archives of oral biology 59 (2014) 735–740

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Roles of notch signalling in mandibular condylar

cartilage

Maria J. Serrano *, Sarah So, Robert J. Hinton

Department of Biomedical Sciences, Texas A&M University Baylor College of Dentistry, 3302 Gaston Ave, Dallas, TX
75246, USA

article info abstract

Article history: Importance: Notch proteins are cell surface transmembrane spanning receptors which
Received 23 October 2013 mediate critically important cellular functions through direct cell–cell contact. Interactions
Received in revised form between Notch receptors and their ligands regulate cell fate decisions such differentiation,
9 April 2014 proliferation and apoptosis in numerous tissues. We have previously shown using immu-
Accepted 12 April 2014 nohistochemistry that Notch1 is localized primarily to the prechondroblastic (chondropro-
Available online genitor) layer of the mandibular condylar cartilage (MCC).
Objective: To test if Notch signalling changes patterns of proliferation and differentiation in
Keywords: the MCC and to investigate if Notch signalling acts downstream of Fibroblast Growth Factor
Notch 2 (FGF-2).
Fibroblast growth factor Methods: Condylar cartilage explants were cultured over serum-free DMEM containing
Gene array either 0 or 50 nM DAPT, a Notch signal inhibitor. Explants were used for RNA extraction
Condylar cartilage and immunohistochemistry.
Temporomandibular joint Results: Analysis of gene array data demonstrated that the perichondrial layer of the MCC is
rich in Notch receptors (Notch 3 and 4) and Notch ligands (Jagged and Delta) as well as
various downstream facilitators of Notch signalling. Disruption of Notch signalling in MCC
explants decreased proliferation (Cyclin B1 expression) and increased chondrocyte differ-
entiation (Sox9 expression). Moreover, we found that the actions of FGF-2 in MCC are
mediated in part by Notch signalling.
Conclusion: These data suggest that Notch signalling contributes to the regulation of prolif-
eration and differentiation in the MCC.
# 2014 Elsevier Ltd. All rights reserved.

conditions, it is these undifferentiated cells, rather than the

1. Introduction
The mandibular condylar cartilage (MCC) is a secondary
cartilage that differs structurally from both limb growth plate
and articular cartilage. This difference is most pronounced in its
superficial layers, which comprise a perichondrium in which
undifferentiated (prechondroblastic) cells secrete a matrix rich
in type I collagen rather than the type II collagen matrix
characteristic of chondrocytes.1,2 Under normal functional
chondrocytes in deeper layers, that proliferate and differentiate
to effect growth at the MCC.3 The Notch family of trans-
membrane receptors has been implicated as cell fate mediators
in many tissues.4–6 In a context-dependent manner, Notch
signalling promotes or suppresses proliferation, cell death,
acquisition of specific cell fates and activation of differentiation
programs during development and during maintenance of self
renewing adult tissues.7 Because of their critical role in many
fundamental processes and in a wide range of tissues, signalling

* Corresponding author. Tel.: +1 214 828 8976.

