DEVELOPMENTAL DYNAMICS 243:864–874, 2014

DOI: 10.1002/DVDY.24130

REVIEW

Genes that Regulate Morphogenesis and Growth

of the Temporomandibular Joint: A Review
a

Robert J. Hinton*

Department of Biomedical Sciences, Texas A&M Baylor College of Dentistry, Dallas, Texas

Compared with the joints of the limbs, our understanding of the genes that regulate development and growth in the tempo-
romandibular joint (TMJ) is fairly limited. Because the morphogenesis of the secondary cartilage and other intra-articular struc-
tures in the TMJ occurs later and in a different manner than in the limbs, the genetic control of TMJ development might
reasonably be assumed to differ from that in the limbs. However, studies of the specific genes regulating TMJ morphogenesis
and growth have only begun to appear in the literature within the last decade. This review attempts to survey and interpret
the existing knowledge on this topic and to suggest fruitful avenues of investigation for the future. Studies to date using
knockout and over-expression of candidate genes suggest that a developmental hierarchy of joint structures exists, with
DEVELOPMENTAL DYNAMICS

condyle development primary. A hierarchy of gene expression also exists: Runx2 and Sox9 expression is critical for condylar
cartilage formation. Several of the other genes discussed in this report may regulate TMJ morphogenesis by affecting
Sox9 and Runx2 expression and control the ihh-PTHrP axis by means of these genes. Developmental Dynamics 243:864–874,
2014. VC 2014 Wiley Periodicals, Inc.

Key words: temporomandibular joint; development; gene expression; condylar cartilage; mandibular fossa; articular disc

Submitted 30 January 2014; First Decision 11 March 2014; Accepted 17 March 2014; Published online 26 March 2014

Introduction cartilage. They are found, transiently in some instances, at many

The temporomandibular joint (TMJ) is an important contributor
to the growth of the mandible in length and height, resulting
from endochondral ossification at the deep surface of the man-
dibular condylar cartilage (MCC). The growth at the MCC appears
to occur largely in response to that of the midface, which is
mostly driven by the cartilages of the cranial base and nasal cav-
ity (Petrovic et al., 1975). This ”adaptive” growth, which enables
mandibular growth to remain synchronous with that of the mid-
face, is facilitated by unique features of the MCC that relate to its
unusual developmental history.
Because its morphogenesis occurs late in prenatal develop-
ment, the mandibular condylar cartilage has been designated as a
secondary cartilage in contradistinction to primary cartilages of
the limbs and cranial base (Beresford, 1981). Unlike these carti-
lages, the mandibular condylar cartilage arises either from alka-
line phosphatase-positive cells of likely periosteal origin (Shibata
et al., 2002) or as a condensation separate from the developing
bone (Vinkka, 1982; Anthwhal et al., 2008); it develops adjacent
to the intramembranous bone of the mandible, distinct from
Meckel’s cartilage (Vinkka, 1982; Radlanski et al., 2003). Second-
ary cartilages such as the MCC are fibrocartilages that are struc-
turally distinct from both the limb growth plate and the articular
*Correspondence to: Robert J. Hinton, Department of Biomedical Sciences,
Texas A&M Baylor College of Dentistry, Dallas, TX 75246.
E-mail: bhinton@bcd.tamhsc.edu
Grant sponsor: NIH; Grant number: DE015401
locations in the developing craniofacial complex (Vinkka, 1982)
and continue postnatally in regions such as the mandibular con-
dyle, angular process of the mandible, and intermaxillary suture
(Petrovic, 1972; Hinton, 1988). Because of its persistence and role
in the growth of the mandible, the MCC is one of the most impor-
tant secondary cartilages. During the evolution of the TMJ as the
multi-bone reptilian jaw changed in mammals to a single bone
housing a specialized dentition, the periosteum of formerly intra-
membranous bones was transformed into a perichondrium in
force-bearing areas where the “new” TMJ would develop (Cromp-
ton, 1985). In the mammalian TMJ, the seamless transition from
a periosteum on the ramus to a perichondrium in the force-
bearing mandibular condyle is reflected in the spatial continuity
of the fibrous and osteogenic layers of the periosteum with the
articular and prechondroblastic layers of the perichondrium (Fig.
1). In another reflection of its evolutionary origins, the TMJ is
formed by three separate mesenchymal condensations (condylar,
temporal, and disc) that grow toward each other during develop-
ment (Sperber, 2001); in contrast, the joints involving primary
cartilaginous articulations develop within a single block of
mesenchyme.
A secondary cartilage, such as the mandibular condylar carti-
lage, differs from primary cartilages most clearly in its superficial
layers, comprising a perichondrium in which the cells that are
Article is online at: http://onlinelibrary.wiley.com/doi/10.1002/dvdy.
24130/abstract
C 2014 Wiley Periodicals, Inc.
V

864

DEVELOPMENTAL DYNAMICS
Fig. 1. Continuity of perichondrium in mandibular condylar cartilage
with periosteum of adjacent ramus in growing (postnatal) rat. Note how
the fibrous layer of the periosteum (long arrows) on the condylar neck
is continuous with the articular layer of the condylar cartilage (long
arrows). Similarly, note how the osteogenic layer of the periosteum
(short arrows) is continuous with the prechondroblastic layer of the
condylar cartilage (short arrows). Alcian blue stain.
relatively undifferentiated (prechondroblastic) (Fig. 2) secrete a
matrix rich in type I collagen rather than the type II collagen
matrix secreted by chondrocytes (Silberman et al., 1987; Mizogu-
chi et al., 1990). It is these relatively undifferentiated cells of the
prechondroblastic zone of the perichondrium, not the chondro-
cytes in deeper layers, that proliferate and mature to effect
growth at the mandibular condylar cartilage (Petrovic et al.,
1975; Carlson et al., 1980). Unlike the proliferative chondrocytes
of primary cartilaginous joints (inset, Fig. 2), the prechondrocytes
in the condylar cartilage exhibit a dual potential, forming either
cartilage or bone, depending on the mechanical forces impinging
on the tissue (Glineberg et al., 1982; Lydiatt and Davis, 1985).
This phenotypic lability is underscored by the expression of both
Sox9 and Runx2 RNA (markers for the chondrogenic and osteo-
genic pathways, respectively) in the mesenchymal cell condensa-
tion and later in the prechondroblastic cells of the developing
MCC (Shibata et al., 2006) and by the preferential localization of
the cell fate mediators Notch and Twist to these cells (Serrano
et al., 2011). Because the localized transformation of a periosteum
to a perichondrium is thought to occur in regions subjected to
biomechanical forces or low oxygen tension (Hall, 1972), second-
ary cartilages can shrink or be replaced by bone if the evoking
stimulus is reduced or eliminated. Numerous examples of this
lessening or loss of the cartilaginous phenotype have been docu-
mented for the MCC in response to lack of movement (Carlson
GENES AND TEMPOROMANDIBULAR JOINT 865
Fig. 2. Histology of the growing (postnatal) mandibular condylar carti-
lage. A, articular layer; P, prechondroblastic layer; C, chondroblastic
layer; H, hypertrophic layer; B, zone of endochondral bone formation.
Coronal section, Attwood’s stain. Inset: Cranial base synchondrosis, an
example of a primary cartilage. Note that all cells are chondrocytes,
and that they are oriented in vertical columns (palisades) in contrast to
the more multi-directional orientation in the condylar cartilage. Sagittal
section, hematoxylin and eosin stain.
et al., 1980; Glineberg et al., 1982), reduced loading (Bouvier and
Hylander, 1984; Hinton, 1998), and placement in a nonfunctional
environment (Duterloo and Wolters, 1971; Petrovic et al., 1975;
Englesma et al., 1980).
In light of this unusual manner of development and the dis-
tinctive characteristics of the dividing cells in the MCC, it would
not be surprising if the molecular determinants of development
and growth of the TMJ were somewhat different from those in
primary cartilaginous joints. However, studies of the specific
genes regulating TMJ morphogenesis and growth have only
begun to appear in the literature within the last decade, although
many of these developmental and structural differences have
been known for years. To a significant degree, this recognition
may reflect a belated examination of the TMJ in existing trans-
genic and gene knockout models, prompted by a realization of
the peculiarities of this “dental” joint. The present study is an
attempt to synthesize what we have learned from these models
and to reflect on what we still need to know.
Genes Affecting Morphogenesis of the TMJ
The formation of the temporomandibular joint in the mouse (Fig.
3) largely resembles the sequence that occurs in humans: the
appearance of mesenchymal condensations that will become the
condyle, mandibular fossa, and articular disc (embryonic day [E]
13.5), followed by the differentiation of chondrocytes in the
deeper layers of the MCC (E15.0) and bone formation in the man-
dibular fossa. The process finishes with the formation of the artic-
ular disc and the upper and lower joint cavities (E17.5) (Shibata
et al., 1996; Gu et al., 2008). The inactivation of several genes
has been shown to result in the absence or major diminution of
structures at each of these developmental stages.
 866 HINTON

