From the Functional Genomics Unit and Gene Targeting
Facility, National Institute of Dental and Craniofacial Research,
National Institutes of Health, Bethesda, Maryland 20892 and the
§ Department of Pediatric Dentistry, School of
Dentistry, University of North Carolina,
Chapel Hill, North Carolina 27599
Received for publication, November 20, 2000, and in revised form, December 12, 2000
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ABSTRACT |
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Transforming growth factor (TGF)- Mammalian development is a complex and highly orchestrated process
that involves intricate cross-talk between growth factors and other
regulatory molecules. These molecules interact with each other to
induce specific molecular and cellular changes leading to
organogenesis. Interactions between epithelium and mesenchyme are
particularly crucial during the initiation of development of key organs
such as teeth, skin, hair, mammary gland, and prostate (1). Tooth
development is initiated by epithelial-mesenchymal interactions in the
first branchial arch, and several transcription factors and growth
factors are known to be expressed by dentin extracellular matrix
(DECM)-producing1
odontoblasts and enamel-producing ameloblasts during tooth development (2-5). Transforming growth factor- To gain more insights into the specific in vivo roles of
TGF- TGF- Microradiography--
Transgenic and wild type mice were
euthanized by cervical dislocation, and the heads were dissected out
and sliced sagittally into two symmetrical halves. The mineral density
of teeth was analyzed by microradiographic technique using x-ray
imaging with a standard setting of 120 s × 15KV (model MX20,
Faxitron x-ray Corporation, Wheeling, IL). Images were scanned and
quantified using a computerized National Institutes of Health image system.
Preparation of Tissue Sections, Histological Analysis, and
Immunohistochemistry--
Whole jaws from the transgenic and wild type
mice were dissected under a stereomicroscope and fixed in 10% buffered
formalin overnight. The tissues were decalcified in EDTA-sodium
phosphate buffer for 10-15 days, dehydrated, and embedded in paraffin
wax, and 5-micron-thick sections were cut and collected onto silanated microscope slides. Immunostainings for TGF- Northern Blot Analysis--
The incisors and molars were
dissected out from wild type and transgenic mice, and total RNA was
prepared using a RNA STAT-60TM kit according to the
manufacturer's recommendations (Tel-Test, Inc., Friendswood, TX).
Total RNA (10 µg) was electrophoresed on a 1% formaldehyde gel and
transferred onto a nylon membrane. The membrane was hybridized with
32P-labeled dspp probe (pSX1.7 Exon IV) (18).
Autoradiographs were exposed to Kodak x-ray film (Eastman Kodak Co.)
for 24 h at In Situ Hybridization--
Templates for antisense and sense
riboprobes for dspp gene were generated by digesting pSX1.7
containing exon IV with SacI and XbaI, and an
in vitro transcription assay was carried out to incorporate
digoxigenin-11-dUTP with T7 and T3 RNA polymerases according to the
manufacturer's recommendations (Roche Molecular Biochemicals). Frozen
sections (15 microns) were cut, air dried, and fixed in 4%
paraformaldehyde for 10 min at 4 °C. The sections were rinsed with
phosphate-buffered saline and treated with 0.2 M HCl, 1 µg/ml proteinase K, 0.25% acetic anhydride in 0.1 M
triethanolamine buffer 5 min each with brief rinses in DEPC water in
between the treatments. In situ hybridization and signal
detection were carried out according to Roche Molecular Biochemicals
nonradioactive In Situ Hybridization Application Manual.
Slides were counter stained with hematoxylin and mounted with Crystal
Mount (Biomedia, Foster City, CA) for photography.
