RESEARCH ARTICLE DEVELOPMENT AND STEM CELLS
Development 140, 1424-1432 (2013) doi:10.1242/dev.089599
© 2013. Published by The Company of Biologists Ltd
Sox2 marks epithelial competence to generate teeth in
mammals and reptiles
Tissue renewal requires cells that can produce differentiating
progeny. Many organs, including skin, hair and intestine, renew
throughout the life of an individual. Interestingly, some organs, such
as teeth, differ in their regenerative capacities between species. For
example, reptiles replace their teeth continuously throughout life,
whereas in mammals tooth replacement is restricted to one round
(Fig. 1A). The numbers and shapes of teeth also vary between
species. Dentitions of reptiles are generally homodont (all teeth have
a similar shape) whereas mammals have a heterodont dentition
(differently shaped teeth that belong to several tooth classes:
incisors, canine, premolars and molars) (Fig. 1A). During
mammalian evolution, replacement capacity has been reduced,
whereas complexity of tooth shapes has increased. Most
mammalian species replace their deciduous teeth (incisors, canine
and premolars) once. The permanent molars form posterior to the
deciduous teeth and are part of the primary dentition (Osborn,
1893), but they are not replaced in any mammal (Fig. 1A).
The main features of tooth morphogenesis have been conserved
throughout evolution. All teeth form from surface oral epithelium
and underlying neural crest-derived mesenchyme. Interactions
between the tissues, mediated by conserved signaling pathways,
control epithelial morphogenesis and cell differentiation (Tummers
and Thesleff, 2009). In all vertebrates, the first sign of tooth
development is the formation of an epithelial primary dental lamina.
It can be recognized as a horseshoe-shaped thickening and as a
localized band of gene expression in the embryonic oral cavity or
pharynx that marks the future tooth rows (Fraser et al., 2009). In
mammals, development of the primary dentition starts from
epithelial placodes forming within the dental lamina. Knowledge
concerning the role of placodes in the initiation of different types of
teeth is still limited because they have been studied mainly in mice,
which have only a single continuously growing incisor and three
molars in each jaw quadrant (Fig. 1A).
Likewise, the mechanisms of tooth replacement have remained
poorly understood because mouse teeth are not replaced. The single
replacement of teeth in other mammals and the continuous
replacement in some reptiles share similar morphological and
molecular features (Järvinen et al., 2009; Leche, 1895; Ooë, 1981;
Richman and Handrigan, 2011; Smith et al., 2009b). In both cases,
the replacement teeth develop successionally from epithelium
associated with the preceding tooth, and are initiated during an early
stage of morphogenesis of the preceding tooth. In most vertebrates,
the enamel organs of deciduous tooth germs are connected with a
dental lamina running on the lingual side and linking the tooth
germs to oral epithelium (Fig. 1B). The individual replacement teeth
are initiated as an extension of the dental lamina, which is called
the successional dental lamina (Järvinen et al., 2009; Leche, 1895;
Ooë, 1981; Philipsen and Reichart, 2004; Richman and Handrigan,
2011; Smith et al., 2009b) (Fig. 1B). The capacity for tooth
replacement is believed to reside in the dental lamina and
1Institute of Biotechnology, Developmental Biology Program, University of Helsinki,
00014 Helsinki, Finland. 2Departments of Orofacial Sciences and Pediatrics and
Program in Craniofacial and Mesenchymal Biology, UCSF, San Francisco, CA 94143-
0442, USA. 3Life Sciences Institute, Department of Oral Health Sciences, University
of British Columbia, Vancouver, BC V6T 1Z3, Canada. 4Department of Pathology,
University of Southern California, Los Angeles, CA 90033, USA. 5Department of
Oral and Maxillofacial Surgery, Institute of Dentistry, University of Turku and Turku
University Hospital, 20014, Turku, Finland. 6Howard Hughes Medical Institute and
Department of Stem Cell and Regenerative Biology, Harvard University and Harvard
Medical School, Cambridge, MA 02138, USA. 7General Hospital Cancer Center and
Center for Regenerative Medicine, Boston, MA 02114, USA. 8Harvard Stem Cell
Institute, Cambridge, MA 02138, USA.
*These authors contributed equally to this work
‡Author for correspondence (email@example.com)
Accepted 17 January 2013
Tooth renewal is initiated from epithelium associated with existing teeth. The development of new teeth requires dental epithelial
cells that have competence for tooth formation, but specific marker genes for these cells have not been identified. Here, we analyzed
expression patterns of the transcription factor Sox2 in two different modes of successional tooth formation: tooth replacement and
serial addition of primary teeth. We observed specific Sox2 expression in the dental lamina that gives rise to successional teeth in
mammals with one round of tooth replacement as well as in reptiles with continuous tooth replacement. Sox2 was also expressed in
the dental lamina during serial addition of mammalian molars, and genetic lineage tracing indicated that Sox2+cells of the first
molar give rise to the epithelial cell lineages of the second and third molars. Moreover, conditional deletion of Sox2 resulted in
hyperplastic epithelium in the forming posterior molars. Our results indicate that the Sox2+dental epithelium has competence for
successional tooth formation and that Sox2 regulates the progenitor state of dental epithelial cells. The findings imply that the
function of Sox2 has been conserved during evolution and that tooth replacement and serial addition of primary teeth represent
variations of the same developmental process. The expression patterns of Sox2 support the hypothesis that dormant capacity for
continuous tooth renewal exists in mammals.
