cultures of whole tooth germs. After 2 days
of culture in control medium, the mesenchymal cells had aligned under the
enamel epithelium and a distinct PAS-positive basement membrane was
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I. THESLEFF AND R. M. PRATT
B
•,
Inhibition of tooth morphogenesis by tunicamycin
201
observed (Fig. 3 A). After 7 days predentin was deposited and the ameloblasts
had polarized (Fig. 3C). The extent of the normal cusp pattern could not be
assessed in these explants (compare Fig. 1C with Fig. 3 A). TM prevented
differentiation of odontoblasts, and the effect was dose-dependent: 0-01 /tg/ml
of TM had no effect, and predentin was secreted in all five explants after 6
days of culture; 005/tg/ml of TM prevented odontoblast differentiation in
three offiveexplants, whereas 0-1 and 0 2 fig/raX of TM prevented differentiation
in all 19 explants. TM at 2/ig/ml was toxic and therefore the concentration of
0-15/ig/ml of TM was used in all subsequent studies. After 2 days of culture
in the presence of TM, no alignment of the mesenchymal cells was observed
and the basement membrane appeared irregular and was not uniformly PASpositive as in the controls (Figs 3A and B). After 7 days the TM-treated
explants looked similar to those after 2 days of culture, and although these
explants were smaller than the controls, there was no obvious cell death (Figs
3CandD).
The effect of TM was reversible when the explants were transferred to control
medium after 3 days of culture (Fig. 3E). However, if they were cultured with
TM for 4 days, only one out of four explants recovered. None of the seven
explants recovered when cultured for 7 days in the presence of TM prior to
transfer into control medium. TM did not prevent secretion of predentin,
polarization of ameloblasts or enamel secretion if the explants were first grown
in control medium for 4 days. At this time the odontoblasts had already begun
to secrete predentin. After 7 days of culture in the presence of TM more
FIGURE 2
Photomicrographs illustrating the effect of tunicamycin (TM, 0-15/<g/ml) on the
development of molar tooth germs from day-16 mouse fetuses. Mallory's phosphotungstic acid-hematoxylin stain.
(A) After 7 days in control medium well-developed cusps are seen, and odontoblasts
(O) have secreted predentin (PD).
(B) After 7 days in the presence of TM, cuspal morphogenesis is inhibited. This is
one of the four explants where no predentin was detected. In the remaining two
explants some predentin had been secreted in the cuspal area. Dental epithelium (E),
dental mesenchyme (M).
(C) A tooth germ cultured first in the presence of TM for 4 days and then for 10
days in control medium. Predentin has been secreted and cuspal morphogenesis
has advanced, indicating partial recovery from TM. However, the explant is smaller
than those cultivated in control medium for 14 days.
(D) A tooth germ cultured for4 days in control medium and then for 10 days in the
presence of TM. TM has not prevented secretion of predentin (PD) and enamel
matrix (EM).
(E) A tooth germ incubated in 2-25 % trypsin-0-75 % pancreatin to remove the
basement membrane, and then cultured for 7 days in control medium. Welldeveloped cusps are seen; odontoblasts (O) have secreted predentin (PD).
(F) A tooth germ similar to the one in Fig. 2E, but cultured in the presence of TM.
Cuspal morphogenesis has been prevented; mesenchymal cells (M) have not
differentiated into odontoblasts.
202
I. THESLEFF AND R. M. PRATT
Inhibition of tooth morphogenesis by tunicamycin
203
predentin had been secreted and the ameloblasts had polarized (Fig. 3F) and
occasionally secreted enamel matrix.
Autoradiographic examination of incorporation of radioactive precursors
The effect of TM on the incorporation of [3H]mannose and [3H]fucose,
precursors of glycoproteins, and [35S] sulfate, a precursor of sulfated glycosaminoglycans, was examined. Explants of enzyme-treated whole tooth germs of
day-16 fetuses were incubated for 6 h with the radioactive precursors on the
third day of culture. The incorporation of [3H]fucose was particularly high at
the basement membrane region in the control explants (Fig. 4A). In the TMtreated explants, very little incorporation was seen in the basement membrane
area, although no clear decrease in the overall incorporation between the
controls and the TM-treated explants was detected (Fig. 4B). TM significantly
decreased the incorporation of [3H]mannose in the mesenchymal and epithelial
tissues (Fig. 4C, D). A deposition of silver grains from [35S]sulfate was also
seen in control cultures in the basement membrane area. TM substantially
decreased the number of grains observed in this region, but had no effect on the
overall incorporation (Fig. 4E, F). It is evident (Fig. 4) that the cell density in
the TM-treated explants is higher than that observed in the controls, which
suggests that TM reduced the amount of extracellular matrix material in the
mesenchymal tissue.