Generation of dTGF- Novel Tooth Phenotype of dTGF-
In dTGF- Increased Levels of Dentin Extracellular Matrix Proteins in
dTGF-
The phenotypic characterization and histological analysis along with
the radiographic profile of dTGF- Regulation of Dentin Sialophosphoprotein Gene Expression by
dTGF- To analyze in vivo functions of the multi-functional
growth factor TGF- The dTGF- Although dspp regulatory sequences are odontoblast-specific,
an intense staining for TGF- Interestingly, calcium, a major component of hydroxyapatite crystals in
the teeth, was apparently elevated in the transgenic mice. The
distribution in the dentin appeared to be patchy and reduced or absent
in certain regions. In addition to the collagen trimers and calcium
ions, inorganic phosphates are essential for proper mineralization of
teeth. The growth and proper organization of hydroxyapatite crystal
formation resulting from tri- or hexa-calcium phosphate provides the
strength to the dentin. Hence, we examined the expression of DSPP, a
highly phosphorylated tooth-specific phosphoprotein, in the teeth of
these mice. Additionally, the dspp gene has been mapped to
the DGI locus and is implicated as a potential candidate gene for
DGI-II (20). The dspp gene product also has been
demonstrated to function as a nucleator in hydroxyapatite crystal
formation in dentin (29). Our studies demonstrate for the first time a
significant reduction in the levels of dspp mRNA in the
presence of high levels of TGF-1 is
expressed in developing tooth from the initiation stage through
adulthood. Odontoblast-specific expression of TGF-
1 in the tooth
continues throughout life; however, the precise biological functions of
this growth factor in the odontoblasts are not clearly understood.
Herein, we describe the generation of transgenic mice that overexpress
active TGF-
1 predominantly in the odontoblasts. Teeth of these mice
show a significant reduction in the tooth mineralization, defective
dentin formation, and a relatively high branching of dentinal tubules.
Dentin extracellular matrix components such as type I and III collagens
are increased and deposited abnormally in the dental pulp, similar to
the hereditary human tooth disorders such as dentin dysplasia
and dentinogenesis imperfecta. Calcium, one of the crucial inorganic
components of mineralization, is also apparently increased in the
transgenic mouse teeth. Most importantly, the expression of dentin
sialophosphoprotein (dspp), a candidate gene implicated in
dentinogenesis imperfecta II (MIM 125420), is significantly
down-regulated in the transgenic teeth. Our results provide in
vivo evidence suggesting that TGF-
1 mediated expression of
dspp is crucial for dentin mineralization. These findings
also provide for the first time a direct experimental evidence
indicating that decreased dspp gene expression along with
the other cellular changes in odontoblasts may result in human
hereditary dental disorders like dentinogenesis imperfecta II (MIM
125420) and dentin dysplasia (MIM 125400 and 125420).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 (TGF-
1), a prototype of the
TGF-
superfamily, is a multi-functional growth factor expressed in a
wide variety of developing tissues from the early stages. The
regulation of cell proliferation, differentiation, embryonic development, and apoptosis by TGF-
1 is well established (6-8). During mouse tooth development, TGF-
1 is expressed initially in the
oral epithelium at embryonic day 13, and later its expression extends
into the mesenchymal compartment and then gets restricted to the
ectomesenchymal layer (odontoblasts). The odontoblast-restricted expression of TGF-
1 persists throughout life in the mice (9). Odontoblasts produce DECM from embryonic day 16 and subsequently mineralize in an orderly manner. TGF-
1 has been shown earlier to
have mitogenic effects in tooth explant cultures (10) and to induce
secretion of DECM components. Although it has been suggested that
TGF-
1 plays a crucial role in dental tissue repair processes by the
induction of reactionary (11) and reparative dentinogenesis (12), the
precise in vivo functions associated with its continued expression are not clearly understood. Interestingly, subtle changes such as attrition and reduced mineralization of the teeth along with
inflammation were observed in TGF-
1 knockout mice (13). However, the
maternal transfer of active TGF-
1, multi-focal inflammation, and
neonatal lethality in these mice further complicate the clear understanding of the precise role of TGF-
1 in tooth development (14).