KEY WORDS: Dental lamina, Ferret, Mouse, Reptile, Stem cells, Successional tooth formation, Tooth replacement
Emma Juuri1,*, Maria Jussila1,*, Kerstin Seidel2, Scott Holmes3, Ping Wu4, Joy Richman3,
Kristiina Heikinheimo5, Cheng-Ming Chuong4, Katrin Arnold6,7,8, Konrad Hochedlinger6,7,8, Ophir Klein2,
Frederic Michon1and Irma Thesleff1,‡
Development ePress. Posted online 5 March 2013
successional dental lamina. Recently, label-retaining putative stem
cells have been localized in the successional dental lamina in species
with life-long tooth replacement, the leopard gecko (Eublepharis
macularius) (Handrigan et al., 2010) and American alligator
(Alligator mississippiensis) (P.W. and C.-M.C., unpublished).
Successional formation of teeth is observed, in addition to tooth
replacement, when new primary teeth are added within a tooth class.
This process is characteristic of the heterodont dentitions of
mammals, which usually have more than one incisor, premolar and
molar (Fig. 1A). In general, the primary teeth within one class erupt
in a specific sequence. However, their early development has not
been described in detail. The three mouse molars offer a model
system for the analysis of successional tooth formation, as the
second (M2) and third molars (M3) are added posteriorly along the
tooth row in the back of the jaw (Fig. 1A; Fig. 5A). A dissected first
molar (M1) tooth germ gives rise to M2 and M3 when transplanted
to the anterior chamber of the eye, but the mechanism of the
initiation of M2 and M3 has remained elusive. It appears that all
successionally forming teeth, including replacement teeth and
serially added teeth in tooth classes, develop from dental epithelium
associated with preceding teeth, which has maintained the
competence for tooth development (Järvinen et al., 2006).
Understanding the mechanisms of successional tooth formation
requires the molecular characterization of the tooth-forming
Sox2 in tooth formation
epithelium and identification of progenitor cells. Specific markers
for epithelial tissue with tooth-forming capacity are so far unknown.
We recently discovered that Sox2 marks the epithelial stem cells
in the continuously growing mouse incisor and demonstrated that
Sox2-positive (Sox2+) stem cells give rise to all epithelial cell
lineages of the incisor (Juuri et al., 2012). These findings led us to
explore whether Sox2expression is associated with tooth renewal in
general. In the present study, we have localized Sox2 expression
during the two modes of successional tooth formation in different
species: tooth replacement in ferret, human and five reptiles; and
serial posterior addition of molars in mouse and ferret. The results
indicate that Sox2+progenitors are already present in the primary
dental lamina during tooth initiation, and later reside in the dental
lamina in both modes of successional tooth formation. Genetic fate
mapping of Sox2-expressing cells of mouse M1 showed that they
give rise to the sequentially developing M2 and M3. Conditional
deletion of Sox2 resulted in expansion of dental lamina epithelium
associated with M2 and M3. Our results indicate that Sox2 marks
the epithelial competence to generate teeth and suggest that it acts
as a negative regulator of successional tooth formation.
MATERIALS AND METHODS
Animals and tissue processing
Wild-type NMRI mice were used at various embryonic stages. Plug day
was counted as embryonic day (E) 0 and embryos were staged according to
morphological criteria. Sox2-GFP mice (D’Amour and Gage, 2003) were
a kind gift from Fred H. Gage (Salk Institute, CA, USA). For genetic fate
mapping, Sox2CreERT2 (Arnold et al., 2011) and R26RlacZ(Soriano, 1999)
mice were crossed and genotyped as described previously.
Shh::GFPCre;Sox2fl/flmouse line (Sox2cKO) was generated by crossing
Shh::GFPCre (Harfe et al., 2004) with Sox2fl/flmice (Smith et al., 2009a)
(The Jackson Laboratory, stock 013093). For histology, radioactive in situ
hybridization and immunohistochemistry, mouse tissues were fixed in 4%
paraformaldehyde (PFA), dehydrated, and embedded in paraffin. Sections
from 4.5-µm to 7-µm thickness were cut in frontal and sagittal planes. All
aspects of mouse care and experimental protocols were approved by the
National Board of Animal Experimentation.
Pregnant ferret females (gestation period is 42 days) were euthanized
according to the guidelines for fur animals in fur farming research station
of Agricultural Research Centre of Finland (Kannus, Finland) and embryos
were collected at E28, E32 and E34. Heads were fixed in 4% PFA and the
E34 heads were decalcified in Morse’s solution for one week before paraffin
embedding. Sections of 7-µm thickness were cut in frontal plane.
Human jaw sections from 13-gestational-weeks-old fetuses were
obtained from the collection owned by the University of Turku, Finland.
The samples had been fixed in 4% PFA, decalcified, and embedded in
paraffin. The ethical approval was obtained from the Joint Authority for the
Hospital District of Southwest Finland Ethics Committee, Turku, Finland
(1/11 March 2007) and from the National Supervisory Authority for Welfare
and Health of Finland (VALVIRA 648/32/300/05).
Corn snake, ball python and gecko embryos were provided by Triple-R-
Corns (Aldergrove, BC, Canada). Gecko embryos were decalcified in 7%
EDTA in 2% PFA for 8 weeks whereas snakes were fixed in 4% PFA
overnight, embedded in paraffin and sectioned. Animals were sacrificed
according to procedures approved by the University of British Columbia
animal ethics committee, protocol number A11-0352.
Fertilized alligator eggs were collected in the Rockefeller Wildlife
Refuge, Louisiana. Eggs were incubated at 30°C and staged according to
Ferguson (Ferguson, 1985). One-year-old iguanas were from a local vendor
and kept in the University of Southern California (USC) animal facility.