Mandibular Condylar Cartilage

The genes Runx2 (essential for bone formation) and Sox9 (needed
for cartilage formation) are both expressed in the mesenchymal
condensation that initiates the formation of the mandibular con-
dyle. As zonal differentiation in the MCC progresses by E15, both
genes are expressed in the prechondroblastic zone, as well as in
the newly formed cartilage (Shibata et al., 2006).
Runx2 (2/2) mice, which do not form bone at all, exhibited no
disruption of primary cartilage formation (e.g., in the limbs and
Meckel’s cartilage), while the MCC and other secondary cartilages,
such as the angular and coronoid, were completely absent (Shibata
et al., 2004). A condylar condensation that was alkaline phosphatase
(AP) -positive and exhibited weak Col 1 expression was present, but
reduced in size at E18.5, compared with the wild-type condensation
at E14. Because Runx2 is also an important contributor to the matu-
ration of the mesenchymal condensation (Nakashima et al., 2002), it
is possible that its absence may have accounted for the smaller con-
densate size. The mesenchymal condensations that also formed at
DEVELOPMENTAL DYNAMICS

the muscle attachment sites on the mandible were inter-connected.

However, no genetic markers of secondary cartilage formation (e.g.,
Col2 or aggrecan) were evident, suggesting that differentiation had
been arrested in the preosteoblastic stage. A subsequent study
showed that Runx2 (2/2) condylar explants could be induced to
undergo chondrogenic differentiation (but not hypertrophy) by the
addition of exogenous BMP-2 (Fukuoka et al., 2007), suggesting
that the secretory products of osteoblasts may contribute to second-
ary cartilage formation.
Inactivation of Sox9 in cranial neural crest cells (Fig. 4) also
resulted in the complete agenesis of the condylar cartilage (Mori-
Akiyama et al., 2003; Wang et al., 2011). In addition, mandibular
fossa formation was greatly abbreviated, and the articular disc
appeared more diffuse than in the controls, with incomplete joint
cavity formation by E18.5 (Fig. 4L). However, Runx2 expression
was still present in the mandibular ramus and early on in the man-
dibular fossa condensation of the null mice. Meckel’s cartilage was
absent, as were any bones formed by endochondral ossification.

Mandibular Fossa
Only two studies have reported a malformed or absent mandibu-
lar fossa. One of these is the Sox9 knockout described above
(Wang et al., 2011). Taking advantage of the fact that the man-
dibular fossa is derived from cranial neural crest cells but does
not express Sox9, Wang et al. were able to examine fossa devel-
opment in the absence of the mandibular condyle. Although the
mesenchymal condensation for the fossa formed normally at
E14.5, it experienced a delay in ossification (expression of
Runx2) and began to gradually regress, so that only a small bit of
bone remained laterally at day E18.5 (Fig. 4J). Thus, the mandib-
ular fossa was not congenitally absent but rather failed to sustain
its development and actually regressed over time. The authors
were able to further test this relationship by using two mouse
models that were “natural experiments”: one in which the devel-
oping condyle becomes dislocated from the developing mandibu-
lar fossa and one in which a greatly enlarged Meckel’s cartilage
displaces the condyle from its normal position relative to the
mandibular fossa. Dislocation of the mandibular condyle from
the mandibular fossa (mimicking the absence of the condyle)
once again resulted in the arrested development of the fossa,
while the presence of Meckel’s cartilage in articulation with the
Fig. 3. A: Temporomandibular joint at blastematic stage showing con-
dylar (CO) and temporal (T) blastemas separated by less dense mesen-
chyme that will form the future disc. Mandibular bone, MB. Mouse,
embryonic day 15. Hematoxylin and eosin, sagittal section. B: Temporo-
mandibular joint at cavitation stage illustrating chondro-differentiation of
condylar cartilage and formation of joint spaces in progress. Superior
joint space is evident (upper right), but inferior joint space is not yet
formed. CC, condylar cartilage; AD, articular disc; LPM, lateral pterygoid
muscle; TB, temporal bone. Mouse*, embryonic day 17. Hematoxylin
and eosin, sagittal section. *The superior joint space forms earlier in a
mouse, opposite the human condition. C: Temporomandibular joint at
maturation stage. Both inferior and superior joint spaces are fully
formed, lateral pterygoid muscle is attached to condylar neck and articu-
lar disc begins to resemble postnatal form. CC, condylar cartilage; AD,
articular disc; LPM, lateral pterygoid muscle; TB, temporal bone. Mouse,
embryonic day 19. Hematoxylin and eosin, sagittal section.
fossa permitted normal fossa development to continue. These
experiments suggest that normal fossa development depends on
normal condyle development.

