DEVELOPMENT AND STEM CELLS
RESEARCH REPORT 3545
Development 137, 3545-3549 (2010) doi:10.1242/dev.052415
© 2010. Published by The Company of Biologists Ltd
Identification of putative dental epithelial stem cells in a
lizard with life-long tooth replacement
Gregory R. Handrigan, Kelvin J. Leung and Joy M. Richman*
SUMMARY
Most dentate vertebrates, including humans, replace their teeth and yet the process is poorly understood. Here, we investigate
whether dental epithelial stem cells exist in a polyphyodont species, the leopard gecko (Eublepharis macularius). Since the gecko
dental epithelium lacks a histologically distinct site for stem cells analogous to the mammalian hair follicle bulge, we performed a
pulse-chase experiment on juvenile geckos to identify label-retaining cells (LRCs). We detected LRCs exclusively on the lingual side
of the dental lamina, which exhibits low proliferation rates and is not involved in tooth morphogenesis. Lingual LRCs were
organized into pockets of high density close to the successional lamina. A subset of the LRCs expresses Lgr5 and other genes that
are markers of adult stem cells in mammals. Also similar to mammalian stem cells, the LRCs appear to proliferate in response to
gain of function of the canonical Wnt pathway. We suggest that the LRCs in the lingual dental lamina represent a population of
stem cells, the immediate descendents of which form the successional lamina and, ultimately, the replacement teeth in the gecko.
Furthermore, their location on the non-tooth-forming side of the dental lamina implies that dental stem cells are sequestered
from signals that might otherwise induce them to differentiate.
KEY WORDS: Squamate, Dental lamina, Regeneration, Gecko, Wnt signaling
Department of Oral Health Sciences, Faculty of Dentistry, University of British
Columbia, Vancouver, BC V6T 1Z3, Canada.
*Author for correspondence ([email protected])
Accepted 25 August 2010
Some authors have suggested that epithelial stem cells regulate
tooth replacement in vertebrates as they do hair renewal in
mammals (Huysseune and Thesleff, 2004; Smith et al., 2009).
Here, we test this hypothesis using a novel animal model, the
leopard gecko Eublepharis macularius. The gecko replaces its teeth
continuously throughout life and is amenable to postnatal
experimentation. We present pulse-chase and gene expression data
that imply that the gecko dental lamina houses cells that resemble
mammalian adult stem cells in terms of cell cycling, gene
expression and molecular regulation.
MATERIALS AND METHODS
BrdU pulse-chase of leopard geckos
Juvenile geckos (see Fig. 1A) were reared at 30°C (Thorogood and
Whimster, 1979). Once a gecko reached a mass of 3 g, we administered
5-bromo-2⬘-deoxyuridine (BrdU; 1 mg/ml) twice daily for 1 week by
squirting the solution into the mouth. BrdU was also added to drinking water
(1 mg/ml). Two geckos were collected immediately after the pulse to gauge
the initial degree of BrdU saturation. Three other BrdU-treated animals were
chased for 4, 9 or 20 weeks prior to euthanasia. A segment of the right
maxillary tooth row of the 20-week-chased gecko was digitally reconstructed
from frontal sections using WinSurf software (Buchtová et al., 2008).
Immunohistochemistry and gene expression analyses
Paraffin sections were steamed in 10 mM sodium citrate for 10 minutes,
incubated with antibodies against either BrdU (GE Healthcare) or mouse
PCNA (1:50; Vector Laboratories), and visualized using Alexa Fluor 488
(1:200). Radioactive in situ hybridization was performed as described
(Buchtová et al., 2008) using probes against E. macularius Lgr5, Igfbp5,
Dkk3, Tcf3 and Tcf4 (see Table S1 and Figs S1-S4 in the supplementary
material). cDNAs were amplified by degenerate RT-PCR (see Table S2 in
the supplementary material) and cloned into pGEM-T Easy (Promega).
Gecko explant culture
Dental tissues from stage 39 gecko embryos (Wise et al., 2009) were grown
in Trowell-type culture (see Handrigan and Richman, 2010) for 5 days. The
GSK3b inhibitor 6-bromoindirubin-3⬘-oxime (BIO; EMD Biosciences)
was added to experimental cultures (20 M) to activate Wnt signaling
DEVELOPMENT
INTRODUCTION
Most vertebrate species, from fish to humans, can replace lost teeth.
