REvIEWS

Role of thyroid hormones in

craniofacial development
Victoria D. Leitch 1,2
, J. H. Duncan Bassett 1
* and Graham R. Williams 1
*
Abstract | The development of the craniofacial skeleton relies on complex temporospatial
organization of diverse cell types by key signalling molecules. Even minor disruptions to these
processes can result in deleterious consequences for the structure and function of the skull.
Thyroid hormone deficiency causes delayed craniofacial and tooth development, dysplastic
facial features and delayed development of the ossicles in the middle ear. Thyroid hormone
excess, by contrast, accelerates development of the skull and, in severe cases, might lead to
craniosynostosis with neurological sequelae and facial hypoplasia. The pathogenesis of these
important abnormalities remains poorly understood and underinvestigated. The orchestration
of craniofacial development and regulation of suture and synchondrosis growth is dependent
on several critical signalling pathways. The underlying mechanisms by which these key pathways
regulate craniofacial growth and maturation are largely unclear, but studies of single-​gene
disorders resulting in craniofacial malformations have identified a number of critical signalling
molecules and receptors. The craniofacial consequences resulting from gain-​of-function and
loss-​of-function mutations affecting insulin-​like growth factor 1, fibroblast growth factor
receptor and WNT signalling are similar to the effects of altered thyroid status and mutations
affecting thyroid hormone action, suggesting that these critical pathways interact in the
regulation of craniofacial development.

Craniosynostosis
Premature fusion of the fibrous
calvarial sutures.
Facial hypoplasia
Reduced growth of features in
the midface, which results in an
abnormal facial appearance.
1
Molecular Endocrinology
Laboratory, Department of
Metabolism, Digestion and
Reproduction, Imperial
College London, London, UK.
2
Royal Melbourne Institute
of Technology (RMIT) Centre
for Additive Manufacturing,
RMIT University, Melbourne,
VIC, Australia.
*e-​mail: d.bassett@
imperial.ac.uk;
graham.williams@
imperial.ac.uk
https://doi.org/10.1038/
s41574-019-0304-5
The development of the craniofacial skeleton is complex
and relies on the temporospatial organization of diverse
cell types by key signalling molecules. Even minor dis-
ruptions to these intricate and integrated processes can
result in deleterious consequences for the structure and
function of the skull.
The mechanisms of thyroid hormone action in bone
and cartilage, and the consequences of thyroid hor-
mone deficiency and excess on the skeleton, have been
thoroughly reviewed1. Nevertheless, and even though
the skull is exquisitely sensitive to changes in thyroid
hormone availability, the effects of thyroid hormones
on craniofacial development have not been considered
systematically. Thyroid hormone deficiency causes
delayed craniofacial and tooth development, dysplastic
facial features and delayed development of the ossicles
in the middle ear. By contrast, high levels of thyroid
hormone accelerate development of the skull and can
lead to craniosynostosis , neurological sequelae and
facial hypoplasia in severe cases. The pathogenesis of
these important abnormalities is poorly understood and
underinvestigated.
In this Review, we first discuss the processes of
craniofacial bone formation via intramembranous and
endochondral ossification and the signalling pathways
involved. We then consider the physiological control of
thyroid status by the hypothalamic–pituitary–thyroid
(HPT) axis and the regulation of thyroid hormone
(3,5,3′-l-​triiodothyronine, T3) action in bone. In subse-
quent sections, we review the consequences of thyroid
hormone deficiency and excess and the effect of genetic
disorders that alter T3 action on the development of the
craniofacial skeleton in mouse models and human dis-
ease. Finally, we discuss current understanding of the
cellular and molecular mechanisms of T3 action in bone
and cartilage in the context of craniofacial development.
Craniofacial development
Development of the craniofacial skeleton begins by
4 weeks of gestation in humans, with the formation
of distinct condensations of mesoderm-​derived and
neural crest-​derived mesenchyme, and continues into
adulthood2,3. The skull is uniquely formed by a com-
plex integration of bones that have diverse origins and
develop from both intramembranous and endochondral
ossification (Fig. 1a; Table 1).
Intramembranous ossification. Intramembranous ossifi-
cation is the osteoblast-​driven process that forms the vis-
cerocranium and cranial vault, together with the scapula,

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Key points and posterior regions of the supraorbital ridge, respec-

• Thyroid hormone deficiency during development results in delayed intramembranous
and endochondral ossification in the skull, which manifests as patent or persistent
fontanelles, patent sutures, delayed dentition, wormian bones and deafness.
• Thyroid hormone excess during development results in advanced intramembranous
and endochondral ossification in the skull, which manifests as premature fusion of
the calvarial sutures and cranial base synchondroses.
• These characteristic craniofacial malformations indicate that thyroid hormones have a
pivotal role in development and growth of the craniofacial skeleton and demonstrate
that the skull is exquisitely sensitive to changes in thyroid status.
• Thyroid hormone-​induced changes in fibroblast growth factor receptor, insulin-​like
growth factor 1 and WNT signalling in osteoblasts and chondrocytes indicate
that these pathways are intricately involved in the regulation of craniofacial
development by T3.
ilium and lateral two-​thirds of the clavicle4,5. The process
begins with aggregation of mesenchymal cells to form
dense, tightly packed condensations. Cell-​surface hepa-
ran sulfate proteoglycans (HSPGs), neural cell adhesion
molecule, N-​cadherin, other proteoglycans, hyaladher-
ins, versican and tenascin all contribute to the cell–cell
adhesions that develop during mesenchyme conden-
sation6–8. At the centre of these condensations, mes-
enchymal cells differentiate directly to pre-​osteoblasts
and then osteoblasts that express runt-​related tran-
scription factor 2 (RUNX2) and secrete type I collagen
(COL1A1) and other extracellular matrix proteins,
including osteocalcin, bone morphogenetic protein 2
(BMP2), transforming growth factor-​β (TGFβ) and
insulin-​like growth factor 1 (IGF1)9. Calcium phos-
phate is then deposited on the extracellular matrix to
form mineralized bone2,6,10 (Fig. 1b).
tively, to form the primordia of the frontal and parietal
bones16,17 (Fig. 1d). The frontal and parietal bone pri-
mordial cells begin to express bone-​specific markers at
E10.5–12.5. Mineralization of these rudiments begins
at E14.0–14.5 via intramembranous ossification17–20.
Formation of the occipital bone during embryogene-
sis involves five distinct rudiments: four of these develop
via endochondral ossification within the cranial base,
whereas the interparietal section develops by intramem-
branous ossification to form part of the cranial vault3.
Like the occipital bone, the temporal bone comprises
five components that include the bones of the inner ear.
The squamous and tympanic sections of the temporal
bone form from neural crest cells and develop by intra­
membranous ossification, while the remainder form via
endochondral ossification2. The interparietal occipital
bone is largely mesodermal in origin, with additional
areas of neural crest-​derived tissue18. The interparietal
occipital bone develops from two, or sometimes three,
ossification centres that fuse together to form a single
bone, with the midline of the two ossification centres
housing the neural crest-​derived cells18,21. Occasionally,
extra centres of intramembranous ossification form
in the calvaria and eventually become wormian bones3.
The reason for wormian bone formation is unknown,
but it has been postulated to correct or compensate for
increased suture or fontanelle width in conditions in
which development of the skull is delayed22. Continued
growth of calvarial bones occurs via proliferation of
osteoprogenitor cells at the osteogenic front. When the
osteogenic fronts meet, they either merge and ossify
the margin, or form a suture3.

Calvaria
The upper part of the skull that
surrounds the brain.
Wormian bones
Additional bones that form
between sutures in the skull.
Endochondral ossification. Some bones of the cranial
base, the temporal bones and the bones of the middle
ear develop by endochondral ossification. This process
also occurs in the long bones and throughout the rest
of the skeleton. During endochondral ossification, cells
at the centre of mesenchyme condensations differentiate
into chondrocytes, which secrete a cartilaginous extra-
cellular matrix rich in type II collagen (COL2A1) that
forms an anlage, or template, for the developing bone.
These chondrocytes undergo proliferation, maturation
and hypertrophic differentiation, ultimately secreting
type X collagen (COL10A1), alkaline phosphatase (ALP)
and angiogenic factors, including vascular endothelial
growth factor (VEGF)11,12. Hypertrophic chondrocytes
might apoptose or transdifferentiate to become osteo-
blasts, and additional osteoblast and osteoclast progen-
itor cells arrive via newly formed blood vessels13–15. The
cartilage anlage mineralizes, forming a template for bone
formation and, finally, remaining cartilage is resorbed
by osteoclasts to leave mineralized bone matrix (Fig. 1c).
Intramembranous cranial vault. The cranial vault com-
prises paired frontal and parietal bones, the interpari­
etal section of the occipital bone and the squamous
sections of the temporal bones (Fig. 1a). Development of
the cranial vault begins between embryonic day (E) 8.0
and E9.5 in mice, with migration of neural crest and
mesodermal cells from the mid-​hindbrain to the anterior
Sutures. Sutures comprise two osteogenic fronts and a
region of fibrous mesenchyme encased by the pericra-
nium and dura mater (Fig. 1b). Importantly, sutures con-
tain cells at various stages of differentiation, including
osteoprogenitors, osteoblasts, osteoclasts, fibroblasts and
osteocytes3. At birth, the calvarium has paired coronal
and lambdoid sutures, a sagittal suture and a metopic
suture, together with an anterior and posterior fontanelle
where the sutures intersect. Like the craniofacial bones,
the mesenchymal cells contained within the suture
derive from either the neural crest or mesoderm; these
developmental origins influence the pathogenesis of
suture disorders such as the craniosynostosis syndromes.
The sutures normally remain patent (that is, open and
capable of growth) during neonatal and early develop-
ment to enable rapid expansion of the brain during the
postnatal period. The fontanelles are the first regions to
fuse and ossify, with closure of the posterior and ante-
rior fontanelles in humans completed by 3 months and
2 years of age, respectively. The age at which calvarial
suture fusion occurs, however, is variable, although it
is generally agreed that the metopic suture fuses dur-
ing childhood in humans, whereas the coronal, sagittal
and lambdoid sutures remain patent into adulthood3,16.
Similarly, the posterior frontal suture in mice, which
corresponds to the metopic suture in humans, fuses by
postnatal day (P) 40, whereas the sagittal, coronal and
lambdoid sutures are still patent at P250 (ref.23).