DEVELOPMENTAL DYNAMICS
Fig. 4. Inactivation of Sox9 in the cranial neural crest cells results in an
absence of condylar cartilage and temporomandibular joint (TMJ) malfor-
mation. A,B: Skeletal preparations of mandibles from postnatal day (P) 0
wild-type control (A) and Wnt1Cre;Sox9f/f mice (B) reveal an absence of
cartilage elements in the condyle and angular process in the mutant.
The mutant mandible appears shorter and also lacks the coronoid pro-
cess. C,D: Coronal sections through the TMJ forming region of embry-
onic day (E) 14.5 wild-type (C) and Wnt1Cre;Sox9f/f embryos (D) show
formation of the glenoid fossa condensation. In the wild-type control, the
Meckel’s cartilage and condylar condensation are evident (C); however,
in the mutant, the mandibular ramus condensation is observed instead,
and the Meckel’s cartilage is also absent (D). E–J: Coronal sections
through the TMJ forming site of wild-type and mutant mice at various
stages reveal normal processes of TMJ development (E,G,I) and regres-
sion of the glenoid fossa in Wnt1Cre;Sox9f/f mice (F,H,J). K: Higher
magnification of the TMJ from E18.5 wild-type mice shows a compact
normal articular disc structure with flatted cells (arrow). L: Higher magni-
fication of the disc-like structure from E18.5 Wnt1Cre;Sox9f/f mice
shows a less compact tissue with irregular cell shape (arrow). c, condyle;
m, Meckel’s cartilage; gf, glenoid fossa; mr, mandibular ramus; agp,
angular process; crp, coronoid process; lpm, lateral pterygoid muscle.
Scale bars ¼ 1 mm in A,B; 200 mm in C–J. Wang et al., Dev Dyn
2011;240:2466–2473. Used with permission of the author.
The mandibular fossa also fails to develop in mice deficient in
both the genes Sprouty 1 and 2 (Spry1(2/2); Spry2 (/)),
which encode intracellular inhibitors of the receptor tyrosine
GENES AND TEMPOROMANDIBULAR JOINT 867
kinase signaling pathways, including those triggered by fibro-
blast growth factors (Purcell et al., 2012). Spry1 and Spry2 were
expressed in the lateral pterygoid and temporalis muscles adja-
cent to the TMJ structures, while the fibroblast growth factor
receptors Fgfr1, Fgfr2, and Fgfr3 were expressed differentially in
the periosteum, perichondrium, and MCC. The overall effects of
the Spry deletion were specific to muscle: Meckel’s cartilage was
unaffected but the temporalis and lateral pterygoid muscles were
48% and 69% larger, respectively, than in the control embryos.
The articular disc formed normally, and the condyle, although
histologically normal, was somewhat smaller than wild-type con-
dyles. However, no condensation was observed at E14.5 for the
mandibular fossa, and the fossa was absent except for a small lat-
eral portion, similar to that seen in the study by Wang and col-
leagues (2011). Individual inactivation of either Spry1 or Spry2
had no effect on the TMJ phenotype, nor did inactivation of
Spry1 and/or Spry2 in bone or cartilage. The authors suggested
that the enlarged temporalis muscle may have impeded the con-
densations of the temporal bone from fusing to form the mandib-
ular fossa in the mutant animals.
Articular Disc and Joint Cavities
The articular disc forms within a third mesenchymal condensa-
tion situated between the condensations that will become the
MCC and the mandibular fossa. By E16.5, a condensation of cells
in this intermediate region is discernible and gradually assumes a
disc shape. Simultaneously, small clefts in the mesenchyme
appear between the forming disc and the temporal bone. As these
clefts coalesce, the superior joint cavity (space) takes shape. By
E17.5, the inferior joint space between the disc and condyle is
present (Gu et al., 2008). The earliest studies describing the
absence or incomplete formation of the articular disc featured
mice lacking the Indian hedgehog (ihh) gene (Shibukawa et al.,
2007) or one of its downstream effectors such as Smoothened
(Smo) or the Gli family of transcription factors (Purcell et al.,
2009). Ihh is recognized as an important regulator of long bone
development and growth, including intramembranous bone collar
formation, chondrocyte proliferation and maturation, and endo-
chondral ossification (Koyama et al., 2007). Mice in which ihh
was inactivated failed entirely to develop a disc or to undergo
cavitation (Fig. 5), leaving a thin layer of mesenchyme separating
the condyle and mandibular fossa. Disruption of downstream
effectors of ihh signaling resulted in disc formation but incom-
plete cavitation (Purcell et al., 2009). The authors concluded that
ihh signaling is required not only to initiate disc morphogenesis
but also to promote disc maturation and cavitation at a later
stage of development.
Another model in which the disc failed to form or was delayed
involves a gene that interacts with ihh. Mice deficient in Trps1, a
gene coding for a transcription factor mutated in human Tricho-
Rhino-Phalangeal syndrome, show a nearly identical phenotype
to that in ihh (2/2)) mutants—i.e., the complete absence of a
disc and joint cavities (Michikami et al., 2012). Like ihh, Trps1
regulates various aspects of growth plate proliferation and matu-
ration, as well as the intramembranous ossification of the peri-
chondrium (Napierala et al., 2008). In perichondrial ossification,
Trps1 interacts with ihh/Gli3 signaling (Wuelling et al., 2009). In
addition, Trps1, expressed in the perichondrium and chondro-
cytes of the developing MCC at E15.5 (Michikami et al., 2012), is
known to be a strong repressor of Runx2 (Napierala et al., 2008).
 868 HINTON
DEVELOPMENTAL DYNAMICS
Fig. 5. Histological analysis of the effect of ihh inactivation on articular
disc formation. A–F: Serial sections from embryonic day (E) 18.5 wild-
type (A,B). ihh / (C,D), and ihh //Gli3/ (E,F) embryos were
stained with hematoxylin and eosin or processed for in situ hybridiza-
tion. The articular disc is indicated by an arrowhead, the upper joint
cavity by an arrow, and the lower joint cavity by a double arrow. Lubri-
cin expression was given a computer-assisted artificial color. Scale
bar ¼ 100 mm. Shibukawa et al., Dev Dyn 2007;236:426–434. Used with
permission from the author.
Because Trps1 is also expressed in the mesenchyme where the
presumptive disc will form (Michikami et al., 2012), it is perhaps
not surprising that Trps (2/2) mice share a disc agenesis pheno-
type with ihh (2/2) mice.
The implication of these models—that ihh-related signaling
may be critical for disc formation—can be evaluated in other
models displaying TMJ disc agenesis or malformation. For exam-
ple, the condylar agenesis in Sox9 (2/2) mice is accompanied by
a poorly formed disc and no definitive joint cavities (Wang et al.,
2011), which could be attributed to the absence of the ihh nor-
mally present in the absent condyle. In another model by the
same group, mice in which the ectopic activity of canonical Wnt
signaling occurs due to stabilized b-catenin exhibit a wide open
mouth due to abnormal skin development (Wang et al., 2011).
This circumstance disrupts the normal relationship between the
developing condyle and mandibular fossa and leads to a regres-
sion of the mandibular fossa and no articular disc. The authors
postulated that the increased distance between the condyle and
the disc condensation occasioned by the open mouth posture
could inhibit diffusible factors that would normally mediate disc
formation.
Conditional inactivation of the homeobox gene Shox2 in cra-
nial neural crest cells results in a delayed appearance of TMJ
structures, including the articular disc. At E17.5, at which time an
articular disc had formed in wild-type mice, the Shox2 (2/2)
mice had no discernible disc primordium. A disc had finally
formed in the newborn Shox2 (2/2) mice, but was adherent to
both the mandibular fossa and condyle, indicating incomplete
formation of joint cavities (Gu et al., 2008). The Shox2 (2/2)
mice, which showed a considerably reduced expression of both
Runx2 and Sox9, exhibited reduced ihh expression in the MCC
but the ectopic expression of ihh in the mandibular fossa. In the
context of the previous models, it could be argued that the
reduced ihh in the condyle plays a role in the delayed and mal-
formed disc and cavities.
Genes Affecting Growth at the TMJ
For several genes, inactivation results in few if any effects on the
morphogenesis of joint structures, but instead exerts a significant
impact on growth, primarily at the mandibular condyle. The pre-
viously discussed effects on articular disc formation in ihh (2/2)
mice are accompanied by inhibited proliferation and reduced
expression of Sox9 and PTHrP (a critical component of the ihh
regulatory loop) in the MCC, resulting in a severely shortened
mandible (Shibukawa et al., 2007). In tamoxifen-induced condi-
tional ihh knockouts at postnatal days 4, 7, 14, and 56, disrup-
tions in the zonal architecture of the MCC were also evident, with
greatly reduced zones of prechondroblastic and early chondro-
cytic cells (Ochiai et al., 2010). Partial ankylosis of the disc to the
condylar surface was also present, suggesting that ihh may be
important for the postnatal growth of the TMJ. In limb cartilage,
PTHrP forms a negative feedback loop with ihh, with PTHrP
maintaining the chondrocytes in a proliferative, less differenti-
ated state. This feedback loop appears to work in the prechondro-
blasts of the MCC as it does in proliferative chondrocytes of the
growth plate (Shibukawa et al., 2009). Mice with the targeted
expression of a constitutively active PTHrP receptor in cartilage
also show postnatal TMJ abnormalities, but in this instance, the
condylar cartilage is mostly composed of immature chondrocytes
and fibroblastic cells, with only a few islands of hypertrophic car-
tilage (Tsutsui et al., 2008). Another study analyzed the effect on
the TMJ of Ndst1 inactivation, which catalyzes the constituent
sulfation of heparan sultate proteoglycans (HS-PGs). Although
severely affected Ndst (2/2) mice lacked a TMJ, mildly affected
mutants at E18.5 exhibited a thicker prechondroblastic cell layer
with increased proliferation compared with wild-type animals
(Yasuda et al., 2010). Although the ihh expression was unaffected
in mutants, the topography of ihh signaling activity, as indicated
by its receptor Ptch, was much broader than in wild-type animals.
The authors suggest that this finding reflects a more extensive
field of ihh diffusion allowed by the lack of ihh binding to the
defective HS-PGs in the mutant condyles.
Transforming growth factor-beta (TGF-b) signaling also plays
an important role in regulating the growth of the MCC, as indi-
cated by the consequences of conditional inactivation of one of
its receptors, Tgfbr2, in cranial neural crest cells (Oka et al.,
2007). This receptor is specifically required for proliferation in
both osteoprogenitor and chondroprogenitor cells. Although a
mesenchymal condensation for the condyle was present in Tgfbr2
mutant mice at E14.5 (Fig. 6F), metachromatic staining of the
cartilage was greatly diminished in the mutant condyles by E16.5
(Fig. 6D). When joint formation was complete in wild-type ani-
mals at E18.5, chondrogenesis of the condylar process remained
greatly reduced compared with mutant condyles, and the hyper-
trophic zone was entirely absent (Fig. 6B). Thus, although mor-
phogenesis of the condyle was not completely abolished, the
resulting structure had only a vestigial cartilaginous component.
A subsequent study by this group (Oka et al., 2008) demonstrated
a greatly reduced proliferation in the condylar cartilage of
mutant animals as well as a diminished expression of Sox9.

