Transcription Factor FoxO1 Is Essential for Enamel
Biomineralization
Ross A. Poché1, Ramaswamy Sharma3, Monica D. Garcia1, Aya M. Wada1, Mark J. Nolte4, Ryan S. Udan1,
Ji-Hye Paik5, Ronald A. DePinho6, John D. Bartlett3, Mary E. Dickinson1,2*
1 Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas, United States of America, 2 Program in Developmental Biology, Baylor
College of Medicine, Houston, Texas, United States of America, 3 Department of Cytokine Biology, Forsyth Institute, and Department of Developmental Biology, Harvard
School of Dental Medicine, Cambridge, Massachusetts, United States of America, 4 Department of Genetics, University of Texas M. D. Anderson Cancer Center, Houston,
Texas, United States of America, 5 Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, New York, United States of America,
6 Departments of Medical Oncology, Medicine, and Genetics, Belfer Institute for Applied Cancer Science, Dana-Farber Cancer Institute and Harvard Medical School,
Boston, Massachusetts, United States of America
Abstract
The Transforming growth factor b (Tgf-b) pathway, by signaling via the activation of Smad transcription factors, induces the
expression of many diverse downstream target genes thereby regulating a vast array of cellular events essential for proper
development and homeostasis. In order for a specific cell type to properly interpret the Tgf-b signal and elicit a specific
cellular response, cell-specific transcriptional co-factors often cooperate with the Smads to activate a discrete set of genes in
the appropriate temporal and spatial manner. Here, via a conditional knockout approach, we show that mice mutant for
Forkhead Box O transcription factor FoxO1 exhibit an enamel hypomaturation defect which phenocopies that of the Smad3
mutant mice. Furthermore, we determined that both the FoxO1 and Smad3 mutant teeth exhibit changes in the expression
of similar cohort of genes encoding enamel matrix proteins required for proper enamel development. These data raise the
possibility that FoxO1 and Smad3 act in concert to regulate a common repertoire of genes necessary for complete enamel
maturation. This study is the first to define an essential role for the FoxO family of transcription factors in tooth
development and provides a new molecular entry point which will allow researchers to delineate novel genetic pathways
regulating the process of biomineralization which may also have significance for studies of human tooth diseases such as
amelogenesis imperfecta.
Citation: Poché RA, Sharma R, Garcia MD, Wada AM, Nolte MJ, et al. (2012) Transcription Factor FoxO1 Is Essential for Enamel Biomineralization. PLoS ONE 7(1):
e30357. doi:10.1371/journal.pone.0030357
Editor: Rebecca Berdeaux, University of Texas Health Science Center at Houston, United States of America
Received September 1, 2011; Accepted December 14, 2011; Published January 24, 2012
Copyright: ß 2012 Poché et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: These studies were supported by funding from the National Institutes of Health R01EB005173 (to MED) and P20EB0070769 (to MED) as well as
Institutional Core Grant #CA16672 High Resolution Electron Microscopy Facility, University of Texas, M.D. Anderson Cancer Center. The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: mdickins@bcm.edu
ultimate thickness of the mature enamel layer. Next, during the
transition stage, the ameloblasts reduce their deposition of
extracellular matrix proteins and convert from the columnarshaped secretory ameloblasts to the more shortened maturation
ameloblasts. It is during the maturation stage that minerals are
deposited on the sides of the fully elongated crystallites resulting
in an increase in thickness and width. This structure is further
elaborated until the thickening parallel crystals come into contact
with adjacent crystals [8,9].
Genetic studies of amelogenesis imperfecta patients and mice
with mutations in the genes encoding the enamel matrix proteins
show that these proteins are critical for the proper growth and
maturation of enamel crystals [10,11,12,13,14,15,16,17]. However, these proteins are not part of the mature enamel structure.
To complete mineralization of the enamel matrix, in which
enamel rods grow firmly against one another and become
mechanically interlocked, the extracellular matrix components
must be digested and reabsorbed by the ameloblasts [8]. In the
mouse, both Matrix metalloproteinase 20 (Mmp-20) and
Kallikrein 4 (Klk4) have been shown to have enamel matrix
protein protease activity in vivo and both Mmp-20 and Klk4
Introduction
The process of biomineralization is observed throughout
metazoans and results in the generation of biologically important
tissues such as shells, carapaces, spicules, bones and teeth [1].
The biomineralization of the mammalian tooth is a particularly
striking case as dental enamel contains less than 1% organic
matter by weight and is predominantly composed of a highly
ordered lattice of calcium hydroxyapatite (Ca10(PO4)6(OH)2)
crystals making it the hardest mineralized tissue known [2]. Prior
to the development of the mature protein-free enamel structure,
enamel formation consists of specific cellular events termed the
secretory, transition and maturation stages [3,4]. During the
secretory stage, specialized, ectodermally-derived cells called the
ameloblasts deposit a complex extracellular matrix composed of
amelogenin, ameloblastin, enamelin and other proteins [5,6,7].
These enamel matrix proteins are thought to promote the
formation and elongation of thin ribbons of hydroxyapatite
crystallites which lengthen parallel to one another while not
growing in width [8]. At the end of the secretory stage, the
enamel ribbons have reached their full-length thus defining the
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FoxO1 Regulates Biomineralization
knockout mice exhibit malformed enamel [18,19]. Also, mutations in MMP-20 and KLK-4 were reported in amelogenesis
imperfecta patients [20,21,22,23,24]. Interestingly, Mmp-20 is
expressed and deposited within the extracellular matrix by the
ameloblasts during the secretory stage coincident with Amelogenin, Ameloblastin and Enamelin. It is believed that Mmp-20
cleavage of the extracellular matrix permits hydroxyapatite
crystal elongation rather than thickening [8]. Klk4 is expressed
beginning at the transition stage and into the maturation stage;
however, it has not been detected in secretory ameloblasts [19].