Snakes and lizards, for example, generally form multiple sets of teeth
throughout life (polyphyodonty), whereas mammals form only two.
Despite being a topic of study for over a century (Bolk, 1922;
Edmund, 1960; Leche, 1895; Woerdeman, 1919), the cellular basis of
tooth replacement is poorly understood. Two reasons account for this.
First, tooth replacement occurs postnatally and so is challenging to
study. Second, the mouse, the predominant animal model for studying
tooth development, does not actually replace its teeth. Tooth renewal
in mice is limited to the continuous growth of incisor teeth. Extra teeth
can be induced in mice by genetic manipulation, particularly of Wnt
pathway genes (Järvinen et al., 2006; Wang et al., 2009), but these
experiments reveal little about tooth replacement as it naturally occurs.
Compared with tooth replacement, a great deal more is known
about the renewal of the mammalian hair follicle. Hair is continually
renewed by a population of stem cells located in the bulge, an area
near the base of the follicle. Bulge stem cells were first located by a
pulse-chase strategy, whereby retention of tritiated thymidine revealed
the presence of cells with exceedingly slow cell cycling times
(Cotsarelis et al., 1990). Bulge stem cells have since been shown to
express many of the same genes as other adult stem cells (Blanpain
et al., 2004; Morris et al., 2004; Tumbar et al., 2004). Lgr5, for
instance, marks adult stem cells of the intestine (Barker et al., 2007),
the mouse incisor (Suomalainen and Thesleff, 2010) and the hair
follicle (Jaks et al., 2008). Likewise, mammalian adult stem cells
share a requirement for Wnt pathway signaling. When the pathway is
active, stem cells divide to reconstitute a tissue, and when inactive,
stem cells remain undifferentiated (Haegebarth and Clevers, 2009).
3546 RESEARCH REPORT
Development 137 (21)
(Sato et al., 2004); controls received DMSO. Three hours prior to
collection, cultures were treated with BrdU (10 M) for proliferation
assays.
RESULTS AND DISCUSSION
Tooth replacement in the leopard gecko
Juvenile geckos bear ~100 erupted marginal teeth and a greater
number of replacement teeth organized into tooth families in series
along the jaw margins (Fig. 1B). At hatching, each family
comprises up to three generations of teeth connected by the dental
lamina (Fig. 1B-D; see Movie 1 in the supplementary material).
Replacement teeth form lingual to the erupted tooth and increase
in size until they ultimately erupt and displace the predecessor
tooth, a process that takes ~3-4 months (Edmund, 1960). All
replacement teeth initiate from the successional lamina, an
outgrowth of the outer enamel epithelium of the predecessor tooth
(Fig. 1C-E). The lamina consists of lingual and labial layers and,
in between them, loose interstitial cells (Fig. 1F).
The gecko dental lamina houses slow-cycling cells
on its lingual face
The successional lamina makes an obvious candidate for the site of
dental stem cells as it gives rise to all replacement teeth in amniotes
and in amphibians. However, we showed previously that the
successional lamina of the gecko and other squamates comprises a
population of proliferative cells (Handrigan and Richman, 2010).
This defies a key criterion of adult stem cell populations: that they
turnover slowly (Fuchs, 2009). It is unclear, however, where else
dental stem cells might be; we found no structure in the gecko
dental epithelium that resembles the mouse hair follicle bulge or
the distended cervical loop of the mouse incisor.
To determine whether the gecko dental epithelium contains
slow-cycling cells, we performed the first BrdU pulse-chase
experiment on a non-avian reptile. In geckos fixed immediately
following the pulse (0 week), BrdU had been incorporated into
~57% of dental epithelial cells (Fig. 2A; see Table S3 in the
supplementary material). Similarly, Cotsarelis et al. achieved less
than 100% saturation of slow-cycling cells in the hair follicle when
they pulsed mice for 7 days (Cotsarelis et al., 1990). They reasoned
that this was because not all hair follicles cycled during the pulse.
