Original Article
Caspase-7 participates in differentiation of cells forming
dental hard tissues
Eva Matalova,1,2
* Herve Lesot,3,4
Eva Svandova,2
Tom Vanden Berghe,5,6
Paul T.
Sharpe,7
Christopher Healy,7
Peter Vandenabeele5,6
and Abigail S. Tucker7
1
Department of Physiology, University of Veterinary and Pharmaceutical Sciences, 2
Institute of Animal Physiology and
Genetics CAS, v.v.i., Brno, Czech Republic; 3
INSERM UMR 1109, Team “Osteoarticular and Dental Regenerative
NanoMedicine”, 4
Faculte de Chirurgie dentaire, Universite de Strasbourg, Strasbourg, France; 5
Department for Molecular
Biomedical Research, VIB, 6
Department of Molecular Biology, Ghent University, Ghent, Belgium; and 7
Department of
Craniofacial Development and Stem Cell Biology, King′s College London, London, UK
Apoptosis during tooth development appears dependent on the apoptotic executioner caspase-3, but not caspase-7.
Instead, activated caspase-7 has been found in differentiated odontoblasts and ameloblasts, where it
does not correlate with apoptosis. To further investigate these findings, the mouse incisor was used as a model.
Analysis of caspase-7-deficient mice revealed a significant thinner layer of hard tissue in the adult incisor. Micro
computed tomography scan confirmed this decrease in mineralized tissues. These data strongly suggest that
caspase-7 might be directly involved in functional cell differentiation and regulation of the mineralization of dental
matrices.
Key words: ameloblast, apoptosis, caspase, cell differentiation, odontoblast.
Introduction
Caspase-7 is a cystein protease involved in apoptosis
and inflammation. Caspase-7 has often been considered
to be redundant to caspase-3, another member
of the death trio of executive caspases. Both of these
enzymes become activated by cleavage, mediated by
a cascade initiated by a death ligand–receptor interaction
or by apoptosome formation after cytochrome-c
release from the mitochondria (Lamkanfi & Kanneganti
2010). In addition, particularly caspase-7 has been
linked to inflammatory responses (Martinon & Tschopp
2007). Recently, non-apoptotic and non-inflammatory
functions of caspases have been suggested, such as
a role in tissue regeneration, cell fate determination,
neural activation and cell differentiation (Kuranaga
2012). Caspase-3, the most studied caspase, was
demonstrated to be a putative gatekeeper in stem
cells, playing a role in non-apoptotic events in several
stem cell derived lineages (Fujita et al. 2008). Lack of
caspase-3 causes disorders also in bone turnover due
to decreased osteogenic differentiation of bone marrow
stem cells (Miura et al. 2004).
Morphogenesis of tooth germs during odontogenesis
is accompanied by proliferation, differentiation, adhesion,
migration, and also apoptosis (Peterkova et al.
2003; Matalova et al. 2004). Caspases become activated
during the course of tooth development (Matalova
et al. 2006; Setkova et al. 2007), particularly in
relation to apoptotic events (Matalova et al. 2012a).
Notably, non-apoptotic cleavage of caspase-3 was
reported in normal and neoplastic odontogenic epithelial
cells in human patients (Kumamoto et al. 2001).
Moreover, cleaved caspase-7 was found in mouse
molars in the primary enamel knot undergoing apoptosis
but also in non-apoptotic areas (Matalova et al.
2012b). Attempts were thus made to further investigate
the possible non-apoptotic role of caspase-7, by
moving to the mouse incisor as a model.
The mouse incisor grows throughout the animal’s life
with the population of hard tissue producing cells (odontoblasts
and ameloblasts) renewed by differentiation
of cells derived from populations of stem cells located
at the apical base of the tooth. The epithelial stem
*Author to whom all correspondence should be addressed.
Email: matalova@iach.cz
Received 3 December 2012; revised 12 March 2013;
accepted 2 April 2013.