Klk4 is thought to catalyze the proteolytic degradation of the
residual extracellular matrix thereby providing additional free
space for the elongated enamel crystals to expand in width,
contact adjacent crystals and interlock [19]. Overall, the process
of enamel biomineralization requires tight spatial and temporal
control of numerous genes which also interact post-translationally. Thus, we hypothesized that an ameloblast-specific mechanism of transcriptional regulation likely coordinates this highly
specialized developmental process.
Previously, the Smad3 transcriptional co-factor was implicated
in the regulation of biomineralization in vivo, as mice with a
targeted mutation in Smad3 exhibit a hypomineralized tooth
phenotype [25,26]. Given the well-documented role of Smad3
(together with the co-Smad, Smad4) as an intracellular molecule
that mediates signaling from the Transforming growth factor-b
(Tgf-b) receptor, we reasoned that Tgf-b signaling might activate a
repertoire of genes necessary for the coordination of biomineralization [27]. Consistent with this idea, many studies have already
implicated Tgf-b signaling as being important for proper
craniofacial development, including tooth formation [28]. However, due to various functional redundancies between Tgf-b
signaling molecules, receptors, Smads and Smad transcriptional
co-factors, a complete picture of Tgf-b signaling as it relates to
specific cellular events during tooth development and maturation
remains elusive. Further complicating the issue is the multifunctional role Tgf-b signaling can serve in any given tissue. Thus,
targeted deletion of Tgf-b components in mice often results in
early embryonic lethality or other pleiotropic effects which make it
difficult to assign specific functions to specific components of the
pathway.
When considering the broad transcriptional changes which
generally occur in response to Tgf-b, how is the Tgf-b signal
interpreted by differentiated ameloblasts as a cue to coordinate
the expression of specific genes essential for the completion of
such specialized developmental processes as enamel biomineralization? Due to studies suggesting that the Smad complex
alone is usually insufficient for target gene binding and activation
[29], transcription target specificity likely depends on the
association with specific Smad transcriptional co-factors expressed coincidentally within the ameloblasts. Members of the
forkhead box O family of transcription factors, which serve
diverse roles such as cellular growth and proliferation, development, metabolism and longevity [30], have been shown to
function as Smad co-factors in vitro [31,32,33]. Here, via a
conditional knockout approach, we show that ameloblast-specific
loss of Forkhead box O transcription factor 1 (FoxO1) results in
an enamel hypomaturation phenotype that is reminiscent of the
Smad3 mutant [25]. Furthermore, we also show that both FoxO1
and Smad3 mutants exhibit a reduction in the expression of a
similar cohort of genes encoding enamel matrix proteins. These
data raise the possibility that FoxO1 acts as a Smad co-factor,
within an ameloblast-expressed transcriptional complex, to
regulate a specific set of genes required for proper enamel
biomineralization.
PLoS ONE | www.plosone.org
Results
Ameloblast-specific loss of FoxO1 results in a white tooth
phenotype
In an independent study, we aimed to address the role of FoxO1
in retinal development by crossing the FoxO1 floxed allele [34] to
the Rx-Cre recombinase line [35]. The Rx-Cre+/tg; FoxO flox/flox mice
were born at a normal Mendelian frequency and were viable and
fertile. However, the retinae in these mice had normal thickness
and the cellular composition was indistinguishable from controls
(data not shown). Upon further examination of the mutant mice,
we found that 100% of the FoxO1 conditional knockouts exhibited
a chalky, white tooth phenotype that was first apparent by 1
month postnatally and more pronounced by 3 months (Figure 1A,
1C). Extracted maxillary incisors showed that the proximal,
immature enamel of the un-erupted mutant incisor appeared
yellowish-brown in color similar to controls (Compare Figure 1B
to 1D, arrows). However, in the more distal incisor where the
tooth has erupted, we noticed that the FoxO1 mutants were
progressively whiter toward the incisal edge (Compare Figure 1B
Figure 1. Gross phenotype of the FoxO1 mutant teeth. Rx-Cre
and K14-Cre-mediated knockout of FoxO1 results in viable, fertile mice
exhibiting abnormally white, chalky incisors (compare A to C and E to
G). Extracted incisors had intact enamel on the un-erupted surface of
the incisor that appeared similar to controls (arrows in B, D, F and H).
However, the most distal, erupted region of the mutant incisors was
white in appearance suggesting enamel attrition due to occlusal forces
(arrowheads in B, D, F and H). FoxO1 is expressed in differentiated
postnatal ameloblasts (Am) (I). qrtPCR analysis of postnatal conditional
knockout whole incisors confirmed the expected reduction of FoxO1
transcript (J).
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to 1D, arrowhead). This finding suggested that the enamel on
FoxO1 mutant teeth experienced extensive attrition due to
chewing. Importantly, we also analyzed two control groups, RxCre+/tg; FoxO+/flox and Rx-Cre+/tg mice and they were found to be
phenotypically normal (data not shown).