E-mail address: mserrano@bcd.tamhsc.edu (M.J. Serrano).
http://dx.doi.org/10.1016/j.archoralbio.2014.04.003
0003–9969/# 2014 Elsevier Ltd. All rights reserved.
736 archives of oral biology 59 (2014) 735–740
via Notch receptors has been linked to human disorders and
developmental syndromes.8 In articular cartilage, Notch 1 is
expressed in progenitor cells that exhibit phenotypic plasticity;
blockage of Notch signalling decreases proliferation in these
cells.9 Notch receptors are also expressed in the early stages of
chondrogenic differentiation, becoming confined to the peri-
chondrium as differentiation proceeds.10 We have previously
localized Notch immunohistochemically to the prechondro-
blastic and upper chondroblastic layers of the MCC11 and shown
that FGF-2 and Tgfb1 can upregulate and downregulate,
respectively, Notch 1 expression in the MCC.12 It is known that
FGF-2 regulates proliferation and differentiation of growth-
plate chondrocytes but the mechanisms underlying growth
regulation in secondary cartilages such as the mandibular
condyle have been less studied.12 The goals of this study were:
(1) to assess gene expression related to Notch signalling in the
MCC perichondrium; (2) to test whether disruption of Notch
signalling alters proliferation or differentiation in MCC; and (3)
to investigate whether Notch signalling lies downstream of FGF-
2 signalling.
2. Materials and methods
2.1. Gene array
The mandibular condyle and adjacent ramus were dissected
from CD1 mice at embryonic day 17 (E17). Under a dissecting
microscope, the perichondrium (PC) was gently teased away
from the underlying cartilage and the cartilage (C) was
separated from the bone. The PC and C samples were then
snap frozen in liquid nitrogen. RNA was extracted from pooled
samples of approximately 50 tissues using the RNEasy Micro
RNA Isolation Kit (Qiagen, Valencia, CA). The quantity and
quality of mRNA were measured by an Agilent 2100 Bioana-
lyzer. The RNA samples were analysed using the Mouse Notch
Signalling Pathway RT2 ProfilerTM PCR Array (PAMM-059,
SABiosciences, Frederick, MD), which profiles the expression
of 84 genes involved in Notch signalling including binding and
receptor processing genes as well as genes that cross-talk with
Notch. Genes were considered to be differentially expressed if
they were expressed at least two times higher in either the PC
or C sample.
2.2. Condylar cartilage explant culture
Condylar cartilage explants were placed on a triangular piece
of Poretics polycarbonate disc filter and metal grid suspended
over 1 mL of serum-free DMEM (3 mM sodium phosphate,
0.1 mM non-essential amino acids, 2 mM L-glutamine,
ITS + TM Premix (BD Biosciences), 500 mg/mL gentamicin,
100 mg/mL of ascorbic acid) containing either 0 or 50 nM
DAPT, a Notch signal inhibitor (Sigma Cat # D5942-5MG) in a
60  15 mm centre well organ culture dish. After 3 days of
culture, the mandibular condylar cartilages were separated
from the bone under a dissecting microscope, and snap frozen
in liquid nitrogen for RNA extraction. Five to six explants in
each group were used for immunohistochemistry.
In a second set of experiments, other explants (5–6 per
group) were incubated with media alone, media containing
50 nM DAPT, or media containing 50 nM DAPT plus 3 ng/mL of
FGF-2. After 3 days of incubation, RNA was extracted for gene
expression analysis.
2.3. Statistical analysis
Means and standard deviations were calculated for the
relative quantities of gene expression in the control and
experimental explant samples. Statistical differences were
evaluated using the Kruskall–Wallis test to identify differences
among the samples and the Mann–Whitney test to analyse
specific sample pairs for significant differences.
2.4. Quantitative real-time PCR
Primers were designed using the SciTools software (Integrated
DNA Technology). Each primer was evaluated in a primer matrix
to determine what combination of sense (S) and anti-sense (AS)
primer concentrations maximized the fluorescence response for
real-time PCR. SYBR1 Green Real-Time PCR Master Mix
(Stratagene) was used for fluorescence. The primer sequences
used were: Cyclin B1, (S: ACA GGG TCG TGA AGT GAC TGG AAA;
AS: CTT GGG CAC ACA ACT GTT CTG CAT); Delta like 1 (S: ATG
GAG CCGAGA AGA GCA GCT TTA; AS: ACT TGG TGT CAC GTT
TGC TGT GTG); HES1, (S: AGC CTA TCA TGG AGA AGA GGC GAA;
AS: TGG AAT GCC GGG AGC TAT CTT TCT). Samples along with
primers and SYBR Green Master Mix were loaded in an eight tube
strip and the reaction was run in the MX4000 Multiplex
Quantitative PCR System (Stratagene). 18s II was used as an
internal control. The primer sequences for 18s II were: S: CCG AAG
CGT TTA CTT TGA; AS: GCC GTC CCT CTT AAT CAT.
The standard curve method was used to calculate the
efficiency of the target genes. Serial dilutions of cDNA (1:10)
were made for the calibration curve and loaded into an eight
strip tube. Correlation coefficients for all standard curves were
0.92 or higher.
2.5. Immunofluorescent staining
Condyles were fixed in 4% neutral buffered paraformaldehyde,
decalcified in 0.5 M EDTA, processed for routine histology, and
embedded in paraffin. 6-mm sections cut in the sagital plane
were taken for immunohistochemical staining. Immunohis-
tochemistry was done by incubating at 4 8C overnight with
primary antibody to Notch 1 (Santa Cruz) at 1:75 dilution. After
three 5 min washes in 2% serum, the sections were incubated
in Alexa Fluor 546 (Molecular Probes) goat anti-rabbit (Notch)
secondary antibody for 1 h in the dark at RT. Finally, the
sections were washed four times in PBS, incubated in To Pro
1:500 for 5 min, cover-slipped with slow fade mounting
medium (Invitrogen), and examined using a Leica TCS SP2
confocal microscope.
3. Results
3.1. Notch-related gene expression in MCC perichondrium
To examine if differential expression of Notch pathway genes
exists in the perichondrium, we analysed gene expression in
 archives of oral biology 59 (2014) 735–740 737