ª 2013 The Authors
Development, Growth & Differentiation ª 2013 Japanese
Society of Developmental Biologists
Develop. Growth Differ. (2013) 55, 615–621 doi: 10.1111/dgd.12066
The Japanese Society of Developmental Biologists
cells are housed in a bulge at the end of the labial
cervical loop, while the mesenchymal stem cells have
been suggested to reside distally near from the cervical
loop (Harada et al. 1999; Lapthanasupkul et al.
2012). The mouse incisor forms a tooth bud around
embryonic day (E) 13, enters the cap stage (E14–15)
and forms a bell structure by E16 (Kieffer-Combeau
et al. 2001). During embryonic development, the tooth
germ elongates in a posterior direction due to cell proliferation.
The elongation then proceeds anteriorly and
allows tooth eruption and lifelong compensation for the
progressive abrasion at the incisor tips. The labial and
lingual parts of the incisor differ in histology, as only
the labial side is covered by enamel, allowing the
upper and lower incisors to self-sharpen on contact.
The mouse incisor, therefore, represents a unique
model for addressing questions of growth and differentiation,
allowing several stages of cell differentiation to
be visualized as gradients on a single sagittal section.
This work aims to investigate the possible non-apoptotic
functions of caspase-7 in cell differentiation during
odontogenesis by evaluation of the cleavage
(activation) pattern of caspase-7, and analysis of the
phenotype of loss of caspase-7 on formation of the
continuously growing mouse incisor.
Materials and methods
Samples
CD1 mice were killed according to the experimental
protocol approved by the Laboratory Animal Science
Committee of the IAPG CAS, v.v.i., Brno, Czech
Republic. Mouse heads or mandible quadrants (at
postnatal stages) were immediately fixed in 4% buffered
paraformaldehyde and decalcified in buffered
ethylenediaminetetraacetic acid (EDTA). Samples were
dehydrated through a graded series of ethanols, treated
with xylene, paraffin embedded and cut in 4-lm
serial sections. Prenatal (embryonic) stage E17.5, postnatal
(P) stages P0, P5 and P10 were used in the
study. Caspase-7À/À
mice that had been backcrossed
on the C57BL/6 genetic background over at least 10
generations were provided by Professor P. Vandenabeele
(Duprez et al. 2011) and samples processed as
for wild type animals.
Immunohistochemistry
Immunohistochemistry was performed as described in
Matalova et al. (2012b). Briefly, deparaffinized and rehydrated
histological sections were pre-treated (only
anti-caspase-7 samples) in citrate buffer (pH = 6.0)
10 min/98°C and in 3% hydrogen peroxide in PBS
(5 min/RT). The primary antibody (anti-caspase-7: Cell
Signaling 9491, anti-amelogenin: Abcam ab59705,
anti-PCNA: Santa Cruz sc-7907) was diluted (anti-caspase-7:
ready-to-use solution 50x, anti-amelogenin:
ready-to-use solution 400x, anti-PCNA: 1009), and
applied overnight/4°C. Peroxidase-conjugated streptavidin-biotin
system (Vectastain, S7100) and chromogen
substrate diaminobenzidine (DAB, Dako, K3466) reactions
were used to visualize positive cells as brown.
Slides were counterstained by hematoxylin to clearly
distinguish the cell nuclei. Negative control was performed
by omitting the primary antibody. Specificity of
the selected anti-caspase-7 antibodies was verified
using caspase-7 deficient tissues.
MicroCT
Specimens for microCT (computed tomography) were
scanned using a GE Locus SP microCT scanner. The
specimens were immobilized using cotton gauze and
scanned to produce 14 lm voxel size volumes, using
an X-ray tube voltage of 80 kVp and a tube current of
80 lA. An aluminum filter (0.05 mm) was used to
adjust the energy distribution of the X-ray source. The
specimens were characterized further by making threedimensional
isosurfaces, generated and measured
using Microview software (GE). Mineral measurements
were carried out using the Advanced Bone Module of
Microview. Scan data was reconstructed and rendering
carried out using the Microview 2.2 software package.
Dentin and enamel was segmented using Microview’s
auto-thresholding facility and isosurfaces were reconstructed
separately and loaded into the software using
the Geometry overlay feature. Statistical significance
was determined using a t-test and F-test (P > 0.05).