Given the documented forebrain and retinal-specific activity of
Rx-Cre [35], a tooth phenotype was surprising. Since the Rx-Cre is
expressed within the forebrain early in development, it is possible
that Cre expression is broader than previously appreciated,
encompassing precursors of the enamel producing ameleoblasts.
To show that FoxO1 is required in ameloblasts, we crossed the
FoxO1 floxed allele to mice carrying the Keratin14 (K14)-Cre
transgene, which has been shown to drive Cre activity within
ectodermally-derived tissues including the ameloblasts [36,37]. As
expected, K14-Cre+/tg; FoxO1flox/flox mice also exhibited a white
tooth phenotype similar to the Rx-Cre+/tg; FoxO1flox/flox mutants
(Compare Figure 1E to 1G, arrows). These mutants also showed
the same progressive proximal to distal whitening of the incisor
surface (Compare Figure 1F to 1H).
To further confirm a role for FoxO1 in the mouse ameloblasts,
we labeled cryosections of postnatal day 7 (P7) incisors with antiFoxO1 antibodies and 49,6-diamidino-2-phenylindole (DAPI)
nuclear dye. Upon confocal microscopy, we found that FoxO1
was expressed ubiquitously throughout the mouse incisors and
molars and co-localized with the DAPI signal (Figure 1I and data
not shown). The nuclear expression of FoxO1 was consistent with
the known role of FoxO1 as a transcription factor. Furthermore,
we performed quantitative reverse transcriptase polymerase chain
reaction (qrtPCR) on mRNA isolated from adult FoxO1 conditional mutant and control incisor enamel organs and found that
the FoxO1 conditional mutants exhibited a 65% reduction
(P,0.001) in FoxO1 mRNA levels relative to the controls
(Figure 1J). Given the broad expression of FoxO1, the residual
FoxO1 mRNA observed in the conditional mutants is likely due to
the presence of cells outside the ameloblast layer which did not
undergo Cre-mediated recombination.
For final confirmation that the Rx-Cre+/tg; FoxO1flox/flox mutant
phenotype was due to specific loss of FoxO1 within the ameloblasts,
we crossed the Rx-Cre line to mice expressing the ROSA26+/lacZ
Cre reporter [38], thus generating Rx-Cre+/tg; ROSA26R+/lacZ
progeny. Postnatal day 7 heads were cryosectioned and
ROSA26+/lacZ Cre reporter activity was assessed via 5-bromo-4chloro-3-indolyl-b-D-galactopyranoside (X-gal) staining. As expected, we observed lacZ activity in a wide array of tissues within
the head and facial regions including hair follicles, retina, skin, oral
epithelium, tongue, bone and teeth (Figure 2A and data not
shown). Within the teeth, lacZ expression was confined predominantly to the ameloblast layer and we did not observe expression
within the odontoblasts (Figure 2B–C). Furthermore, the ameloblasts of the incisors exhibited uniform expression of lacZ whereas
the molars had a variable mosaic pattern of expression in which
regions of the ameloblast layer were negative for lacZ (Compare
Figure 2C to 2E, arrows). The basis for the mosaic Cre activity in
the molars is not known.
Figure 2. Within the tooth, Rx-Cre activity is confined to the
ameloblast layer. Rx-Cre+/tg mice were crossed to the ROSA26+/lacZ Cre
reporter line and Cre activity was assessed via X-gal staining. Rx-Cre
exhibited broad activity throughout the anterior head in such tissues as
the retina, tongue, skin, hair follicles, and teeth (A). Closer inspection of
the incisors (B and C) and the molars (D and E) revealed that Cre activity
was confined to the ameloblast layer (Am) and completely absent from
the odontoblast layer (Od). The Cre activity in the molars was more
mosaic than the incisors (arrows in E).
doi:10.1371/journal.pone.0030357.g002
exhibited dramatic attrition of the enamel surface such that the
cusps of the molars were smooth in appearance as compared to
controls (Figure 3A–D). We also examined the surface of the
mandibular incisors and found that the FoxO1 mutants had a
rough appearance in stark contrast to the smooth surface of the
control incisors (Compare Figure 3E to 3F). Analysis of maxillary
incisors of 3 month old mutants also revealed a similar defect
(Figure 3G and 3H). Upon close inspection, regions of the enamel
surface appeared to erode away thereby creating the appearance
of valleys (Figure 3H, arrows). We also observed small, discrete
holes within the enamel surface ranging from approximately 0.5 to
2.0 microns in diameter (Figure 3H, arrowheads). Taken together,
these data suggest that the FoxO1 mutant tooth enamel is weaker
than the control mice and erodes away over time. However,
subsequent imaging of mutant enamel layer cross-sections in
fractured incisor preparations revealed that the thickness of the
enamel layer as well as the decussating pattern of the enamel rods
appeared similar to the controls (Figure 4A–D). This finding
suggested that the secretory stage ameloblasts of the FoxO1
mutants are at least partially functional. However, despite
FoxO1 mutant tooth enamel is softer than controls
Given the white tooth phenotype of the FoxO1 mutants and
activity of Rx-Cre and K14-Cre within the ameloblasts, we reasoned
that FoxO1 deletion within the ameloblasts led to hypomaturation
of the enamel layer. In order to examine the enamel structure in
greater detail, control and FoxO1 mutant maxillae and mandibles
were removed and the periradicular bone was dissected away. The
exposed molars and incisors were analyzed via scanning electron
microscopy (SEM). At 15 months, the FoxO1 mutant molars
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Figure 4. FoxO1 mutant teeth deposit an enamel matrix and
form enamel rods. Scanning electron microscopy of fractured 15
month old FoxO1 mutant incisors revealed that, in regions where
enamel had not chipped away, the mutant teeth initially had an enamel
thickness that was comparable to their littermate controls (compare A
and B). Higher magnification revealed that the mutant enamel also
exhibited the typical decussating pattern of enamel rods (compare C
and D).