1 during tooth development, we targeted the overexpression of active TGF-
1 to odontoblasts, starting from embryonic day 17 using
the upstream regulatory sequences of the dentin sialophosphoprotein (dspp) gene (15). These animals develop a novel phenotype
that resembles hereditary dental disorders such as dentinogenesis
imperfecta II (DGI; MIM 125420) and dentin dysplasia (MIM 125400 and
125420). We present here a detailed analysis of this phenotype and
molecular mechanisms leading to this phenotype and discuss the role of
TGF-
1 in dentinogenesis and the tooth disorders.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 Transgenic Construct--
The transgenic construct
consisting of a 6-kilobase dspp upstream regulatory sequence
(15, 16) and a 1.5-kilobase active porcine TGF-
1 cDNA (17) with
a SV40 poly(A) sequence (see Fig. 1a) was microinjected into
fertilized FVB/N eggs to generate transgenic mice. Mice were genotyped
for the presence of the transgene by Southern analysis of the tail DNA
using a SV40 poly(A) probe. The dspp-TGF-
1 transgenic
(dTGF-
1) mouse lines varied in the copy number of
integrated transgenes (data not shown). Mice were housed in a
pathogen-free facility and fed ad libitum with dough diet
(Bio Serv, Holton Industries Co., Frenchtown, NJ).
1, Collagen (Col) I, II,
and III were performed using antibodies at 1:400 dilution. Immunohistochemical analysis was performed using a commercial kit
according to the manufacturer's suggestions (Vectastain ABC Kit,
Vector Laboratories Inc., Burlingame, CA). The sections were counterstained with hematoxylin and eosin and were photographed under
light microscopy. Anti TGF-
1 antibody was a gift of Dr. Kathy
Flanders (NCI, National Institutes of Health). Collagen I and III
antisera were kindly provided by Dr. Larry Fisher (NIDCR, National
Institutes of Health).
70 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 Transgenic Mice--
Five founder mice
were generated from the microinjections of the dspp/TGF-
1 construct (Fig. 1a) into
fertilized FVB/N mouse eggs. All of the founders were established as
independent lines based on independent integrations of the transgenic
construct in the genome. Mouse tail DNA preparations were subjected to
restriction enzyme digestion, and the presence of the transgene was
determined by Southern analysis using the whole transgene fragment as a
probe. Copy number in each line was analyzed by using the endogenous dspp gene as an internal control. Transgenic mice were
maintained as heterozygotes and mated with either wild type FVB/N or
with the transgenic heterozygotes for further analysis. The lines were classified as low, medium, and high expressors, and these lines displayed a general correlation between the level of TGF-
1
expression and severity of the tooth phenotype (data not shown). One of
the high expressor lines was further analyzed in detail.
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Fig. 1.
Strategy for generation of
dTGF- 1 mice and gross tooth
abnormalities in these mice. a, schematic
representation of the TGF-
1 transgenic construct. The transgenic
construct was constructed by fusing a 6-kilobase dentin
sialophosphoprotein (dspp) upstream regulatory sequence with
active porcine TGF-
1 cDNA in which Cys223 and
Cys225 codons are replaced with serine codons.
b, normal maxillary and mandibular incisors in wild type
mice. c, loss of maxillary incisors (arrowhead)
and discolored and fractured mandibular incisors (arrows) in
dTGF-
1 mice. d, normal opacity in the
craniofacial region of 15-day-old wild type mice as measured by
radiographic analysis. e, significantly reduced
opacity in the incisors (arrows) and molars
(arrowheads) of dTGF-
1 mice.
1 Mice--
All the transgenic
mouse lines displayed tooth-specific phenotypes with varying degrees of
severity. The dTGF-
1 mice were born with no apparent
defects and grew normally on the dough diet. However, from the age of 2 weeks, the dTGF-
1 mice displayed progressive discoloration of teeth (Fig. 1, b and c).
Initially, both the mandibular and maxillary incisors of the
dTGF-
1 mice appeared opaque, turned chalky white, and
fractured, leaving behind stumps. The high resolution radiographic
images of the incisors and molars of the dTGF-
1 mice
exhibited remarkably reduced mineralization (Fig. 1, d and
e). The quantitation of dTGF-
1 teeth by x-ray image analysis indicated a reduction of opacity by about 90% in the
incisors (wild type, 145.5 ± 15.3 au (arbitrary units);
dTGF-
1, 14.8 ± 4, au; n = 6, p < 0.001) and 62% in molars (wild type, 229.8 ± 16.2 au; dTGF-
1, 87.3 ± 15.7, n = 6; p < 0.001).