Eight adult specimens were analyzed, four wild type
(WT) mice and four knock-out (KO) mice.
Results
Cleaved caspase-7 during incisor development
At E17.5, the incisor tooth germ has reached the bell
stage. At this stage the differentiation of functional odontoblasts
proceeds as a gradient from the apex (cervical
loops), while the ameloblasts are just starting to
differentiate (Fig. 1A). Positive staining for cleaved caspase-7
was apparent particularly in the cytoplasm of
the differentiating ameloblasts, cells of the stratum intermedium
and also the polarized odontoblasts
(Fig. 1B). Staining was detected on the inner portion of
the epithelium on the labial part of the cervical loop
(Fig. 1C). In contrast, no expression was observed on
the lingual part of the cervical loop (data not shown).
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Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists
616 E. Matalova et al.
At P0 (Fig. 1D1,2), functional ameloblasts and enamel
were covering the labial part of the tooth germ,
although only in its anterior portion (Fig. 1D1). Staining
for cleaved caspase-7 was found in the odontoblasts
and ameloblasts (Fig. 1E), whereas the neighbor cells
of stratum intermedium were caspase-7 negative at
this stage (Fig. 1E). In the labial part of the cervical
loop, caspase-7 positive cells were detected on the
inner portion of the epithelium (Fig. 1F), while the outer
epithelium in contact with the peridental mesenchyme
remained unstained (Fig. 1C,F).
Later on, at P10 (Fig. 1G), the active form of caspase-7
was detected in the cytoplasm of ameloblasts
(Fig. 1H,J) and odontoblasts (Fig. 1I, K) while nuclei in
(A) (B) (C)
(D1)
(D2)
(E) (F)
(G) (H) (I)
(J) (K) (L)
Fig. 1. Cleaved caspase-7 in incisor development. (A,D,G) Hematoxylin/Eosin stained tissue. (B,C, E, F, H, I) Immunohistochemistry of
cleaved caspase-7. (A–C) Embryonic day (E) 17.5. (D–F) Postnatal day (P) 0. (G–I) P10. (A): Sagittal section, with clearly formed cervical
loop. Scale bar = 100 lm. (B) Caspase-7 positive ameloblasts, odontoblasts and stratum intermedium cells. Scale bar = 100 lm. (C)
Cervical loop at apex of tooth with positive label in the inner epithelium on the labial side. Scale bar = 50 lm. (D1) Sagittal section. Anterior
region with differentiated ameloblasts and odontoblasts. (D2) Apical part of the tooth with the cervical loop. Hematoxylin/Eosin. Scale
bar = 100 lm. (E) Caspase-7 positive ameloblasts and odontoblasts. Scale bar = 100 lm. (F) Caspase-7 localized to the inner epithelium
of the labial cervical loop. Scale bar = 100 lm. (G) Frontal section. Enamel covers the labial part of the tooth (top) while the lingual
side (bottom) remains enamel free. Scale bar = 100 lm. (H) Detailed look at the localization of cleaved caspase-7 at P10 with positive
ameloblasts and negative stratum intermedium cells. Scale bar = 50 lm. (I) A border is evident between the caspase-7 positive ameloblasts
and odontoblasts and negative periodontal mesenchyme and dental pulp. Scale bar = 50 lm. (J) Detailed look at the localization
of cleaved caspase-7 at P10 with positive ameloblasts and negative stratum intermedium cells. Scale bar = 50 lm. (K) Detailed look at
the localization of cleaved caspase-7 at P10 with positive odontoblasts. Scale bar = 50 lm. (L) A border is evident between the caspase-7
positive ameloblasts and odontoblasts and negative periodontal mesenchyme and dental pulp. Scale bar = 50 lm. AM,
ameloblasts; b, bone; CL, cervical loop; dn, dentin; DP, dental papilla; en, enamel; OD, odontoblasts; pAM, preameloblasts; PDM,
peridental mesenchyme; pdn, predentin; SI, stratum intermedium; lin, lingual side; lab, labial side.