doi:10.1371/journal.pone.0030357.g004
Figure 3. FoxO1 mutant teeth suffer from severe enamel
attrition. Scanning electron microscopy revealed that 15 month old
FoxO1 mutant molars displayed a pronounced wearing of the enamel
layer to the extent that the molar cusps had almost completely eroded
away (compare A and B and high magnification views in C and D). The
15 month old mutant incisors had a similar phenotype in which the
labial surfaces of both the maxillary (shown) and mandibular (not
shown) incisors had a dramatic chipping of the enamel from the dentin
layer giving the incisors a rough appearance (compare E to F). This
phenotype was also seen as early as 3 months (compare G to H) and
higher magnification revealed discrete valleys (arrows in H) and holes
(arrowheads in H) in the enamel surface.
doi:10.1371/journal.pone.0030357.g003
to transition between different stages of amelogenesis that could
explain the resulting weakened enamel structure. To assess
ameloblast cytoarchitecture, we dissected and decalcified maxillary
and mandibular incisors from 15 month old control and FoxO1
mutant littermates and performed hematoxylin and eosin-Y (H&E)
staining on paraffin sections. Interestingly, in control and mutant
mice, both the secretory and maturation stage ameloblast layers
appeared very similar in terms of overall cell density, organization
and polarization (Figure 6 A–D). Thus, it is unlikely that the
observed FoxO1 mutant soft enamel phenotype is due to defects in
ameloblast genesis and/or differentiation. Interestingly, Smad3
mutants show a very similar white tooth phenotype yet also fail to
substantial enamel matrix deposition, our data indicated that the
FoxO1 mutant enamel does not undergo normal maturation
resulting in compromised enamel strength.
In order to confirm our interpretation of the FoxO1 mutant
SEM data as indicating an enamel hypomaturation phenotype, we
next attempted to determine whether the FoxO1 mutant enamel is
softer than controls. To do this, we subjected littermate control
and mutant teeth to an enamel microhardness test. Microindentations were created on maxillary incisors from 9 week old
FoxO1 mutant and control mice (n = 3 per group). The average
hardness for control enamel was found to be 631.4 VHN (SEM 6
17.78) whereas the FoxO1 mutant enamel was 547.7 VHN (SEM
6 17.28). The difference was statistically significant (p = 0.0279)
and the results demonstrated that the mature mutant enamel was
approximately 13.3% softer than control enamel (Figure 5). We
attempted the same analysis on 15 month old teeth, but we were
unable to identify large enough, intact enamel surfaces on which to
perform the test.
FoxO1 loss-of-function does not affect gross ameloblast
development or differentiation
Figure 5. FoxO1 mutant enamel is softer than controls. The
Vickers microhardness test revealed that the adult FoxO1 mutant teeth
are significantly softer than their littermate controls.
doi:10.1371/journal.pone.0030357.g005
Since our data support a requirement for FoxO1 in ameloblasts,
we next sought to determine whether the FoxO1 mutants exhibited
a decrease in ameloblast density, failure of polarization or a failure
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(compare Figure 8A to 8B and Figure 8C to 8D). While
immunofluorescence provides spatial information regarding gene
product expression, it cannot be reliably used as quantitative
assessment. Thus, we next performed qrtPCR analysis of adult
FoxO1 mutant and control incisors. Given the previously
documented role of FoxO transcription factors as Smad
transcriptional co-activators [31,32,33], the expression of FoxO1
and Smads within endogenous ameloblasts [41,42,43] and the
white tooth phenotype of the FoxO1 and Smad3 mutant mice
(Figure 1 and Figure 7) [25], we decided to include Smad3 mutant
teeth in our qrtPCR analysis. We extracted adult maxillary and
mandibular incisors from FoxO1 mutant, Smad3 mutant and
control mice and purified mRNA for subsequent qrtPCR analysis.
It should be noted that littermate controls were used in every case
so that genetic background was consistent with that of the
respective mutant. Upon analysis of transcript levels, we found
that Ameloblastin (Ambn), Amelogenin (Amel), Enamelin (Enam), Mmp20
and Klk4 mRNA levels were all significantly reduced in both the
FoxO1 and Smad3 mutant mice as compared to controls with the
enamel matrix proteins (including amelogenin) showing the
greatest reduction (Figure 9A–B). Furthermore, the relative
reduction of Ameloblastin, Amelogenin, Enamelin, Mmp20 and Klk4
expression had a very similar trend in both FoxO1 and Smad3
mutants with Enamelin being the most reduced and Klk4 being the
least reduced. Since FoxO1 loss-of-function in other tissues during
development has been shown to cause a dramatic reduction in the
expression of Connexin-37 (Cx37) and Connexin-40 (Cx40) mRNA
levels (47), we included these genes in our analysis [44]. We also
included Connexin-43 (Cx43) as Cx43 mutants exhibit an enamel
maturation defect [45,46,47]. Loss of FoxO1 or Smad3 has no effect
on the levels of Cx37 or Cx40 mRNA (Figure 9A–B). The level of
Cx43 was modestly down-regulated in the Smad3 mutants but not
significantly reduced in the FoxO1 mutants. Finally, we determined
that Smad3 is normally expressed in the FoxO1 mutants and FoxO1
is normally expressed in the Smad3 mutants. Taken together, these
data suggest that FoxO1 and Smad3 potentially regulate a
common set of genes necessary for enamel formation and
maturation.