DEVELOPMENTAL DYNAMICS
Fig. 6. TGF-b signaling is required for proper development of the con-
dylar process. Histological analysis of the coronoid, condylar and
angular processes in control and Tgfbr2fl/fl;Wnt1-Cre mice. A,B: Hema-
toxylin and eosin staining shows clear zones of endochondral ossifica-
tion in the condylar and angular processes in the control at E18.5. The
insets show X-gal staining of the condyle and angular processes. The
endochondral ossification of the condylar process is diminished in the
Tgfbr2fl/fl;Wnt1-Cre mutant at E18.5. C–F: Safranin-O staining of the
condylar process at E16.5 (C,D) and at E14.5 (E,F). At E16.5, the three
zones, articular, intermediate and hypertrophic, are visible in the con-
trol, but chondrogenesis of the condylar process is dramatically dimin-
ished and the hypertrophic zone is not detectable in the mutant
(arrows). At E14.5, the condylar cartilage matrix is visible in control
(arrows), but not in the Tgfbr2fl/fl;Wnt1-Cre mice (asterisk). Scale
bar ¼ 200 mm in panels A–F. Cd, condylar process; Cr, coronoid pro-
cess; Ang, angular process; az, articular zone; iz, intermediate zone;
hz, hypertrophic zone. Oka et al., Dev Biol 2007;303:391–404. Used
with the permission of the author.
Runx2 expression, however, increased in mutant condyles, as did
the expression of Dlx5, a transcription factor important for osteo-
blast differentiation. The authors suggest that TGF-b signaling
influences cell fate in the dividing cells of the MCC by regulating
Sox9 expression, which in turn represses Runx2 expression. In a
follow-up to these experiments, half mandibles from E13.5 and
E14.5 mice exposed in explant culture to an inhibitor of the
downstream signaling of type I TGF-b receptors that partner with
Tgfbr2 showed a complete loss of secondary cartilage not seen in
vehicle-treated explants, and pre-existing secondary cartilages in
E15.5 explants disappeared after 4 days in culture (Anthwal
et al., 2008). These results indicate that TGF-b is necessary for
initiation of secondary cartilages in the mandible. However, stud-
ies of mice lacking all striated musculature show that muscular
influences are critical for the maintenance of secondary cartilages
in the mandible. The most pronounced effects were seen in the
muscle-associated angular and coronoid cartilages with a lesser
effect on the condylar cartilage, where the authors speculate that
GENES AND TEMPOROMANDIBULAR JOINT 869
articular forces may also contribute to maintenance of the sec-
ondary cartilage (Rot-Nikcevic et al., 2007).
Fibroblast growth factors (FGFs) signal by means of four recep-
tors (fgfr1–4) to regulate a variety of cellular processes, including
cartilage growth and endochondral bone formation (Ornitz,
2005). One of those receptors, Fgfr3, is considered to inhibit
chondrocyte proliferation and negatively regulate endochondral
ossification in long bones, with Fgfr3 disruption leading to over-
growth (Colvin et al., 1996) and constitutive activation to short-
ening (Deng et al., 1996). Three of the FGF receptors (fgfr1, fgfr2,
and fgfr3) are expressed in different layers of the condylar carti-
lage (Molteni et al., 1999; Purcell et al., 2012). Mice with a
knock-in mutation in Fgfr3 that greatly enhances the receptor’s
affinity to its ligands display a set of alterations in the TMJ struc-
ture and growth beginning at postnatal day 21 (Yasuda et al.,
2012). The overall size of the condyle, as well as total cartilage
thickness, was less in the mutant mice, with reductions in prolif-
eration, Sox9 and Col10 expression, and a diminished trabecular
bone network underlying the cartilage. The secondary cartilage
lining the temporal bone was also reduced. The treatment of
mutant explants with ligands for Fgfr3 resulted in reduced prolif-
eration and down-regulation of Ptch, the receptor for ihh, as well
as other components of the ihh feedback system, suggesting that
Fgfr3 and ihh may exert opposing actions on proliferation and
chondrocyte maturation in the MCC. The Sprouty genes Spry1
and Spry2 also encode intracellular signaling inhibitors that
affect FGF signaling. In the Spry1(2/2)/Spry2 (2/2) mice
described earlier as exhibiting regression of the developing man-
dibular fossa, the condylar cartilage was described as having a
normal histological structure, albeit 50% smaller in length and
25% smaller in width compared with wild-type animals (Purcell
et al., 2012).
The Shox2 (2/2) mice, which exhibited a delayed develop-
ment of joint structures, also displayed reduced bone formation
in the condyle, as reflected by a greatly diminished osteocalcin
expression, as well as thinner layers in the condylar cartilage and
less hypertrophy (Gu et al., 2008). Both Sox9 and Runx2 expres-
sion were reduced in the mutant condyles, but the overall effect
on the condylar cartilage structure and growth appeared consid-
erably less than in the TGF-b model.
Although I am not aware of published TMJ phenotypes from
any other transgenic mice models, there is suggestive evidence
that other genes may affect TMJ morphogenesis and growth.
While not fully explored, two studies demonstrate that signaling
by BMPs, like that of TGF-b, may affect condylar cartilage mor-
phogenesis and growth. Deletion of the BMP type 1 receptor Alk2
in neural crest tissues has been shown to result in a much smaller
mandible with no development of any secondary cartilages in the
mandible (Dudas et al., 2004). Moreover, conditional deletion of
BMPr1A in cartilage of early postnatal mice significantly reduces
mandibular length and drastically attenuates the extent of the
condylar cartilage as well as cell proliferation and Sox 9 immu-
noreactivity in 4-week-old mice (Jing et al., in press).
Another likely possibility is the Notch family of receptors
(Notch 1–4) that have been identified as cell fate mediators in
many tissues (Curry et al., 2005; Purow et al., 2005). In cartilage,
there is a growing understanding that Notch signaling is context-
dependent, with disparate effects in developing and fully differ-
entiated chondrocytes (Kohn et al., 2012; Chen et al., 2013).
Although these studies have focused on limb cartilage, there is
evidence that Notch signaling may also be important in the
 870 HINTON
perichondrial cells of the TMJ. Examination of the gene expres-
sion in the perichondrial (PC) and underlying cartilaginous (C)
portions of the MCC micro-dissected from 2-day-old mouse con-
dylar cartilages using a gene array permits the identification of
genes that are preferentially expressed in the perichondrium
(Hinton et al., 2009). Use of an array profiling 84 genes related to
the Notch signaling pathway indicated that the perichondrium of
the MCC was enriched in many Notch signaling pathway genes
(Serrano et al., in press). Genes with considerably higher expres-
sion in the PC sample compared with the C sample included
Notch 3 and Notch 4 (5, 3.5), their ligands Jagged 1 and 2
(4, 5), and other regulators of Notch ligand specificity and
downstream signaling, such as MFNG (7), Deltex (13), and
presenilin enhancer 2 (4). Another notch receptor, Notch1, has
been shown using immunohistochemistry to be localized in the
superficial layers of the MCC, most prominently in the prechon-
droblastic layer (Serrano et al., 2011). Moreover, Notch1 expres-
sion in the MCC explants from E17 mice was up-regulated by
DEVELOPMENTAL DYNAMICS
increasing concentrations of exogenous FGF-2 and down-
regulated by increasing concentrations of exogenous TGF-b2
(Serrano et al., in press).
The Wnt/b-catenin pathway is still another potential regulator
of TMJ morphogenesis and growth. b-catenin signaling has been
shown to be an important determinant of cell fate in osteoproge-
nitor cells, and in its absence, these cells develop into chondro-
cytes (Hill et al., 2005). Indeed, the only study using b-catenin
signaling to examine the TMJ demonstrated that transgenic mice
with stabilized b-catenin had complete agenesis of the condylar
cartilage (Wang et al., 2011). Two other studies have character-
ized the effect of the b-catenin levels on cells similar to the divid-
ing cells of the MCC. The first study focused on cranial sutures in
which b-catenin is critical for both the proliferation and differen-
tiation of osteogenic precursors such that the inhibition of b-
catenin results in suture abnormalities (Mirando et al., 2010).
Like the prechondroblastic cells of the MCC, the preosteogenic
cells of a cranial suture are continuous with the osteogenic layer
of the periosteum and can exhibit a secondary cartilaginous phe-
notype in certain sutures that is reversible in response to
decreased loading (Hinton, 1988). Goodnough and colleagues
(2012) showed that the transcription factor Twist1 is directly acti-
vated by b-catenin in osteoprogenitor cells and that Twist1 binds
Sox9 to suppress chondrogenesis. Twist has been localized to the
preosteogenic cells of cranial sutures (Rice et al., 2000), as well as
to the prechondroblastic layer of the MCC (Serrano et al., 2011)
where it is joined by another cell fate determinant gene Notch. It
has also been shown that the prechondroblastic (chondroprogeni-
tor) cells of the MCC, similar to the preosteogenic (osteoprogeni-
tor) cells of developing sutures (Iseki et al., 1999), express Fgfr2
receptors (Fuentes et al., 2002; Molteni et al., 1999). Fgfr2 expres-
sion and bromodeoxyuridine (BrdU) immunoreactivity are pres-
ent in the same cell layer (proliferating osteoprogenitor cells) of
developing sutures, with Fgfr1 expressed as these cells differenti-
ate into osteoblasts (Iseki et al., 1999). These findings parallel the
observed distribution in the MCC of Fgfr2, BrdU immunoreactiv-
ity in prechondroblasts, and Fgfr1 in chondroblasts, the differen-
tiated phenotype (Fuentes et al., 2002). Thus, in sutures and in
MCC, Twist has an overlapping distribution with Fgfr2, making it
plausible that Twist activated by b-catenin contributes to the
regulation of cell fate in both tissues. Our study of genes differen-
tially expressed at higher levels in the perichondrium of the MCC
indicates that two mediators of Wnt signaling, PPAR -g (6) and
Frizzled 4 (8), are highly expressed in the perichondrium (Ser-
rano et al., in press).
An additional indication that b-catenin signaling affects
cells similar to MCC prechondroblasts arises from its effect on
superficial zone (SFZ) cells in the articular cartilage. These cells
have gene expression and phenotypic profiles distinct from
those of the underlying articular chondrocytes and exhibit
chondro-progenitor properties with a strong capacity for prolif-
eration over time (Yasuhara et al., 2011). The activation of b-
catenin signaling caused a strong increase in proliferation and
lubrican expression in these cells, whereas the ablation of b-
catenin signaling produced the opposite effects. Although the
SFZ cells still retain the properties of articular chondrocytes, it
is interesting that they represent a chondro-progenitor cell pop-
ulation phenotypically labile in response to varying levels of b-
catenin signaling. Moreover, these progenitor cells express the
Notch1 receptor, which mediates their proliferation (Dow-
thwaite et al., 2004).
Implications
Although changes in TMJ morphogenesis or growth occur in
response to inactivation or constitutive activation of numerous
genes, some generalizations are possible regarding (1) the devel-
opmental hierarchy of TMJ structures and (2) the hierarchy of
gene expression affecting TMJ morphogenesis and growth.
Developmental Hierarchy of TMJ Structures
Four genes are expressed very early in the mesenchymal conden-
sation that will become the mandibular condyle: Sox9, ihh,
Runx2, and Osterix (Shibata et al., 2006; Gu et al., 2008). Inacti-
vation of either Sox9 (Mori-Akiyama et al., 2003; Wang et al.,
2011) or Runx2 (Shibata et al., 2004) resulted in complete agene-
sis of the condylar cartilage. Although not described in the
Runx2-null mice, the mandibular fossa condensation occurred in
Sox9-null mice; bone formation was initiated but the majority of
the fossa regressed, leaving only a slight remnant on the lateral
aspect (Fig. 4). In light of their experiments in which the fossa
failed to form when physically displaced from the developing
condyle or maintained by an enlarged Meckel’s cartilage (Noggin
(2/2) mice), Wang and colleagues (2011) postulated that man-
dibular condyle formation (or a surrogate such as Meckel’s carti-
lage) is prerequisite for the formation of the mandibular fossa.
The experiment in which fossa agenesis occurred following an
open mouth posture that created greater displacement between
the fossa and condylar condensations suggested to them that
some sort of diffusible signal originating in the condyle must be
necessary for proper joint development. Interestingly, the only
other model in which fossa agenesis occurred (the Sprouty 1 (/
) Sprouty2 (/) mice) featured an intact, albeit smaller, con-
dyle; however, condyle-fossa continuity was disrupted by a
greatly enlarged temporalis muscle. The common factor in both
models appeared to be the requirement for signals emanating
from the condyle to the fossa, such that a lack of fossa formation
could result from either agenesis of the condyle or from disrup-
tion/ blocking of the signal due to distance or an intervening
structure.
Several models displayed abnormalities of the articular disc
and/or the joint cavities surrounding it. The complete absence of
disc and joint cavity formation was observed in ihh (/) mice

