Embryology of The Head and Neck: Martin Catala

Embryology of the Head and Neck
Martin Catala
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
The Skull . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
The Vault of the Skull . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Migration of the Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Tissue Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Control at the Genetic Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
The Base of the Skull . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Face and Neck . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
The Face . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
The Neck . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Abstract
The region of the head and neck is complex according to its anatomy. Its
development is particularly difficult to unravel because many interactions are
playing important roles, these processes are only beginning to be revealed. It is
easy to split this chapter into four: vault, base of the skull, face and neck. The
bone forming the vault of the skull derive from membranous ossification. The
different bones of the vault are derived either from neural crest or mesoderm.
These bones are being established gradually receiving information of the neural
M. Catala (*)
Fédération de Neurologie, Groupe Hospitalier Pitié-Salpêtrière, Paris, France
Laboratoire de biologie du développement, UMR 7622, CNRS and université Pierre et Marie Curie,
Paris, France
e-mail: martin.catala@upmc.fr
# Springer-Verlag Berlin Heidelberg 2016 1

A. Rossi (ed.), Pediatric Neuroradiology,
DOI 10.1007/978-3-662-46258-4_59-1
2 M. Catala
tube, the surface ectoderm and the dura. The area separating two bones constitutes
the suture, a real organ whose anomaly leads to the formation of various cranio-
synostosis. The main genes explaining human craniosynostosis are MSX, FGF
receptors, TWIST and EphA4. Skull base follows endochondral ossification.
Schematically one can distinguish three regions: prechordal area, chordal region
and otic capsule. Sonic hedgehog is a morphogen very involved in the formation
of the skull base. The otic capsule is directly induced by the otic vesicle. Finally,
the regulation of the supra-occipital is radically different from the rest of the base.
The face results from merging of five buds (a frontonasal, two maxillary and two
mandibular). The interactions playing a role in the development of the face are
just beginning to be elucidated. Nerve and sensory structures (optic vesicle and
olfactory mucosa) have a major role in these processes. Neck results from the
formation and evolution of branchial arches. These arches are made of endoderm,
mesoderm, mesectoderm and surface ectoderm. Many molecules account for the
interactions between these tissues that are necessary to build normal anatomical
structures.
Introduction
The skeleton of the head and neck represents a very complex set of bones whose
development is both complicated and precise. It is by now impossible to write a
precise and thorough chapter to account for the tremendous amount of new data
gained by experimental embryology. So, I will focus my chapter on (i) some
elements of descriptive embryology that are mandatory to give the readers land-
marks for the human development, (ii) the origin of the cells that are fated to form
these structures and their subsequent development, (iii) tissue interactions that could
account for the malformative associations that can be observed in humans, and
(iv) genetic controls of these processes.
The bones of the face and neck are constructed according to two different
modes. Either they differentiate directly in the mesenchyme realizing the
so-called membrane ossification, or they replace a cartilage template following
the process of endochondral ossification. The skeleton of the head and neck may be
divided into four compartments, which are quite separated and follow different
regulative mechanisms. Thus, it is tempting to separate the vault of the skull, the
base of the skull, the face, and the neck. Whatever the compartment of these
structures, it is important to notice that the skeleton develops after the development
of neural and visceral structures. This could explain why the shape of the skeleton
is controlled by the neural and visceral elements. This scenario also explains why
an absence or a hypoplasia of a bony canal or foramen is always secondary to a
maldevelopment of another structure. For example, the carotid canal is absent or
hypoplastic in case of primary developmental defect of the carotid artery (Kubis
et al. 1996).
Embryology of the Head and Neck 3
Fig. 1 Head of a human

embryo at the eighth week of
gestation showing the
ossification in progress. Note
that the sole calvarial bone
visible at that stage is the
frontal bone. The mandible,
maxillary, and malar bones are
already present in the face

embryo at the ninth week of
ossification in progress. Both
frontal and interparietal bones
enlarge, while the alisphenoid
and squamous part of the
temporal are now developing.
Note that the supraoccipital
and the exoccipital are
ossifying from the
cartilaginous scaffold
4 M. Catala

embryo at the tenth week of
ossification in progress. In the
vault, the parietal is now
expanding. The bones of the
face are developing and the
nasal bone is now present
Fig. 4 Aspect of the parietal

bone of a human fetus at
12 weeks of gestation (Alcian
Blue and alizarine red
staining). The bones of the
vault of the skull arise directly
from the mesenchyme
(membranous ossification).
Note that the center of the
bone is made up of a network
of bony structures, whereas
the periphery shows spicules
that extend radially
Fig. 5 The vault of the skull

of a human fetus (12 weeks of
gestation). Removing the
scalp shows the dermal bones
that constitute the vault of the
skull. The frontal bone (FB)
and the parietal one (PB) are
separated by the coronal
suture (CS). The anterior
fontanel (aF) is largely patent.
no nose
Fig. 6 Histological section

from a human fetus (19 weeks
of gestation) showing the
frontoparietal suture
separating the frontal bone
from the parietal one
The Skull
The Vault of the Skull
The vault of the skull has sometimes received different meanings according to the
specialists who treat the subject. Nevertheless, it seems appropriate to limit the
definition to the skull bone located subcutaneously and in contact with brain hemi-
spheres. Thus, the infratentorial portion of the squamous part of the occipital bone is
not part of the cranial vault according to this definition. This is in keeping with the
fact that this bone undergoes radically different embryological changes than the vault
of the skull.
The skull vault is composed of different bones whose anatomical status is not
univocal. Thus, the parietal bone is the only one that is an integral part of the vault.
The frontal bone is involved in the formation of both the vault of the skull and the
6 M. Catala
Fig. 7 Dorsal view of a chick

embryo at 8-somite stage. The
neural tube (NT) is located on
the midline. Its most rostral
part corresponds to the
prosencephalon (Pro) with the
still open anterior neuropore
(aN). The paraxial mesoderm
is divided into the cephalic
mesoderm (CM) and the
somites (So)
roof of the orbit. Sphenoid, temporal, and occipital bones consist of two compo-
nents: (i) one that enters into the formation of the vault (lamina ascendens of the
greater wing of the sphenoid or alisphenoid, squamosal part of the temporal and
supratentorial part of the occipital bone also known as the interparietal bone) and
(ii) one that participates in the formation of skull base.
Descriptive Embryology
The bones that constitute the vault of the skull are dermal bones produced by
membranous ossification. In humans, ossification begins around the eighth to ninth
week of gestation. The first bones of the vault to ossify are the frontal bones (Fig. 1)
followed by the interparietal ones, the alisphenoids, and the squamous part of the
temporal (Fig. 2) and at last the parietal bones (Fig. 3). It is interesting to note that the
bones of the skull vault grow according to very peculiar features: they form a
network of bone separated by non-ossified areas. Bony spicules are visible on the
periphery with radial extension (Fig. 4). This mode of growth concerns neither the
alisphenoid nor the squamous part of the temporal bone (Fig. 3). Cell and molecular
regulations of this phenomenon are completely unknown.
The surface of these bones gradually increases so that their free edges become
closer and closer. However, they do not join them, leaving an unossified connective
tissue, namely, the suture (Figs. 5 and 6). The latter eventually occludes after birth
with the end of the growth of the skull vault. Three sutures play a major role in
human craniosynostosis affecting the vault: the metopic suture lies between the two
primordia of the frontal bones and its involvement leads to trigonocephaly, the
sagittal suture lies between the two parietal bones whose dysfunction generates
scaphocephaly, and the coronal suture between the frontal and parietal bones is
impaired in brachycephaly. Furthermore, the lambdoid suture located between the
parietal bone and the supraoccipital separates the vault from the skull base.
The most widely used model in the academic literature explaining the growth of
bones of the vault refers to accretion. Sutural cells proliferate and progressively
differentiate into bone progenitors as they approach the free edges of already formed
bone. There they constitute the osteogenic front, and then they integrate into the
bone matrix. Such a model must be questioned because of the experimental results
that begin to accumulate.
Embryonic Origin and Subsequent Development