Figure 6. The morphology of FoxO1 mutant ameloblasts
appears unaffected. Hematoxylin and EosinY staining of paraffin
sections of decalcified control and FoxO1 adult mutant teeth showed
that mutant secretory stage (A and B) and maturation stage (C and D)
ameloblasts (Am) appeared structurally similar to controls.
doi:10.1371/journal.pone.0030357.g006
exhibit obvious defects in the morphology of the ameloblast layer
(Figure 7) [25].
FoxO1 and Smad3 mutants exhibit dysregulation of a
similar cohort of genes necessary for biomineralization
When considering the known role of FoxO1 as a transcription
factor, we next sought to address whether FoxO1 regulates a set of
genes that might provide insight into the enamel hypomaturation
phenotype. Since the FoxO1 mutants failed to exhibit an obvious
change in ameloblast specification and differentiation, we decided
to focus our analysis predominantly on genes that have been
directly implicated in the process of enamel biomineralization.
Since Amelogenin proteins constitute 90% of the ameloblast
extracellular matrix and are essential for normal enamel
organization and maturation [11,39,40], we began our analysis
by assessing Amelogenin protein expression in FoxO1 mutant and
control teeth. Using antibodies against Amelogenin, we performed
immunofluorescent analysis of cryosections from 7 day and 5
month old FoxO1 mutant and control incisors. Confocal
microscopy suggested that the FoxO1 mutant Amelogenin protein
expression levels were reduced as compared to littermate controls
In silico analysis of the enamel matrix proteins revealed
conserved FoxO and Smad binding elements
Next, using a method similar to previous studies [31,32,33], we
performed in silico analysis to map putative FoxO and Smad
binding elements within the genes that showed a transcriptional
decrease in the FoxO1 and Smad3 mutants (Figure 10). As a positive
control, to ensure our search method was comparable to previous
methods, we included searches of regions upstream of p21Cip1 and
p15Ink4a as these have been previously mapped and were
validated experimentally [31,32,33]. Also, since its binding sites
are occasionally located within FoxO/Smad binding site clusters
[32,33], we searched for elements recognized by the transcription
factor C/EBPb (CBEs). For this analysis, we used mouse genomic
sequence from 4 kb upstream of the transcription start site to the
stop codon of each gene. FoxO binding elements (also know as
forkhead-binding elements or FHBEs) [(G/A)(T/C)AAA(T/A)A]
and Smad binding elements (SBEs) [AGAC] (within 100
nucleotides upstream and downstream of the FoxO elements)
were then identified using the dual site matching program.
Putative FoxO and Smad elements were localize upstream of the
transcription start site of Ameloblastin, Amelogenin and Enamelin.
Furthermore, Amelogenin contained a single C/EBPb element
within the same FoxO/Smad cluster. Ameloblastin and Enamelin
contained additional sites within the first exon and intron. Mmp20
contained only a single FoxO/Smad cluster and it was localized to
Figure 7. Smad3 mutants suffer from a white, chalky tooth
phenotype reminiscent of the FoxO1 mutants.
doi:10.1371/journal.pone.0030357.g007
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Figure 8. FoxO1 mutant incisors exhibit an apparent reduction in amelogenin protein expression. Immunofluorescent analysis of
cryosections derived from P7 and 5 month old FoxO1 and littermate control mice revealed that FoxO1 mutants exhibit a qualitative reduction in
Amelogenin protein expression. This reduction in fluorescent signal was observed in both the ameloblasts (Am) and the enamel matrix (EM).
doi:10.1371/journal.pone.0030357.g008
transcriptional start sites of Ameloblastin, Amelogenin and Enamelin.
Interestingly, in the FoxO1 and Smad mutants, these three genes
also showed a greater reduction in expression levels as compared
to Mmp20 and Klk4 proteases which did not contain upstream
FoxO or Smad binding elements. These data raise the possibility
that a FoxO1/Smad3 complex may directly regulate a subset of
genes that must be expressed in an overlapping temporal and
spatial manner and serve a similar role during enamel development. However, unequivocal demonstration of such a complex
directly regulating the expression of enamel matrix proteins in
ameloblasts in vivo awaits FoxO1/Smad3 co-immunoprecipitation
and chromatin immnoprecipitation analyses of adult mouse
incisors.
Surprisingly, loss-of-function studies of genes encoding the
enamel matrix proteins (Ameloblastin, Amelogenin, and Enamelin) exhibit a phenotype which is more severe than the FoxO1 or
Smad3 mutants. These mutants have dramatic defects in the
development of the enamel in which the normal prismatic enamel
structure is severely disrupted or the enamel completely fails to
form [10,11,14,48,49]. In the FoxO1 mutant teeth, the enamel
prisms form but fail to become fully mineralized resulting in a
weakened structure. Furthermore, while the Ameloblastin, Amelogenin, and Enamelin mutant ameloblasts show morphological
abnormalities such as loss of cell polarity, cyst-like structures and
detachment from the extracellular matrix [10,11,14,48,49], the
FoxO1 and Smad3 ameloblasts appear morphologically normal
[25]. The relatively mild defects observed in the FoxO1 and Smad3
mutants are likely explained by the finding that these mutants still
express appreciable levels of the major enamel matrix proteins.