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a Metopic suture Ethmoid bone Fronto-

ethmoidal
Frontal synchondrosis
bone
Spheno-frontal
Frontal synchondrosis
Frontal bone
bone Sphenoid Spheno-
Parietal bone Coronal bone
suture occipital
synchondrosis
Anterior-
intraoccipital
Temporal synchondrosis
bone Parietal
bone Sagittal Posterior-
suture intraoccipital
synchondrosis
Occipital
Occipital bone bone
Lambdoid
suture Occipital
bone

Suture – intramembranous ossification Synchondroses – endochondral ossification

Osteoblast HZ PZ RZ PZ HZ
Osteoprogenitor
Mesenchymal
cell

Osteocyte Osteoid

Chondrocyte Osteoblast

Mesoderm Neural crest

Mixed mesoderm and neural crest

Fig. 1 | craniofacial development. a | Schematic representation of the skull showing the bones of the cranial vault and
cranial base together with the associated sutures and synchondroses. Intramembranous ossification in the sutures of
the cranial vault and endochondral ossification in the synchondroses of the cranial base are also shown. Purple cells
represent osteoblasts and the blue cells are chondrocytes at different stages of maturation. b | Mesodermal and neural
crest origin of the bones of the skull. HZ, hypertrophic zone; PZ, proliferative zone; RZ, resting zone.

Endochondral cranial base. The cranial base com-
prises the basioccipital, sphenoid and ethmoid bones,
together with part of the temporal bone3 (Fig.  1a) .
These bones derive from mesenchymal condensations
at the anterior (neural crest-​derived) and posterior
(mesoderm-​derived) regions of the cranial base24 (Fig. 1d).
Chondrocyte differentiation occurs at the centre of the
condensations and the newly differentiated chondro-
cytes secrete extracellular matrix rich in COL2A1 and
proteoglycans to form the cartilage template11,12,25,26.
By E16 in mice, the 14 cartilage plates of the cranial base
have fused to form one large cartilaginous structure,
the chondrocranium, which covers the entire base of the
skull24,27,28. Ossification centres form in the chondrocra-
nium and undergo endochondral ossification to form
the bones of the skull base. Between these bones, areas
of cartilage persist. Like the sutures between the intra­
membranous bones, these regions, the synchondroses,
are critical for continued growth during postnatal matu­
ration. Along with the cranial base, the ossicles of the

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middle ear (malleus, incus and stapes) and parts of the
mandible also develop by endochondral ossification29,30.
Synchondroses. Chondrocytes align within synchon-
droses to form structures that are analogous to the epi-
physeal growth plates in long bones. The organization of
proliferating and differentiating chondrocytes in these
regions drives orientation and the anterior–posterior
elongation of the cranial base28 (Fig. 1c). The three syn-
chondroses responsible for the majority of the growth
of the cranial base are the spheno-​occipital, spheno-​
frontal and fronto-​ethmoidal. There are contradictory
reports in the literature regarding the age at which the
synchondroses fuse, although the consensus indicates
that the spheno-​frontal and fronto-​ethmoidal synchon-
droses start to fuse around 7 years of age3. By contrast,
the spheno-​occipital synchondrosis remains patent up to
between 14 and 25 years of age31,32. In mice, the spheno-​
occipital synchondroses start to fuse at approximately
P28 and the process is completed by P84 (ref.33).
Abnormal craniofacial development
Both genetic and environmental factors influence the
processes of craniofacial formation and can cause serious
deleterious effects to the developing and growing cranio-
facial skeleton. Premature fusion of the calvarial sutures,
craniosynostosis, is one of the most common malfor-
mations, occurring in approximately 1 in 2,500 births16.
Fusion can occur in one or multiple sutures and results
in compensatory growth in other areas of the skull.
This premature fusion can also result in complications,
including raised intracranial pressure with neurological
and cognitive developmental delay16. Delayed growth
at the suture manifests as large or persistent fontanelles
and is a feature of a number of disorders, including
cleidocranial dysplasia, achondroplasia, Down syndrome
and rickets34.
Premature fusion of the synchondroses can change
the angle of the cranial base, which might cause retrogn­
athia, malocclusion, midface hypoplasia or cleft palate35,36.
The incidence of these malformations has not been
thoroughly investigated, but patients with syndromic
craniosynostosis are at increased risk of accompanying
Table 1 | Timing of key craniofacial events in humans and mice
developmental stage or event Age in humans Age in mice
Neural crest migration 21 days pc E8.0
Mesodermal and neural crest mesenchymal cell 28 days pc E12.5
condensations
Deciduous tooth buds begin to form 40 days pc E13.0
Ossification centre of mandible body appears 6–7 weeks pc E14.0
Ossification centres of frontal, parietal, occipital 8 weeks pc E14.0
(interparietal) and nasal bones appear
Formation of the chondrocranium begins 40 days pc E16.0
Metopic suture fusion (posterior frontal in mouse) 2 years P10
Dental eruption (first molar) 6–7 years P20
Spheno-​occipital synchondrosis fusion 12–14 years P28
Coronal, lambdoid and sagittal suture fusion Adulthood After P250
E, embryonic day; P, postnatal day; pc, post coitum.
cranial base abnormalities37, which might also be accom-
panied by serious comorbidities, including the Chiari
malformation at the base of the brain38.
Key signalling pathways
The orchestration of craniofacial development and
regulation of suture and synchondrosis growth is depen­
dent on several critical signalling pathways (Fig. 2). The
underlying mechanisms by which these key pathways
regulate craniofacial growth and maturation are largely
unclear, but in single gene disorders resulting in cranio­
facial malformations, a number of critical signalling
molecules and receptors have been identified.
Fibroblast growth factor signalling. Mutations in the
genes that encode fibroblast growth factor (FGF) recep-
tors (FGFRs) cause over 20 craniofacial syndromes,
the most common of which are FGFR2-related cranio-
synostoses39. Humans express 18 FGF ligands and four
FGFRs, with each ligand binding to one or more of the
receptors. Specific FGFs can act as either endocrine
or paracrine factors binding to FGFRs in a complex
combined with heparin and/or heparan sulfates and
β-​Klotho that results in an FGF–FGFR co-receptor with
increased β-​glucuronidase activity40. FGFRs are tyro­
sine kinase receptors that span the cell membrane; after
binding FGFs a cascade of intracellular signalling events
is initiated, including activation of the phosphoinositide
3-kinase (PI3K), signal transducer and activator of tran-
scription protein (STAT) and mitogen-​activated protein
kinase (MAPK) pathways40.
The FGFRs are highly homologous, with three of the
four isoforms (FGFR1, FGFR2 and FGFR3) expressed
in the skull. Of the 18 FGF ligands in humans, six have
been identified in the skull. FGF2, FGF4 and FGF9 are
expressed in the suture mesenchyme, FGF8 and FGF18
are expressed in osteoblasts lining the bones of the cal-
varia and FGF10 is expressed in the palate during early
embryogenesis41–43. FGF2, FGF4 and FGF8 can bind all
three receptor isoforms expressed in the skull, FGF9
and FGF18 can only bind to FGFR2 and FGFR3, and
FGF10 binds FGFR1 and FGFR2 (ref.44).
FGFR1 is expressed in the suture mesenchyme, in
osteoblasts at the osteogenic fronts and in chondrocytes
in the cranial base. FGFR1 gain-​of-function mutations
lead to Pfeiffer (OMIM 101600) and Jackson–Weiss
(OMIM 123150) syndromes, which are both charac-
terized by premature fusion of the calvarial sutures
and, in some cases, shortening of the cranial base35,41.
Fgfr1 gain-​of-function mutations in mice also result in
craniosynostosis of both the calvaria and cranial base,
via upregulation of Runx2 (ref.45). Conversely, loss-​
of-function mutations in FGFR1 are rare, but can result
in hypomineralization of calvarial bones, micrognathia
and dental agenesis46,47.
FGFR2 is expressed in proliferating osteoprogen-
itors, differentiating osteoblasts and chondrocytes at
the cranial base. FGFR2 gain-​of-function mutations
cause a variety of craniosynostosis syndromes including
Crouzon (OMIM 123500), Pfeiffer (OMIM 101600),
Apert (OMIM 101200) and Jackson–Weiss (OMIM
123150) syndromes. Gain-​of-function mutations in
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Mesenchymal condensation Fig. 2 | Key signalling pathways in craniofacial