1 animals, the teeth displayed irregular dentin
formation with a significant number of cellular inclusions (Fig. 2, b, c,
e, and f). Compared with the wild type (Fig. 2,
a and d), the transgenic mice displayed a highly
disorganized odontoblast layer and irregular dentinal tubules all along
the dentinal layer (Fig. 2, b and c). The
dentinal tubules were short in length and sparsely distributed (Fig. 2,
c and f). Electron microscopic analysis of wild
type mouse teeth showed normal dentin architecture with dentinal
tubules coursing from the dentin-enamel junction in a parallel
organization toward the dental pulp (Fig.
3, a and c), whereas dTGF-
1 mouse incisors showed a thin layer of
relatively normal mantle dentin and markedly abnormal dentin with
reduced numbers of dentinal tubules (Fig. 3, b and
d). The coronal area of the transgenic tooth pulp was
obliterated with a disorganized dentin, similar to the structural
abnormalities observed in the incisors.
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Fig. 2.
Histological analysis of
dTGF- 1 mouse teeth.
a, representative sagittal section of the molar of
15-day-old wild type mouse stained with hematoxylin and eosin.
b, molar of 15-day-old transgenic mice with a low TGF-
1
expression. c, molar of 15-day-old transgenic mice with
higher TGF-
1 expression. d-f are the high magnifications
of a-c, respectively. Normal organization of odontoblast
layer and orientation of dentinal tubules are seen in the wild type
molars. Irregular odontoblast layer and dentinal tubules with cellular
inclusions (arrow) are present in dTGF-
1
molars. de, dentin; od, odontoblasts;
p, pulp.
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Fig. 3.
Ultrastructural analysis of
dTGF- 1 mouse teeth.
Ground sections of incisors from wild type (a and
c) and dTGF-
1 (b and d)
mice. The arrow indicates dentin-enamel junction. Note the
highly abnormal and irregular dentin in b. c and
d, higher magnifications of dentin in a and
b, respectively. The arrow indicates regular
dentinal tubules in c and highly disorganized tubules with
voids in d. Note the abnormal deposition of DECM with void
spaces (arrow) in d. de, dentin;
dt, dentinal tubules; en, enamel; md,
mantle dentin.
1 Mice--
To confirm whether the observed phenotype was due
to the increased TGF-
1 levels in teeth of the transgenic mice, we
performed immunohistochemical analysis on the cross-sections of the
incisors from 1-day-old mice using anti-TGF-
1 antibodies (Fig.
4, a and e). High
levels of TGF-
1 were detected in the dentin matrix, around the
odontoblasts and also in the dental pulp (Fig. 4e). Transgenic TGF-
1 was also detected transiently in the ameloblasts similar to the endogenous dspp (data not shown). DECM
components are among the most prominent molecules that are regulated by
TGF-
1. Increased and abnormal accumulation of DECM was detected in
the teeth of transgenic mice by Masson's trichrome staining (Fig. 4,
b and f). Further, we examined the expression of
collagens I and III in the dentin and the dental pulp by
immunohistochemistry. Increased levels of collagens I and III were
observed in the tooth pulp of dTGF-
1 mice (Fig. 4,
g and h). However, in the dentin, the staining of
collagen I appeared to be either unchanged or slightly reduced, whereas
in the dental pulp the expression was increased (Fig. 4, c
and g). The expression of collagen was not uniformly
distributed in the dental pulp. Interestingly, the collagen III level
was increased in both dentin and dental pulp (Fig. 4, d and
h). It has been reported that the collagen III levels are elevated in osteogenesis imperfecta dentin and also in DGI dentin, suggesting the incomplete differentiation or maturation of the odontoblasts (19).
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Fig. 4.
Immunohistochemical analysis of
TGF- 1 and collagenous components of DECM in
dTGF-
1 mice. Incisors
from 1-day-old wild type (a) and transgenic (e)
mice were cross-sectioned and immunostained with anti-TGF-
1
antibodies. a, the arrow indicates TGF-
1
staining in the odontoblasts. e, the arrows
indicate intense staining of TGF-
1 in the dental pulp of
dTGF-
1 mice. b and f, Masson's
trichrome staining for DECM components. b, blue
staining of DECM (arrow) is noticeable in dentin of the wild
type mouse incisors. f, massive deposition of DECM is seen
as blue stain (arrows) in the dental pulp and
dentin region of dTGF-
1 mice. Immunostaining of collagen
I (c and g) and collagen III (d and
h) in DECM. Collagen I staining in the control animals
(c) follows a similar pattern as DECM staining seen in
b. Incisors of the dTGF-
1 mice show excessive
collagen I staining (arrows) in the dental pulp and dentin
region (g). Collagen III staining in the control group is
meager (d), whereas in the dTGF-
1 group, there
is increased staining (arrows, h). am,
ameloblasts; de, dentin; od, odontoblasts;
p, pulp.