However, the levels are significantly reduced from wild type. It is
worth noting that the Enamelin knockout mice exhibit an
autosomal dominant enamel phenotype that appears less severe
the first intron. Klk4 did not contain any putative FoxO, Smad or
C/EBPb elements. These data suggest that at least a subset of the
genes down-regulated in the FoxO1 and Smad mutants may be
direct targets of the FoxO/Smad transcriptional complex that has
been previously identified in epithelial cells [31,32,33] and that, in
some cases, C/EBPb may provide additional regulation.
Discussion
Here, we have uncovered a novel role for FoxO1 in enamel
maturation as well as a molecular entry point from which to
further elucidate the mechanism of transcriptional control over
mammalian biomineralization. While the FoxO1 mutants exhibit
soft tooth enamel which undergoes severe attrition over time, we
did not observe any obvious dysplasia of the ameloblasts. This
phenotype is strikingly similar to that of the Smad3 mutant mice
[25]. Based on such a close phenocopy between these two mutants,
we hypothesized that these genes play similar roles in enamel
development. Indeed, we demonstrated that the FoxO1 and Smad3
mutants exhibit down-regulation of a similar cohort of genes
known to be important for enamel development and maturation.
Previously, in in vitro studies of epithelial cells, it was shown that
FoxO and Smad proteins exist in a TGF-b-dependent transcriptional complex which regulates the expression of a common set of
genes termed a synexpresison group [31,32,33]. These genes
comprise a functionally diverse group including mediators of
cellular stress and cystostatic responses. This finding raises the
possibility that a similar transcription factor complex functions in
ameloblasts (the epithelial component of the tooth) to regulate
enamel matrix protein expression. Consistent with this idea, in
silico mapping of FoxO and Smad binding sites uncovered clusters
of conserved regulatory elements located upstream of the
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Figure 9. Loss of FoxO1 and Smad3 results in the down-regulation of a common set of genes. Quantitative rtPCR was performed on mRNA
isolated from adult FoxO1 mutant (A) and Smad3 mutant (B) incisors and compared to control littermates. Both mutants exhibited a similar trend in
the down-regulation of genes known to be necessary for proper enamel development and maturation (*p,0.05, **p,0.01, ***p,0.001).
doi:10.1371/journal.pone.0030357.g009
than the FoxO1 and Smad3 mutants [13]. The mandibular incisors
of these mice were white and chalky in appearance and exhibited
attrition of the erupted portions. However, as in the FoxO1 mutant
mice, the enamel prisms looked normal. These data suggests that
the Enamelin heterozygous mutant enamel forms but is softer than
normal. Interestingly, among all the putative FoxO/Smad target
genes analyzed in this study, Enamelin mRNA levels showed the
greatest reduction. Thus, it is formally possible that the reduction
of Enamelin expression is the main contributing factor to the FoxO1
and Smad3 mutant enamel phenotypes.
In a recent study, FoxO1 was shown to directly induce the
expression of transcription factor Runx2 (Cbfa1) as well as
physically associate with Runx2 protein to regulate genes
essential for osteoblast differentiation and skeletogenesis [50].
Of further significance is the finding that Runx2 has the ability to
bind to regions of the Ameloblastin promoter and possibly
PLoS ONE | www.plosone.org
participate in the control of Ameloblastin transcription [51]. Also,
Runx2 was shown to be endogenously expressed in secretory and
maturation ameloblasts, and loss of Runx2 expression results in
severely hypoplastic teeth lacking definitive odontoblast and
ameloblast differentiation [52,53,54,55]. Thus, it is possible that
FoxO1 and Runx2 may be functionally related during enamel
maturation. However, we performed qrtPCR to assess the levels
of Runx2 expression in FoxO1 mutant incisors and found that
Runx2 (and Runx1) levels were the same as controls (not shown). It
is also important to recognize that the documented interaction of
FoxO1 and Runx2 during osteoblast differentiation occurs in
mesenchymal cells [50] while enamel formation is driven by the
ameloblasts, which are epithelial cells. Thus, one can envision
dramatic differences in terms of Runx2 function depending on
the specific cellular context. Indeed, it has been shown that TGFb signaling to osteoblasts, via Smad3, has the ability to repress the
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FoxO1 Regulates Biomineralization
Figure 10. Mapping of putative FoxO/Smad genomic binding sites. In silico analysis of putative FoxO, Smad, and CEBPb binding elements,
conserved between mice and humans and residing within several genes down-regulated in the FoxO1 and Smad mutants (see text for further details).
The colored numbers indicate additional SBEs or CBEs that, due to scaling, could not be represented on the gene tracks.
doi:10.1371/journal.pone.0030357.g010
differentiation, we do not believe this scenario to be likely. The
K14-Cre transgene has been shown by several groups to be active
at the E12 tooth bud stage which is several days before
ameloblast differentiation occurs [60,61] and K14-Cre-mediated
conditional knockouts of other genes have been shown to disrupt
ameloblast differentiation and organization [61,62]. Furthermore, it is well-known that rodent incisors contain a stem cell
niche called the cervical loop from which ameloblasts continuously renew throughout the lifetime of the animal [63,64]. It is
also known that K14-Cre and Rx-Cre (shown in our study,
Fig. 2B) are active in the cervical loop [36]. Thus, the ameloblast
stem cell population would be expected to lose FoxO1 expression
thereby giving rise to FoxO1-deficient ameloblasts in the adult.