Mesenchymal cell
development. FGF–FGFR, IGF1–IGF1R, BMP and WNT
signalling pathways regulate key stages in chondrocyte and
osteoblast specification, proliferation and differentiation.
Green boxes indicate stages where the listed growth
factors promote the process and red boxes indicate stages
• SOX5 • RUNX2 where they prevent it. BMP, bone morphogenetic protein;
WNT • SOX6 • TWIST1 BMP
FGF, fibroblast growth factor; FGFR, fibroblast growth
• SOX9 • MSX2
factor receptor; IGF1R, insulin-​like growth factor 1
• WNT receptor; MSX2, msh homeobox 2; RUNX2, RUNX family
• FGFR2 transcription factor 2; SOX, SRY-​box transcription factor;
• IGF1R
TWIST1, Twist family bHLH transcription factor 1; WNT,
Osteoprogenitor WNT family member.

RUNX2 in hypomineralization of calvarial bones, micrognathia

and dental agenesis46,47.
Chondroprogenitor
• WNT1 FGFR3 is expressed in the cranial base and at low
• WNT4 levels in osteoblasts at the osteogenic front of calvar-
• WNT16
ial sutures42. FGFR3 gain-​of-function mutations cause
Crouzon syndrome (OMIM 612247), Muenke syndrome
• WNT (OMIM 602849) and achondroplasia (OMIM 100800).
FGFR2 • FGFR1
FGFR3 IGF1R • IGF1R Features of these syndromes include craniosynosto-
sis, hydrocephaly, hearing loss and frontal bossing
Osteoblast (protuberance of the frontal bones)49–51. Fgfr3 gain-of-
function mice have craniosynostosis and midface hypo-
plasia owing to decreased chondrocyte proliferation and
maturation52.
• FGF8
• FGF18 Ultimately, FGFR2 stimulates the proliferation of
Proliferating
osteoblast progenitor cells, while FGFR1 promotes
chondrocyte differentiation and FGFR3 regulates chondrocyte pro-
liferation and differentiation. The reason for the allelic
heterogeneity of the FGFR-​b ased craniosynostosis
• IGF1R
FGFR3
• WNT
syndromes is unknown but might be a consequence
of the activation of convergent downstream signalling
pathways in response to ligand binding. The actions of
the receptor are dependent on receptor dimerization,
autophosphorylation of the kinase domain and bind-
ing of specific adaptor proteins for each of the down-
stream pathways53. Thus the cellular, and consequently
Hypertrophic Osteocyte clinical, outcome of mutations can differ depending on
chondrocyte which functional domain within the FGFR contains
the mutation.

IGF signalling. The IGF signalling pathway includes

• IGF1 • WNT6 two ligands (IGF1 and IGF2) and three receptors (IGF1
• IGF2 • WNT7A receptor (IGF1R), insulin receptor and IGF2 receptor
• WNT1 • WNT7B
• WNT3A (IGF2R)). IGF1 is the predominant functional ligand
Surrounding tissue during the postnatal period, when it also mediates some
of the actions downstream of growth hormone signal-
ling, whereas IGF2 acts mainly during intra-​uterine
FGFR1 FGFR2 FGFR3 IGF1R development. Both ligands bind with high affinity to
IGF1R, but only IGF2 displays high-​affinity binding
for IGF2R and the insulin receptor. IGF1R is a tyrosine
kinase receptor; after ligand binding, IGF1R activates
Fgfr2 in mice also lead to ectopic cartilage formation the PI3K, MAPK and extracellular signal-​related kinase
in the calvarial sutures and increased amounts of carti- (ERK) pathways. By contrast, IGF2R is a signalling
lage in the cranial base48. Although they display fusion antagonist that regulates the actions of IGF2 by seques-
of the coronal suture, Fgfr2-overexpressing mice also tering the ligand, preventing its binding to and activation
have characteristic defects in the sagittal suture, where of IGFR1 signalling54.
increased FGFR2 signalling leads to an accumulation of IGF1 and IGF2 are expressed in the dura mater that
pre-​osteoblastic cells with reduced numbers of differenti- surrounds the suture mesenchyme at the time of calvar-
ated osteoblasts and decreased ossification. Like FGFR1, ial suture fusion. In patients with craniosynostosis, IGF1
loss-​of-function mutations in FGFR2 are rare, but result is upregulated in the fused and fusing suture tissue55.

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Administration of recombinant IGF1 to rat cranial sutures
also induces fusion56,57. Homozygous loss-of-function
mutations in IGF1 (OMIM 608747) cause microcephaly,
delayed bone age and deafness, and heterozygous loss-
of-function mutations in IGF2 (OMIM 616489) results
in macrocephaly, frontal bossing, micro­gnathia, tria­
ngular face, low-set ears and delayed bone age58,59. In
osteo­blasts, IGF1 regulates DNA binding of the master
transcription factor RUNX2 via the PI3K and ERK1/2
pathways60. Thus, IGF1 regulates expression of RUNX2
target genes, inducing upregulation of Ocn and bone
sialoprotein (Bsp)61. In chondrocytes, IGF1 drives pro-
duction of COL2A1, aggrecan and SOX9 (refs62,63). The
consequences to the cranial base in Igf1−/− mice have
not been reported, but the growth plates of long bones
of these mice have impaired hypertrophic chondrocyte
differentiation64. The craniofacial phenotype in Igf2−/−
mice has also not been reported, although decreased
BMD throughout the appendicular skeleton and cranial
dysmorphology indicates that a defect in growth of the
skull is present65.
IGFR1 is expressed in both chondrocytes and osteo­
blasts. Heterozygous loss-​of-function mutations in
IGFR1 (OMIM 270450) cause intrauterine growth retar-
dation, microcephaly, short stature, delayed bone age
and increased serum levels of IGF66–68. Both Igf1−/− and
Igfr1−/− mice recapitulate this phenotype, with decreased
ossification in the calvaria and viscerocranium69,70.
Overall, the IGF–IGFR signalling pathway pro-
motes intramembranous and endochondral ossification
in the skull, acting via PI3K and MAPK–ERK signal-
ling pathways to stimulate osteoblast and chondrocyte
differentiation.
RUNX2. RUNX2 is a transcription factor expressed in
osteoprogenitor cells in response to TGFβ and BMP2
signalling via the SMAD, p38 and ERK1/2 pathways71,72,
where it drives osteoblast differentiation and expres-
sion of Ocn, Osteopontin (Opn), Bsp, Alp and COL1A1
(ref.73). It is indispensable for osteoblast formation, as
shown by the lack of osteoblasts and bone formation
in Runx2 knockout mice74,75. RUNX2 also controls cell
proliferation in pre-​hypertrophic chondrocytes and is
expressed in terminally differentiated hypertrophic
chondrocytes76,77. RUNX2 regulates expression of Fgfr1,
Fgfr2 and Fgfr3 in the calvaria to enhance pre-​osteoblast
proliferation, as well as the expression of Ihh and the
hypertrophic chondrocyte-​specific marker Col10a1
(refs77–80). Expression of Runx2 is also tightly regulated,
with MSX2 and TWIST1 downregulating its expression
in the calvarial sutures to prevent osteoblast differentia-
tion and maintain suture patency81,82. Heterozygous loss-​
of-function mutations of RUNX2 result in cleidocranial
dysplasia (OMIM 119600), a condition characterized by
delayed closure of the anterior fontanelle and metopic
suture, decreased intramembranous ossification in the
cranial vault, abnormal dental development and clavi­
cular hypoplasia. These abnormalities are recapitulated
in Runx2 knockout mice74,75.
Overall, RUNX2 has critical roles in both intramem-
branous and endochondral ossification. RUNX2 therefore
controls the timing of suture formation and closure and
regulates the timing of synchondrosis development and
maturation during growth at the skull base.
WNT. The WNT family of signalling molecules contains
19 proteins, which bind Frizzled receptors and the LDL
receptor-​related protein 5 (LRP5) and LRP6 co-​receptors
at the cell surface. WNT signalling is either dependent on
(canonical signalling) or independent of (non-​canonical
signalling) the transcription factor β-​catenin. The exact
mode of action of the WNT ligands has not been deter-
mined, but in most tissues signalling occurs locally via
paracrine actions between adjacent cells83. Evidence sug-
gests that the WNT protein remains tethered to the cell
membrane and acts at its site of expression84. Calvarial
pre-​osteoblasts express WNT1, WNT4 and WNT16, and
surrounding tissues produce WNT1, WNT3a, WNT6,
WNT7a and WNT7b85,86. WNT signalling is important
at all stages of bone development and growth. Its initial
role is as the fate-​determinant of mesenchymal con-
densations in the craniofacial region. The presence or
absence of β-​catenin dictates whether the condensa-
tion will become intramembranous or endochondral,
demonstrating that β-​catenin is a key molecular switch
that distinguishes ossification fate87.
Mutations in WNT ligands and their receptors result
in a variety of systemic bone diseases, but those with
specific malformations affecting the skull include tetra-​
amelia syndrome (WNT3; OMIM 273395), Robinow
syndrome (WNT5A; OMIM 180700), tooth agenesis
and hypodontia (WNT10A; OMIM 150400, 257980;
WNT10B; OMIM 617073; LRP6; OMIM 616724) and
Van Buchem disease (LRP5; OMIM 607636). During
bone growth, reduced WNT signalling caused by loss-​
of-function mutations in the WNT co-​receptor LRP5
causes osteoporosis–pseudoglioma syndrome (OPPG),
which is associated with reduced calvarial thickness88.
Furthermore, reduced WNT signalling caused by LRP6
deficiency in Lrp6−/− mice causes midface hypoplasia and
cleft palate89. By contrast, gain-​of-function mutations in
LRP5 result in increased WNT signalling and cause the
LRP5 high bone mass phenotype (LRP5–HBM) that is
associated with craniosynostosis and is further character-
ized by increased calvarial thickness, foramen magnum
stenosis and thickening of the mandible90,91. These pheno­
types result from WNT-​regulated actions in both osteo-
blasts and chondrocytes. In osteoblasts, canonical WNT
signalling induces Alp and Runx2 expression in response
to BMP2 (refs92,93). In chondrocytes, canonical WNT
signalling inhibits cell differentiation by downregulating
Sox9 and drives chondrocyte hypertrophy87,94.
Thus, WNT signalling promotes osteoblast pro-
liferation and differentiation, stimulates chondrocyte
hypertrophy and determines the intramembranous or
endochondral ossification developmental fate of cranial
mesenchyme.
Thyroid hormones
The HPT axis. Thyroid hormones are essential homeo-
static regulators of numerous developmental and meta­
bolic processes. Circulating levels of the prohormone T4
(also known as thyroxine) and the active hormone T3 are
tightly controlled by the HPT axis, which forms a classic
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 Reviews