1 teeth suggested a defect in the mineralization. Therefore, we examined the calcium levels
in the undemineralized ground sections from the wild type and
transgenic mice teeth by von Kossa's staining (Fig.
5A). Uniform distribution of
calcium was detected in the mineralized dentin and also in enamel of
wild type mice (Fig. 5A, panel a). Interestingly, in dTGF-
1 mouse teeth, the overall expression of calcium
appeared to be elevated and unevenly distributed (Fig. 5A,
panel b).
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Fig. 5.
Tooth calcification studies on
dTGF- 1 mice.
A, undemineralized sections from 1-month-old wild type
(panel a) and dTGF-
1 (panel b)
molars stained by von Kossa's method. en, enamel;
de, dentin. B, analysis of expression of
dspp; Northern blot analysis of total RNA from teeth of wild
type and dTGF-
1 mice. The dspp mRNA level
was significantly reduced in teeth of dTGF-
1 mice as
compared with controls, whereas the GAPDH level remained unaffected.
dspp and GAPDH probes were as described earlier (18).
C, expression of dspp mRNA by in
situ hybridization. Frozen cross-sections of the tooth from
1-week-old animals were processed and probed with a
digoxigenin-labeled dspp ribo-probe for in
situ hybridization. Distinct dark staining pattern for the
presence of dspp transcripts is observed in the incisors
(panel a) and molars (panel c) of the wild type
animal. Significant reduction in dspp transcripts is
observed in the transgenic incisors (panel b) and molars
(panel d).
1 in the Teeth--
Because the dTGF-
1 tooth
phenotype resembles dentin dysplasia and DGI, we examined the
expression of the dspp gene that has been implicated in the
etiology of the DGI II subtype (28). Northern analysis of tooth RNA
using dspp exon-IV DNA as a probe revealed a significant
reduction in the levels of dspp transcripts in
dTGF-
1 mice (Fig. 5B). Furthermore, we also
examined the odontoblast specific expression of the dspp
gene by in situ hybridization using the same dspp
riboprobe. The expression of dspp mRNA was detected only
in the odontoblasts of both incisors and molar teeth of the wild type
mice (Fig. 5C, panels a and c). The
odontoblast specific expression of dspp gene (Fig.
5C, panels b and d) was significantly
reduced in the transgenic mouse teeth, confirming the reduction seen in
the Northern analysis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 in tooth development, we generated transgenic
mice overexpressing active TGF-
1 from embryonic day 17 in the teeth. We achieved the tissue-specific expression by driving the transgene with mouse dspp gene regulatory sequences, which were well
characterized for the presence of tooth-specific expression in both
in vitro and in vivo model systems (16).
1 teeth displayed a gradual discoloration of
teeth, finally resulting in an opalescent appearance. The teeth were worn progressively or fractured, leaving short stumps. These changes were associated with significantly decreased mineralization of teeth
and abnormal dentin formation. Defective mineralization has been
identified in human autosomal tooth disorders such as DGI (MIM 125490 and 125500), and dentin dysplasia (MIM 125400 and 125420). These
disorders are generally characterized by discoloration and fractures of
teeth associated with poor mineralization of DECM. Also, mutations in
the Col1A1 and Col1A2 genes encoding collagen I
that result in increased deposition and altered assembly of collagen
fibers, major components of DECM, have been described for DGI-I
associated with osteogenesis imperfecta (MIM 166240) (21-23).