To determine the fate of an embryonic primordium, it is necessary to mark the cells
of the latter in order to follow in their destiny. Thus, such studies can be carried out
only under experimental conditions and cannot be performed in humans. I can only
ask readers to be as cautious as possible before affirmations of authors claiming to
have followed the fate of human cells by conducting studies of embryos or fetuses at
different ages. In higher vertebrates, different techniques have been developed to
answer the question of the embryonic origin of the bones of the skull vault.
An avian fate map is available for the skull vault using the quail-chick chimera
technique described by Le Douarin in 1969 (Le Douarin 1969). This technique is
based upon the differential properties of quail and chick cells. It is indeed possible to
recognize the cells from these two birds by using either a histochemical staining or a
monoclonal antibody. So, if one performs a graft of quail cells into a chick host, it is
possible to follow the fate of the grafted cells. This technique allows one to construct
fate maps of embryonic territories. Consequently, Couly et al. (1993) showed that the
vault of the skull has two distinct embryonic origins: the cephalic mesoderm
(corresponding to the paraxial mesoderm located rostrally to the first somite)
(Fig. 7) yields the supraoccipital, postorbital, orbitosphenoid, and pleurosphenoid,
while the rest of the vault derives from the mesencephalic neural crest. In this study,
the origin of the interparietal bone (a very tiny structure in birds located between the
parietal and the supraoccipital bones) has not been reported. Furthermore, the cells of
the mesencephalic neural crest differentiate into meninges, smooth muscle fibers of
the media of the brain and cephalic vessels, and dermal cells.
More recently, the group of Noden (Evans and Noden 2006) worked on the chick
embryo and marked cells using retroviruses that cannot replicate and which enable
the integration of the gene encoding beta-galactosidase. Their results are slightly
different from those of Couly et al. (1993). The mesoderm gives rise to the
supraoccipital as found by Couly but also to the parietal, the part of frontal belonging
to the vault, the pleurosphenoid, and a small portion of the squamous part of the
temporal. Only a large part of the squamous part of the temporal is derived from the
neural crest according to these authors.
One of the first differences between birds and mice is the topography of the neural
crest cells that participate in the formation of the frontonasal migratory stream. In
birds, no neural crest cells migrate from the forebrain. In contrast, in mice, neural
crest cells detach from the forebrain neural plate with the exception of its most rostral
8 M. Catala
Fig. 8 The embryonic origin

of the bones of the skull in
human postulated from
experimental data (profile
view). The bones derived
from the mesoderm are
represented in dark gray,
while those derived from the
neural crest are represented in
light gray
extremity. Cells delaminate even before the closure of the neural tube and are
involved in the formation of the nasofrontal bud and, probably, of the skull bones
(Osumi-Yamashita et al. 1994). Moreover, the potential for differentiation of these
murine cells differs in vitro. Indeed, cells from the forebrain generate pigment cells
and few neurons, but not cells expressing type II collagen. In contrast, cells from the
mesencephalon give rise to more neurons and cells expressing type II collagen, but
never to pigment cells (Chareonvit et al. 1997). This difference in developmental
potentials is interesting to know, although its relevance in human disease remains to
be discovered. The fate of neural crest cells in mouse embryos has been proposed
recently using a very precise genetic strategy. The bacterial gene Cre (encoding for
the recombinase enzyme) is driven by the promoter sequence of the Wnt1 gene. This
gene is expressed by some cells of the neural tube and by neural crest cells. By
crossing this transgenic line with the R26R line, it is possible to generate mice in
which the lacZ gene (coding for beta-galactosidase) is activated only in cells that
express Wnt1. Revealing galactosidase activity allows to follow the fate of neural
crest cells. This technique was applied to the study of the origin of the skull vault by
Jiang et al. (2002). In mice, neural crest cells give rise to the nasal, alisphenoid,
squamosal, frontal, and center of the interparietal bones. In contrast to the results
published for birds by Couly et al. (1993), parietal bones do not arise from neural
crest cells in the mouse.
Some of the discrepancies between the results gained by these two studies can be
easily explained. The orbitosphenoid participates in the formation of the vault in
birds, whereas it only gives rise to the lesser wing of the sphenoid in mammals. The
postorbital bone does not exist in mammals. The pleurosphenoid is not homologous
to the alisphenoid, which is the mammalian component of the vault. The alisphenoid
derives from the palatoquadrate (the reptilian epipterygoid bone). This structure
develops from neural crest cells of the first branchial arch. So, one of the differences
between the results obtained in birds and mice derives from the variation in the
Fig. 9 Mesencephalic neural

crest cells (Mes NCC) migrate
around the rostral part of the
neural tube and will
eventually form the vault of
the skull. In contrast,
rhombencephalic neural crest
cells (rhom NCC) migrate and
populate the branchial arches
constitution of the skull in both species. Thus, it seems much more reasonable to
disregard the results established in the chicken and not to enter into the controversy
between different authors. Since the constitution of the head skeleton in humans is
probably much more similar to that of mice than of birds, we propose to extrapolate
these results to human beings (Fig. 8).
A very interesting result obtained through the use of these genetic markers (Jiang
et al. 2002; Yoshida et al. 2008; Deckelbaum et al. 2012) corresponds to the origin of
the sutures. The coronal suture derives from the mesoderm (Yoshida et al. 2008;
Deckelbaum et al. 2012), while the sagittal suture is yielded by the neural crest in
mice (Yoshida et al. 2008). Such a result is at least curious. Indeed, the sagittal suture
lies between the two parietal bones whose origin is the mesoderm. Thus, this suture
does not have the same embryonic origin as bones that are supposed to be derived.
Similarly, the coronal suture is situated between the frontal bone (which is derived
from the neural crest) and the parietal bone (derived from mesoderm). Thus, the
coronal suture that is supposed to participate in the growth of both frontal and
parietal bones does not have the same origin as the frontal bone. This result led to
reservations about the growth model of the skull that we have described previously.
The growth pattern of the skull has been completely reexamined in mice due to
the results that contradict with the classical model. The marking of the anlage of the
frontal or parietal bone from its formation in mice shows that the subsequent growth
of these bones is predominantly ensured by the labeled cells (Yoshida et al. 2008).
The mesenchymal tissue away from the bone primordium does not contribute to the
subsequent formation of bone (Yoshida et al. 2008). During later stages, cells located
at the center of the suture do not contribute to bone formation, while the latter comes
10 M. Catala
Fig. 10 Section through the

head of a human embryo
(Carnegie stage
16, corresponding to 37 days
postfertilization). The
mesenchyme (M ) deriving
from neural crest cells is
located between the surface
ectoderm (SE) and the wall of
the telencephalon (Te). Note
that a dense vascular
perineural plexus (VP) can be
observed around the
neural tube
from the differentiation of cells in the periphery of the developing bone (Lana-Elola
et al. 2007). Thus, a new growth model of the skull can be offered. The suture is
constituted by two cell regions, which become radically different. Peripheral cells
form the osteogenic primordium and have the same embryonic origin as the devel-
oping bone. In contrast, central cells lack osteogenic potentials; they constitute an
essential inhibitory area necessary for the maintenance of bone growth.
Migration of the Cells
This problem is crucial for mesencephalic neural crest cells that arise from the dorsal
part of the neural tube and have to migrate to populate the future area of the skull
vault (Fig. 9). The problem is quite complex, since the mode of migration is different
according to the studied species. In chick embryos, mesencephalic neural crest
migrates using a subectodermal pathway (Noden 1975), whereas in rodents (both
mouse and rat) the stream of migration passes through the mesenchyme (Serbedzija
et al. 1992). Nothing is known on this subject for the human embryo.
Neural crest cells migrate along the pathways determined by the molecules of the
extracellular matrix (e.g., fibronectin, laminin, etc.). These cells recognize the
extracellular molecules, thanks to membrane receptors belonging to the family of
integrins. This system allows a general flow of migration. It is likely that