(Shibukawa et al., 2007). Most other instances of disc malforma-
tion or delay, often accompanied by failure of the joint cavities
to fully form, occurred in three groups of models: (1) those
involving genes in the ihh signaling pathway (Smo, Gli); (2) those
involving genes that interacted with elements of the
ihh signaling pathway (Trps1); or (3) models that exhibited
reduced or no ihh in the MCC (Shox2, Sox9, respectively). The
Spry1 (2/2)Spry2(2/2) mice with mandibular fossa agenesis,
but in which the MCC was present, still exhibited disc formation.
Taken together, these studies present a strong argument that sig-
nals originating within the condyle are critical for initiating disc
formation and cavitation. The fact that the articular disc failed to
form in the experiment in which Meckel’s cartilage maintained
fossa formation (Wang et al., 2011) lends further complexity to
the signals required.
Hierarchy of Gene Expression Affecting TMJ
Morphogenesis and Growth
DEVELOPMENTAL DYNAMICS
This catalog of TMJ phenotypes resulting from inactivation or
alteration of homeobox genes and growth factors has clearly
demonstrated that multiple genes contribute to TMJ growth, and
to a lesser extent, to TMJ morphogenesis. However, two genes,
Runx2 and Sox9, figure prominently in many of the models and
are the only genes known to date that if inactivated, produce
agenesis of the MCC. Both are expressed very early (at E13.5–
E14) in the mesenchymal cell condensation that will become the
condyle (Shibata et al., 2006; Shibata and Yokohama-Tamaki,
2008). In limb bud development, Sox9-expressing osteo-
chondroprogenitors give rise to Runx2-expressing osteogenic
cells (Akiyama et al., 2005). However, studies in avian (Buxton
et al., 2003) and mammalian (Shibata and Yokohama-Tamaki,
2008) secondary cartilages demonstrated that the reverse may
occur in secondary cartilage where chondrocytes form by means
of an up-regulation of Sox9 in a precursor population expressing
Runx2. If Sox9 is expressed downstream of Runx2 in the TMJ
instead of the situation found in the limbs, the Runx2 and Sox9
knockout phenotypes are explicable and would account for the
continued expression of Runx2 in Sox9 knockout mice (Wang
et al., 2011).
The actions of several of the other genes outlined in this
report may regulate TMJ morphogenesis by affecting Sox9 and
Runx2 expression, and, by means of these genes, control the
ihh-PTHrP axis. Oka and colleagues (2007, 2008) demonstrated
that the inactivation of TGF-b signaling drastically reduces
Sox9 expression, resulting in a defective condylar cartilage at a
very early stage that retards chondrocyte differentiation. In
addition, condyles in mice with inactivated Tgfbr2 signaling
had a slightly increased Runx2 expression and enhanced
expression of Dlx5, a transcription factor important in osteo-
blast differentiation (Oka et al., 2008). Because cartilage is not
lost entirely in the Tgfbr2-inactivated mice, other genes must
affect Sox9 expression. One possibility may be Shox2, because
both Runx2 and Sox9 were reduced in Shox2 null mice (Gu
et al., 2008). However, Sox9 is also a downstream transcrip-
tional target of Notch signaling, which in turn is activated by
FGF signaling (Nakanishi et al., 2007). Although Notch recep-
tors are present on mesenchymal cells (Nakanishi et al., 2007)
and indeed on prechondroblastic cells of the MCC (Serrano
et al., 2011), over-expression studies in pellet culture (Grogan
et al., 2008) and gain-of-function studies in vivo (Chen et al.,
GENES AND TEMPOROMANDIBULAR JOINT 871
Fig. 7. Cartoon of genes that may regulate cell fate, proliferation, and
differentiation of mandibular condylar cartilage cells based on the stud-
ies described in this review.
2013) make it clear that Notch signaling inhibits chondrogenic
differentiation in committed chondrocyte progenitors. More-
over, TGFb-mediated chondrogenesis is facilitated by reducing
the Notch repression of chondrogenesis (Grogan et al., 2008)
and by down-regulating Dlx5 expression (Oka et al., 2008).
Finally, studies in cranial bone osteo-progenitors (Goodnough
et al., 2012) have demonstrated that activated b-catenin signal-
ing inhibits Sox9 expression and chondrogenesis in vivo by
means of Twist1 expression, which binds Sox9. Conversely,
Goodnough and colleagues showed that Twist1 conditional
deletion in the cranial mesenchyme before fate specification
induces chondrogenesis, suggesting that down-regulating
Twist1 or its upstream progenitor b-catenin may also allow for
Sox9 expression in the MCC. Adenoviral introduction of Runx2
in Runx2 (/) chondrocyte cultures strongly induces the
expression of ihh (Yoshida et al., 2004), which (by means of its
feedback loop with PTHrP) clearly mediates the rate of hyper-
trophic chondrocyte differentiation, as well as the proliferation
in MCC (along with TGFb and perhaps Notch), as shown in the
ihh-null mice (Shibukawa et al., 2007). Possible regulators of
TMJ cell fate, proliferation, and differentiation are depicted in
Figure 7.
Conclusions
Although chondrocytes in the MCC secrete typical cartilage-
specific products such as Col2, aggrecan, and Col10 (Shibata
et al., 1997, 2006), the MCC differs from primary cartilages of the
limbs in the perichondrium containing its dividing cells. In some
respects (e.g., their responses to ihh), it appears that these pre-
chondroblasts behave similarly to the proliferating chondroblasts
in a growth plate. However, these models and other studies have
documented clear-cut differences in gene expression between the
MCC and limb cartilages for which the significance is currently
unknown. These differences include: the rapid chondrocyte
hypertrophy in the MCC as reflected in the greatly overlapping
expression of Col1, Col2, and Col10 (Shibata et al., 1997); the
absence in the MCC of Gdf5 & Gdf6 proteins seen in the joint
cavities of the developing limb (Purcell et al., 2009); differences
in osteopontin and bone sialoprotein expression (Gu et al., 2008);
 872 HINTON
and qualitatively different responses to gene inactivation in MCC
and limbs (Yasuda et al., 2010). In addition, the nature of the pre-
chondroblastic cells of the MCC perichondrium has not been well
characterized. Although they were formerly identified as
“mesenchymal stem cells” (Stutzmann and Petrovic, 1982), current
evidence suggests that these prechondrocytes have already differ-
entiated into Runx2-expressing periosteum-like cells that are bipo-
tent between osteogenic and chondrogenic lineages (Shibata et al.,
2006). Yet gene arrays that target isolated preparations of MCC
perichondrium show that vascular endothelial growth factor-B or
VEGF-B, a member of a family of angiogenic agents (Breen, 2007),
is expressed at levels twice as high in the perichondrial sample as