ª 2013 The Authors
Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists
Caspase-7 and cell differentiation 617
both cell types were negative. A clear border was
observed between the cleaved caspase-7 positive odontoblasts/ameloblasts
and the cells of the peridental
mesenchyme, next to these ameloblasts, which was
negative (Fig. 1L). Caspase-7 positive odontoblasts
were detected also in the root-analog of the incisor
(data not shown), whereas the stratum intermedium
cells remained caspase-7 negative (Fig. 1H). Dental
pulp was in general caspase-7 negative at all investigated
stages. Few scattered, slightly positive cells
were found in the marginal parts beneath the forming
odontoblast layer.
Analysis of caspase-7 knock-out incisor phenotype
In the adult caspase-7 deficient mice, the incisors
erupted as in wild type littermates (Fig. 2A,D). Despite
normal amelogenin expression in the WT and KO
(Fig. 2B,E), the histological analysis suggested some
differences in the thickness of the hard tissue with less
mineralized matrix laid down in the knock-out compared
to the wild type (Fig. 2C,F). There were no histologically
apparent differences in the stratum
intermedium cells of the wild type and deficient mice
(Fig. 2G1,H1). Based on PCNA expression in the adult
mice, in both, the wt and deficient mice, the lines of differentiated
odontoblasts and ameloblasts were negative;
few scattered positive cells were detected in the mesenchymal
tissue surrounding the labial part of the incisor
(Fig. 2G2,H2) and the mesenchyme around the interface
of the enamel and enamel-free region of the tooth
(Fig. 2G3,H3). MicroCT (Fig. 2I) and statistical analyses
(Fig. 2J) confirmed a significant decrease in the
amount of mineralized tissue in caspase-7 KO mice.
Discussion
Proteins known for their specific roles in apoptosis
have recently been reported as associated with a wide
range of non-apoptotic functions such as cell cycle
progression, differentiation, self-renewal, metabolism
or autophagy (Galluzzi et al. 2012). Caspases, as the
key proteins/enzymes in the apoptotic machinery
(Feinstein-Rotkopf & Arama 2009), are the hottest candidates
for such diverse roles.
Dental apoptosis is caspase dependent (Matalova
et al. 2012a); however, so far only caspase-3 (Matalova
et al. 2006) and caspase-9 (Setkova et al. 2007) have
been shown to be essential in molar development as
their deficiency leads to absence of apoptotic cells.
Caspase-7 was found expressed in the apoptotic
regions of the molar tooth signaling centers, the enamel
knots, but its absence (knock-out) did not cause any
inhibition or prevention of apoptosis (Matalova et al.
2012a). Notably, in the molars cleaved caspase-7 was
present in non-apoptotic, differentiated cells, particularly
odontoblasts and ameloblasts. Despite this
expression, deficiency of caspase-7 did not alter molar
development in a distinct way (Matalova et al. 2012a).
Therefore, we turned to the incisor, which is permanently
growing in the mouse, and differentiation of cells
producing dental hard tissues occurs continuously.
Immunohistochemistry confirmed the presence of
cleaved caspase-7 in incisor ameloblasts and odontoblasts,
in a similar pattern to the molars (Matalova
et al. 2012a). In the incisor a difference in expression
was shown between the labial and lingual parts of the
cervical loop. The epithelial cells in contact with the
basement membrane on the lingual side of the incisor
were caspase-7 negative. Since these cells do not
produce enamel, the absence of caspase-7 cleavage
on the lingual side and strong cleavage on the labial
side may suggest a role of caspase-7 in formation of
hard tissues, unrelated to apoptosis. The expression
indicates that caspase-7 may play a role in the fate of
dental cells that are producing extracellular matrix and
undergoing mineralization. Speculatively, the scattered
slightly positive cells in the marginal part of the dental
pulp mesenchyme at early stages of development may
mirror different fate of pulp located cells as described
in molar teeth (Diep et al. 2009; Rothova et al. 2012).
Furthermore, the caspase-7 deficient incisors displayed
a significant decrease in the amount of mineralized
matrix. However, the loss of caspase-7 did not
cause an absence of enamel in the knock-out but
rather delayed mineralization and/or hypomineralization.