chemoattraction also plays a role by promoting the direction of this flow of migra-
tion. The chemotactic action of many molecular systems was identified on cells of
the mesencephalic neural crest. However, these studies did not attempt to separate
the different migratory streams of the cephalic region of the embryo. It remains to be
shown whether such systems act on all cells (frontonasal migration and migration in
the branchial arches). It is possible that these systems have a unique effect on all cells
or that they otherwise play a regionalized role. FGF2 and FGF8 promote migration
of neural crest cells originating from the mesencephalon (Kubota and Ito 2000).
Such is also the case for the SDF1-CXCR4 system (Rezzoug et al. 2011).
Another pivotal goal associated with the process of migration is to know the
factors involved in the regulation of the arrest of migration. Thorogood (1988)
proposed that a molecular cue exists in the environment that is mandatory to stop
the migration of the cells. This model has been named the “flypaper model” to
illustrate that the involved molecule acts as a sort of glue that stops the cells.
Thorogood proposed that such a role could be played by type II collagen. However,
a precise look of the distribution of type II collagen in the amphibian, Xenopus
laevis, leads to the conclusion that this molecule is expressed only after completion
of neural cell migration (Seufert et al. 1994). An alternative molecule that could be
involved in such a control is serotonin. Indeed, migrating neural cells express the
5-HT1A receptor, and the specific antagonist NAN-190 leads to arrest of neural crest
cell migration (Moiseiwitsch and Lauder 1995). This result shows that the flypaper
model is a valid model even though the exact molecules involved in the stop signal
are still to be discovered.
Tissue Interactions
One of the most important questions to answer in order to describe the correct steps
of normal development is the problem of tissue interactions. Precursor cells of the
bones of the vault are primarily located between the neural tube and the surface
ectoderm (Fig. 10). The salient feature of vault ossification is that it proceeds
according to membranous ossification, i.e., the bones directly arise from a connec-
tive scaffold without formation of cartilage.
Three different embryonic tissues play a seminal role in the development of the
vault mesenchyme. The first one is the neural tube itself. Schowing (1961, 1968a, b, c),
in a series of very elegant experiments in the chick embryo, showed that the different
bones of the skull are dependent upon signals coming from the different regions of the
brain. Thus, the frontal bone develops after interaction with both the prosencephalon
and the mesencephalon in the chick embryo. The parietal bone depends on interactions
from both the mesencephalon and the rhombencephalon. The occipital bone is depen-
dent upon a signal coming from the rhombencephalon. This experiment illustrates that
a correct development of neural structures is mandatory for the normal development of
the bones. In other words, it is thus obvious that an abnormal development of the brain
will lead to a secondary skull malformation.
12 M. Catala
The second tissue involved in skull mesenchyme development is the superficial

ectoderm. Tyler (Tyler 1983) demonstrated that the mesenchyme alone is unable to
give rise to bone structures when cultured in isolation. In case of co-cultures of brain
tissue and skull mesenchyme, the latter differentiates into cartilage. In contrast, in
case of co-cultures of superficial ectoderm and skull mesenchyme, the latter adopts a
bony fate. The canonical Wnt system acts through β-catenin. The activation of this
system results in inhibition of chondrogenesis promoting osteogenesis in the mes-
enchyme (Day et al. 2005). Its inhibition gives opposite results (Day et al. 2005).
Thus, this family of secreted molecules represents good candidates to account for
Tyler’s observations. Indeed, many molecules of the canonical Wnt family are
produced by the surface ectoderm of the cranial region in the mouse embryo
(Goodnough et al. 2014). If one genetically disrupts the expression of various
Wnts selectively in the surface ectoderm of mice, cranial ossification is abolished
and replaced by chondrification (Goodnough et al. 2014). This defect is associated
with impairment of the development of the dermis (Goodnough et al. 2014). The
Wnts are secondarily expressed by the mesenchyme itself, but their role is weaker
since they only control late ossification (Goodnough et al. 2014). Thus, the family of
canonical Wnts plays a major role in the interactions between surface ectoderm and
bone of the cranial vault.
The third tissue involved in the development of the skull mesenchyme is the dura
mater. In the mouse before birth, the presence of the dura is mandatory to maintain
the suture as an active center (Kim et al. 1998). Such an effect of the dura is not
crucial after birth in the mouse (Kim et al. 1998). Both BMP4 (a member of the
TGFβ superfamily) and TGFβ RII are expressed by the dura mater (Kim et al. 1998;
Ito et al. 2003). The invalidation of the gene coding for TGFβ RII is lethal very early
during development. However, it is possible to construct a conditional knockout of
this gene in which TGFβ RII is selectively not expressed in neural crest cells and
their derivatives. In this case, the meninges develop poorly and the skull vault is
severely impaired, with no frontal bone and a very rudimentary parietal bone (Ito
et al. 2003). This bone phenotype is due to an impairment of osteogenic cell
proliferation. The selective invalidation of BMP R1A (one of the receptors of the
BMPs) in neural crest cells leads to facial anomalies and reduced ossification of the
frontal bones, in turn causing enlargement of the anterior fontanel (Saito et al. 2012).
All these results demonstrate the role played by the system of the BMPs in the
control of skull bone formation. Thus, it is not surprising that BMPs are commonly
used now to promote bone formation in surgical techniques developed for curing
calvarial defects.
Control at the Genetic Level
This chapter has gained more and more importance nowadays. This may be
explained by both the development of molecular biology and the development of
human genetics.
The Genetic Control of the Osteoblastic Lineage

Two genes encoding for transcription factors play a crucial role in the establishment
of the osteoblastic lineage: Runx2 (formerly Cbfa1) and Osterix (Osx or SP7). In
mice, the invalidation by homologous recombination of one of these two genes
generates homozygous mice that die in the immediate postnatal period (Komori
et al. 1997; Otto et al. 1997; Nakashima et al. 2002). These mutants are devoid of
osteoblasts, showing that the presence of either of these genes is essential for the
differentiation of the lineage and that no compensation exists between them. This
lack of compensation is explained by the fact that the mouse lacking Osx expresses
Runx2, while the mouse that is invalidated for Runx2 does not express Osx
(Nakashima et al. 2002). Thus, Runx2 is located upstream to Osx. In addition,
RUNX2 binds to the Osx promoter and directly activates the gene transcription
(Nishio et al. 2006). The mouse invalidated for Runx2 in the heterozygous state has a
bone phenotype characterized by partial lack of ossification of the skull, absence or
hypoplasia of the clavicles, and other bone defects (Otto et al. 1997). This phenotype
resembles that of human cleidocranial dysplasia due to a heterozygous mutation
affecting the RUNX2 gene and resulting in loss of function of the encoded protein
(Mundlos et al. 1997; Lee et al. 1997). No homozygous mutant for human RUNX2
has been described so far. By comparing the results obtained in heterozygotes and
homozygotes, we can conclude that some bones are very sensitive to the dose of
RUNX2. This is particularly the case of the skull vault. A recessive mutation in the
gene encoding Osterix was demonstrated in a boy with osteogenesis imperfecta
(Lapunzina et al. 2010). This mutation leads to the formation of an abnormal protein
with 18 novel residues preceding a truncated region. Even if the authors did not
specifically study the functional consequences of this mutation, we can assume that it
leads to a hypomorphic protein since bones were not totally absent in the proband.
The Genes Involved in the Development of the Vault

Other systems involved in the control of the development of the vault are the genetic
pathways that control the development of the sutures.
Msx Genes and Their Role in the Maintenance of Undifferentiated Cells