it is in the cartilage sample. In addition, the VEGF receptors Flt-1
(3) and kinase insert domain receptor/ Flk-1-KDR (4) are ele-
vated to an even greater extent (Hinton et al. 2009). Although the
functions of VEGF-B are not well understood, a recent study (Liu
et al., 2012) suggests it may affect the differentiation of osteopro-
genitors by regulating the Runx2 and peroxisome proliferator acti-
DEVELOPMENTAL DYNAMICS
vated receptor-gamma (PPAR-c), which is enriched three-fold in
the perichondrium (Hinton et al., 2009). Even more unclear is the
nine-fold enrichment of myogenic factor 6 (Myf6) in the perichon-
drial sample. Myf6 is an important transcription factor in the spec-
ification and differentiation of skeletal muscle myotubes during
embryogenesis (Maak et al., 2006). This relatively high expression
of genes such as myogenic factor 6 and VEGF-B may indicate a
degree of unsuspected plasticity in this bipotent cell population
derived from an osteogenic lineage.
It is abundantly clear from this review that our knowledge of
the genes that regulate the morphogenesis and growth of the TMJ
is itself embryonic. One clear need is for TMJ phenotypes involv-
ing conditional inactivation of additional genes, especially relat-
ing to Notch or Wnt/ b-catenin signaling. In view of its
sensitivity to mechanical loading (Case et al., 2008; Premaraj
et al., 2011; Mantila-Roosa et al., 2011), b-catenin signaling is of
particular interest for the MCC, because the cartilaginous pheno-
type of the MCC, unlike that of primary cartilages, has been
shown to be readily influenced by mechanical forces (Duterloo
and Wolters, 1971; Petrovic et al., 1975; Carlson et al., 1980). In
addition, further experiments or models must address the identity
of the “diffusible signaling molecule(s)” (Wang et al., 2011) that
coordinate the development of the condyle, mandibular fossa,
articular disc, and joint cavities. Finally, transgenic mice with
alterations in the malleus-incus joint in the middle ear also
exhibit major abnormalities of the lower or upper jaws as well as
in Meckel’s cartilage (Anthwal et al., 2012). This suggests that the
“primary jaw joint” in the middle ear and the normal dissolution
of Meckel’s cartilage, if disrupted, may have effects on TMJ mor-
phogenesis. It is only through more detailed knowledge of the
structural and developmental differences of this unique joint and
its surrounding structures that we will be able to better under-
stand how congenital TMJ anomalies occur and what constraints
are important for fashioning replacement joint tissues for dis-
eased or malformed joints.
Acknowledgments
I thank Drs. Eiki Koyama, Kyoko Oka, and Yipeng Chen to allow
me to use images from their work in this review. The research per-
formed in my lab was supported by a NIH grant, with able techni-
cal assistance from Dr. Maria Serrano. Finally, I thank Dr. Jerry
Feng for encouraging me to undertake this review.
References
Akiyama H, Kim J-E, Nakashima K, Balmes G, Iwai N, Deng JM,
Zhang Z, Martin JF, Behringer RR, Nakamura T, de Crombrugghe
B. 2005. Osteo-chondroprogenitor cells are derived from Sox9
expressing precursors. Proc Natl Acad Sci U S A 102:14655–
14670.
Anthwal N, Chai Y, Tucker AS. 2008. The role of transforming
growth factor-b signaling in the patterning of the proximal proc-
esses of the murine dentary. Dev Dyn 237:1604–1613.
Anthwal N, Joshi L, Tucker AS. 2012. Evolution of the mammalian
middle ear and jaw: adaptations and novel structures. J Anat
222:147–160.
Beresford WA. 1981. Chondroid bone, secondary cartilage, and
metaplasia. Baltimore, MD: Urban and Schwartzenberg; 454 p.
Bouvier M, Hylander WL. 1984. The effect of dietary consistency
on gross histologic morphology in the craniofacial region of
young rats. Am J Anat 170:117–126.
Breen EC. 2007. VEGF in biological control. J Cell Biochem 102:
1358–1367.
Buxton PG, Hall B, Archer CW, Francis-West P. 2003. Secondary
chondrocyte-derived Ihh stimulates proliferation of periosteal
cells during chick development. Development 130:4729–4739.
Carlson DS, McNamara JA Jr, Graber LW, Hoffman DL. 1980.
Experimental studies of growth and adaptation of TMJ. In: Irby
WB, editor. Current advances in oral surgery. St. Louis: Mosby; p
28–77.
Case N, Ma M, Sen B, Xie Z, Gross TS, Rubin J. 2008. b-catenin
levels influence rapid mechanical responses. J Biol Chem 283:
29196–29205.
Chen S, Tao J, Bae Y, Jiang M-M, Bertin T, Chen Y, Yang T, Lee B.
2013. Notch gain of function inhibits chondrocyte differentiation
via Rbpj-dependent suppression of Sox9. J Bone Miner Res 28:
649–659.
Colvin JS, Bihne BA, Harding GW, McEwen DG, Ornitz DM. 1996.
Skeletal overgrowth and deafness in mice lacking fibroblast
growth factor receptor 3. Nat Genet 12:390–397.
Crompton AW. 1985. Development and adaptation of the temporo-
mandibular joint. In: Carlson DS, McNamara JA Jr, Ribbens KA,
editors. Developmental aspects of temporomandibular joint dis-
orders. Ann Arbor MI: University of Michigan, Center for Human
Growth and Development. p 1–18.
Curry CL, Reed LL, Golde TE, Miele l, Nickoloff BJ, Foreman KE.
2005. Gamma secretase inhibitor blocks Notch activation and
induces apoptosis in Kaposi’s sarcoma tumor cells. Oncogene
24:6333–6344.
Deng C, Wynshaw-Boris A, Zhou F, Kuo A, Leder P. 1996. Fibro-
blast growth factor receptor 3 is a negative regulator of bone
growth. Cell 84:911–921.
Dowthwaite GP, Bishop JC, Redman SN, Khan IM, Rooney P,
Evans DJR, Haughton L, Bayram Z, Boyer S, Thomson B, Wolfe
MS, Archer CW. 2004. The surface of articular cartilage contains
a progenitor cell population. J Cell Sci 117:889–897.
Dudas M, Sridurongrit S, Nagy A, Okazaki K, Kaartinen V. 2004.
Craniofacial defects in mice lacking Alk2 in neural crest cells.
Mech Dev 121:173–182.
Duterloo HS, Wolters JM. 1971. Experiments on the significance of
articular function as a stimulating chondrogenic factor for the
growth of secondary cartilages of the rat mandible. Trans Eur
Orthod Soc 103–115.
Engelsma SO, Jansen HWB, Duterloo HS. 1980. An in vivo trans-
plantation study of growth of the mandibular condyle in a func-
tional position in the rat. Archs Oral Biol 25:305–311.
Fuentes MA, Opperman LA, Bellinger LL, Carlson DS, Hinton RJ.
2002. Regulation of cell proliferation in rat mandibular condylar
cartilage in explant culture by insulin-like growth factor-1 and
fibroblast growth factor-2. Archs Oral Biol 47:643–654.
Fukuoka H, Shibata S, Suda N, Yamashita Y, Komori T. 2007. Bone
morphogenetic protein rescues the lack of secondary cartilage in
Runx2-deficient mice. J Anat 211:8–15.
Glineberg RW, Laskin DM, Blaustein DI. 1982. The effects of immo-
bilization on the primate temporomandibular joint. A histologic
and histochemical study. J Oral Maxillofac Surg 40:3–8.