a b T4
T4
Hypothalamus T4 T3 T4
T3 T4 T4
TRH
MCT8, MCT10 MCT8, LAT1
or LAT1 or LAT2

T3
T3
T4
T4 T4 T3
Pituitary T3 T4 T4 T4
gland
T3 DIO3 DIO2 DIO3
TSH
T2 T3 T2 rT3
T3 rT3
T3

Thyroid Co-activator Co-activator

T3 T3
RXR TRα1 RXR TRα1
T4 T3

TRE TRE
Bone

Chondrocyte Osteoblast

Fig. 3 | Thyroid hormone action in chondrocytes and osteoblasts. a | The thyroid gland secretes T4 and T3, and
circulating levels are maintained by the hypothalamic–pituitary–thyroid (HPT) axis, which is a classic endocrine negative
feedback loop. b | Chondrocytes (blue) express the MCT8, MCT10 and LAT1 thyroid hormone transporters. Intracellular T3
supply is regulated by the activity of the inactivating enzyme DIO3, and the actions of T3 are mediated by TRα1. Osteoblasts
(purple) express the MCT8, LAT1 and LAT2 thyroid hormone transporters. Intracellular T3 supply is regulated by the relative
activities of the activating enzyme DIO2 and the inactivating enzyme DIO3, and the actions of T3 are mediated by TRα1.
DIO2, iodothyronine deiodinase type 2; DIO3, iodothyronine deiodinase type 3; LAT, L-​type amino acid transporter; MCT,
monocarboxylate transporter; rT3, reverse T3; RXR, retinoid X receptor; TRα1, thyroid hormone receptor α1; TRE, thyroid
response element; TRH, thyrotropin-​releasing hormone; TSH, thyroid-​stimulating hormone.

negative feedback loop. The axis originates in parvo­
cellular neurosecretory neurons of the hypothalamic
paraventricular nucleus, which secrete thyrotropin-​
releasing hormone (TRH)95–97. TRH passes through the
pituitary-​portal vasculature and binds to G protein-​
coupled TRH receptors on the surface of anterior pitui-
tary gland thyrotrophs, which stimulates synthesis and
secretion of TSH98–101. In the pituitary, TSH then acts
via G protein-​coupled TSH receptors (TSHR) on thy-
roid follicular cells to stimulate their growth and the
synthesis and secretion of T3 and T4 (ref.102). Binding
of the active hormone T3 to nuclear thyroid hormone
receptors (TRs) in the hypothalamus and pituitary inhib-
its production and secretion of TRH and TSH, respec-
tively103,104 (Fig. 3a). The HPT axis set point maintains the
physiological reciprocal relationship between circulating
levels of T4, T3 and TSH and is genetically determined,
with considerable variation between individuals105,106.
Thyroid hormone entry into target cells is mediated
by specific membrane transporters, including mono­
carboxylate transporters 8 and 10 (MCT8 and MCT10),
L-type amino acid transporters 1 and 2 (LAT1 and
LAT2), organic anion transporting polypeptide 1C1
(OATP1C1) and the solute carrier SLC17A4 (refs106–109)
(Fig. 3b). Mct8, Mct10, Lat1 and Lat2 are expressed in
the skeleton, although currently only MCT8 is known to
be essential for thyroid hormone transport in bone110–112.
The thyroid hormone transporters facilitate entry
of both T4 and T3, with each protein displaying differ-
ing affinities for the two ligands. The prohormone T4
must be converted to the active hormone T3 in target
cells to mediate its physiological actions113,114. Activation
of T4 to T3 occurs via removal of the 5′-iodine atom by
either iodothyronine deiodinase type 1 (DIO1) or DIO2
(refs114,115). A third deiodinase (DIO3) irreversibly inac-
tivates T4 and T3 by removal of the 5-iodine atom, which
converts them to reverse T3 (rT3) or T2, respectively116,117
(Fig. 3b). Mitochondrial aminoadipate aminotransferase
(AADAT) has been identified as an additional thyroid
hormone-​inactivating enzyme. AADAT facilitates
removal of the alanine side chain of T4 and T3, forming
TK4 and TK3, respectively106. Expression patterns of
DIO1, DIO2 and DIO3 differ between tissues and stages
of development, which suggests that these enzymes
have specific roles at different developmental stages and
in different tissues. For instance, DIO1 is not expressed in
the skeleton, DIO2 is present in mature osteoblasts and
DIO3 activity is present in chondrocytes, osteoblasts
and osteoclasts118. Thus, the action of thyroid hormone
in target tissues is dependent on the intracellular con-
centration of T3, which is locally regulated by the relative
expression of DIO2 and DIO3.
Ultimately, the cellular actions of thyroid hormone
are mediated by genomic (type 1) and non-​genomic