Similar incomplete impairment in enamel formation
was reported in other transgenic animals
(Yoshioka et al. 2011). There was no obvious difference
between localization of amelogenin in the wild
type and caspase-7 knock-outs, but it is possible that
the levels of such enamel-associated proteins were
reduced but this was not quantified in our analysis. MicroCT
and histology showed variations in the amount
of mineralized matrix. All together, the set of observations
reported here supports the hypothesis that active
caspase-7 could be involved in the modulation of
ameloblast functional differentiation/maturation and not
in an on/off regulation. Very complex mechanisms regulate
dentinogenesis and amelogenesis (Caton &
Tucker 2009; Simmer et al. 2010) and the data presented
here suggest that caspase-7 may take part in
these networks. The hypothesis that caspase-7 is likely
to play a role in the differentiation/mineralization of
hard tissue/cells is supported by the fact that cleaved
caspase-7 can be observed not only in odontoblasts
and ameloblasts but also in osteoblasts forming the
bone hard tissue (Matalova et al. 2012a).
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Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists
618 E. Matalova et al.
Although it has been shown that some caspases,
such as caspase-3, are required for differentiation of a
variety of cell types (Larsen et al. 2010), the mechanism
allowing caspases to influence cell-fate decision
and regulate differentiation, survival or proliferation
remains unclear.
Subcellular localization of cleaved caspase-7 seems
to correspond with its particular function. During
apoptosis, activated caspases becomes translocated
into the nucleus (Lamkanfi & Kanneganti 2010),
whereas cleaved caspase-7 in non-apoptotic areas of
the incisor was found exclusively in the cytoplasm.
(A) (B) (C)
(D) (E) (F)
(G) (G1) (G2)
(H) (H1) (H2) (H3)
(I1) (I2) (J)
(G3)
Fig. 2. Analysis of caspase-7 deficient incisors. (A–C) Wild type adult incisor. (D–F) Caspase-7 knock-out mouse. (A) Macroscopic view
of fully erupted adult wild type incisor. Scale bar = 3 mm. (B) Immunohistochemistry of amelogenin in the adult wild type incisor. Scale
bar = 100 lm. (C) Detailed view at dentin (and odontoblasts) and enamel (and ameloblasts) structure and deposition in the adult wild
type mouse. Scale bar = 50 lm. (D) Macroscopic view of fully erupted adult incisor of the caspase-7 deficient mouse. Scale
bar = 3 mm. (E) Immunohistochemistry of amelogenin in the adult type incisor of the caspase-7 deficient mouse. Scale bar = 100 lm.
(F) Detailed view at dentin (and odontoblasts) and enamel (and ameloblasts) structure and deposition in the adult caspase-7 deficient
mouse. Scale bar = 50 lm. (G) Hematoxylin/Eosin stained sagittal section of the wild type incisor, indicating regions magnified in G1,2,3.
(G1) Detailed view at the stratum intermedium cells of the wild type incisor. (G2) Proliferation (PCNA staining) in the mesenchymal cells
surrounding the labial part of the wild type incisor. (G3) Proliferation (PCNA staining) in the mesenchymal cells surrounding the enamel
and enamel-free region interface of the wild type incisor. (H) Hematoxylin/Eosin stained sagittal section of the caspase-7 deficient incisor,
indicating regions magnified in H1,2,3. (H1) Detailed view at the stratum intermedium cells of the caspase-7 deficient incisor. (H2) Proliferation
(PCNA staining) in the mesenchymal cells surrounding the labial part of the caspase-7 deficient incisor. (H3) Proliferation (PCNA
staining) in the mesenchymal cells surrounding the enamel and enamel-free region interface of the caspase-7 deficient incisor. (I)
MicroCT of the adult incisor in the wild type (I1) and caspase-7 deficient mice (I2) confirmed decreased mineralization in the caspase-7
incisors. Scale bar = 2 mm. (J) Statistical evaluation of the microCT results. Both, the enamel area and volume display significantly lower
value in the knock-out than in the wild type. AM, ameloblasts; dn, dentin; DP, dental papilla; en, enamel; OD, odontoblasts; pAM, preameloblasts;
PDM, peridental mesenchyme; SI, stratum intermedium.