Goodnough LH, Chang AT, Treloar C, Yang J, Scacheri PC, Atit
RP. 2012. Twist1 mediates repression of chondrogenesis by
b-catenin to promote cranial bone progenitor specification.
Development 139:4428–4438.
Grogan SP, Olee T, Hiraoka K, Lotz MK. 2008. Repression of chon-
drogenesis through binding of Notch signaling proteins HES-1
and HEY-1 to N-box domains in the COL2A1 enhancer site.
Arthritis Rheum 58:2754–2763.
Gu S, Wei N, Yu L, Fei J, Chen YP. 2008. Shox-2-deficiency leads
to dysplasia and ankylosis of the temporomandibular joint in
mice. Mech Dev 125:729–742.
Hall BK. 1972. Immobilization and cartilage transformation into
bone in the embryonic chick. Anat Rec 173:391–404.
Hill TP, Spater D, Taketo MM, Birchmeier W, Hartmann C. 2005.
Canonical Wnt/b-catenin signaling prevents osteoblasts from dif-
ferentiating into chondrocytes. Dev Cell 8:727–738.
Hinton RJ. 1988. Response of the intermaxillary suture cartilage to
alterations in masticatory function. Anat Rec 220:376–387.
Hinton RJ, Serrano M, So S. 2009. Differential gene expression in
the perichondrium and cartilage of the neonatal mouse temporo-
mandibular joint. Orthod. Craniofac Res 12:168–177.
Iseki S, Wilkie AOM, Morriss-Kay GM. 1999. Fgfr1 and Fgfr2 have
DEVELOPMENTAL DYNAMICS
distinct differentiation- and proliferation-related roles in the
developing mouse skull vault. Development 126:5611–5620.
Jing JJ, Hinton RJ, Mishina Y, Liu Y, Zhou X, Feng JQ. Critical role
of Bmpr1a in mandibular condyle growth. Connect Tissue Res
(in press).
Kohn A, Dong Y, Mirando AJ, Jesse AM, Honjo T, Zuscik MJ,
O’Keefe RJ, Hilton MJ. 2012. Cartilage-specific RBPjk-
dependent and –independent Notch signals regulate cartilage
and bone development. Development 139:1198–1212.
Koyama E, Ochiai T, Rountree RB, Kingsley DM, Enomoto-
Iwamoto M, Iwamoto M, Pacifici M. 2007. Synovial joint forma-
tion during mouse limb skeletogenesis. Ann N Y Acad Sci 1116:
100–112.
Liu Y, Berendsen AD, Jia S, Lotinun S, Baron R, Ferrara N, Olsen
BR. 2012. Intracellular VEGF regulates the balance between
osteoblast and adipocyte differentiation. J Clin Invest 122:3101–
3113.
Lydiatt DD, Davis LF. 1985. The effects of immobilization on the
rabbit temporomandibular joint. J Oral Maxillofac Surg 43:188–
193.
Maak S, Neumann K, Swalve HH. 2006. Identification and analysis
of putative regulatory sequences for the MYF5/MYF6 locus in
different vertebrate species. Gene 379:141–147.
Mantila-Roosa SM, Liu Y, Turner CH. 2011. Gene expression pat-
terns in bone following mechanical loading. J Bone Miner Res
26:100–112.
Michikami I, Fukushi T, Honma S, Yoshioka S, Itoh S, Muragaki Y,
Kurisu K, Ooshima T, Wakisaka S, Abe M. 2012. Trps1 is neces-
sary for normal temporomandibular joint development. Cell Tis-
sue Res 348:131–140.
Mirando AJ, Maruyama T, Fu J, Yu HM, Hsu W. 2010. b-catenin/
cyclin D1 mediated development of suture mesenchyme in cal-
varial morphogenesis. Dev Biol 10:116–127.
Mizoguchi I, Nakamura M, Takahashi I, Kagayama M, Mitani H.
1990. An immunohistochemical study of localization of type I
and type II collagens in mandibular condylar cartilage compared
with tibial growth plate. Histochemistry 93:593–599.
Molteni A, Modrowski D, Hott M, Marie PJ. 1999. Differential
expression of fibroblast growth factor receptor-1, 2, and 3
and syndecan-1, 2, and 4 in neonatal rat mandibular condyle
and calvaria during osteogenic differentiation in vitro. Bone 24:
337–347.
Mori-Akiyama Y, Akiyama H, Rowitch DH, de Crombrugghe B.
2003. Sox9 is required for determination of the chondrogenic cell
lineage in the cranial neural crest. Proc Natl Acad Sci U S A 100:
9360–9365.
Nakanishi K, Chan YS, Ito K. 2007. Notch signaling is required for
the chondrogenic specification of mouse mesencephalic neural
crest cells. Mech Dev 124:190–203.
Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer
RR, de Crombrugghe B. 2002. The novel zinc finger-containing
GENES AND TEMPOROMANDIBULAR JOINT 873
transcription factor osterix is required for osteoblast differentia-
tion and bone formation. Cell 8:17–29.
Napierala D, Sam K, Morello R, Zheng Q, Munivez E, Shivdasani
RA, Lee B. 2008. Uncoupling of chondrocyte differentiation and
perichondrial mineralization underlies the skeletal dysplasia in
tricho-rhino-phalangeal syndrome. Hum Mol Genet 17:2244–
2254.
Ochiai T, Shibukawa Y, Nagayama M, Mundy C, Yasuda T, Okabe
T, Shimono K, Kanyama M, Hasegawa H, Maeda Y, Lanske B,
Pacifici M, Koyama E. 2010. Indian hedgehog roles in post-natal
TMJ development and organization. J Dent Res 89:349–354.
Oka K, Oka S, Sasaki T, Ito Y, Bringas P, Nonaka K, Chai Y. 2007.
The role of TGF-b signaling in regulating chondrogenesis and
osteogenesis during mandibular development. Dev Biol 303:391–
414.
Oka K, Oka S, Hosokawa R, Hosokawa R, Bringas P, Brockhoff
HC, Nonaka K, Chai Y. 2008. TGF-b-mediated Dlx5 signaling
plays a crucial role in osteo-chondroprogenitor cell lineage deter-
mination during mandible development. Dev Biol 321:303–309.
Ornitz DM. 2005. FGF signaling in the developing endochondral
skeleton. Cytokine Growth Factor Rev 16:205–2013.
Petrovic AG. 1972. Mechanisms and regulation of mandibular con-
dylar growth. Acta Morphol Neerl Scand 10:25–34.
Petrovic AG, Stutzmann JJ, Oudet CL. 1975. Control processes in
the postnatal growth of the condylar cartilage of the mandible.
In: McNamara JA Jr, editor. Determinants of mandibular form