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Reviews
Mineral apposition
The deposition of mineral-​
containing bone matrix during
new bone formation.
Thyroid gland dysgenesis
Abnormal or
underdevelopment of the
thyroid gland.
Iodine organification
The incorporation of iodine
into the thyroglobulin protein
during the synthesis of
thyroid hormones by thyroid
follicular cells.
Dyshormonogenesis
The inability to concentrate
iodine or synthesize thyroid
hormones in thyroid follicular
cells.
(types 2 and 3) actions of nuclear TRα and TRβ119,120.
The genomic actions are mediated by binding of TR (as a
homodimer, or heterodimer with retinoid X receptors
(RXRs)) to thyroid response element sequences (TREs)
located within target gene promoters (Fig. 3b). In the
absence of T3, the TR complex is bound to DNA and
interacts with co-​repressor proteins that inhibit target
gene transcription121. In the presence of T3, the TR
undergoes a conformational change, promoting release
of the co-​repressor, facilitating binding of a co-​activator
protein and stimulating target gene transcription122–124.
Non-​genomic actions involve phosphorylation of the TR
and might be mediated via indirect binding of the TR to
DNA or via second messenger signalling, including the
PI3K pathway125,126.
Like the deiodinases, the TRs display distinct tem-
porospatial patterns of expression. TRα is the main
isoform expressed in bone, central nervous system and
heart, while the liver expresses mainly TRβ1,127. Skeletal
responses to T3 during bone development are medi-
ated by transcriptional activation of T3 target genes by
genomic (type 1) actions of TRα119.
Thyroid hormone action in bone. Thyroid hormones are
essential for bone development and growth, and consid-
erable progress has been made in defining the actions of
T3 in the appendicular skeleton. In osteoblasts, T3 pro-
motes proliferation, differentiation, nodule formation
and miner­a lization 128–130, acting via the FGF, IGF,
MAPK and WNT signalling pathways104,131–133. In vivo,
T3-induced proliferation and differentiation of osteoblasts
results in increased mineral apposition and bone forma-
tion during skeletal development134,135. The osteoblastic
response to thyroid hormone results in increased expres-
sion of several genes that have important roles in cranio­
facial development, including Alp, Ocn, Osx, Igf1, Fgf1,
Fgf2, Fgf3, Fgfr1, Runx2 and Mmp9 (refs128,129,132,136,137). In
chondrocytes, T3 inhibits cell proliferation and induces
differentiation and hypertrophy138–140, acting via the FGF
and WNT signalling pathways104,141. In vivo, the increased
chondrocyte hypertrophy in response to T3 leads to the
hypertrophic zone in the growth plate increasing in
size, advanced development of secondary ossification
centres and promotion of transdifferentiation to osteo-
blasts15,139. A number of T3 target genes in chondrocytes
have been identified, including Col10a1, Alp, Fgfr1 and
Fgfr3 (refs138–141). In osteoclasts, the evidence is less clear.
Although excess levels of thyroid hormone stimulate
osteoclastic bone resorption117,118, there is conflicting
evidence about whether this response is dependent on
the presence of osteoblasts117,142. Thus, it is not known
whether T3 acts directly in osteoclasts, or whether the
thyroid hormone-​stimulated increase in osteoclast
activity results from an indirect mechanism following
T3 action in another cell type.
Overall, thyroid hormone promotes osteoblastic bone
formation and terminal hypertrophic differentiation in
chondrocytes. These effects are demonstrated by the
increased bone formation and growth plate maturation
seen in the appendicular skeleton in hyperthyroidism
and the decreased bone formation and impaired growth
plate maturation characteristic of hypothyroidism1.
The skeletal consequences of abnormal thyroid hor-
mone levels in bone and cartilage during development
and in adulthood have been reviewed comprehen-
sively1,143,144. Briefly, hypothyroidism causes delayed skel-
etal development during childhood that is characterized
by growth retardation, delayed tooth eruption and short
stature owing to impaired endochondral ossification1.
Conversely, childhood thyrotoxicosis results in accel-
erated growth and advanced bone age but ultimately
results in short stature owing to premature fusion of the
growth plates1. In adults, hypothyroidism inhibits bone
turnover, which results in increased bone mass and
mineralization. By contrast, hyperthyroidism stimu­
lates increased bone turnover with a greater effect on
bone resorption than formation, which results in bone
loss and osteoporosis1.
Effects of thyroid hormone deficiency
Congenital hypothyroidism. Congenital hypothyroidism
has a global incidence of between 1 in 1,400 and 1 in
2,800 (ref.145) and results from thyroid gland dysgenesis,
defects of iodine organification, dyshormonogenesis, mater-
nal iodine insufficiency, maternal TSHR antibodies or
genetic mutations145. Genetic causes include mutations
of thyroid peroxidase (TPO), dual oxidase 2 (DUOX2),
paired box gene 8 (PAX8), TSHR and selenocysteine
insertion sequence binding protein 2 (SECISBP2)1,145–150.
TPO (OMIM 606765) and DUOX2 (OMIM 606759)
mutations inhibit normal thyroid hormone synthesis,
resulting in low circulating concentrations of T4 and T3
and increased levels of TSH146,147. Mutations in PAX8
(OMIM 167415) cause thyroid gland dysgenesis, result-
ing in low to normal circulating levels of T4 and mark-
edly increased levels of TSH148. Loss-​of-function TSHR
mutations (OMIM 275200) result in either impaired
binding of TSH to its receptor or reduced downstream
signalling responses, which lead to high levels of TSH
but low levels of T4 (ref.149). SECISBP2 loss-​of-function
mutations (OMIM 607693) prevent the insertion of
the amino acid selenocysteine into 25 different human
proteins, including the three deiodinase enzymes.
Selenocysteine is critical to the enzymatic activity of the
deiodinases and SECISBP2 loss-​of-function mutations
impair their ability to activate and inactivate thyroid
hormones150,151. Consistent with this finding, SECISBP2
loss-​of-function mutations result in normal to high
levels of T4, low levels of T3 and high circulating levels
of TSH150.
The clinical craniofacial phenotypes for all forms of
congenital hypothyroidism are similar and include a
widened anterior fontanelle (50.3% incidence) and per-
sistent posterior fontanelle (47.2% incidence)152. Other
common craniofacial abnormalities include delayed
dental eruption, deafness, facial clefting, widened or per-
sistent sutures, supernumerary cranial bones (wormian
bones), microcephaly and micrognathia152–158. In severe,
untreated hypothyroidism, the delay in fontanelle clo-
sure can persist into the teenage years and even adult-
hood159. Treatment of congenital hypothyroidism with
levothyroxine replacement therapy is effective in pro-
moting longitudinal growth and dental eruption153; how-
ever, the predicted final height might not be achieved,
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particularly if the diagnosis of hypothyroidism is delayed
and if treatment is not commenced immediately follow-
ing diagnosis160. In the case of individuals with SECISBP2
mutations, levothyroxine can normalize TSH concentra-
tions but, owing to impaired conversion of T4 to T3 by
DIO1 and DIO2, liothyronine treatment is necessary to
achieve physiological euthyroidism and effectively treat
the consequences of hypothyroidism150,158,161.
Pregnant rats treated with 2-mercapto-1-methyl imi-
dazole or radioiodine give birth to hypothyroid offspring
that model congenital hypothyroidism and display
characteristic abnormalities, including microcephaly,
blunt snouts, abnormal facial features and deafness due
to abnormal ossicle morphology162,163. These animals
respond well to levothyroxine treatment, recovering
to achieve normal growth that is equivalent to their
wild-​type counterparts164. Genetically modified mice
with congenital hypothyroidism also recapitulate the
craniofacial phenotype of the human condition1. Thus,
Tshr−/− mice display thinning of the calvaria and enlar­
ged ossicles with delayed ossification165,166. Although
Secisbp2 −/− mice have been generated, their skele-
tal phenotype has not yet been analysed167, whereas
Tpo-mutant, Pax8−/− and Hyt/Hyt mice with congen-
ital hypothyroidism have not been investigated for
craniofacial abnormalities or defective skull devel-
opment168,169. The molecular mechanisms that cause
craniofacial abnormalities in congenital hypothyroid-
ism have also not been characterized, although delayed
calvarial ossification in hypothyroid rodents is accom­
panied by decreased IGF1 signalling and osteocalcin
production66,170.
Zebrafish are a useful model to study the effects of
congenital hypothyroidism in the craniofacial complex.
Fish that have undergone thyroid ablation have an over-
all decrease in bone mineralization and an increase in
the amount of cartilage with malformation of the bones
of the skull171. In these fish, the frontal and parietal
bones fail to fuse, leaving a void in the apex of the skull
similar to the enlarged fontanelle seen in humans and
mice with congenital hypothyroidism169. At the base
of the skull, the amount of cartilage in the synchon-
droses is increased and ossification and mineralization
are decreased171. The bones of the Weberian apparatus
(analogous to the auditory ossicles in humans and mice)
are considerably malformed and disorganized, with
abnormal secondary ossification centres and regions
of low mineralization171. Craniofacial development of
flatfish is also reliant on thyroid hormone, with anti-​
thyroid treatment of Senegalese sole larvae resulting in
inhibition of the normal asymmetrical migration of the
eye during thyroid hormone-​dependent metamorpho-
sis172. This change is accompanied by abnormal ossi-
fication of the ethmoid bone and pseudomesial bone
(a bone unique to flatfish) and decreased expression of
osteonectin and matrix gla protein172.
Overall, the distinct craniofacial phenotype obser­
ved in congenital hypothyroidism provides evidence of
important developmental roles for thyroid hormone dur-
ing the prenatal period that affect intramembran­ous ossi­
fication of the cranial vault as well as endochondral
ossification of the facial bones and middle ear.
Nature Reviews | Endocrinology
Reviews
Juvenile hypothyroidism. Primary hypothyroidism
in the perinatal, postnatal or childhood periods had a
reported incidence of approximately 69 in 100,000 in
a community-​based study in Scotland, UK173. Juvenile
hypothyroidism results from autoimmune disease
(Hashimoto disease; OMIM 140300), thyroidectomy
following surgical treatment of malignancy or could
be idiopathic 173. Juvenile hypothyroidism presents
with growth retardation and delayed bone age, which
is frequently accompanied by persistent fontanelles
and wormian bones in the skull174,175. The age at which
juvenile hypothyroidism first presents has a consider­
able effect on the form and severity of these craniofacial
manifestations. Fontanelle closure occurs by approxi­
mately 24 months of age, and thus hypothyroidism
occurring after this age is not characterized by the com-
mon complication of persistent fontanelles, which might
result in delayed diagnosis. Levothyroxine treatment is
effective in accelerating skeletal maturation; however,
short stature can persist unless treatment is commenced
immediately following early diagnosis175–177.
The effects of juvenile hypothyroidism on craniofacial
development have been studied in animal models follow-
ing thyroidectomy or radioiodine ablation. In juvenile
sheep, thyroidectomy results in delayed tooth eruption,
defects in the anterior maxilla, frontal and orbital bones
and a prominent jaw178,179. In rabbits, shortening of the
facial bones occurs, which results in decreased length,
width and height of the face and cranium, and the devel-
opment of the mandible is retarded180. Hypothyroid
juvenile rats have wide skulls, a decreased anterior–
posterior cranial base length, sunken nasal bridge and an
altered relationship between the position of the calvaria
and foramen magnum181. In starlings, juvenile hypo-
thyroidism results in delayed formation of the palate
and patent cranial sutures182. In dogs, hypothyroidism
results in anterior–posterior shortening of the skull, with
reduced bone growth in both the calvarial sutures and
synchondroses of the cranial base183.
Overall, juvenile hypothyroidism provides extensive