Msx is a multigene family of vertebrate homologs of the drosophila gene, muscle
segment homeogene (msh). These genes code for transcription factors. MSX 1 and
2 proteins play a role in the maintenance of an undifferentiated state of the devel-
oping cells. These genes are highly expressed by the cells of the cranial sutures and
the dura and their expression is downregulated as soon as these cells are incorporated
into the developing bones (Kim et al. 1998; Jabs et al. 1993). The cells expressing
Msx1 or Msx2 are thus mitotically active, and they account for the growth of the
suture and the subsequent growth of the bones (Satokata et al. 2000). The crucial role
played by MSX proteins in the control of the development of the vault of the skull
has been demonstrated by constructing knockout mice for these genes. Msx1 /
mice display a deficiency of growth of both the frontal and the parietal bones
14 M. Catala
(Satokata and Maas 1994). Msx2 / mice develop a large midline foramen involv-
ing the two frontal bones (Satokata et al. 2000). Furthermore, the double knockout
Msx 1 / /Msx2 / is characterized by a major skull phenotype; indeed, no bony
structures of the vault are formed, whereas the chondrocranium is normally devel-
oped (Satokata et al. 2000; Han et al. 2007). The same skull phenotype is observed
for the Dlx5 / /Dlx6 / knockout mice (Robledo et al. 2002). These two couples
of genes (Msx1/Msx2 and Dlx5/Dlx6) are thus mandatory for the correct formation of
the vault. MSX2 acts through protein-protein interactions (Newberry et al. 1997),
and MSX2 and DLX5 form heterodimers (Newberry et al. 1998). Thus, the complex
MSX-DLX is necessary for the formation of the calvaria. However, the situation is
far from simple. It is possible to selectively invalidate Msx1 and 2 genes only in the
cells of the neural crest. The loss of a progressive number of alleles leads to a more
and more serious reduction of the frontal bone (Roybal et al. 2010). However, the
loss of the four alleles is accompanied by a curious phenotype: the frontal bone is
reduced, but supernumerary bones are lodged in the enlarged anterior fontanel
(Roybal et al. 2010). These bones originate from the mesenchymal cells that
normally are not fated to form bones. It is therefore necessary in the future to
study the different roles played by these proteins according to the type of cells
where they are expressed.
In humans, a mutation in the gene coding for MSX2 was found in one Bostonian
family presenting an autosomal dominant craniosynostosis (Jabs et al. 1993). This
mutation leads to an enhancement of the binding affinity of MSX2 for the DNA
(Ma et al. 1996). It is possible to reproduce such a syndrome, constructing a
transgenic mouse either by overexpressing the wild-type Msx2 murine gene or the
human mutated allele (Liu et al. 1995). This indicates that the mutation acts through
a gain of function. Furthermore, a functional haploinsufficiency of MSX2 has been
described in human autosomal dominant cases of enlarged parietal foramina (Wilkie
et al. 2000). Even if the exact mechanism at the molecular level played by MSX2 is
not unraveled, it is tempting to propose that MSX2 plays a promoting role on bone
formation in the skull of the developing human.
The Receptors of FGF Are Involved in the Control of Suture Development

Numerous syndromic craniosynostoses are due to a mutation in a gene coding for a
receptor of FGF (FGFR1, 2, or 3) (Lajeunie et al. 1999). These receptors belong to
the tyrosine kinase family of transmembrane receptors. Their extracellular region
comprises three immunoglobulin-like domains (I, II, and III). An alternative splicing
of the exon coding for the IgIII leads to the formation of two different isoforms of the
membrane-bound receptors IIIb and IIIc. These isoforms differ in their ligand
activity and their biological effects. In case of mutation of FGFR2, the effect of
these mutations is likely to be a gain of function generating an autoactivation of the
receptor (Galvin et al. 1996). Fgfr2c is expressed by mesenchymal cells of the
osteogenic fronts (Kim et al. 1998; Iseki et al. 1997, 1999; Rice et al. 2000), whereas
Fgfr1c is expressed by immature osteoblasts of the osteoid layer (Iseki et al. 1999). It
is important to note that FGFR2 regulates cell proliferation, whereas FGFR1 regu-
lates osteogenic differentiation (Iseki et al. 1999; Mansukhani et al. 2000;
Fig. 11 The base of the

human skull as it stands at
12.5 weeks of gestation.
Anterior, middle, and
posterior fossae (AF, MF, and
PF) are evident. ST sella
turcica. Notice the insertion of
the tentorium (arrows)
Eswarakumar et al. 2002). These functions are consistent with the observed pattern
of expression of these two genes. In human craniosynostosis, the FGF receptor
mutations are activating. This suggests that the bone phenotypes are due to increased
proliferation and/or differentiation of the osteoblast causing premature closure of the
suture. It is interesting that S252W FGFR2 protein, which results from mutations
observed in Apert syndrome, does not lead to premature suture closure when
expressed by mesenchymal cells of the suture (Heuzé et al. 2014). The coronal
suture closes prematurely when this protein is expressed by cells of the neural crest
or mesoderm. This is easily understandable with the new growth model of the skull,
in which the osteogenic front plays a major role contrarily to the central mesenchyme
of the suture.
Twist Is a Transcription Factor Preventing the Differentiation of Sutural Cells

into Osteoblasts
The EphA4 molecule belonging to the ephrin (Eph) families of membrane receptors
is a target of Twist. This family of receptors is very well known for its function in
boundary formation and maintenance. Saethre-Chotzen syndrome is due to the
mutation of the gene coding for Twist (a transcription factor that belongs to the
basic helix-loop-helix family) (El Ghouzzi et al. 1997; Howard et al. 1997). Such a
human disease is mimicked by the mouse line Twist /+, suggesting that (i) this gene
plays a similar role in both mouse and human and (ii) the human condition is due to
haploinsufficiency of this gene. In the mouse, Twist is expressed by the sutural cells
and preosteogenic cells but not by mature osteoblasts (Rice et al. 2000). Further-
more, overexpression of Twist in osteoblasts leads to their change into fibroblasts.
16 M. Catala
Fig. 12 Sagittal section of a

human skull base at 10 weeks
of gestation. The hypophysis
(A adenohypophysis,
N neurohypophysis) develops
between the basi-postphenoid
(B post Sp) and the basi-
presphenoid (B pre Sp). DS
dorsum sellae. Notochordal
remnants (arrows) are still
visible at this stage
Fig. 13 Sagittal section of

the skull base in a human fetus
at 10 weeks of gestation. Sphe
sphenoid, ST sella turcica, cl
clivus. Note the ossification
center of the basioccipital
bone (c)
Fig. 14 Sagittal section of

the skull base in a human fetus
at 20 weeks of gestation. Note
ossification centers of the
basi-presphenoid (a), basi-
postsphenoid (b), and
basioccipital (c) bones
These results indicate that Twist is an inhibitor for osteoblast differentiation. In case
of haploinsufficiency, the sutural cells will differentiate earlier into osteoblasts
Fig. 15 The two ossification

centers of the basisphenoid are
separated by cartilage forming
a so-called synchondrosis
leading to premature closure of the sutures. This scenario has the advantage of being
simple, but it cannot fully describe reality. Indeed, some patients with Saethre-
Chotzen syndrome also have a parietal foramen indicating that the same mutation
may result both in an acceleration of ossification and a bony defect (Ishii et al. 2003).
The foramen is due to a defect of proliferation and differentiation of cell that are
fated to generate the bones of the skull vault (Ishii et al. 2003). It is interesting to note
that Msx2 haploinsufficiency leads to a very similar phenotype (Ishii et al. 2003) and
that the two genes act in synergy (Ishii et al. 2003). In the future, precise networks of
molecular interactions are mandatory to interpret the eventual phenotype. Recently,
the cause of craniosynostosis in Saethre-Chotzen syndrome has been unraveled. In
the absence of one allele of Twist, cells from the frontal and parietal bones are
actively mixing because of a defect in boundary maintenance (Merrill et al. 2006;
Ting et al. 2009). It is particularly interesting to note that EPHA4 mutations have
been evidenced in humans with craniosynostosis (Merrill et al. 2006; Ting
et al. 2009). Furthermore, mutations of TCF12, a partner of TWIST, have also been
found in patients presenting coronal craniosynostosis (Sharma et al. 2013).
In the next future, protein interactions will be very important to analyze in order
to better understand the phenotypes and to try to propose specific therapies with
good molecular targets that could restore the defect.
The Base of the Skull
The base of the skull (or chondrocranium) is laid down according to endochondral
ossification, meaning that the initial cartilaginous scaffold is progressively replaced by
the bony structures. In humans, the first evidence of cartilaginous differentiation of the
base of the skull appears as soon as the sixth week of gestation (M€uller and O’Rahilly
1980). The cartilaginous cells are tightly associated with the notochord. The progression
of chondrification is very rapid, and the total base of the skull is chondrified as soon as
the eighth week of gestation (Kjaer and Fischer-Hansen 1995; Fig. 11). The notochord
18 M. Catala
Fig. 16 The embryonic