Animals were sacrificed according to the procedure approved by the
Institutional Animal Care and Use Committee (IACUC) at USC.
E14.5 Sox2-GFPmouse molars were dissected and cultured in Trowell type
organ culture (Sahlberg et al., 2002). The medium contained DMEM and
Fig. 1. Variations in tooth replacement in reptiles and mammals, and
serial addition of molars in mammals. (A) Schematics of dentitions of
different vertebrates showing the left half of the lower jaws. Replacement
teeth form at the lingual side of the dental arch. Mammalian molars are
added serially in the posterior direction. Most reptiles have a homodont
dentition, which is continuously replaced. Humans and ferrets represent
typical mammals with a heterodont dentition composed of incisors, one
canine, premolars and molars, and all teeth except molars are replaced
once. Mice have one continuously growing incisor, a toothless diastema
region and three molars. Mouse teeth are not replaced. (B) Reconstruction
of human deciduous tooth germs illustrating their connection by the
continuous dental lamina and the initiation of replacement tooth
formation by budding of the successional dental lamina. Lingual view of
anterior tooth germs in the lower jaw. A similar dental lamina is present in
most other mammals and squamate reptiles. C, permanent canine; dC,
deciduous canine; dI, deciduous incisor; dP, deciduous premolar; I,
permanent incisor; M, molar; P, permanent premolar.
1426 RESEARCH ARTICLE
Development 140 (7)
F12 (Ham’s Nutrient Mix: Life Technologies) (1:1) supplemented with 10%
fetal calf serum (PAA Laboratories), 150 mg/ml ascorbic acid, glutamine
and penicillin-streptomycin and the medium was changed every second day.
In situ hybridization and immunohistochemistry
Radioactive in situ hybridization on paraffin sections was carried out
according to standard protocols (Wilkinson and Green, 1990). [35S]-UTP
(Amersham)-labeled RNA probes were used to detect expression of mouse
Sox2 (Ferri et al., 2004). Whole-mount in situ hybridization was performed
on mouse lower jaws fixed with 4% PFA using InSituPro robot (Intavis
AG). BM Purple AP Substrate Precipitating Solution (Boehringer
Mannheim) was used to visualize the digoxigenin-labeled probe. For
immunostaining, sections were rehydrated and heated in a microwave in
10 mM sodium-citrate buffer (pH 6.0). Immunostaining was performed
using the Ultravision Large Volume Detection System Anti-Rabbit, HRP
Kit (Thermo Scientific) and the DAB Peroxidase Substrate Kit (Vector
Laboratories, SK4100) using rabbit anti-Sox2 antibody (1:500-1:2000,
Millipore) or goat anti-Sox2 antibody (R&D Systems).
Genetic fate mapping
For genetic fate mapping of Sox2+cells, 10 mg tamoxifen (Sigma T-5648;
Sigma-Aldrich) in corn oil was given by oral gavage to pregnant females
carrying Sox2CreERT2;R26RlacZembryos and control embryos lacking the
Cre-driver at E13. Whole-mount X-Gal staining was performed as
previously described (Seidel et al., 2010). Tissues were processed into
paraffin, sectioned at 4.5-µm thickness and counterstained with Fast Red
Sox2 localizes to the primary dental lamina and to
lingual dental epithelium during mouse molar
Sox2 is expressed in the mouse incisor during morphogenesis and
becomes gradually restricted to the epithelial stem cell niche (Juuri et
al., 2012). Here, we examined Sox2 expression during the initiation
of mouse dentition and during morphogenesis of the mouse molar,
which does not grow continuously and lacks the epithelial stem cell
niche found in the incisor. The primary dental lamina gives rise to
teeth in all vertebrates and thus probably represents the origin of all
dental epithelia. In mouse, both incisor and molar placodes form from
the dental lamina. We localized Sox2 expression by whole-mount in
situ hybridization specifically to the dental lamina in mouse lower
jaw at E11 (Fig. 2A). At E12, Sox2 was strongly expressed at the
lingual side of the molar and incisor placodes, but the expression had
decreased between placodes (Fig. 2B).
We next localized Sox2 expression by radioactive in situ
hybridization in frontal sections of E12-E16 mouse M1 (Fig. 2C-E).
Throughout this period, Sox2 was expressed in the oral epithelium,
and faint or no expression was observed in mesenchymal tissues.
At E12, Sox2 expression was strongest at the lingual aspect of
placode epithelium (Fig. 2C). Subsequently, the dental epithelium
undergoes morphogenesis into a cap-shaped enamel organ
composed of distinct cell layers, including outer and inner enamel
epithelium (OEE and IEE, respectively) surrounding a core of
stellate reticulum (SR) cells. The enamel organ is connected to the
oral epithelium by a dental cord, which becomes disrupted during
later stages. At E14 and E16, Sox2 was expressed in the oral
epithelium at the lingual side of the tooth germ and continued from
there to the lingual cells of the dental cord and enamel organ,
including OEE and SR cells (Fig. 2D,E). Faint expression was also
detected in the cervical loops at E16 (Fig. 2E). The OEE on the
buccal aspect of the tooth was negative for Sox2. Similar expression
patterns were recently reported (Zhang et al., 2012).
For comparison, we detected Sox2 protein from E12 to E16
(Fig. 2F-H) and found expression in a similar pattern to Sox2
mRNA at all stages. At E16, an epithelial protrusion expressing
Sox2 was seen at the junction between the dental cord and OEE at
the lingual aspect of the molar (Fig. 2H). This small lingual bud can
also be observed in frontal sections of E17 and E18 mouse molars
(data not shown). This bud of the dental lamina is reminiscent of
the initiation of replacement tooth formation in the ferret (Fig. 3A)
and might represent the rudiment of the successional dental lamina
for molar replacement teeth, which mammals lost >200 million
years ago in the course of evolution (Kielan-Jaworowska et al.,
2004; Ungar, 2010).