FIGURE 3
Photomicrographs illustrating the effect of TM (015 /*g/ml) on explants of dental
epithelium and mesenchyme that were enzymically separated and recombined for
culture. Periodic acid-Schiff stain (PAS).
(A) An explant grown for 2 days in control medium. A distinct PAS-positive
basement membrane is present between the epithelium (E) and the mesenchyme (M),
and mesenchymal cells have begun to be aligned under the basement membrane.
(B) After 2 days of culture in the presence of TM, the epithelial-mesenchymal
interface is irregular and the basement membrane (arrows) is not uniformly PASpositive as in the controls (Fig. 3 A).
(C) An explant grown for 7 days in control medium. Odontoblasts (O) have differentiated and secreted predentin (PD), and the ameloblasts (A) are polarizing.
(D) In the presence of TM differentiation of the mesenchymal (M) cells into
odontoblasts has been prevented. The explant is similar to those after 2 days of
culture (Fig. 3B).
(E) An explant grown first for 3 days in the presence of TM and then for 7 days in
control medium. Predentin (PD) has been secreted by the differentiated odontoblasts
(O), suggesting recovery from TM treatment.
(F) An explant grown first in control medium for 4 days and then in the presence of
TM for 7 days. TM has not prevented the secretion of predentin (PD) or ameloblast
(A) polarization.
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I. THESLEFF AND R. M. PRATT
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Inhibition of tooth morphogenesis by tunicamycin
205
DISCUSSION
Tunicamycin (TM) has been shown to inhibit differentiation in a number of
developing systems. It blocks sea-urchin embryogenesis at gastrulation
(Schneider, Nguyen & Lennarz, 1978; Heifetz & Lennarz, 1979), and interferes
with compaction and blastocyst formation in early mouse embryos (Surani,
1979; Atienza-Samols, Pine & Sherman, 1980). It also inhibits kidney tubulogenesis (Ekblom et al. 1979) and myoblast fusion (Olden, Law, Hunter &
Parent, 1980). The biochemical effect of TM is specific and has been well
established. It inhibits the formation of dolichol-diphospho-N-acetylglucosamine thereby preventing the glycosylation of asparagine residues of glycoproteins (Takatsuki et al. 1975; Tkacz & Lampen, 1975). In all of the studies
where TM prevented embryonic differentiation, its action appeared to result
from alterations in cell-surface glycoproteins.
Our results show that TM inhibited morphogenesis in cultured mouse molar
tooth germs. TM also prevented differentiation of odontoblasts with subsequent
inhibition of predentin secretion, ameloblast polarization and enamel secretion.
This effect of TM was observed in tooth germs from early developmental stages
or from later stages providing the dental basement membrane was enzymically
removed before cultivation. Furthermore, our results indicate that TM did not
prevent deposition of predentin, ameloblast polarization or enamel secretion
if it was introduced after the time that odontoblasts had begun predentin
secretion.
In the TM-treated tooth germs examined in this study, cuspal morphogenesis
was inhibited and the tooth germs were smaller than the controls. It is likely
that the reduction in size was due to a decrease in secretion of extracellular
FIGURE 4
Autoradiograms illustrating the effect of tunicamycin (TM, 0-15/tg/ml) on
incorporation of labeled precursors in day-16 molars that had been incubated in
trypsin-pancreatin prior to cultivation. The explants were exposed to the precursor
(50 ywCi/ml) for 6 h on the third day of culture. Periodic acid-Schiff stain.
(A) [3H]fucose incorporation into explant grown in control medium. Intense
incorporation is seen in the basement membrane area between dental mesenchyme
(M) and epithelium (E).
(B) [3H]fucose incorporation into explant grown in the presence ot TM. No
accumulation of silver grains is seen in the basement membrane (arrows).