origin of the bones of the base
of the skull in human
postulated from experimental
data (superior view). The
bones derived from the
mesoderm are represented in
dark gray, while those derived
from the neural crest are
represented in light gray
terminates at the caudal border of the dorsum sellae (M€uller and O’Rahilly 1980;
Fig. 12). Notochordal remnants are still visible at 10 weeks of gestation (Fig. 12);
they could explain the occurrence of chordomas arising from the base of the skull. The
most anterior extension of the notochord makes it possible to divide the base of the skull
into two halves: a prechordal and a chordal part. The first elements to ossify are the
frontal and the squamosal bones (ninth to tenth week of gestation), then the basioccipital
(Fig. 13) and the greater wing of the sphenoid (12th week of gestation), the basi-
postsphenoid (15th week of gestation), the basi-presphenoid (16.5th week of gestation)
(Fig. 14), and the ethmoid bone (21st to 22nd week of gestation) (Kjaer 1990; Bach-
Petersen and Kjaer 1993). Ossification centers of the skull base are separated by
cartilage forming the so-called synchondrosis (Fig. 15).
The sequence of cartilage differentiation of the base of the skull has not been
described in humans. A recent description was published for the mouse (McBratney-
Owen et al. 2008). The salient results of this study may be summarized as follows.
The first cartilages to develop in the mouse belong to the caudal domain
(parachordal, occipital arch, and canalicular part of the auditory capsule). Then,
the anterior cartilages develop (trabecular and paranasal). Lastly, the two compo-
nents fuse together. Since cartilage represents the scaffold that serves to construct the
skull base, such data should be very valuable for humans strengthening the impor-
tance of gathering data on human embryos and fetuses.

The embryonic origin of the cells involved in the formation of the skull base has long
been ignored in mammals due to the lack of suitable techniques. The only available
fate map was obtained in birds by using the technique of quail-chick chimeras
(Couly et al. 1993). In birds, the basioccipital bone derives from the somitic
mesoderm. The basi-postsphenoid bone is produced by the cephalic mesoderm,
whereas the basi-presphenoid arises from the mesencephalic neural crest cells.
This fate map thus shows that the skull base, like the vault, has a triple embryonic
origin. It is also interesting to note that the otic capsule also has a triple origin in the
chick from the mesencephalic neural crest, the cephalic mesoderm, and the somitic
mesoderm (Couly et al. 1993).
The results obtained in mice with genetic tracing experiments invalidated all
these data that cannot be used to extrapolate to humans. Rathke’s pouch, namely,
the primordium of the adenohypophysis, marks the transition between neural
crest mesodermal contributions during embryonic life (McBratney-Owen
et al. 2008). On postnatal day 1 (P1), the mesoderm contributes to basioccipital,
exoccipital, and supraoccipital bones, auditory capsule including the tympanic
ring, and hypochiasmatic cartilage (McBratney-Owen et al. 2008). Neural crest
cells populate the nasal capsule, ethmoid bone, basi-presphenoid, basi-
postsphenoid (except for a limited contribution from the mesoderm),
alisphenoid, and orbitosphenoid (McBratney-Owen et al. 2008). It is important
to notice that the spheno-occipital synchondrosis is of mixed origin at P1, but
the cells coming from neural crest are totally replaced by mesodermal cells at
P10 (McBratney-Owen et al. 2008). This last result shows that the limits may be
variable with development, and readers should be very cautious in interpreting
fate maps. The hypochiasmatic cartilage is the sole structure that derives from
the mesoderm located rostrally to the notochord. This cartilage is thought to
contribute to preoptic and postoptic roots of the orbitosphenoid in humans. It is
also important to note that the basi-postsphenoid of mouse is, in fact, a
prechordal bone (McBratney-Owen et al. 2008), whereas it is a chordal bone
in humans. Taking into account all these comparative data, I propose to extrap-
olate these results to human beings (Fig. 16).

The cells of both the occipital somites and the cephalic mesoderm are located near
the notochord, and they enwrap the latter during their differentiation. The migra-
tion of these cells is thus very limited, with their final destination very close to
their initial origin. These cells participate in the formation of the mesodermal or
chordal basicranium. In contrast, the di- and mesencephalic neural crest cells that
will eventually give rise to the basi-presphenoid have to migrate before reaching
their final destination and forming the so-called prechordal basicranium. Their
exact pathway of migration is largely unknown in mice. Recently, this question
has been addressed in chick embryos (Wada et al. 2011). The prechordal
basicranium derives from two main streams of migrating cells. The preoptic
stream arises from more rostral levels of the neural tube, migrates and constitutes
the frontonasal bud, and gives rise to the nasal cartilage and the ethmoid. The
postoptic stream migrates with the mandibular bud and produces the basi-
presphenoid.
20 M. Catala
Fig. 17 The aspect of the

axial mesoderm of a chick
embryo. The notochord is
indicated by arrowheads. The
most anterior part of the axial
mesodermal domain is
represented by the prechordal
plate (arrow). OVoptic vesicle
Tissue Interactions
The chordal part of the skull base is dependent upon the influence of the notochord.
If one removes the sole anterior notochord in a chick embryo (Schowing 1968b), two
bones are hypoplastic: the basi-postsphenoid and basioccipital. These two bones are
thus notochord-dependent, like the ventrolateral parts of the vertebrae. It is quite
tempting to consider that this skull base region represents the most anterior part of
the somitic system. This interpretation is reinforced by the results of the knockout of
the Bapx1 gene in mouse. Bapx1 is a vertebrate homolog of the drosophila bagpipe
gene, coding for a transcription factor of the NK2 family. Bapx1 is expressed in the
sclerotome, splanchnic mesoderm, limb, and Meckel’s cartilage (Lettice et al. 1999).
Bapx1 / mouse shows abnormal development of the axial skeleton and also
hypoplasia of both the basioccipital and basi-postsphenoid bones (Lettice
et al. 1999). In contrast, the prechordal elements of the skull base are normal.
These results indicate that the genetic regulations of the chordal part of the base
differ from those controlling the prechordal basicranium and are similar to those
acting to control the development of the axial skeleton.
In contrast, the tissues acting on the development of the prechordal part of the
skull are largely represented by the prechordal plate (Fig. 17) (the most anterior part
of the axial mesoderm) (Pera and Kessel 1997). This region is independent of the
Fig. 18 In situ hybridization

in a chick embryo revealing
the expression of Sonic
hedgehog. The gene is
expressed by both the
notochord (arrowheads) and
the prechordal plate (arrow).
OV optic vesicle, NT
neural tube
presence of both the neural tube and the notochord. It is interesting to note that a
defect in the development of the prechordal plate has been associated with
holoprosencephaly (Pera and Kessel 1997). Furthermore, holoprosencephaly is
associated with developmental defects of the basi-presphenoid, while the develop-
ment of the basi-postsphenoid is normal (Arnold et al. 1988; Cannistrá et al. 2001;
Kjaer et al. 2002; Arnold and Meiselbch 2009).
The otic capsule is formed after an induction of the mesenchyme by the otic
vesicle (that will eventually differentiate into the inner ear) (Van de Water and
McPhee 1987; Frenz and Van de Water 1991). It is interesting to note that the
mesenchymal tissue located immediately in contact to the otic vesicle does not
give rise to cartilage but to a loose tissue that secondarily generates the perilymphatic
spaces (Van de Water and McPhee 1987). Murine mutants for Shh develop no base
structure of the skull except the otic capsule, showing that regulation of the differ-
entiation of these structures is independent (Balczerski et al. 2012). The mode of
interactions between the otocyst and mesenchyme explains the malformation of the
otic capsule induced by a maldevelopment of the otic vesicle. Chondrogenesis of the
otic capsule is promoted by the secreted protein BMP4 (Chang et al. 2002; Liu
et al. 2003).
22 M. Catala