Table 1 – Genes showing a higher expression in the MCC Table 2 – Genes showing a higher expression in the MCC
perichondrium (PC) than in the cartilage (C). Only genes cartilage (C) than in the perichondrium (PC). Only genes
with a PC/C ratio of 2.0 or greater were included. with a C/PC ratio of 2.0 or greater were included. A
previous study17 using this dissection technique to
Gene Expression in PC/Expression in C
separate PC and C demonstrated the expected differen-
Aes 4.3 tially higher gene expression in the C sample for
Delta 1 3.5 cartilage-specific genes such as Sox9 (4T), Col XI (14T),
Deltex 1 13 Col X (33T), and aggrecan (11T).
Frizzled 4 8.0
Hes 1 2.6 Gene Expression in C/Expression in PC
Hey 1 2.0 Hairless 4.5
IIL7b 8.5 Gli 1 7.0
Jagged 1 4.0 Shh 2.8
Jagged 2 5.3 Fzd 1 3.3
Lmo2 7.5
Mfng 7.0
Mtap 1b 13 of the most highly expressed genes in the PC sample were for
Mycl1 16
genes not obviously related to Notch signalling: V-Myc1
Notch 3 5.3
(16), Microtubule-associated protein 1b (13), Interleukin
Notch 4 4.3
PPAR-g 6.1 17b (9), and LIM domain only 2 (8). Among the genes
Presenilin 2 3.7 expressed more highly in the cartilaginous portion of the
Presenilin-Enhancer2 2.0 condylar cartilage were the Notch antagonist hairless (5),
Sonic hedgehog (3) and Gli1 (7), a mediator of hedgehog
signalling (Table 2).
manually-dissected perichondrial (PC) and cartilage (C)
samples using a gene array focused on genes related to 3.2. Gene expression in response to exogenous DAPT
Notch signalling. Expression of genes involved in Notch
signalling was differentially higher in the perichondrial To assess the effect of blocking Notch signalling, we used an
sample compared to the cartilage portion of the condylar organ culture system in which some explants were incubated
cartilage (Table 1). Expression of Deltex 1 was 13 times higher with media containing the Notch inhibitor, DAPT. We chose
in the PC sample, with Notch 3 and 4, Jagged 1 and 2, and specific genes that were upregulated in the array data or were
Aes higher by 3.5 to 5 times. Downstream mediators of involved in proliferation or chondrogenesis. The expression of
Notch signalling ranged from 7 higher (MFNG) to 2–4 times Notch ligand Delta-1 (2 fold), Notch transcription factor Hes 1
(Hes1, Hey1, Presenilin 2 and Presenilin enhancer 2). Two (3 fold), and Cyclin B1 (9 fold) were decreased in MCC tissue
genes instrumental in Wnt signalling, PPAR-g (6) and treated with DAPT. In contrast, expression of Sox 9 was
Frizzled 4 (8), were also enriched in the PC sample. Several increased 2 fold (Fig. 1).

Fig. 1 – Expression of Delta1, Hes 1, Cyclin B1, and Sox 9 in MCC explants following 3 day treatment with 50 mM DAPT. Bars
indicate SD. Differences between means are indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001.
738 archives of oral biology 59 (2014) 735–740