Since, we only observed an enamel maturation defect in the
mutant mice, this would argue against a role for FoxO1 in
ameloblast differentiation.
Our study of the role of FoxO1 in mouse ameloblasts has
established a critical molecular entry point which will allow
researchers to delineate novel genetic pathways regulating the
process of biomineralization. FoxO1 and Smads, either directly or
indirectly, likely control the expression of numerous genes, possibly
in addition to enamel matrix proteins, which are essential for the
completion of tooth mineralization. Some of these genes may also
transcription of Runx2 as well as Runx2 protein activity [56,57].
However, whether this particular function of Smad3 is FoxOdependent was not addressed. Other transcription factors
potentially functioning in concert with FoxOs or Smads in
enamel maturation are C/EBPb and related family members.
One member, C/EBPa, was shown to be a transcriptional
activator of the mouse Amelogenin gene in vitro. Interestingly,
ameloblast specific ablation of C/EBPa did not result in an
enamel phenotype or loss of Amelogenin expression and this was
shown to be due to redundancy with C/EBPd [58,59]. By
employing future bioinformatic experiments to map FoxO/Smad
binding elements, we should be able to uncover more putative
target genes. By combining this approach with subsequent
conditional knockout of target genes and functional validation
of direct binding sites, we will gain increasingly better insight into
the process of enamel formation and maturation in vivo.
Our data show that FoxO1 mutant ameloblasts are morphologically normal yet the enamel is weaker than controls. This
suggests to us that FoxO1 likely does not function in ameloblast
development, but rather in the production of mature enamel.
While it is formally possible that low or mosaic Cre activity at
early tooth developmental stages might preclude the discovery of
a subtle, earlier function of FoxO1 in ameloblast specification and
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FoxO1 Regulates Biomineralization
H&E staining
be dysregulated in human diseases such as amelogenesis
imperfecta.
Maxillary and mandibular incisors from control and FoxO1
mutant littermate mice were fixed overnight in Zinc-Formalin,
decalcified in 5% formic acid, dehydrated and embedded in
paraffin. For staining, sections were re-hydrated and stained with
Hematoxylin and EosinY.
Materials and Methods
Mouse Strains
FoxO1flox/flox [34], Rx-Cre flox/flox [35], K14-Cre flox/flox [65], and
ROSA26R+/lacZ [38] mice were maintained on a mixed C57BL/6J,
FVB/NJ background. Smad32/2 mice were maintained on a
BALB/c background [26]. PCR genotyping was performed as
described in the references indicated above. All animal research
was conducted according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Baylor
College of Medicine (assurance number 3823-01).
X-gal staining and immunofluorescence
Whole postnatal day 7 mouse heads were fixed in 4%
paraformaldehyde for 3 hours at 4uC. After fixation, the heads
were washed in 16 phosphate-buffered saline (PBS, pH 7.3) 3
times for 10 minutes at 4uC. Next, the samples were cryoprotected
by immersing in 15% and then 30% sucrose until the tissue sank to
the bottom of the tubes. Subsequently, the tissue was immersed in
a 1:1 solution of 30% sucrose and OCT medium and left at 4uC
for a couple of hours. Then, the tissue was embedded in OCT on
dry ice and stored at 280uC prior to sectioning. Cryosections were
cut at 20 mm on a cryostat and mounted on Superfrost Plus slides
(VWR Brand, Westchester, PA). X-gal staining was performed at
37uC for 6 hours as previously described [66]. For immunofluorescence of cryosections, slides were post-fixed in 4% PFA for
10 minutes and then washed in 16 PBS-T (PBS+0.1% TritonX100) 3 times for 10 minutes at room temperature. Next, the slides
were blocked in 2% normal donkey serum diluted in 16 PBS for
1 hour at room temperature. anti-FoxO1 (sc49437, Santa Cruz) or
anti-Amelogenin (sc32892, Santa Cruz) primary antibodies were
diluted (1:200) in the same blocking solution and incubated on the
slides overnight at 4uC in a humid chamber. Next, the slides were
washed 4 times at room temperature in 16 PBS. Labeling with
donkey, anti-rabbit Alexa Fluor 488 secondary antibodies (1:400)
(Molecular Probes) was performed using the same blocking
solution (2% donkey serum in 16 PBS) and slides were incubated
for 1 hour at room temperature. Slides were then stained with
DAPI (1:500) and mounted with FluoroMount-G (Southern
Biotech).
Scanning Electron Microscopy
Mandibular and maxillary incisors from 3 animals at each stage
and genotype were mounted whole or were fractured and then
mounted on aluminum stubs to expose the fractured plane.
Samples were sputter-coated for analyzing enamel structure on a
scanning electron microscope (JEOL).
Microhardness Test
Erupted portions of maxillary incisors from 3 FoxO1 mutant and
3 control littermates were fixed overnight in 100 mg/ml
formaldehyde, 10 g/L zinc sulfate, washed and dehydrated with
grade alcohol and acetone. Incisors were embedded sagittally in
hard-formulation epoxy embedding medium (EpoFix, EMS).
Samples were ground and polished to 0.25 mm with a diamond
suspension (EMS). The polished samples were tested for enamel
microhardness on a Leco M 400 HI testing machine (Leco).
Testing was performed with a load of 25 g for 5 sec with a Vickers
tip. Twenty-five indents per sample were measured for hardness.
Statistical significance was determined by one-way student’s t-test
(GraphPad Prism 5).