Sox2 expression marks the dental lamina during
replacement tooth formation in ferret and human
The finding that Sox2 was expressed during mouse molar
morphogenesis in the lingual aspect of dental epithelium, which is
the location of replacement tooth initiation in other species,
Fig. 2. Sox2 is localized to primary dental lamina
and to lingual dental epithelium during mouse
molar development. (A,B) Whole-mount in situ
hybridization showing expression of Sox2 mRNA
(purple) in mouse lower jaw at E11 in the primary
dental lamina (A), and at E12 in oral epithelium and
lingual to the tooth placodes (B, dashed circles).
(C-H) The expression of Sox2 mRNA (red, C-E) and
protein (brown, F-H) in the lower molar from E12 to
E16 is gradually restricted to lingual dental epithelium
(arrows). Arrowheads in E point to Sox2 expression in
E16 cervical loops. Arrow in H points to budding of
dental epithelium at the lingual side of molar at E16
(inset shows higher magnification). dc, dental cord;
Lab, labial; Lin, lingual; OEE, outer enamel epithelium;
SR, stellate reticulum; T, tongue. Scale bars: 100 μm.
prompted us to examine Sox2 expression during replacement of
ferret and human teeth. Ferret (Mustela putorius furo) dentition
resembles that of human (Fig. 1A). We have previously described
the process of tooth replacement in ferret embryos (Järvinen et al.,
2009) and demonstrated that replacement is initiated from the free
edge of the dental lamina on the lingual side of each deciduous tooth
(Fig. 3A). We selected three stages of canine and third premolar
development to investigate the localization of Sox2 protein during
replacement: (1) before the initiation of replacement tooth
development, (2) during the splitting of the successional dental
lamina from the deciduous tooth enamel organ and (3) after the
initiation of replacement tooth morphogenesis.
At all three stages studied, Sox2+cells were observed on the lingual
aspect of the dental epithelium both in the deciduous canine (dC) and
in the deciduous third premolar (dP3) (Fig. 3C-H). As the
successional dental lamina detached from the OEE and started to
grow down from the deciduous tooth to form the permanent canine
(C) and the third premolar (P3), the free end of the budding lamina
was negative for Sox2 (Fig. 3D,H). When the permanent canine had
reached early cap stage, continuous lingual Sox2 expression extended
from the oral epithelium to the successional dental lamina connecting
the dC and C, and continued in the OEE of C to its cervical loop
(Fig. 3E). A similar pattern of Sox2 expression persisted in the bell
stage enamel organ of C (data not shown). The dental lamina between
the deciduous tooth germs (dP3 and dP4) expressed Sox2 in the
lingual epithelial cells (Fig. 3I-J). The free end of the dental lamina
was, however, negative for Sox2. A similar lingual localization of
Sox2 was observed during replacement of the dP2 and dP4 and in the
dental lamina between all teeth (data not shown). We detected Sox2
also in the cervical loops and IEE of each tooth, as well as in the oral
epithelium, but not in mesenchymal cells.
Sox2 in tooth formation
As is typical of mammalian teeth, human teeth are replaced
once. We studied Sox2 localization during the initiation of
permanent premolars in sections of a human fetus at 13 weeks of
gestation. Sox2 was expressed in the lingual side of the dental
lamina of a deciduous premolar (supplementary material Fig. S1).
The free end of the successional dental lamina of the budding
permanent premolar was negative for Sox2, similar to the ferret
Sox2 localizes to the dental lamina during
continuous tooth replacement in reptiles
Continuous tooth replacement in reptiles has been characterized at
both the morphological and the molecular level (Osborn, 1974;
Richman and Handrigan, 2011), and putative stem cells have been
localized in the dental lamina of the leopard gecko and American
alligator (Handrigan et al., 2010) (P.W. and C.-M. C., unpublished).
We used three lizards, American alligator, green iguana (Iguana
iguana) and leopard gecko, and two non-venomous snakes, ball
python (Python regius) and corn snake (Elaphe guttata), as models
of continuous tooth replacement to study Sox2 localization.
In all species, Sox2 was expressed in the dental lamina
connecting the teeth to the oral epithelium (Fig. 4). In alligator, Sox2
was detected in the dental lamina as well as in the lingual side of the
developing first-generation tooth (surface tooth) (Fig. 4A). The
forming successional dental lamina showed strong Sox2 expression
on its lingual side (Fig. 4B). Lingual Sox2 expression continued in
the OEE of the second-generation tooth (submerged tooth) (Fig. 4C,
2°). The successional dental lamina further detaches from the OEE
of the second-generation tooth, and Sox2+cells were detected in the
whole successional dental lamina except the free end (Fig. 4D). A
similar Sox2 expression pattern was observed in the successional
Fig. 3. Sox2 expression localizes to dental
lamina and successional dental lamina during
ferret tooth replacement. (A) Schematic of
frontal sections showing development of the
ferret permanent canine (C), which will later
replace the deciduous canine (dC). (B) Schematic
sagittal and buccal view of the developing ferret
tooth row showing the deciduous canine (dC),
deciduous second (dP2), third (dP3) and fourth
premolar (dP4), and first molar (M1) connected
by the dental lamina (dl). Dashed lines in B
indicate the sites of sections in C-E (dC), F-H (dP3)
and I,J (dl) and in Fig. 5D (M1). (C-J) Localization
of Sox2 protein (brown) during ferret tooth
replacement. Arrows point to Sox2 expression in
lingual dental epithelium. Asterisks indicate the
Sox2-negative free end in the successional dental
lamina, and in the dental lamina between
deciduous teeth. Sox2 localizes also to the
cervical loops and inner enamel epithelium (C-H,
arrowheads). A and C-J are frontal sections,
lingual to the right; B is a sagittal view, posterior
to the right. Scale bars: 100 μm.
dental lamina extending from the second-generation tooth of the
juvenile iguana (Fig. 4E-G) as well as in the successional dental
lamina extending from the second-generation teeth of the post-
hatching juvenile gecko (Fig. 4H-J).