evidence of a key role for thyroid hormones during post-
natal craniofacial development that is conserved across
species. The craniofacial abnormalities are similar to
those caused by congenital hypothyroidism, with defects
affecting both intramembranous and endochondral
ossification (Table 2).
THRA dominant-​negative mutations. Mutations in
THRA were first described in 2012 and cause resis­tance
to thyroid hormone-​α (RTHα), with 23 independent
mutations currently identified (OMIM 614450)184.
Most mutations occur in the ligand-​binding domain of
TRα at the C-​terminus and result in a dominant-​negative
receptor that binds persistently to the co-​repressor
NCoR, which prevents activation of T3 target gene tran-
scription185,186. The severity of the RTHα syndrome is
variable as the resulting phenotype correlates with the
differing interaction affinities of individual mutant
TRs for the transcriptional co-​repressor NCoR187. Thus,
the presentation of patients with RTHα is variable, but the
clinical features are consistent with the consequences of
severe congenital hypothyroidism and include impaired
Reviews
Table 2 | craniofacial phenotypes resulting from thyroid hormone deficiency
causes craniofacial phenotype
Human
Congenital: cause unknown Persistent anterior fontanelle, large posterior
or dyshormonogenesis fontanelle, persistent metopic suture, delayed
dentition, microcephaly and micrognathia
Juvenile: autoimmune Patent anterior fontanelle
thyroiditis
Genetic: mutations in PAX8, Large and/or persistent posterior fontanelle,
TSHR, SECISBP2 or THRA persistent anterior fontanelle, persistent sutures,
microcephaly or macrocephaly with thickened
calvaria in patients with THRA mutations, delayed
dental eruption and wormian bones
Rodents
Congenital: maternal anti-​ Blunt snout, impaired cranial ossification and widened Decreased bone length, impaired
thyroid treatment and/or persistent sutures
Juvenile: thyroidectomy, Reduced calvarial length, reduced cranial base length, Decreased bone length
radioiodine delayed dental eruption and shortened mandible
Genetic: mutations in Tshr, Thra Thinning of calvaria, large and/or persistent anterior
fontanelle and wide and/or persistent sutures
growth, delayed bone age and cognitive deficits185. The
craniofacial malformations that result from RTHα are
also consistent but vary in severity according to the
potency of individual THRA mutations1,187,188.
Truncating mutations, lacking all or part of the
C-​terminal alpha helix of TRα, delete the domains criti­
cal for co-​activator recruitment185. Additionally, the
deletion exposes the hydrophobic cleft that accom-
modates NCoR185. Together, these changes result in a
strongly dominant-​negative TRα1 receptor. Examples
of these truncating mutations are THRAE395X, THRAE403X
and THRAC380fs387X (refs184,185,188). The phenotypic conse-
quences resulting from expression of potent dominant-​
negative truncated receptors include macrocephaly,
delayed fusion of the cranial sutures, patent anterior
fontanelle, wormian bones, delayed dentition and
tooth eruption and thickening of the calvaria185,188–190.
Missense mutations, such as THRAL274P, which do not
result in truncation of the receptor, might also have
potent dominant-​negative activity188 and cause a severe
phenotype that includes facial dysmorphia, delayed
dentition, delayed anterior fontanelle closure, wormian
bones during childhood and increased calvarial thickness
during adolescence190.
Many missense mutations, however, result in milder
phenotypes than those caused by THRAL274P because the
expressed mutant TRα1 exerts only moderate dominant-​
negative activity. The point mutations in these instances
cause a morphological change at the T3 binding site of
TRα1 that still allows ligand binding at a lower affinity
than normal190. Examples include THRAR384H, THRAA263S,
THRAD211G, THRAA263V (refs188,190,191) and the resulting
phenotypes comprise delayed dentition, persistence of
the anterior fontanelle, wormian bones, thickening
of the calvarial bones and macrocephaly during child-
hood, with the macrocephaly and calvarial thickening
persisting during adulthood188. For some missense
mutations, for example THRAD211G, the phenotype is
even milder and manifests only as a broad and flattened
other abnormalities refs
Short stature, delayed bone age, absent 153,159,160
or delayed secondary ossification centre
and intellectual disability
Short stature and delayed bone age 174
Short stature, delayed bone age, absent 155,156,158,161,183,
or delayed secondary ossification centres, 184,188,191,193,194,234
intellectual disability, hearing loss and
macroglossia
164,170,171
ossification and reduced bone strength
178,180,182
Decreased bone length and bone mineral 165,195,196,198,199
density and delayed ossification
nasal bridge, but normal head circumference191. Finally,
a further mutation (THRAN359Y) has been reported in a
single patient and affects both TRα1 and TRα2 (ref.192).
The mutant TRα1 has reduced binding affinity for
T3 and a moderate dominant-​negative effect but the
consequences to TRα2 are unknown as the physiolo­
gical role of TRα2 has not been characterized192. The
THRAN359Y mutation is also accompanied by a micro­
deletion and results in a unique phenotype that includes
micrognathia, macro­cephaly, persistent fontanelles,
absent clavicles and an abnormal rib cage192. The cause
of this fairly severe and unusual skeletal dysplasia
remains unclear, but it is unlikely to result solely from
the dominant-​negative effects of the mutant TRα1.
Taking these results together, the consistent cranio­
facial features associated with RTHα include macroce­
phaly, persistent fontanelles, delayed dentition, wormian
bones and thickening of the calvaria. Owing to vary-
ing potencies of the individual THRA mutations, the
effectiveness of treatment with thyroid hormone dif-
fers depending on the mutation present. Some clinical
features are improved following levothyroxine adminis-
tration, including constipation, energy levels185,193,194 and
growth velocity195; however, effects on predicted final
height might be variable193,195.
A variety of mouse models with Thra mutations have
been generated, and all present with distinct craniofacial
phenotypes. TRα1PV/+ mice, which are euthyroid with
reduced T3 action, have normal-​sized skulls, but wid-
ened and persistently patent cranial sutures and fonta-
nelles with more porous frontal and parietal bones196,197.
TRα1PV/+ mice also have enlarged ossicles in the middle
ear that result from delayed ossification and are associ-
ated with deafness166. TRα1R384C mutant mice are mildly
hypothyroid during early life and have a less severe
resistance to T3 than TRα1PV/+ mice but also exhibit
delayed intramembranous ossification and delayed
tooth eruption198,199. Furthermore, cell-​specific expres-
sion of a dominant-​negative TRα1L400R mutant receptor
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in chondrocytes results in malocclusion, shortening of
the snout and delayed mineralization of endochondral
ossification-​derived bones of the cranial base200.
Overall, these data demonstrate a clear and important
role for TRα1 in both intramembranous and endochon-
dral ossification during craniofacial development that is
conserved in humans and mice. The phenotype in RTHα
shares features with congenital hypothyroidism and var-
ies in severity depending on the potency of the THRA
dominant-​negative mutation.
Effects of thyroid hormone excess
Juvenile hyperthyroidism. Juvenile hyperthyroidism is
rare, although there are reports suggesting that the global
incidence is between 3 and 14 per 100,000 people and
that its incidence is increasing201,202. This condition can
be a consequence of Graves disease, genetic disorders or
pituitary tumour (TSHoma), or an adverse effect of over-
treatment with levothyroxine. Genetic causes include
gain-​of-function mutations in TSHR (OMIM 609152),
which result in constitutive activation of the recep-
tor and increased systemic thyroid hormone levels203,
and McCune–Albright syndrome (OMIM 174800),
which results from mutations in GNAS, which encodes
a component of the TSHR downstream intracellular
signalling cascade.
The most common craniofacial manifestation of
juvenile hyperthyroidism is premature fusion of the
sutures, or craniosynostosis204–206. Fusion of the syn-
chondroses at the cranial base and advanced dental
eruption have also been reported207,208. The incidence of
craniosynostosis in those with childhood thyrotoxico-
sis is high, although the severity and number of sutures
affected varies between patients and is related to the time
of disease onset204. Craniofacial manifestations of TSHR
gain-​of-function mutations include frontal bossing,
premature closure of the fontanelles and craniosynos-
tosis203,209,210. Anti-​thyroid drugs might be effective in
preventing further advancement in bone age or suture
fusion206,210,211. However, if craniosynostosis has already
occurred, surgical intervention might be required211,212.
Modelling juvenile hyperthyroidism in animals by
administration of liothyronine or levothyroxine results
in a number of morphological abnormalities of the
skull, including increased ossification in the sutures,
Table 3 | craniofacial phenotypes resulting from thyroid hormone excess
causes craniofacial phenotype
Human
Juvenile: cause unknown Craniosynostosis, microcephaly,
or Graves disease advanced dental age and
malocclusion
Genetic: mutations Premature anterior fontanelle closure
in TSHR or THRB and craniosynostosis
Rodents
Juvenile: levothyroxine Advanced dentition, reduced
treatment suture width, premature suture and
synchondrosis fusion and microcephaly
Genetic: mutations Reduced fontanelle size and reduced
in Thrb suture width
Nature Reviews | Endocrinology
Reviews
accelerated tooth eruption and microcephaly213–215.
The cranial base is also affected, with decreased bone
growth and reduced width and shortening and prema-
ture fusion of the synchondroses205,207,216. A mouse model
of the human TSHRD633H mutation has been developed
that accurately recapitulates the features of the human
disease, but the detailed skeletal consequences have
not yet been reported217. Similarly, genetically modi-
fied mice with the equivalent of the human TSHRV509A
and TSHRI486F mutations have been generated but their
craniofacial phenotypes have not been reported218.
In the zebrafish model of hyperthyroidism, an overall
increase in ossification in the craniofacial complex has
been reported171. Specifically, the increased ossification
results in increased overlap at the junctions between the
frontal and parietal bones, a reduction in cartilage and
increased fusion rates in the synchondroses at the base
of the skull171. The bones of the Weberian apparatus in
hyperthyroid zebrafish are also malformed171.
The advanced intramembranous and endochondral
ossification that occurs in response to excess thyroid
hormone during craniofacial development highlights the
important role of T3 (Table 3). The contrast between
the phenotypes resulting from juvenile hypothyroidism
and hyperthyroidism indicates that the skull is exquisitely
sensitive to the level of available thyroid hormone.
THRB dominant-​negative mutations. Dominant-​
negative mutations of THRB had an incidence of approx-
imately 1 in 40,000 in a study of Spanish patients, and
they result in RTHβ (OMIM 188570)219. Mutations
in THRB that lead to RTHβ are located mainly in the
ligand-​binding domain of TRβ and result in reduced
affinity for T3, RXR or co-​activator proteins that inter-
act with TR–RXR complexes at the TRE168,220–222. The
consequence is impaired negative feedback of the HPT
axis, resulting in increased circulating levels of T3 and T4
and normal or high levels of TSH1. Thus, individual T3
target tissues might display phenotypes of thyroid hor-
mone deficiency or excess depending on whether they
predominantly express and respond to TRα or TRβ,
respectively1. RTHβ is thus a complex condition that
causes a variety of phenotypes in different tissues, and
its heterogeneity is further confounded by treatment of
individuals with anti-​thyroid drugs and thyroidectomy
other abnormalities refs
Advanced bone age, goitre, 205–207,211,
hyperactivity and intellectual 212,235–237
disability
Advanced bone age, goitre, irritability, 209,210,220,
intellectual disability and diarrhoea 238–241
Advanced skeletal development 174,216,217,
and increased bone size 218,238,239
Advanced bone age and advanced 226,234
growth plate maturation
Reviews