The skull base or chondrocranium can be divided into three major regions. The
prechordal region depends on the prechordal plate, the chordal region is tightly
associated with the notochord, and the supraoccipital bone responds to a radically
different regulation. The first two regions are homologous to the ventral region of the
vertebrae, and in these two regions, Sonic hedgehog plays a major role. The
supraoccipital bone is closest to the dorsal region of the vertebrae.
Sonic Hedgehog and the Basicranium

The bones constituting the base of the skull are formed by chondrification of the
mesenchyme under the influence of Sonic hedgehog, a secreted protein produced by
the notochord, the prechordal plate, and the floor plate of the neural tube (Fig. 18).
The role of Sonic hedgehog signaling in the control of head and brain formation has
been reinforced by human genetics. Indeed, hypomorphic mutations of the Sonic
hedgehog gene have been described associated with autosomal dominant forms of
holoprosencephaly (Roessler et al. 1996). Sonic hedgehog acts on Patched, a
transmembrane receptor. In humans, haploinsufficiency of Patched leads to Gorlin
syndrome, characterized by cleft palate and odontogenic cysts in the mandible
(Gailani et al. 1992, 1996). The hedgehog signaling is mediated through the cyto-
plasm by homologs of the drosophila cubitus interruptus. In vertebrates, there are at
least three homologs: Gli1, Gli2, and Gli3. The partial loss of GLI3 functions in
humans has been associated with Grieg’s syndrome, in which facial maldevelopment
is characterized by a wide forehead and nasal bridge (Vortkamp et al. 1991). In
conclusion, the molecular pathways involved for Sonic hedgehog signaling are
probably very complex, since the human phenotypes observed after Sonic hedgehog
hypomorphic mutation, haploinsufficiency of Patched, and partial loss of function of
GLI3 are different. This illustrates the complexity of the molecular interactions that
play a role in this signaling. In mice, haploinsufficiency of Sonic hedgehog does not
impair the correct development of the brain and skull, unlike in humans where it
generates holoprosencephaly and prechordal skull base defect. The homozygous null
mutation of Sonic hedgehog has never been described in humans, and it should
probably be lethal very early in development. In mice, it causes a complete absence
of the sphenoid and basiocciput.
Transcription Factors
In mouse, knockout experiments with genes coding for transcription factors high-
light the role of some of these factors in the control of the morphogenesis of the base
of the skull. It is obviously beyond the scope of this chapter to review extensively all
these genes, and interested readers will find a thorough review in Francis-West
et al. (1998). However, from the analysis of literature, we have to point out that a
single gene mutation leads to abnormal formation of different bones that are not
located in the same skull compartment. Some genes, like Otx2 and Lim1, control the
development of both the basi-presphenoid and the basi-postsphenoid (i.e.,
Fig. 19 Dorsal view of a

chick embryo at 8-somite
stage. The neural tube is
divided into the prospective
rhombencephalon (Rh) and
spinal cord (s). CM cephalic
mesoderm. The somites 1–4
are the occipital somites. The
cervical somites are located
caudally
components of both the prechordal and chordal parts of the skull base). In these
cases, however, all structures located rostral to the level of the third rhombomere are
deleted, indicating a more global role of these genes in the control of head develop-
ment. In contrast, Dlx2 controls the development of the basi-postsphenoid and not
the basi-presphenoid, while Pax6 controls the basi-presphenoid and not the basi-
postsphenoid. These results indicate that the control at the genetic level is likely to be
very complex. In the next future, it would be very important to address the exact role
played by these genes during morphogenesis of the skull.
The Genes Involved in the Development of the Supraoccipital Bone
Homeotic Genes in Drosophila

Drosophila is a quite simple organism in which genetic studies could be performed
easily. At the end of the nineteenth century, William Bateson (1894) described
strange transformations affecting the shape of an organism: a petal into a stamen,
an eye into an antenna in crustaceans, or an antenna into a foot in insects. These
changes can be interpreted as transformation of one corporeal segment into another.
24 M. Catala
Fig. 20 The pathways of

migration of neural crest cells
that will eventually form the
face represented in a profile
view of a human embryo
(fourth week of gestation).
The rostral stream (light gray)
is separated into two halves by
the eye vesicle. The caudal
stream migrates to populate
the first branchial arches
Bateson coined the term “homeosis” to define all these kinds of transformation. Such
is the case of the transformation of the cephalic antenna into a leg (the so-called
antennapedia transformation) or the acquisition of extra wings by transformation of
an abdominal segment into a thoracic one (the so-called bithorax transformation).
These modifications have been named homeotic transformations. The next step was
gained by genetic analyses of these transformations. It was found that the mutation
of a single gene is responsible for the observed phenotype, implying that one gene is
able to control the identity of a body segment in drosophila. These genes were named
“homeotic genes.” After development of molecular biology, the genes responsible
for these mutations were shown to encode for transcription factors belonging to the
family of homeodomain-containing proteins. The function of these genes in dro-
sophila is to code for positional identity of body segments. In the absence of one of
these genes, the involved segment will adopt a new positional identity, leading to a
homeotic transformation.
Homeotic Genes in Vertebrates

The first homeotic gene was cloned in 1983 in Gehring’s lab (Garber et al. 1983).
Then, the comparison of the sequence of three homeotic genes identified a DNA
sequence of 60 homologous amino acids in all these proteins (McGinnis
et al. 1984a). This DNA sequence codes for the homeodomain and is called the
“homeobox.” This sequence is conserved in other species (McGinnis et al. 1984b)
including vertebrates (Shepherd et al. 1984; Carrasco et al. 1984). One family of
Fig. 21 The different cell

streams involved in face
formation, represented in a
face view of a human embryo
at the beginning of the fifth
week of gestation. The face
develops from the frontonasal
bud (dark gray), the maxillary
buds (light gray), and the
mandibular ones
(intermediate gray)
these vertebrate genes is called the Hox (for homeobox-containing genes) family.
Hox genes are grouped into clusters in the chromosomes (a, b, c, and d). In one
cluster, the genes are orientated according to the DNA sequence and named
according to their chromosomal position by a number 1–13.
Hox d4, a Key Gene for Vertebral Identity

The paraxial mesoderm of vertebrates is organized according the rostrocaudal axis.
The cephalic mesoderm is the most rostral part of this domain (Fig. 7). The
remaining paraxial mesoderm is segmented into metameric units extending to the
tip of the embryonic, called somites. The first four somites are called occipital
somites, since they contribute to the formation of the occipital bone (Couly
et al. 1993). The fifth somite is the first cervical somite that participates into the
formation of the first cervical vertebra (Fig. 19).
Hox d4 is normally expressed from the first cervical vertebra caudalwards. If
one forces the expression of Hox d4 in more rostral mesoderm, both the
supraoccipital and the exoccipital bones are transformed into occipital vertebrae
(Lufkin et al. 1992). This experiment indicates that the sole expression of Hox d4
in the paraxial mesoderm is sufficient to modify the rostrocaudal identity of a
vertebra. It is important to note that the rest of the vault is normal in this
experiment.
Mhox (Prx-1) Gene, a Key Regulator for the Formation of the Supraoccipital Bone
Mhox gene codes for a transcription factor whose knockout leads to total absence of
the supraoccipital bone, while the rest of the vault forms normally (Martin
et al. 1995). It is interesting to note that the interparietal bone is not affected by
this mutation, indicating that the regulative networks controlling the development of
26 M. Catala
Fig. 22 Face view of a

human embryo at the end of
the sixth week of gestation.
The frontonasal bud is
separated into two halves by
the developing nasal placode.
This allows the formation of
the lateral nasal and medial
nasal buds
Fig. 23 Fusion of two