In pre-hatching snakes, we examined the mirror image rows of
palatal and marginal teeth to look for the labial-lingual asymmetry
in Sox2 expression that was observed in the ferret and human. In
contrast to mammals, staining was equivalent on both sides of the
dental lamina and was similar in marginal and palatal teeth
(Fig. 4K,L,N). As in other species studied, the free end of the
successional dental lamina was negative for Sox2 (Fig. 4M,O).
In summary, Sox2 was associated with tooth replacement in all
species studied, and the expression patterns showed shared features.
Development 140 (7)
We detected Sox2 expression exclusively in epithelial dental cells
as well as in the oral epithelium in all species, and Sox2 specifically
marked the dental lamina and successional dental lamina in both
mammals and reptiles. The free end of the successional lamina,
which actively proliferates to produce the next-generation tooth,
was negative for Sox2. In addition, regional differences in Sox2
expression appeared in the dental lamina. Its lingual aspect showed
intense Sox2 expression in all species, but the labial expression was
absent in mammals. The lack of this lingual bias in reptiles might
reflect higher competence in their successional dental lamina
Dynamic Sox2 expression is associated with serial
addition of molars in mouse and ferret
Although mice have lost the capacity to replace teeth, their posterior
molars develop in succession, as in other mammals. The M2 and
M3 develop sequentially from M1 along the anterior-posterior axis
of the jaw (Fig. 5A). When M1 reaches the cap stage the dental
epithelium buds from its posterior end. This bud increases in size in
posterior direction and develops into M2. Later, when M2 reaches
the cap stage, the dental epithelium gives rise to a posterior bud,
which will form M3. We used the developing mouse molars as a
model for successional tooth formation and examined the role of
Sox2 in this process.
At E18, M1 and M2 have developed to the bell stage, and the bud
of M3 has been initiated from the posterior end of M2 (Fig. 5B). In
a sagittal section of E18 lower jaw, the continuation of Sox2
expression from the dental cord to the dental lamina connecting M1
and M2 was evident. As at E16, the Sox2+rudimental dental lamina
budding from the OEE epithelium was noticed above M1 (Fig. 2H;
Fig. 5B). Sox2 expression was observed also in the developing M3.
To follow the dynamics of Sox2expression during molar addition,
we dissected E14.5 molars from the Sox2-GFP reporter mouse and
monitored GFP expression in culture (Fig. 5C). Although the Sox2+
oral epithelium had been removed from the dissected M1, some GFP
expression was present on the oral surface, representing the dental
cord. GFP expression was localized in the posterior end of the M1,
which represents the dental lamina generating the M2. After 6 days,
the M1 crown had advanced in morphogenesis and was GFP negative,
whereas the M2 bud had grown in size and was positive for GFP. This
pattern was repeated, and after 9 days the crown of M2 had developed
and was GFP negative, whereas GFP expression was observed
posterior to M2. The GFP+tooth bud of M3 had formed after 14 days.
A domain of GFP+tissue appeared next to the teeth during extended
culture and is likely to represent oral epithelium.
In the ferret, at the posterior end of M1, from where M2 develops,
the majority of dental epithelial cells expressed Sox2 (Fig. 5D),
which is in line with observations of mouse M2 formation. Taken
together, the locations of the most intense Sox2 expression in both
mouse and ferret molars occurred in the dental lamina where new
molars are added.
Genetic fate mapping demonstrates that serially
added molars form from Sox2+cells
To test whether the Sox2+cells of the mouse M1 give rise to M2 and
M3, we utilized Sox2-CreER;R26RlacZmice for genetic fate
mapping (Arnold et al., 2011). We genetically labeled Sox2-
expressing cells by administering tamoxifen in vivo to pregnant
females at E13, collected embryos one day later, and dissected M1s
for organ culture (Fig. 6A). The descendants of the Sox2+cells were
identified after 0.5, 6 and 12 days by detecting lacZexpression using
X-gal staining of whole-mount samples (Fig. 6B-D) and paraffin
Fig. 4. Sox2 expression localizes to dental lamina and successional
dental lamina during continuous tooth replacement in five reptile
species. (A-O) Localization of Sox2 protein (brown) in American alligator
[embryonic stages (Es) 19-24] (A-D), green iguana (juvenile) (E-G), leopard
gecko (post-hatching juvenile) (H-J), 60-day post-oviposition ball python
(K-M) and 30-day post-oviposition corn snake (N,O). Arrows in alligator
and iguana point to Sox2 expression in the lingual dental epithelium
(A,B,C,F). Note that there is no lingual asymmetry in Sox2 expression in
snakes (L,N). Sox2 expression is absent from the free end of the
successional dental lamina (asterisks) in all species except gecko. Staining
in the deposited enamel in gecko is non-specific (I, arrowhead). Boxed
areas in K represent higher magnifications in L and M. Dashed lines
outline the dental epithelium. 1°, first generation tooth; 2°, second
generation tooth; dl, dental lamina; OEE, outer enamel epithelium; sl,
successional dental lamina. Scale bars: 100 μm.
sections (Fig. 6E-H). After 0.5 day, M1 was at the late bud stage
and lacZ+cells were detected in the lingual side of dental epithelium
including OEE and some SR cells (Fig. 6E). X-gal staining
increased towards the posterior end of M1 corresponding to the
areas of the highest Sox2-GFP expression (compare Fig. 6F and
Fig. 5C, 0h). After 6 days, M1 had reached the bell stage of
morphogenesis and the majority of its epithelial cells were lacZ
negative. However, prominent clusters of lacZ+cells were present
Sox2 in tooth formation
in the cervical loop at its posterior end, indicating that E13 Sox2+
cells contribute predominantly to the posterior part of M1 (Fig. 6G).