(C) [3H]mannose incorporation into explant grown in control medium. Intense
incorporation is seen in dental mesenchyme (M).
(D) [3H]mannose incorporation into explant grown in the presence of TM. Incorporation into dental mesenchyme (M) is markedly less than in the control (Fig. 4C).
(E) [^SJsulfate incorporation into explant grown in control medium. Incorporation
is seen in the basement membrane region (arrows) and in dental mesenchyme (M).
(F) PSJsulfate incorporation into explant grown in the presence of TM. The accumulation of silver grains in the basement membrane area (arrows) is not as marked as
in the control (Fig. 4E).
.:
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I. THESLEFF AND R. M. PRATT
matrix material and to a small decrease in cell proliferation. Whether the effect
of TM on cell proliferation was direct or indirect cannot be determined from
our results. In the chick embryo fibroblast, TM has been reported to have only
limited effects on cell proliferation (Olden et al. 1978). In isolated dental
mesenchyme in culture the rate of proliferation is minimal, and it has been
shown that mitosis in this tissue is dependent upon the presence of dental
epithelium (Ruch et al. 1976). Therefore, we suggest that TM decreases proliferation in the dental mesenchyme by impairing the interaction between the
epithelium and mesenchyme rather than by having a direct effect on mesenchymal tissue.
TM inhibited odontoblast differentiation in all cultured tooth germs from
day-14 mouse fetuses but only in 70% of the day-15 and -16 tooth germs.
In day-17 molars, TM did not prevent odontoblast differentiation. However,
TM inhibited odontoblast differentiation in the older tooth germs if the basement membrane was enzymically removed prior to culture. It was shown
earlier that the restoration of the basement membrane in such explants is a
prerequisite for odontoblast differentiation (Thesleff et al. 1978; KarcherDjuricic et al. 1978). One possible mechanism by which the basement membrane
triggers odontoblast differentiation is through stimulation of proliferation in
the pre-odontoblastic cells. It has been suggested that before their terminal
differentiation, these mesenchymal cells must go through a specific number of
cell cycles (Ruch, Karcher-Djuricic & Thiebold, 1976) as has been suggested for
other embryonic cell types (Holtzer, 1970). We have proposed that an additional
function for the basement membrane is to trigger odontoblast differentiation
by serving as a substratum for the alignment and polarization of the preodontoblastic cells (Thesleff, 1978; Thesleff, Stenman, Vaheri & Timpl, 1979).
TM may therefore prevent odontoblast differentiation by altering the composition of the basement membrane and/or the mesenchymal cell surface, thereby
preventing the necessary cell-matrix interaction.
An examination of incorporation of radioactive precursors into control and
TM-treated tooth germs indicated that TM had similar biochemical effects as
reported earlier for other systems (Olden et al. 1978; Surani, 1979). Incorporation of 2-[3H]mannose, which is a specific precursor of glycoprotein biosynthesis, was dramatically reduced by TM. Furthermore, in the basement
membrane of the control explants, particularly high incorporation of
[3H]fucose and [35S]sulfate was observed, and this was dramatically reduced in
the TM-treated tooth germs. This suggests that the basement membrane of the
TM-treated tooth germs contained lesser amounts of glycoproteins and sulfated
glycosaminoglycans than did controls.
Fibronectin, which is a major glycoprotein of the extracellular matrix and
cell surface, is present in dental basement membrane in large amounts at the
time of odontoblast differentiation (Thesleff et al. 1979). One possible function
of fibronectin is to mediate mesenchymal cell alignment and polarization. TM
Inhibition of tooth morphogenesis by tunicamycin
207
prevents glycosylation of fibronectin in various cells in culture (Olden et al.
1978; Duksin & Bornstein, 1977), and it has been shown that proteolytic
degradation of the unglycosylated fibronectin results in decreased amounts of
fibronectin at the cell surface and medium from cultured cells (Olden et al.
1978). It is therefore possible that the TM-induced inhibition of odontoblast
differentiation observed in the present study resulted from reduced amounts of
fibronectin in the basement membrane and at the cell surfaces. All of these
alterations in the basement membrane presumably result in inhibition of the
necessary cell-matrix interaction between the mesenchymal cells and the
basement membrane.
We thank Mrs Frances Cannon for her expert technical assistance.
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{Received 20 November 1979, revised 21 January 1980)