Likewise, only some of the 150 or more replacement teeth
developing in a juvenile gecko are likely to draw on stem cells
during 1 week. BrdU signal in the pulsed geckos was lower in
areas of the dental lamina between tooth buds (see Fig. S5 in the
supplementary material). Presumably, some of these cells have cell
cycling times that exceed 1 week.
Next, we analyzed BrdU retention in geckos chased for 4, 9
and 20 weeks (see Fig. S6 and Table S3 in the supplementary
material). After 4 weeks, overall BrdU saturation of the dental
epithelium had decreased nearly threefold to 21%, but the
distribution of BrdU-positive cells remained unchanged
compared to the 0-week geckos (Fig. 2B). After 9 weeks, overall
DEVELOPMENT
Fig. 1. Tooth replacement in the leopard
gecko Eublepharis macularius. (A)A juvenile
gecko used in this study. (B)Micro-CT scans of
juvenile gecko jaws showing erupted marginal
teeth and immature replacement teeth arranged
as tooth families. (C)Three-dimensional
reconstruction of the right maxillary tooth row of
a juvenile gecko. Insets show the relationship
between a second-generation enamel organ and
the third-generation successional lamina. Red
ovals represent erupted teeth that could not be
reconstructed. (D)Three generations of teeth
connected by the dental lamina within a tooth
family. (E,F)The multilayered successional lamina
projects from the lingual outer enamel epithelium
of a predecessor tooth. Solid white lines mark the
basement membrane; dashed black and red lines
trace the inner enamel epithelium and lamina
interstitium, respectively. 1°/2°/3°, successive
tooth generations; cl, cervical loop; de, dentin; dl,
dental lamina; dp, dental papilla; eo, enamel
organ; iee, inner enamel epithelium; mx, maxilla;
oc, oral cavity; od, odontoblasts; oee, outer
enamel epithelium; sl, successional lamina. Scale
bars: 2 mm in B; 100m in C-F.
Dental stem cells and tooth replacement
RESEARCH REPORT 3547
Fig. 2. The gecko dental lamina houses
slow-cycling, BrdU-retaining cells that are
distributed in a complementary pattern to
proliferating cells. (A-D)Sections of dental
tissues from juvenile geckos chased for 0, 4, 9
and 20 weeks. Solid line, border of dental
epithelium; dashed line, border between the
inner enamel epithelium and the rest of the
enamel organ. BrdU saturation in the dental
epithelium decreases progressively with signal
intensity in each cell, becoming increasingly
granular (insets). Overall percentage saturation
of the dental epithelium and distribution of
BrdU-positive cells for each chase treatment are
presented beneath. The regions A-E are
illustrated alongside. (E)Label-retaining cells
(LRCs, arrowheads) are concentrated on the
lingual layer of the dental lamina. (F)Areas of
high cell proliferation (arrows) in the
successional lamina and cervical loop do not
contain LRCs. (G)Three-dimensional
reconstruction of a maxillary tooth row of a 20week-chased gecko showing the distribution of
label-retaining cells. BrdU-retaining cells are
heterogeneously distributed along the anteriorposterior (a-p) axis of the dental lamina.
(G⬘)Along the aboral-oral (ab-o) axis, BrdUpositive cells are concentrated in regions B and
C of the dental lamina, but are virtually absent
from the successional lamina (region D). Scale
bars: in A, 100m for A-D,F; 10m in E;
100m in G.
tooth row (Fig. 2G,G⬘). In sagittal sections, BrdU-retaining cells
occurred in comparable intertooth areas of the dental lamina that
showed relatively low BrdU uptake in the 0-week-chased geckos
(see Fig. S5 in the supplementary material). In the reconstruction,
BrdU-retaining cells were clustered in spaces between secondgeneration teeth (Fig. 2G; see Movie 2 in the supplementary
material) and were typically found close to, but not in the tip of,
the dental lamina (Fig. 2G⬘).
Label-retaining cells in the dental lamina express
adult stem cell markers
To test the ‘stemness’ of label-retaining cells in the lingual dental
lamina, we scrutinized them for expression of genes that mark hair
bulge stem cells: Dkk3, Igfbp5 and Lgr5 (Morris et al., 2004;
Tumbar et al., 2004). All three genes were expressed in a
restricted domain in the dental lamina, overlapping a subset of
BrdU-retaining cells (Fig. 3A-D). These BrdU-retaining,
Fig. 3. Stem cell marker gene expression coincides with
label-retaining cells in the gecko dental lamina.