M2 development had advanced to the bud stage, and the bud
epithelium, as well as the dental lamina, were composed almost
entirely of lacZ+cells (Fig. 6C,G). After 12 days, M2 had developed
to early bell stage and lacZ+cells were detected in all epithelial
layers of the molar crown: ameloblasts, SR, IEE and OEE cells
(Fig. 6H,H?). At this time, the M3 bud had formed, and it was also
largely composed of lacZ+cells (Fig. 6D; data not shown). Few or
no lacZ+cells were detected at any time point in the mesenchyme.
lacZ-expressing cells were absent in R26RlacZ/+embryos lacking the
Sox2CreER allele, which were used as control (data not shown).
These results demonstrate that Sox2+cells associated with the
M1 at E13 give rise to successional molars, and that Sox2+cells
contribute to all epithelial cell lineages of the mouse molar crown.
Loss of Sox2 leads to abnormal epithelial growth
of successionally developing mouse molars
To investigate the role of Sox2in the dental epithelium, we examined
the morphology of lower molars of Shh::GFPCre;Sox2fl/flmutants
(hereafter called Sox2cKO), in which Sox2is conditionally inactivated
in the epithelium. Because of early postnatal mortality of the mutants,
the analysis was performed between E17 and postnatal day (P) 0. The
epithelial morphology of M2 and M3 in Sox2cKO differed from
littermate controls as the dental cord connecting M2 to oral epithelium
was markedly expanded and hyperplastic (Fig. 7; supplementary
material Fig. S2) and the dental lamina connecting M3 to the oral
epithelium was elongated. No morphological abnormalities were
observed in the mutant M1 (Fig. 7).
We detected no obvious differences in proliferation in the dental
epithelium of Sox2cKOs and controls (data not shown).
Nevertheless, it is possible that very small changes in proliferation
can result in differences in epithelial volume. Sox2 has been shown
to inhibit canonical Wnt signaling (Mansukhani et al., 2005), and
increased Wnt signaling has been linked to supernumerary tooth
formation both in human and transgenic mice (Wang and Fan,
2011). Therefore, we examined expression of the Wnt target gene
Axin2 in control and Sox2cKO embryos, but expression in the
mutant epithelium was unchanged (data not shown).
To assess the efficiency of Sox2inactivation by Cre recombinase,
we examined lacZ expression
Shh::GFPCre;R26RlacZembryos. At E13, the M1 showed a mosaic
pattern of lacZ expression (supplementary material Fig. S3). At P5,
recombinase efficiency was very high in M1 and M2, but a more
mosaic recombination pattern was observed in the dental cord and
M3 (supplementary material Fig. S3). We also detected some
remaining Sox2 protein expression in the Sox2cKO embryos at E17
and E18 (supplementary material Fig. S4). This incomplete deletion
of Sox2 might explain the mild molar phenotype of the mutants.
in lower molars of
We have localized Sox2 in dental epithelia at the sites that are either
known or proposed to possess capacity for tooth renewal in several
mammalian and reptilian species. By genetic fate mapping we
demonstrated that Sox2+cells of the mouse M1 give rise to the
successionally developing M2 and M3. In addition, conditional
deletion of Sox2 indicated that Sox2 regulates the amount of dental
Sox2 was expressed in the primary dental lamina, which marks
the future dental arches in vertebrates and has been proposed to
house stem cells for all dental epithelial tissues (Fig. 8) (Smith et al.,
2009b). Importantly, Sox2was subsequently expressed in the dental
Fig. 5. Sox2 expression is associated with successional formation of
posterior molars in mouse and ferret. (A) Schematic of successional
development of molars (see text for details). (B) Localization of Sox2 protein
(brown) in E18 mouse molars. Sox2 is expressed in the dental cord and
dental lamina connecting M1 to M2, and in the rudimentary dental lamina
bud above M1 (arrow). Sox2 is expressed in the bud, which will form M3.
Arrowheads point to Sox2 expression in M1 cervical loops. (C) Dynamics of
Sox2expression (green) during successional addition of molars. A dissected
E14.5 M1 of a Sox2-GFPreporter mouse gives rise to M2 and M3 during 14
days of culture. The buds of M2 and M3 express Sox2-GFPbut the
completed crowns of M1 after 6 days (6d) and M2 after 9 days (9d) do not
express Sox2-GFP. Arrows point to the GFP+dental lamina, which will give
rise to a new tooth. Dashed lines outline the tooth germs. (D) Localization of
Sox2 protein (brown) in ferret M1 at E34 in frontal sections from anterior (a)
to posterior (c). The planes of sections are shown by dashed lines in Fig. 3B.