Fig. 2 – Effect of DAPT treatment on FGF-2 stimulated gene activity. Bars indicate SD. Statistical analysis was made using
Mann Whitney test. Differences among means are indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig. 3 – Immunolocalisation of Notch after DAPT treatment. Cell nuclei in red, Notch in green. Upper left: Untreated tissues at
low magnification (10T) view showing strong localization of Notch. Upper right: higher power (40T) view of condylar
cartilage showing localization of Notch in prechondroblastic and chondroblastic layers. Lower left: Notch was reduced in
MCC following DAPT treatment, especially in the prechondroblastic layer (lower right).
 archives of oral biology 59 (2014) 735–740
3.3. Gene expression after FGF-2 and DAPT treatment
In order to determine whether Notch signalling was down-
stream of FGF-2, we incubated some FGF-2-treated explants
with DAPT. Compared to explants incubated in control media,
FGF-2 stimulated expression of Notch1 and Cyclin B1 in MCC;
these effects were lost in FGF-2-treated explants also
incubated with DAPT (Fig. 2). Hes 1, downregulated in
response to FGF-2, was further reduced in DAPT-treated
explants. Expression of Sox 9, reduced by FGF-2 treatment,
was unaffected by DAPT.
3.4. Immunohistochemistry
Confocal microscopic images confirmed that Notch was most
prominent in the superficial layers of the MCC. The most
intense immunoreactivity was in the prechondroblastic layer
of cells (Fig. 3 upper). DAPT treatment reduced expression of
Notch in this layer (Fig. 3 lower).
4. Discussion
The MCC perichondrium showed enriched expression for two
Notch receptors (Notch 3 and 4, 5 and 3.5 respectively) and
receptor ligands (Jagged 1 & 2, 4 and 5, respectively and Delta
1, 3.5); the ligands are responsible for the majority of Notch
signalling effects. MFNG (O-fucosylpeptide 3-beta-N acetylglu-
cosaminyltransferase), which modulates Notch signalling by
differentially affecting its ligands,13 was 7 more highly
expressed in the PC than in the C portion of the condylar
cartilage. Other genes more highly expressed in the PC included
those that facilitate Notch signalling downstream of receptor-
ligand interactions: Deltex 1, 13; Hes 1, 3; Hey 1, 2,
presenilin 2, 4. Aes, which is 4 higher in PC, is thought to
be involved in inhibition of cell differentiation following Notch
signalling activation.14 It is also notable that hairless, a Notch
antagonist in Drosophila,15 has relatively reduced expression in
the PC. Regulators of the Wnt pathway (PPAR-g and Frizzled 4),
which interacts with the Notch pathway,16 were also highly
expressed in the PC sample. Thus, numerous genes which are
important for Notch signalling are expressed at higher levels
(from 2 to 13) in the perichondrium of the MCC compared to
its deeper cartilaginous portion. In secondary cartilages such as
the MCC, the dividing cells are prechondroblasts located in the
perichondrium rather than differentiated chondrocytes.17
Thus, Notch signalling genes are most highly expressed in that
portion of the condylar cartilage where cell division takes place.
DAPT is an inhibitor of g-secretase, which catalyses the
cleavage of the Notch receptor to release the Notch intracel-
lular domain (NCID). NCID then moves to the nucleus, where it
activates downstream target genes7 (Fig. 4). The reductions in
Jagged 1, Delta 1 and Hes1 expression, as well as the
attenuated Notch immunoreactivity, confirm that DAPT is
inhibiting Notch signalling. DAPT treatment also decreased
the expression of Cyclin B1, an indicator of cell proliferation,
suggesting that Notch is involved in regulating the prolifera-
tion of prechondroblasts in the MCC. In contrast, the
expression of Sox 9, a transcription factor essential for
differentiation along a chondrocytic pathway,18 was increased
739
Fig. 4 – The Notch-signalling pathway. The Notch receptor
consists of a large extracellular part and an intracellular
part that mediates Notch signal transduction. Binding of
the extracellular part to ligands of the Delta and Jagged
families can be modulated by Fringe proteins. Interaction
of Notch with a ligand induces proteolytic cleavage of
Notch, successively by ADAM metalloprotease and y-
secretase activities, releasing the intracellular part of the
protein (intracellular (NICD-) Notch). NICD-Notch then
translocates to the nucleus and binds to the nuclear
transcription factor CSL. Deltex1 and Numb are
cytoplasmic regulators of Notch signaling. Binding of
NICD-Notch to CSL induces the dislocation of co-
repressors (coR) and recruitment of co-activators (coA)
such as Mastermind, and stimulates the transcription of
Notch target genes. The best known Notch-target gene is
HES1 (Hairy-Enhancer of Split 1).7
by the treatment with DAPT. In view of the widespread
characterization of Notch as a mediator of cell fate, our data
suggest that inhibition of Notch signalling in the MCC
accelerates differentiation of prechondroblasts into non-
proliferating chondrocytes.
A previous study from our lab demonstrated that exoge-
nous application of FGF-2, a known mitogen for MCC,12 caused
a dose-dependent increase in Notch gene expression. Here, we
present evidence that increased proliferation in MCC explants
incubated with exogenous FGF-2 is reversed following DAPT
treatment to inhibit Notch signalling. Several studies in caudal
neural plate,19,20 sensory neurons and glia,21 and vertebrate
somites22 have suggested that Notch signalling lies down-
stream of FGF; our data suggest that is the case in MCC.
These data advance our understanding the role of Notch
signalling in the regulation of proliferation and differentiation
in the MCC by showing (1) an enhanced expression of Notch-
related genes in the perichondrium; (2) a role for Notch
signalling in the regulation of proliferation in this layer; (3)
evidence that Notch signalling in MCC lies downstream to
FGF-2.
Funding
None.
740 archives of oral biology 59 (2014) 735–740
Competing interests
None declared.
Ethical approval
Not required.
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