(A-D)Near-adjacent sections through 20-week-chased gecko
dental tissues. Autoradiographic (A,C,D) and fluorescent (B)
signals have been pseudo-colored red. Lgr5, Dkk3 and Igfbp5
are expressed in a focused domain (arrowhead) in the dental
lamina (solid white line) that coincides with BrdU-retaining
cells. Dashed outline, border of the enamel organ. eo, enamel
organ; LRC, label-retaining cell; sl, successional lamina. Scale
bar: 100m.
DEVELOPMENT
BrdU saturation did not change appreciably compared with 4
weeks (Fig. 2C). However, the fluorescent signal appeared
fragmented, indicating that BrdU had been metabolized
(compare insets in Fig. 2B,C). We detected signal in the labial,
lingual and interstitial cells of the dental lamina.
We extended the chase period to 20 weeks to whittle the BrdU
signal down to only the most quiescent cells. After this lengthy
chase, the percentage of BrdU-labeled cells in the dental epithelium
(regions A-E, Fig. 2D) decreased to ~3%. The few remaining
BrdU-positive cells were disproportionately found in regions B and
C along the oral-aboral axis (Fig. 2D,E; see Fig. S7 in the
supplementary material). There were virtually no labeled cells in
the highly proliferative successional lamina (region D, Fig. 2F).
To determine the spatial distribution of label-retaining cells
along the anterior-posterior axis of the jaw, we analyzed BrdU
labeling in sagittal sections (see Fig. S5 in the supplementary
material) and in a three-dimensional reconstruction of the maxillary
3548 RESEARCH REPORT
Development 137 (21)
Lgr5/Dkk3/Igfbp5-positive cells are likely to be stem cells. We
tested this hypothesis by checking whether the cells share
molecular regulation with mammalian adult stem cells.
Wnt signaling may regulate stem cell fate in the
gecko dental epithelium
The canonical Wnt pathway is sufficient to induce quiescent stem
cells to form transit-amplifying cells in the mouse intestine and hair
follicle (Blanpain et al., 2004; Haegebarth and Clevers, 2009;
Lowry et al., 2005). We characterized the dental expression of Tcf3
and Tcf4, two targets of the Wnt pathway, to determine whether the
dental lamina cells of the gecko are Wnt active. The dental
expression pattern of these genes has not previously been examined
in any species. In the mouse hair follicle, Tcf3 and Tcf4 are coexpressed by bulge stem cells and transit-amplifying cells (Nguyen
et al., 2009). We predicted that the expression domains of the two
genes would overlap with each other and with BrdU-retaining cells
(Fig. 4B) and stem cell marker gene expression (Fig. 4C) in the
dental lamina. Unexpectedly, they did not overlap (Fig. 4D,E): Tcf4
was expressed in the enamel organ and successional lamina
overlapping proliferating cells (regions D, E, Fig. 4E,F), whereas
Tcf3 was expressed throughout the dental lamina, including where
the putative dental stem cells are located (Fig. 4B,D). These
expression data imply that Wnt signals act broadly throughout the
dental epithelium and that Tcf3 and Tcf4 mediate different
functions. Tcf3 might maintain dental lamina stem cells, whereas
Tcf4 might induce them to proliferate. In the mouse, Tcf3 maintains
stem cells in the hair follicle (Nguyen et al., 2006), whereas Tcf4
mediates proliferation in the intestinal crypts (Korinek et al., 1998).
Although neither gene is known to function in mouse tooth
development, a growing body of data implicates canonical Wnt
signaling in promoting tooth renewal in mammals (Järvinen et al.,
2006; Wang et al., 2009).
To determine how canonical Wnt signaling affects the lingual
dental lamina cells, we stimulated the pathway in gecko dental
explants using the GSK3b inhibitor BIO. BIO caused the dental
lamina to thicken at intertooth and tooth levels (Fig. 4G,H). This
phenotype is due to a preferential increase in cell proliferation on
the lingual side of the dental lamina (Fig. 4I-N; see Fig. S8 and
Table S4 in the supplementary material). Interestingly, the effect of
Wnt gain of function on mouse dental cell proliferation has not
been explored (Järvinen et al., 2006; Wang et al., 2009) despite the
presumed requirement for increased cell proliferation in
supernumerary tooth formation.