In silico analysis of FoxO/Smad binding sites
RNA extraction and quantitative rtPCR
Mouse and human genomic sequences were obtained from the
University of California Santa Cruz genome browser (http://
genome.ucsc.edu). Genomic sequence from 4 kb upstream of the
transcription start site to the stop codon of each gene were
extracted from either mouse genome release mm9 (NCBI Build
37, July 2007) or human genome release hg19 (NCBI Build 37,
Feb. 2009). Forkhead binding elements (FHBEs) [(G/A)(T/
C)AAA(T/A)A] and Smad binding elements (SBEs) (AGAC)
within 100 nucleotides upstream and downstream of the FHBE(s)
were then identified in the mouse sequences using the dual site
matching program (http://cbio.mskcc.org/cgi-bin/lash/dualsite).
Binding elements were identified in the dual site program after the
following steps were taken: i) The ‘‘Invert Sites?’’ option marked as
‘‘yes;’’ ii) the known 59-to-39 sequence of the binding element and
its reverse were added into the Primary of Secondary Sites fields
(therefore, noting the SBE sequence to be AGAC, one would enter
this sequence plus the sequence CAGA into the Secondary Sites
field). The identified FHBE-SBE sites detected in the mouse
sequences were BLAST-compared against the homologous human
gene sequences using NCBI bl2seq (http://blast.ncbi.nlm.nih.
gov/Blast.cgi) to confirm conservation between the two species.
Conservation of a FHBE-SBE(s) was only noted on Figure 9 if i)
the FHBE was completely conserved or the conservation was
incomplete by a single nucleotide, ii) if the FHBE conserved
between the two species was not identical by nucleotide character
but exactly matched published FHBE sequences, iii) if the SBE
was completely conserved or the conservation was incomplete by a
Adult control and mutant incisors (n = 3 per genotype), removed
from the surrounding bone and with the enamel organ intact, were
homogenized in liquid nitrogen using a mortar and pestle. The
ground samples were further homogenized in Trizol (Invitrogen)
and total RNA was extracted according to the manufacturer’s
protocol. RNA was subsequently DNAse (Invitrogen) digested to
eliminate genomic DNA, and purified using the RNeasy RNA
clean up kit (Qiagen). The purified RNA was reverse transcribed
using the Superscript III first strand synthesis kit with Oligo(dT)20
and random hexamer priming (Invitrogen). The TaqmanH gene
expression assay from Applied Biosystems was used for qrtPCR,
and primer pairs from Applied Biosystems were used to detect
gene expression: Gja1/Cx43 (Mm00439105_m1), Gja5/Cx40
(Mm01265686_m1), Gja4/Cx37 (Mm01179783_m1), Mmp20
(Mm00600244_m1), FoxO1 (Mm00490671_m1), Klk4 (Mm00517338
_m1), Amelogenin (Mm00711644_g1), Ameloblastin (Mm00477485_m1),
Enamelin (Mm00516922_m1), and Smad3 (Mm00489638_m1).
qrtPCR was performed on an Applied Biosystems’ ABI PRISM
7000 Real time PCR System under the following PCR conditions:
50uC for 2 min., 95uC for 2 min., 40 cycles of 95uC for 15 seconds
and 60uC for 1 minute. To determine relative quantification of gene
expression, qrtPCR was performed at different dilutions (0.1 ng–
10 ng) in quintuplicate between control and mutant cDNA samples.
The data was normalized to a housekeeping gene, Gapdh (Applied
Biosystems Cat #4352932E). For data analysis, the Pfaffl method was
used to determine relative gene expression ratios (Pfaffl MW, 2001).
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FoxO1 Regulates Biomineralization
single nucleotide. The criteria were formulated after determining
the nature of published and functionally validated FHBE-SBEs
[31,32,33]. CBEs [T(G/T)NNG(A/C)AA(G/T)] and SBEs within
100 nucleotides upstream and downstream of the CBE were then
identified in a similar manner. CBEs that were located within
previously identified FHBE-SBE clusters were singled out and the
sequence identity between mouse and human was again detected
using the NCBI bl2seq program. As for the FHBE and SBE
sequences, CBE sequences were only noted if i) the conservation
between mouse and human sequences was complete, ii) incomplete by a single nucleotide, or iii) if the CBE conserved between
the two species was not identical by nucleotide character but
exactly matched published CBE sequences [32,33]. Regions of
$60% or 70% sequence identity larger than the regions
encompassing the FHBE-SBE(s) were determined by comparing
the entire promoter region of each mouse gene to the human gene
using the NCBI bl2seq program. The sequence identity recorded
for the Mmp20 and Cx40 genes come from the dual site program
rather than the NCBI bl2seq program. Sequences for p21CIP1 and
p15INK4b were extracted from releases mm6 and hg17, and mm8
and hg17, respectively, to mimic previously published results. It
should be noted that the binding sites represented for p15INK4b
were acquired from published results and were not obtained using
the methods presented above because of the imperfect conservation of the binding elements between mouse and human.
Acknowledgments
We thank Christina Stuelten for providing Smad3 mutant tissue and Eric
Swindell for providing the Rx-Cre mice. We also thank James Culver for
critical reading of the manuscript.
Author Contributions
Conceived and designed the experiments: RAP MED. Performed the
experiments: RAP RS MDG AMW RSU. Analyzed the data: RAP RS
MDG RSU MJN JDB MED. Contributed reagents/materials/analysis
tools: MJN JP RAD JDB. Wrote the paper: RAP MED.
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