followed by thyroid hormone replacement or use of the regulation of craniofacial development by thyroid
thyroid hormone analogues. hormones (Fig. 4).
Over 170 THRB mutations have been reported in
more than 3,000 patients from about 1,000 families223,224, Fibroblast growth factors. In osteoblasts, thyroid hor-
but the effects on the craniofacial skeleton have been mone excess in TRβ−/− and TRβPV/PV mice or following
incompletely investigated. However, frontal bossing, levothyroxine administration results in increased expres-
scaphocephaly and craniosynostosis have been reported sion of FGFR1, FGF1 and FGF2 (refs128,132,137,199,226,227).
and are mainly compatible with increased circulating Conversely, mouse models of thyroid hormone deficiency
concentrations of thyroid hormone in RTHβ acting (TRα0/0 mice, which lack all TRα transcripts expressed
via the normal TRα expressed in bone. Nevertheless, from the Thra locus, and TRα1R384C/+ and TRα1PV/+ mice,
in patients with potent dominant-​negative truncating which express dominant-​negative forms of the recep-
mutations affecting TRβ, delayed bone age has been tor) show decreased expression of Fgfr1 and Fgfr3 in
reported and, although no specific data are available, it osteoblasts196,225,226. These findings demonstrate a direct
is probable in such cases that the mutant TRβ protein relationship between thyroid hormone concentrations,
interferes with the normal actions of wild-​type TRα that FGF–FGFR signalling and intramembranous ossification.
promote bone maturation and growth. While it is known that FGF–FGFR signalling pro-
In mouse models, the consequences of Thrb muta- motes intramembranous bone formation in the calvaria,
tions are clearer than in humans. Thus, TRβ−/− and the mechanism by which thyroid hormone regulates
TRβPV/PV mice have elevated circulating levels of thy- this process is largely unknown227. In the case of FGFR1,
roid hormone owing to absence of TRβ or expression of T3 stimulation of Fgfr1 mRNA expression requires TRα,
dominant-​negative TRβ in the pituitary and hypothala- and the FGFR1 response to FGF2 is enhanced, with
mus, resulting in disruption of the HPT axis. TRβ−/− mice more rapid and increased autophosphorylation of
display accelerated calvarial bone growth and decreased FGFR1 and activation of the downstream MAPK path-
fontanelle size199,225, and TRβPV/PV mice exhibit prema- way132. Whether similar mechanisms result in activation
ture closure of the anterior fontanelle and coronal suture of FGFR2 signalling by T3 has not been determined.
with accelerated calvarial ossification226. Taken together, Nevertheless, T3 stimulation of FGFR1 and MAPK
studies in mice with Thrb deletion or mutation provide signalling in osteoblasts ultimately results in increased
strong in vivo evidence of a major role for TRα during expression of several genes involved in osteoblast
craniofacial development. differentiation, including ALP and OCN132,228,229.
In chondrocytes, expression of Fgfr1 and Fgfr3 is
Aetiology of the craniofacial phenotype increased in response to thyroid hormone by both gene­
Children with severe thyroid hormone deficiency and tic manipulation (TRβ−/− and TRβPV/PV mice) and treat-
excess present with characteristic craniofacial pheno- ment with liothyronine128. Mouse models of impaired
types (Supplementary Table 1). The recurring features T3 signalling in the skeleton (TRα0/0, TRα1R384C/+ and
of thyroid hormone deficiency in the skull are per- TRα1PV/+ mice) show decreased expression of both Fgfr1
sistent fontanelles and patent or wide sutures, while and Fgfr3 in chondrocytes196,225,226, indicating a direct
those of thyroid hormone excess are craniosynosto- relationship between thyroid hormone concentrations,
sis of the calvarial vault and cranial base sutures and FGFR signalling in chondrocytes and endochondral
synchondroses1. These severe craniofacial phenotypes ossification. Thus, FGFR3 is an important T3 target gene
indicate that, as in the peripheral skeleton, thyroid hor- in chondrocytes in vitro and in vivo. Increased levels of
mone is a critical regulator of both intramembranous FGFR3 in response to T3 result in enhanced activation
and endochondral ossification, and precise control of of MAPK signalling by FGF2 and FGF18 (refs134,141).
T3 action is essential for normal craniofacial growth By contrast, T3 inhibits activation of the STAT1 pathway
and development. mediated by FGF2 and FGF18 and has no effect on FGF9
Juvenile thyroid hormone deficiency results in a signalling132. Overall, T3 differentially regulates chon-
craniofacial phenotype that is similar to that caused drocyte FGFR3 signalling during chondrogenesis, with
by FGFR1, FGFR2, IGF1, IGFR1 and RUNX2 loss-​ the response dependent on the activating ligand and the
of-function mutations46,47,58,59,69,70. By contrast, juvenile downstream signalling pathway affected141.
thyroid hormone excess results in a craniofacial pheno- Furthermore, modulation of FGFR signalling by thy-
type similar to that caused by FGFR1, FGFR2 and LRP5 roid hormones might also involve indirect mechanisms
gain-​of-function mutations39,90. These observations mediated via HSPGs, which interact with FGF–FGFR–
suggest that calvarial intramembranous and endochon- co-​receptor complexes at the cell surface230. Increased
dral ossification are regulated by the actions of thyroid expression of HSPGs in proliferating chondrocytes in
hormone and these actions involve the FGF, IGF1 and growth plate cartilage has been observed in hypothyroid
WNT signalling pathways. mice, and T3 inhibited expression of HSPG core pro-
tein, polymerase and modification enzymes (encoded
Mechanism of action in the skull by Gpc6, Ext1 and Hs6st2) in chondrogenic ATDC5
The craniofacial similarities between gain-​of-function cells230. Thus, the role of T3 in endochondral ossifica-
and loss-​of-function mutations in IGF1, the FGFR tion involves additional interactions with key proteo-
genes, the WNT signalling pathway, patients with hyper­ glycan mediators of FGFR signalling, although such
thyroidism and hypothyroidism and animal models mechanisms have not been investigated in cranial base
of these disorders, suggest these pathways interact in synchondroses during craniofacial development.