ª 2013 The Authors
Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists
Caspase-7 and cell differentiation 619
This observation is supported by the finding that in
developing molars caspase-3 is localized in the
nucleus of apoptotic cells (Matalova et al. 2006); while
it is localized to the cytoplasm of non-apoptotic epithelial
cells (Kumamoto et al. 2001). The cytoplasmic
localization of caspase-7 appears similar in molars and
incisors in non-apoptotic regions of the tooth (Matalova
et al. 2012b), indicating that the localization of
cleaved protein regulates its function.
One hypothesis suggesting the possible mechanisms
of caspase-7 function based on its localization within
the tooth germ would be engagement in differentiation/maturation
of cells by cleavage of proteins typical
for the undifferentiated status of cells. In such cases,
the non-apoptotic mechanism of caspase function
may include the depletion of stem cell markers, OCT4
and NANOG, as demonstrated in cancer cells after
drug treatment (Musch et al. 2010). In the study, caspase-3
cleaved NANOG, whereas caspase-7 was able
to degrade both of these markers. Oct3/4 was
reported to be strongly expressed in the nucleus of
the incisor cervical loop cells starting at E18, persisting
up to late postnatal stages (Li et al. 2011). As there is
no universal marker of the stem cell niche in the incisors
so far, Oct3/4 is one of the molecules expected
in dental stem cells (Li et al. 2011). Caspase-7 was
located in the inner part of the labial cervical loop,
while the outer region was caspase-7 negative. This
caspase-7 negative domain correlates with the region
of the cervical loop where Oct3/4 is present and to the
putative location of stem cells as indicated by Sox2 (Li
et al. 2011). In contrast to the cervical loop, Oct3/4 is
expressed in the cytoplasm in ameloblasts and odontoblasts
(Li et al. 2011) similar to the active form of
caspase-7. These findings could suggest some putative
participation of caspase-7 in the cleavage of Oct4
before/during cell differentiation. Depletion of stem cell
factors was shown to be mediated by cleaved caspases
and OCT4 is a direct in vitro target of active caspase-7
(Musch et al. 2010).
In this context, the expression of Oct4 was previously
reported in relation to different cell populations
during tooth root formation (Nakagawa et al. 2012). A
similar difference was detected between the regions
we show that are caspase-7 positive (ameloblasts) and
negative (stratum intermedium and peridental
mesenchyme) in the incisors. Therefore, the cleaved
form of caspase-7, connected with Oct4, could be a
further marker to distinguish these different cell popu-
lations.
Alternatively, caspase-7 might participate in the activation
of nuclear factor-jB (NF-jB). A link between
these pathways has been demonstrated by the effect
of caspase-3 on osteoclasts (Szymczyk et al. 2006) or
lens (Basu et al. 2012) differentiation. A similar role in
teeth is supported by the importance of tumor
necrosis factor (TNF) signaling in development and
evolution of tooth number, size and shape (Ohazama
& Sharpe 2004). Moreover, Fas receptor, as a member
of the TNF family, was suggested to have a modulatory
function in dental apoptosis (Matalova et al. 2005)
and might play a role in a switch in cell fate, as has
been reported in apoptotic versus non-apoptotic decisions
in other systems (Lavrik & Krammer 2012).
Recent data indicate that non-apoptotic functions of
caspases involve proteolysis exerted by their catalytic
domains as well as non-proteolytic functions exerted
by their prodomains (Lamkanfi et al. 2007).
Confirmation of such hypotheses, as well as the final
matching of caspase-7 into other molecular networks
governing differentiation, maturation and function of
hard tissue cells, is an important area for further analysis.
Acknowledgments
The research was funded by the Grant Agency of the
Czech Republic, P502/12/1285 at the UVPS and
P304/11/1418 at the IAPG. International cooperation
was supported by the Academy of Sciences of the
Czech Republic (project M200451201). The Brno lab
runs under RVO: 67985904.
Conflict of interest
The authors declare that they have no conflict of
interest.
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