Sox2 localizes to the lingual OEE (a, arrow) and cervical loops of M1 (a,
arrowheads). In the posterior end of M1 from where M2 develops, Sox2
localizes both to lingual and labial sides of M1 epithelium (b,c). Lingual is
towards the right. CL, cervical loop; dc, dental cord; dl, dental lamina; IEE,
inner enamel epithelium; M, molar; OEE, outer enamel epithelium; SR,
stellate reticulum. Scale bars: 100 μm.
lamina (Fig. 8), which connects the deciduous tooth germs as an
epithelial sheet and is embedded on their lingual side, i.e. the side
where the replacement teeth form in all animals (Fig. 1B) (Järvinen
et al., 2009; Ooë, 1981; Richman and Handrigan, 2011; Smith et
al., 2009b). This dental lamina is generally assumed to contribute
progenitors for tooth replacement but no markers have been
identified to differentiate the dental lamina from the flanking dental
epithelium. In all species studied, a continuous stripe of Sox2+cells
extended from the oral epithelium through the dental cord and
enamel organ of the first-generation tooth to the successional dental
lamina. This pattern of gene expression is unique and comparison
of replacement of mammalian and reptile teeth indicated that most
aspects of Sox2 expression have been conserved during evolution.
We conclude that Sox2 is the first known gene that marks the dental
lamina and successional dental lamina.
In reptiles, Sox2 was expressed in all successional teeth and in the
successional lamina. Interestingly, in the ferret a stripe of Sox2+
cells continued to the permanent tooth germ, indicating the presence
of a dental lamina at the lingual aspect of the enamel organ.
Although ferret tooth replacement is limited to one round, the dental
lamina appears to be maintained as part of the developing
permanent tooth, similar to reptiles. We did not have human material
of advanced enough stages to see the permanent tooth germs, but a
successional dental lamina extending from the developing human
permanent teeth exists (Ooë, 1981). This, together with our
observations, is in line with the hypothesis that there may be
dormant capacity for further rounds of replacement in the
mammalian teeth (Järvinen et al., 2006; Jensen and Kreiborg, 1990;
Richman and Handrigan, 2011). Additionally, our findings indicate
that even mouse molars might possess the competence for
generation of replacement teeth, although mouse teeth are normally
not replaced and no mammals replace molars. Sox2 was expressed
in mouse in a similar location as that observed in the ferret,
suggesting that mouse molar epithelium might have an embedded
dental lamina. Additionally, a Sox2+epithelial bud was observed
protruding from the cord near the junction to the OEE, perhaps
representing aborted initiation of a replacement tooth.
Development 140 (7)
Serial horizontal addition of primary teeth within a tooth class
represents another example of successional tooth formation. We
observed that dynamic Sox2 expression was repeated when mouse
molars were added to the tooth row, indicating that Sox2 expression
is associated with the posterior extension of dental epithelium where
the addition of new teeth takes place (Fig. 8). By genetic fate
mapping, we demonstrated that the Sox2+cells of the mouse M1 gave
rise to all epithelial cell lineages of the successionally developing M2
and to the bud of M3. Therefore, we propose that the Sox2+cells have
the capacity to form the epithelial component of a new tooth.
Continuous horizontal addition of molars exists in some
mammals, such as the silvery mole rat (Heliophobius
argenteocinereus), and this resembles the continuous tooth
replacement in reptiles (Rodrigues et al., 2011). Supernumerary
posterior molars are occasionally present in humans (Shahzad and
Roth, 2012) and also in wild-type mice (our unpublished
observations). These examples support the notion of a sustained
epithelial competence for tooth formation. Our observations pointed
out striking similarities between the processes of tooth replacement
and serial addition of primary teeth in mammals. First, when mouse
molars and ferret replacement teeth are initiated, the preceding tooth
has reached the early cap or early bell stage of development.
Second, in both cases the new tooth formed successionally from
Sox2+dental epithelial tissue associated with the dental cord and
enamel organ of the preceding tooth, i.e. the dental lamina (Fig. 8).
The two processes differ in the orientation and direction of new
tooth formation: replacement tooth formation is initiated from the
lingual side of the preceding tooth and occurs in a vertical direction,
whereas addition of primary teeth within a tooth class is initiated
from the posterior (sometimes anterior) aspect of the preceding
tooth and takes place horizontally (Fig. 8). The resemblance
between the formation of replacement teeth and posterior molars
has been noted previously (Järvinen et al., 2008; Jensen and
Kreiborg, 1990) and, based on the apparent similarities in
morphology, developmental timing and Sox2 expression patterns,
we suggest that the two modes of successional tooth formation
actually represent variations of the same developmental process.
Fig. 6. Genetic fate mapping demonstrates that
successional molars derive from Sox2+cells.
(A) Timing of tamoxifen administration and analysis of
Sox2Cre-ER;R26RlacZmolars. (B-D) lacZ expression (blue)
in X-Gal-stained whole-mount samples of M1 cultured
for 0.5, 6 and 12 days after tamoxifen administration at
E13.0. Arrow in B points to the posterior end of the M1
from where the M2 develops. Dashed line outlines the
dental epithelium in B and the whole tooth germs in C
and D. (E-H? ?) Histological sections from whole-mount
samples shown in B-D. The sections in E and F were cut
in the frontal plane at positions indicated by black
dashed lines in B, other sections were cut in the
sagittal plane. Dashed lines in E and F mark the border
between epithelium and mesenchyme. Boxed area of
M2 shown at higher magnification in H? shows lacZ
expression in all dental epithelial cell layers. dl, dental
lamina; IEE, inner enamel epithelium; M, molar; OEE,
outer enamel epithelium; SR, stellate reticulum. Scale
bars: 100 μm.