www.nature.com/nrendo
 Reviews

FGF signalling IGF signalling WNT signalling

a Chondrocyte
FGF2 IGF1 GH
FGF18 WNT
LRP5 or
FGFR3 ↓ HSPG IGF1R GHR LRP6 FZD

T3

β-catenin

↑ MAPK ↓ STAT1 ↑ PI3K–AKT ↑ STAT5

T3 T3

Co-activator Co-activator
T3 ↑ Fgfr3 T3
↑ Ghr ↑ Runx2
RXR TRα1 ↑ Fgfr1 RXR TRα1
↓ Ext1 ↑ Igf1 TCF/LEF
↓ Gpc6 ↑ Igf1r
↓ Hs6st2
TRE TRE TRE

b Osteoblast
FGF2 IGF1
WNT
FGFR1
↑ IGF1R
P

↑ MAPK

↑ ALP
↑ OCN ↑ MAPK ↑ AKT

T3 T3 T3

Co-activator Co-activator Co-activator

T3 T3 T3
RXR TRα1 ↑ Fgfr1 RXR TRα1 RXR TRα1
↑ Fgf1 ↑ Igf1
↑ Fgf2
TRE TRE TRE

Fig. 4 | Mechanism of thyroid hormone action in the skull. a | FGF, IGF and WNT signalling in chondrocytes (blue)
in the skull base. T3 induces FGFR expression and enhances MAPK signalling, induces GHR and IGF1R expression and
enhances PI3K–AKT and STAT5 signalling. T3 also enhances canonical WNT signalling and RUNX2 expression. b | FGF,
IGF and WNT signalling in skull vault osteoblasts (purple). T3 induces FGF and FGFR expression and enhances MAPK
signalling, induces IGF1 expression and enhances MAPK and AKT signalling and inhibits WNT signalling. AKT, protein
kinase B; ALP, alkaline phosphatase; Ext1, exostosin 1; FGF, fibroblast growth factor; FGFR, fibroblast growth factor
receptors; FZD, frizzled receptor; GH, growth hormone; GHR, growth hormone receptor; Gpc6, glypican 6; Hs6st2,
heparan sulfate 6-O-​sulfotransferase 2; HSPG, heparan sulfate proteoglycan; IGF1, insulin-​like growth factor 1;
IGF1R, insulin-​like growth factor 1 receptor; LEF, lymphoid enhancer-​binding factor; LRP, low-​density lipoprotein
receptor-​related protein; MAPK, mitogen-​activated protein kinase; OCN, osteocalcin; PI3K, phosphatidylinositol 3-
kinase; Runx2, RUNX family transcription factor 2; RXR, retinoid X receptor; STAT, signal transducer and activator
of transcription; TCF, transcription factor; TRα1, thyroid hormone receptor α1; TRE, thyroid response element; WNT,
WNT family member.

Nature Reviews | Endocrinology

Reviews

Insulin-​like growth factors. Increased expression of Igf1
and Igf1r has been reported in osteoblasts and the cra-
nial sutures in TRβ−/− and TRβPV/PV mice and following
thyroid hormone administration in wild-​type rats and
mice128,216,226,231. IGF1 promotes osteoblast proliferation,
differentiation and mineralization in accordance with
the accelerated intramembranous ossification seen in
animals exposed to increased T3 signalling. Stimulation
of Igf1 expression by T3 in osteoblasts is mediated via
TRα binding to a specific TRE located in the Igf1 pro-
moter133, and increased IGF1 results in activation of the
AKT and MAPK pathways in osteoblasts132. The mecha­
nism by which Igf1r expression is stimulated by T3 in
osteoblasts has not yet been defined.
In chondrocytes, increased expression of growth
hormone receptor (Ghr), Igf1 and Igf1r, and increased
activity of their downstream signalling pathways STAT5
and PI3K–AKT, have been reported in cranial base syn-
chondroses and growth plate chondrocytes in vitro in
response to T3 (refs216,231). Conversely, growth plate chon-
drocytes from TRα0/0 and TRα1PV/+ mice with impaired
T3 signalling exhibit decreased expression of these
genes and their downstream signalling responses196,225.
Overall, analysis of the T3 response in TRα0/0, TRα1PV/+,
TRβ−/− and TRβPV/PV mice is consistent with a model in
which local GH–GHR–IGF1–IGFR mediated STAT5
and PI3K–AKT responses to T3 in chondrocytes are
mediated via TRα.
WNT signalling. The WNT signalling pathway, which
determines the intramembranous or endochondral
fate of cranial mesenchyme during development, is an
important target for thyroid hormones in osteoblasts and
chondrocytes. Investigation of the WNT–β-​catenin path-
way in primary osteoblasts from wild-​type and ThrbPV/PV
mice revealed differential expression of 34 WNT path-
way genes, 12 of which were activated in ThrbPV/PV osteo-
blasts, including the Frizzled co-​receptor Fzd7 and cyclin
D1 cell cycle regulator Ccnd1 (ref.104). Increased activity
of the WNT pathway in ThrbPV/PV mice was demonstrated
by increased expression of Runx2 and decreased expres-
sion of Rankl in trabecular bone and perichondrium
during endochondral ossification, whereas expression
of the activating WNT ligand Wnt4 was decreased, thus
revealing the complex nature of the WNT signalling
response to T3 in vivo104. In vitro studies revealed that
the dominant-​negative mutant TRβPV protein exhib-
its a gain-​of-function activity that stabilizes β-catenin
concentrations in osteoblasts, suggesting a mechanism
to explain the overall increased activation of the WNT
pathway in ThrbPV/PV mice.
Nevertheless, in  vitro studies using the Tcf/Lef
reporter system revealed that T3 activation of wild-​type
TRs actually results in inhibition of WNT signalling
in osteoblasts104, whilst in vivo studies demonstrated
changes in serum concentrations of the WNT inhibi-
tors dickkopf 1 and sclerostin in thyroid-​manipulated
mice rendered hyperthyroid by levothyroxine injection
or hypothyroid by addition of methimazole and sodium
perchlorate to drinking water134. Even so, studies indi-
cate that manipulation of dickkopf 1 in mutant mice
does not notably affect skeletal responses to T3 in adult
mice232. In addition, T3 stimulates WNT signalling in
chondrocytes, resulting in accumulation of β-catenin
and increased expression of Runx2 (refs104,233), find-
ings that are consistent with the stimulatory effects of
RUNX2 gain-of-function mutations during endochondral
ossification231.
Overall, it is evident that thyroid hormones exert
complex regulatory effects on WNT signalling in osteo-
blasts and chondrocytes that might affect the stability and
accumulation of β-​catenin and that could differ between
cell types and during development and adulthood.
Conclusions
Thyroid hormone deficiency results in delayed intra­
membranous and endochondral ossification in the skull,
which manifests as patent or persistent fontanelles,
patent sutures, delayed dentition, wormian bones and
deafness. By contrast, thyroid hormone excess results
in advanced intramembranous and endochondral
ossification in the skull, which manifests as premature
fusion of the calvarial sutures and cranial base synchon-
droses. These characteristic craniofacial malformations
indicate that thyroid hormones have a pivotal role in
development and growth of the craniofacial skeleton,
and demonstrate that the skull is exquisitely sensitive to
changes in available T3. Furthermore, analysis of pheno­
types in TR-​mutant mice indicate that TRα mediates
the major effects of thyroid hormones during skeletal
develop­ment, and the findings in mutant mice recapit-
ulate the craniofacial manifestations of thyroid disorders
in humans. In addition, changes induced by thyroid hor-
mone in FGFR, IGF1 and WNT signalling in osteoblasts
and chondrocytes suggest that these pathways are intri-
cately involved in the regulation of craniofacial develop-
ment by T3. An improved understanding of the role of
thyroid hormones during craniofacial development and
growth might result in novel therapeutic opportunities
for patients with craniofacial disorders.
Published online xx xx xxxx

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Acknowledgements
J.H.D.B. and G.R.W. are funded by a Wellcome Trust Joint
Investigator Award (110141/Z/15/Z and 110140/Z/15/Z).
Author contributions
V.D.L., J.H.D.B. and G.R.W. researched data for the article,
made substantial contributions to discussion of content of
the article, wrote the article and reviewed and edited the
manuscript before submission.
Competing interests
The authors declare no competing interests.
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Supplementary information
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