1 was observed in dental pulp,
indicating apparent secretion of this growth factor from odontoblasts
into the pulp. In agreement with earlier reports on the inductive
effects of TGF-
1 on differentiation of pulpal cells into
odontoblasts (24, 25) and also on vasculogenesis (26), we observed an apparent increase in pulpal cell mass and also differentiation into
odontoblast-like cells in dTGF-
1 teeth. In these
transgenic mice, the dentinal tubules through which the DECM and other
components are secreted to form dentin were disorganized and reduced in
length as a result of improper polarization and alignment of
odontoblasts (data not shown). This improper organization may impair
the orderly secretion and deposition of collagenous and other molecules
into the mineralization front. Increased vasculature in the pulp and around the odontoblasts was also observed in the mice (data not shown). However, unlike the wild type mice, the pulpal cells of dTGF-
1 mice displayed patchy and ocular staining for
collagen I and III, indicating the differentiation of dental pulp into odontoblast-like cells as a result of secreted TGF-
1 protein. Differentiation of dental pulp into collagen producing odontoblast-like cells has been suggested in in vitro and ex vivo
culture systems by addition of exogenous growth factors, either alone
or in combination, to understand the development of odontoblasts (10).
Increased levels of collagen I and III and their abnormal accumulation
in the pulp seen in dspp/TGF-
1 transgenic mice are
reminiscent of similar changes observed in dentinogenesis imperfecta
and dentin dysplasia (27, 28).
1. In situ hybridization studies have confirmed the reduced expression of dspp
mRNA in the odontoblasts of dTGF-
1 mouse teeth.
dspp expression was undetectable in the odontoblast-like
cells in the pulp canal of transgenic mice. This observation clearly
indicates, despite the high levels of calcium and collagen I in the
pulp canal in dTGF-
1 mouse teeth, that no mineralization
in the dental pulp is detected. In addition to the dentin defects, the
transgenic mice also displayed a defect in enamel mineralization.
Because the main components essential for mineralization in the enamel
are calcium and phosphates, the defect in the enamel mineralization may
not be associated with the reduced expression of the dspp
gene. Moreover, dspp expression has been shown to be
transient during early stages of amelogenesis. Therefore, the
transgenic TGF-
1 is expressed transiently, under the control of
dspp gene regulatory sequences in ameloblasts during early
dentinogenesis, and hence, we speculate the involvement of regulatory
molecules other than dspp, in the enamel mineralization. Most importantly, our studies clearly provide direct experimental evidence, suggesting that the reduction in the dssp gene
expression is associated with the tooth phenotype similar to human
hereditary conditions such as DGI. However, the direct correlation
between either a mutation or decreased expression of the
dspp gene in DGI disorders needs to be investigated.
Interestingly, our preliminary studies on the double transgenic mice
(dTGF-
1X dspp-LacZ generated by crossing
dTGF-
1 mice with dspp-LacZ mice (16)) indicate down-regulation of the LacZ expression suggesting the increased levels
of TGF-
1 negatively regulate the dspp promoter
activity.2 However, in
dTGF-
1 mice there is a clear overexpression of TGF-
1 in teeth presumably because of multiple copies of the transgene encoding active TGF-
1. The increased level of TGF-
1 in teeth is
likely to accelerate its signaling cascade resulting in decreased dspp gene expression, which may result in DGI and dentin
dysplasia. A detailed examination of the downstream TGF-
1 signaling
pathway will be important in identifying the molecular events
underlying these dental disorders.
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ACKNOWLEDGEMENTS |
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We thank Drs. Henning Birkedal -Hansen, John Letterio, Anita Roberts, Larry Wahl, Yoshi Yamada, and Mary Jo Danton for critical reading of the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Functional Genomics Unit, NIDCR, NIH, Bldg. 30, Rm. 529, 30 Convent Dr., Bethesda, MD 20892. Tel.: 301-435-2887; Fax: 301-435-2888; E-mail: ak40m@nih.gov.
Published, JBC Papers in Press, December 14, 2000, DOI 10.1074/jbc.M010502200
2 T. Sreenath, T. Thyagarajan, and A. B. Kulkarni, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
DECM, dentin
extracellular matrix;
DGI, dentinogenesis imperfecta;
DSPP, dentin
sialophosphoprotein;
TGF, transforming growth factor;
dTGF-1, dspp-TGF-
1
transgenic.
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REFERENCES |
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