and growth. Ann Arbor, MI: Center for Human Growth and Devel-
opment, University of Michigan. p 101–154.
Premaraj S, Souza I, Premaraj T. 2011. Mechanical loading acti-
vates b-catenin signaling in periodontal ligament cells. Angle
Orthod 81:592–599.
Purcell P, Joo BW, Hu JK, Tran PV, Calicchio ML, O’Connell DJ,
Maas RL, Tabin CL. 2009. Temporomandibular joint formation
requires two distinct hedgehog-dependent steps. Proc Natl Acad
Sci U S A 106:18297–18302.
Purcell P, Jheon A, Vivero MP, Rahimi H, Joo A, Klein OD. 2012.
Spry1 and Spry2 are essential for development of the temporo-
mandibular joint. J Dent Res 91:387–393.
Purow BW, Haque RM, Noel MW, Su Q, Burdick M, Lee J,
Sunderasan T, Pastorino S, Park JK, Mikolaenko I, Maric D,
Eberhart CG, Fine HA. 2005. Expression of Notch-1 and its
ligands, Delta-like-1 and Jagged-1, is critical for glioma cell sur-
vival and proliferation. Cancer Res 65:2353–2363.
Radlanski RJ, Renz H, Klarkowski MC. 2003. Prenatal development
of the human mandible. Anat Embryol (Berl) 207:221–232.
Rice DP, Aberg T, Chan Y-S, Tang Z, Kettunen PJ, Pakarinen L,
Maxson RE, Thesleff I. 2000. Integration of FGF and Twist in calva-
rial bone and suture development. Development 127:1845–1865.
Rot-Nikcevic I, Downing KJ, Hall BK, Kablar B. 2007. Development
of the mouse mandibles and clavicles in the absence of skeletal
myogenesis. Histol Histopathol 22:51–60.
Serrano MJ, So S, Svoboda KK, Hinton RJ. 2011. Cell fate media-
tors Notch and Twist in mouse mandibular condylar cartilage.
Arch Oral Biol 56:607–613.
Serrano MJ, So S, Hinton RJ. Roles of Notch signaling in mandibu-
lar condylar cartilage. Arch Oral Biol (in press).
Shibata S, Suzuki S, Tengan T, Ishii M, Kuroda T. 1996. A histologi-
cal study of the developing condylar cartilage of the fetal mouse
mandible using coronal sections. Arch Oral Biol 41:47–54.
Shibata S, Fukada K, Suzuki S, Yamashita Y. 1997. Immunohisto-
chemistry of collagen types II and X, and enzyme-histochemistry
of alkaline phosphatase in the developing condylar cartilage of
the fetal mouse mandible. J Anat 191:561–570.
Shibata S, Fukada K, Suzuki S, Ogawa T, Yamashita Y. 2002. In
situ hybridization and immunohistochemistry of bone sialoprotein
and secreted phosphoprotein 1 (osteopontin) in the developing
mouse mandibular condylar cartilage compared with limb bud
cartilage. J Anat 200:309–320.
Shibata S, Suda N, Yoda S, Fukuoka H, Ohyama K, Yamashita Y,
Komori T. 2004. Runx2-deficient mice lack mandibular condylar
cartilage and have deformed Meckel’s cartilage. Anat Embryol
(Berl) 208:273–280.
Shibata S, Suda N, Suzuki S, Fukoka H, Yamashita Y. 2006. An in
situ hybridization study of Runx2, Osterix, and Sox9 at the onset
 874 HINTON
of condylar cartilage formation in fetal mouse mandible. J Anat
208:169–177.
Shibata S, Yokohama-Tamaki T. 2008. An in situ hybridization study
of Runx2, Osterix, and Sox9 in the qanlagen of mouse mandibu-
lar condylar cartilage in the early stages of embryogenesis. J
Anat 213:274–283.
Shibukawa Y, Young B, Wu C, Yamada S, Long F, Pacifici M,
Koyama E. 2007. Temporomandibular joint formation and con-
dyle growth require Indian hedgehog signaling. Dev Dyn 236:
426–434.
Silbermann M, Reddi AH, Hand AR, Leapman RD, von der Mark K,
Franzen A. 1987. Further characterisation of the extracellular
matrix in the mandibular condyle in neonatal mice. J Anat 151:
169–188.
Sperber GH. 2001. Craniofacial development. Hamilton, Ontario:
BC Decker.
Stutzmann JJ, Petrovic AG. 1982. Bone cell histogenesis: the skel-
etoblast as a stem-cell for preosteoblasts and secondary-type
prechondroblasts. In: Dixon AD, Sarnat BG, editors. Factors and
mechanisms influencing bone growth. New York: Alan R Liss. p
29–43.
Tsutsui TW, Riminucci M, Holmbeck K, Bianco P, Robey PG. 2008.
DEVELOPMENTAL DYNAMICS
Development of craniofacial structures in transgenic mice with
constitutively active PTH/PTHrP receptor. Bone 42:321–331.
Vinkka H. 1982. Secondary cartilages in the facial skeleton of the
rat. Proc Finn Dent Soc 78(suppl 7):1–137.
Wang Y, Liu C, Rohr J, Liu H, He F, Yu J, Sun C, Li L, Gu S, Chen
YP. 2011. Tissue interaction is required for glenoid fossa devel-
opment during temporomandibular joint formation. Dev Dyn 240:
2466–2473.
Wuelling M, Kaiser FJ, Buelens LA, Braunholz D, Shivdasani RA,
Depping R, Vortkamp A. 2009. Trps1, a regulator of chondro-
cytes proliferation and differentiation, interacts with the activator
form of Gli3. Dev Biol 328:40–53.
Yasuda T, Mundy C, Kinumatsu T, Shibukawa Y, Shibutani T, Grobe
K, Minugh-Purvis N, Pacific M, Koyama E. 2010. Sulfotransfer-
ase Ndst1 is needed for mandibular and TMJ development. J
Dent Res 89:1111–1116.
Yasuda T, Nah HD, Laurita J, Kinumatsu T, Shibukawa Y, Shibutani
T, Minurgh-Purvis N, Pacific M, Koyama E. 2012. Muenke syn-
drome mutation, FgfR3P244R, causes TMJ defects. J Dent Res
91:683–689.
Yasuhara R, Ohta Y, Yuasa T, Kondo N, Hoang T, Addya S, Fortina
P, Pacifici M, Iwamoto M, Enomoto-Iwamoto M. 2011. Roles of
b-catenin signaling in phenotypic expression and proliferation of
articular cartilage superficial zone cells. Lab Invest 91:1739–
1752.
Yoshida CA, Yamamoto H, Fujita T, Furuichi T, Ito K, Inoue K,
Yamana K, Zanma A, Takada A, Ito Y, Komori T. 2004. Runx2
and Runx3 are essential for chondrocyte maturation, and Runx2
regulates limb growth through induction of Indian hedgehog.
Genes Dev 18:952–963.
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