primordia. Two buds grow to
appose each other. Their
epithelia fuse to eventually
form a single structure
the different part of the occipital bone differ. Furthermore, other facial bones are
involved by this knockout, showing that the Mhox gene is not specific for the
supraoccipital bone but participates into other patterning programs affecting the
development of different bones.
Face and Neck
The Face
The human face is formed by the convergence of cell streams formed after emigra-
tion of neural crest cells from the neural plate. These cells merge with those of the
cephalic mesoderm that ingress during gastrulation from primitive streak. Rostrally,
neural crest cells cover the forebrain and are subdivided by the presence of the optic
vesicle, a diencephalic evagination (Fig. 20). The most medial stream of cells forms
the so-called frontonasal process. Caudally, the cells migrate to populate the first
branchial arches leading to two compartments, namely, the maxillary and the
mandibular processes. The face is thus the result of the development of five buds
(the frontonasal process, the two maxillary processes, and the two mandibular ones)
(Fig. 21). The first ever evidence of this differentiation appears as soon as the fourth
week of development (Streeter 1945). At the very beginning of their formation, the
buds that form the embryonic head are largely confluent. There is no clear boundary
between the maxillary and frontonasal buds. Similarly, the maxillary and mandibular
buds are confluent at their proximal region (close to the neural tube). Streeter had
Fig. 24 Merging of
primordia. A unique
primordium is first observed.
Then, mesenchymal
proliferation leads to
development of two buds
united by their base. These
buds appose and their
epithelia fuse to generate a
single structure
28 M. Catala
Fig. 25 Histological aspect

of the branchial arches in a
chick embryo (25-somite
stage). The arch is limited by
both surface ectoderm
(Ec) and endoderm (En). The
core of the branchial arch is
made of mesenchymal cells
(Me) and a branchial arch
artery (BAa). Each branchial
arch is separated from the
others by the obturator
membrane (OMb), in which
surface ectoderm and
endoderm are in close contact
already described such features in the 1940s (Streeter 1945, 1948). This fact is
perfectly illustrated by scanning electron microscopy studies performed on human
embryos (Hinrichsen 1985; Steding 2009). These data contradict some diagrams
published in academic books in which the buds are separated by complete furrows.
The nasal placode is induced in the surface ectoderm covering the frontonasal
region. This placode invaginates and separates the frontonasal process into two
halves, forming both the medial and the lateral nasal processes (Fig. 22). The two
medial nasal processes unite on the midline and eventually form the philtrum and the
medial region of the nose. The two lateral processes form the lateral components of
the nose and merge laterally with the maxillary processes. The latter will eventually
give rise to the cheek and the lateral part of the superior maxillary bone. Lastly, the
two mandibular buds merge on the midline and give rise to the mandibular bone.
Both the malleus and the incus derive from the first branchial arch.
During face morphogenesis, it is important to distinguish between fusion and
merging. Patten, in 1961, was the first to suggest such distinction (Patten 1961).
Fusion concerns two primordia that are separated by a complete cleft constituting a
physical discontinuity. Both primordia grow and approach each other, and their
surface epithelium contacts and fuses allowing their mesenchyme to melt
(Fig. 23). Such is the case for the fusion of palatine shelves during secondary palate
morphogenesis.
In contrast, melting is a process that involves a single primordium. Differential
proliferation leads to the formation of two buds connected by their base. With
growth, these two buds lean against each other allowing the side contact between
their surface epithelium. Then, the epithelium vanishes and their mesenchymal core
can merge (Fig. 24). This mechanism acts during the development of the facial buds.

The frontonasal bud in avian embryos has a double origin (both cephalic mesoderm
and neural crest cells). In birds, the neural crest cells that populate the frontonasal
bud derive exclusively from the mesencephalon (Couly et al. 1993). In mammals, it
is much more complex to follow the fate of cells during the embryonic period since it
is necessary to perform whole embryo culture in vitro. Using this experimental
protocol, Osumi-Yamashita et al. (1994) showed that the mammalian frontonasal
bud receives a double neural crest contribution: both from forebrain and midbrain.
Both the maxillary and the mandibular buds are developed from the first branchial
arches (Fig. 25). The mesenchymal cells that populate the first branchial arches have
two different origins: (i) cells originating from the cephalic mesoderm and (ii) neural
crest cells migrating from the posterior mesencephalon, rhombomeres 1, 2 and a
little contribution from rhombomere 3 (Köntges and Lumsden 1996; Couly
et al. 1998; Fig. 26). The cells that arise from the neural crest populate the periphery
of the branchial arch, whereas the cells from the cephalic mesoderm adopt a central
position, forming the core of the branchial mesenchyme (Trainor and Tam 1995;
Yoshida et al. 2008; Fig. 27). The cells arising from the cephalic mesoderm differ-
entiate into endothelial cells and striated muscle fibers, whereas the other connective
components are of neural crest origin.
Cell Migration
As already mentioned about the cranial vault, the control of the migration of neural
crest cells that form the rostral cephalic stream is poorly understood. However, the
migration of cells that generate the frontonasal bud is disturbed in an autosomal
recessive type of frontonasal dysplasia. This disease is very heterogeneous and
characterized by hypertelorism and a variable degree of midline facial clefting.
Different forms have been associated with mutations of ALX1, ALX3, and ALX4.
The two families with ALX1 mutation are characterized by a very severe form of
frontonasal dysplasia (Uz et al. 2010). An impairment of neural crest migration in the
frontonasal bud has been observed in a zebra fish model of this genetic disease (Dee
et al. 2013). The relevance of this migration defect should be established for the
other types of frontonasal dysplasias.
Tissue Interactions
The development of the face involves numerous tissue interactions that are becoming
better known. These interactions are demonstrated in several animal models: zebra
fish, chicken, and mouse. However, facial anatomy and consequently its
30 M. Catala
morphogenesis vary widely among species (Young et al. 2014). For example, Shh is
expressed by a broad medial domain in the upper beak of chicks, whereas it is
expressed by two separated domains in the upper jaw of mice (Hu and Marcucio
2009a). Another beautiful example is the variation of Bmp4 expression explaining the
different shape of the beak in Darwin’s finches (Abzhanov et al. 2004). Thus, one must
be cautious before transferring some fundamental knowledge from animal models to
humans. Obviously, it is impossible to draw in this chapter a complete list of all
interactions known to date; only a few particular aspects will be illustrated here.
Interactions Between the Facial Buds

Ablation of the medial nasal buds leads to maldevelopment of both the lateral nasal
and the maxillary buds (McCann et al. 1991). This shows that the medial nasal bud
plays an inductive role on both lateral nasal and maxillary buds. This role may
explain the occurrence of eyelid coloboma and hypoplasia of the nasolacrimal duct
associated with lateral proboscis (Garabedian et al. 1999).
Neural Tube, Surface Ectoderm, and Frontonasal Mesenchyme: A Ménage à

Trois
Shh expression is very dynamic during development of the cephalic pole. First, it is
expressed in the chick by the endoderm, prechordal plate, and ventral prosencephalic
vesicle (Marcucio et al. 2005). Then, the prosencephalic expression increases on
either side of the optic recess, namely, in the ventral telencephalon and diencephalon
(Marcucio et al. 2005). At last, the surface ectoderm overlying the telencephalon
expresses Shh. Surface ectoderm in this adjacent region expressing Shh produces
FGF8 in a non-overlapping area (Hu et al. 2003). This double region of surface
ectoderm will form the epidermis covering the upper beak in chick (Hu et al. 2003).
Furthermore, if grafted heterotopically, it induces an extra upper beak
(Hu et al. 2003), indicating that it plays an instructive role in proximo-distal growth
of the beak and in establishing the dorsoventral axis of this structure (Hu and
Marcucio 2009a; Hu et al. 2003). The expression of Shh by surface ectoderm is
induced by Shh, while Bmp produced by underlying tissues (Foppiano et al. 2007;
Hu and Marcucio 2009b; Hu et al. 2015). In conclusion, the telencephalon acts on
surface ectoderm that, in turn, controls both growth and polarity of the frontonasal
mesenchyme. The expression of ectodermal Shh is different in mouse from chick
(Hu and Marcucio 2009a).
This role of Shh on the control of the development of the frontonasal bud in chick
must be related to its possible role in the genesis of some human facial deformities.
Decreased activity of the hedgehog signal would lead to a narrowing of facial
midline structures, whereas an increase would result in the opposite effect
(Brugmann et al. 2010).
The Early Role of Endoderm in Cephalic Patterning