Based on our genetic fate mapping result and on the morphological
and molecular similarities between posterior molar addition and
tooth replacement, we propose that Sox2+cells in the dental lamina,
which contributed to the successive molars, also give rise to
replacement tooth epithelium. This, however, needs to be
experimentally confirmed using a genetic fate mapping approach
in a species with continuous replacement.
Interestingly, supernumerary tooth formation was reported in a
human patient carrying a heterozygous loss-of-function mutation in
the SOX2 gene (Numakura et al., 2010). The supernumerary tooth
phenotype resembles that observed in two other syndromes,
cleidocranial dysplasia (CCD) and the craniosynostosis and dental
anomalies syndrome (CRDSA) (Jensen and Kreiborg, 1990;
Nieminen et al., 2011). Clinical follow-up of the development of extra
teeth in CCD patients indicated that they form in succession as part
of an additional replacement and as supernumerary posterior molars
(Jensen and Kreiborg, 1990), and histological examination of the
tissue associated with the CCD supernumerary teeth revealed an
Sox2 in tooth formation
abundance of dental epithelium (Lukinmaa et al., 1995). Sox2cKO
mice developed hyperplastic dental epithelium associated with the
developing M2 and M3, which is in line with the supernumerary tooth
phenotype of the human patient with SOX2 mutation and indicates
that Sox2 prevents the expansion of dental epithelium. Increased
canonical Wnt signaling induces supernumerary tooth formation in
familial adenomatous polyposis syndrome in human and the capacity
for continuous formation of teeth is unlocked in mice in which the
Wnt pathway is activated by stabilization of β-catenin in the oral
epithelium (Wang and Fan, 2011). Indeed, Sox2 can function as an
inhibitor of canonical Wnt signaling (Mansukhani et al., 2005). Thus,
Sox2 might function as an inhibitor of the formation of replacement
teeth and supernumerary molars by inhibiting epithelial growth. We
did not observe changes in proliferation or Axin2 expression in the
Sox2cKOs, but this may result from an incomplete deletion of Sox2
in the mutants.
Continuous tooth renewal and replacement require a source of stem
cells that can self-renew and produce progeny. In the reptiles studied,
Fig. 7. Conditional deletion of Sox2 leads to
hyperplastic dental epithelium of M2 and M3.
Hematoxylin and Eosin-stained serial frontal sections of
mandibular molars of control and Sox2cKO mice at E18
and P0. Sox2cKO shows no obvious phenotype in M1
(data for P0 not shown), whereas in M2 the dental cord
is expanded (arrows) and dental lamina between M2
and M3 is expanded. M3 is attached to oral epithelium
by an extended sheet of dental lamina (arrowheads). See
also supplementary material Fig. S2. dc, dental cord.
Lingual is towards the right. Scale bars: 100 μm.
Fig. 8. Sox2 expression is associated with epithelial
competence of dental lamina in different modes of
successional tooth formation. Schematic of the
localization of Sox2 (red) in the primary dental lamina in
the lower jaw, and in the dental lamina during different
types of successional tooth formation: mammalian tooth
replacement, continuous replacement and molar addition.
The drawings also illustrate the morphological similarity
between the two modes of successional tooth formation.
1°, first generation tooth; 2°, second generation tooth; A,
anterior; B, buccal; dc, dental cord; dC, deciduous canine;
dl, dental lamina; L, lingual; Le, left; M, molar; P, posterior;
Ri, right; sl, successional dental lamina; t, tongue.
1432 RESEARCH ARTICLE Download full-text
Development 140 (7)
Sox2 expression overlapped the regions in the dental lamina where
putative stem cells have been localized (Handrigan et al., 2010) (P.W.
and C.-M.C., unpublished). Sox2 might play an important role in the
maintenance of the dental lamina, which is likely to be a prerequisite
for successional tooth formation. The termination of successional
tooth formation in mammals might, however, not be the result of
depletion of the Sox2+cells because Sox2 expression was maintained
in ferret secondary tooth germs, which do not give rise to replacement
teeth. One plausible reason for the discontinued successional tooth
formation may be lack of signaling that induces tooth initiation from
the Sox2+cells in the dental lamina. In reptiles, mesenchymal signals
have been suggested to initiate this process (Handrigan and Richman,
2010). The genetic fate mapping experiments reported here
demonstrated that the Sox2+cells contribute to all epithelial cell types
of the successional molars, which is similar to our observations in
incisors (Juuri et al., 2012). Therefore, Sox2 might be a marker for
naive dental progenitor cells, which have the capacity for new tooth
generation. The role of Sox2 in the dental lamina might be to inhibit
proliferation of the progenitors and/or to maintain their progenitor
state. Further investigation with functional experiments is required to
elucidate the specific function of Sox2 in the dental lamina cells.
We thank Merja Mäkinen, Riikka Santalahti, Raija Savolainen and Dong-Kha
Tran for excellent technical help; Fred Gage for the Sox2-GFP mouse strain;
and Marja Mikkola and Jukka Jernvall for their comments on the manuscript.
This work was supported by the Finnish Doctoral Program in Oral Sciences
[E.J.]; Viikki Doctoral Program in Molecular Biosciences [M.J.]; Sigrid Juselius
Foundation [I.T.]; Academy of Finland [F.M. and I.T.]; National Institute of
Biomedical Imaging and Bioengineering [RL9EB008539 to K.S.]; National
Institute of Dental and Craniofacial Research [DP2-OD00719 and R01-
DK095002 to O.K.]; National Institute of Arthritis and Musculoskeletal and
Skin Diseases [42177 to C.-M.C. and P.V.]; and the National Sciences and
Engineering Research Council of Canada [S.H. and J.R.]. Deposited in PMC for
release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
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