Since the lingual dental lamina is the primary location of slowcycling cells, we suggest that Wnt gain of function induced dental
stem cells on that side to proliferate. Tcf4 might have mediated the
mitogenic effect of BIO as its expression was upregulated
throughout the culture, including in the lingual dental lamina (Fig.
4K,L, insets). It is unclear whether BIO acted directly on the
DEVELOPMENT
Fig. 4. The Wnt pathway regulates
proliferation of the lingual dental lamina
cells. (A-F)Near-adjacent sections through 20week-chased gecko dental tissues stained for
histology (A), BrdU-retaining cells (B,
arrowheads), transcripts (pseudo-colored red;
arrowheads in C-E) of Igfbp5 (C), Tcf3 (D) and
Tcf4 (E) and PCNA (F, arrows; to detect
proliferating cells). Tcf3 is expressed in the dental
lamina, whereas Tcf4 expression coincides with
proliferating cells (F) in the successional lamina
and enamel organ. Solid line, boundary of the
dental epithelium. (G-N)Sections (G-L) through
gecko dental tissues treated with BIO or DMSO.
Wnt gain of function causes the dental lamina
to thicken at both tooth (G,H) and intertooth
(insets in G,H) levels owing to an increase in cell
proliferation (I-L, arrows) in the lingual layer of
the dental lamina (M,N) (see also Fig. S9 in the
supplementary material). Dashed line, boundary
of the dental epithelium. Asterisk (G,I) indicates
thickened dental lamina BIO induced ectopic
Tcf4 expression (K,L, insets). (O)Summary of the
data from A-N. Scale bar: 100m.
lingual cells or indirectly via neighboring mesenchymal cells.
Further work is needed to clarify this uncertainty and to dissect
how other pathways (e.g. FGF, BMP, Hh) interact with Wnt to
determine stem cell fate. The sources of the signals that regulate
stem cell fate should also be explored. We suggest that adjacent
tooth buds and neural crest-derived mesenchyme are two potential
sources (see Fig. S9 in the supplementary material).
A model of stem cell-mediated tooth replacement
On the basis of our pulse-chase and gene expression data (Fig. 4O),
we hypothesize that the lingual layer of the gecko dental lamina
bears a population of putative dental stem cells. The lingual dental
lamina constitutes a fitting home for stem cells as it is a quiescent
environment and is not involved in tooth morphogenesis. Thus,
lingual dental lamina cells are sequestered from signals that might
otherwise instruct them to proliferate or differentiate. At the same
time, in the lingual layer of the lamina, stem cells are conveniently
positioned to give rise to the successional lamina. We suggest that
the successional lamina is built from the proliferative descendents of
the stem cells, whereas the stem cells themselves remain fixed in
position and sheltered from molecular signals in the lingual dental
lamina (see Fig. S9 in the supplementary material). The distribution
pattern of BrdU-retaining cells in our reconstruction of the gecko
tooth row implies that stem cells are arranged as discrete plaques or
‘hubs’ along the anterior-posterior axis of the dental lamina. Each
hub may provide progenitors for a single tooth family or a group of
families (see Fig. S9 in the supplementary material). These
hypotheses await experimental confirmation by cell lineage analyses.
Finally, we predict that the possession of dental epithelial stem
cells is a prerequisite for tooth renewal and, furthermore, that it is
a conserved feature of all animals that replace their teeth. Once this
has been confirmed, the molecular network that governs the fate of
dental epithelial stem cells should be worked out in detail. In
particular, more needs to be determined about the signaling that
enables stem cells to self-renew indefinitely in non-mammalian
vertebrates and why this capacity is reduced in mammals.
Acknowledgements
We thank Katherine Fu, Marcia Graves and Arthur Sampaio for help with in
situ hybridization, provision of reagents and micro CT, respectively. Operating
funds from the Natural Sciences and Engineering Research Council (NSERC) to
J.M.R. and postdoctoral fellowships to G.R.H. (Michael Smith Foundation for
Health Research; NSERC) supported this work.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.052415/-/DC1
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DEVELOPMENT
Dental stem cells and tooth replacement