Early during development, cells of the cephalic neural crest migrate in contact with a
rostrally located endodermal recess. After ablation of this recess, the bony structures
of the face do not grow showing that the endoderm plays an early role in the
Fig. 26 The respective contribution of the neural crest coming from the different rhombomeres in
the formation of the mesenchyme of the branchial arches (Redrawn from (Köntges and Lumsden
1996; Couly et al. 1998))
development of cephalic mesenchyme (Couly et al. 2002). In fish, FGFs appear to

play such a role, but this is not known in birds or mammals.
The Orbit Is Formed Through Interactions with the Eyeball

In humans, both anophthalmia and microphthalmia are associated with abnormal
formation of the orbit that is much smaller with a very narrow palpebral fissure
(Gujar and Gandhi 2011). These clinical data suggest that the orbit can form in the
absence of the eyeball, but the latter is necessary for the proper growth of the bone
structure. In chick embryos, early removal of both optic vesicles leads to a consid-
erable reduction in the size of the orbits and interorbital septum (Silver 1962). It is
likely that the rudimentary orbit is due to growth of the maxilla and the frontal bone
under the influence of adjacent normally formed structures. It is interesting to note
that the eyelids are also formed in chick despite the total lack of eye (Silver 1962),
suggesting that this process is independent of the optic vesicle. The subsequent
growth of the eyeball is due to retinal pigment epithelium that induces the formation
of the scleral cartilage in chick (Thompson et al. 2010). It is possible that such
interactions exist in mammals but they have not yet been identified.
The Olfactory Pit Acts on the Lateral Nasal Bud to Induce Cartilage
The removal of the olfactory pit in chick embryos results in a developmental defect
involving lateral nasal processes, whereas median buds are not affected (Szabo-
Rogers et al. 2009). Heterotopic transplantation of olfactory pit resulted in formation
of supernumerary cartilages on the sole condition that the graft is placed in the
cephalic mesenchyme (Szabo-Rogers et al. 2009). This series of interactions could
explain the occurrence of arrhinia, either complete or partial.
32 M. Catala
Fig. 27 Section of a
branchial arch. Cephalic
mesoderm gives rise to cells
that constitute the core of the
arch (gray). The rest of the
mesenchyme (white) is
yielded by neural crest
Dlx Genes Are Expressed According to the Proximo-Distal Polarity of the First
Branchial Arch
Dlx is a family of homeobox-containing genes homologous to the Distal-less gene of
Drosophila. In the first branchial arch, Dlx are expressed according to a proximo-
distal gradient. Dlx-1 and Dlx-2 are expressed by the whole arch (both maxillary and
mandibular buds), whereas Dlx-3, Dlx-5, and Dlx-6 are only expressed by distal
domains (mandibular arch) (Merlo et al. 2000). The expression of Dlx-1, Dlx-2, and
Dlx-5 is induced by FGF8 in the mesenchyme of the first branchial arch (Ferguson
et al. 2000). It is interesting to note that the mutant mice Dlx-1 / , Dlx-2 / , and
Dlx-1/2 / display defects involving the maxillary arch and not the mandibular one
(Qiu et al. 1995, 1997). These results suggest that other Dlx genes can compensate
for the absence of Dlx-1/Dlx-2 in the mandibular arch. However, the strict control of
distal structures by the other genes has been ruled out by the knockout of the Dlx-5
gene. Indeed, this Dlx-5 / mouse displays both proximal and distal defects of the
first branchial arch (Acampora et al. 1999; Depew et al. 1999). The double knockout
Dlx-5 / /Dlx-6 / mutants display a very interesting phenotype: they exhibit a
homeotic transformation of lower jaws into upper jaws (Depew et al. 2002). This
result demonstrates that these two Dlx genes act as selector genes for the distal region
of first branchial arch derivatives.
The Neck
The human neck is formed by the development of the branchial arches, which are
lateral structures that eventually merge on the ventral midline. The first branchial
arches belong to the face and give rise to the lateral part of the maxilla and the
mandible. Furthermore, they generate the malleus, incus, zygomatic bone, and a part
of the temporal bone including the tympanic ring. In humans, it is quite difficult to
distinguish the caudal branchial arches, explaining the discrepancies existing
between authors for numbering the different arches. Thus, I propose to categorize
the cervical branchial arches into the second, the third, and the caudal arches. Each
branchial arch is limited by both surface ectoderm and endoderm. The intermediate
tissue of the arch is made up of an artery (the so-called aortic arch), mesodermal
tissue deriving from the cephalic mesoderm, and mesodermal tissue deriving from
the neural crest (Fig. 25). At the cephalic and caudal borders of each arch, the surface
ectoderm is tightly apposed with the endoderm forming the so-called obturator
membrane.

Each branchial arch is composed of neural crest cells arising from the
rhombencephalic level of the neural tube. The exact rhombomeric origin of these
cells has been extensively studied in the avian embryo (Köntges and Lumsden 1996;
Couly et al. 1998; Fig. 26). The first arch is populated by cells coming from
mesencephalon, r1, r2, and a few cells coming from r3. The second arch receives
cells coming from r3 (the contribution is weak), r4, and r5. The third arch is
populated by cells coming from r5 (weak contribution), r6, and a weak contribution
from r7. The caudal arches (4–6) receive a cellular contribution coming from r6 and
r7. The mesenchyme of branchial arches is of mixed origin. The core derives from
the cephalic mesoderm, whereas the periphery is provided by the neural crest
(Fig. 27).
The development of the arches is still poorly understood. Indeed, it is technically
challenging to mark a sole branchial arch due to the presence of the branchial artery.
For example, transplantation of quail cells into chick host has never been achieved in
such a location. This explains why the fate maps are largely speculative. However,
some general rules can be applied to the derivatives of these structures:
– The surface ectoderm gives rise to epidermal cells (except for Langerhans cells,
Merkel cells, and melanocytes). It is interesting to note that the second branchial
arch develops considerably and covers the caudal arches forming the so-called
cervical sinus, which can sometimes persist leading to the formation of cervical
cysts.
– The cervical mesoderm gives rise to striated muscle fibers and endothelial cells.
It is tempting to propose that striated muscle fibers coming from the same
branchial arch are innervated by the same branchial nerve. Indeed, it is a general
law for all somatic muscles whose innervation is provided by motoneurons of
the spinal segment located at the same transverse level. If one applies this
theoretical law, the first branchial arch is innervated by nerve V, the second
arch by nerve VII, the third by nerve IX, and the fourth to sixth by nerve X. It is
also important to note that the cephalic mesoderm gives rise to endothelial cells
for the neck and the face but also for the brain. This common origin could
34 M. Catala
Fig. 28 Endothelin 1 is
secreted by both the surface
ectoderm and the cephalic
mesoderm (dark gray) and
acts on cells of the neural crest
that express ETA receptor
(light gray). When endothelin
1 binds to its receptor, it
induces the synthesis of
dHAND, which is responsible
for Msx1 synthesis
explain some vascular syndromes associating abnormal vessels in the brain and
the head.
– Neural crest cells give rise to all the other mesenchymal derivatives of the neck.
The exact embryonic origin of the different bones and cartilages of the neck is still
speculative. However, it is classic to consider that neural crest cells of the second
branchial arch give rise to the stapes, the styloid process, the lesser horn, and the
superior rim of the hyoid bone. Neural crest cells of the third branchial arch
differentiate into the greater horn and the inferior rim of the hyoid bone. Lastly,
the cells of branchial arches 4–6 give rise to the laryngeal cartilages.
– Endodermal cells develop and give rise to different glandular organs of the
neck. The endodermal lining of the second arch gives rise to the epithelial sheet
of the amygdala. The endoderm of the third arch yields the epithelial cells of the
thymus and the glandular cells of the inferior parathyroids. The fourth arch
endoderm is responsible for the formation of the glandular cells of the superior
parathyroids.

The migration of neural crest cells in the branchial arches is achieved according to
strictly segregated cell streams. Different families of molecules have been implicated
in the control of migration and segregation of these cell streams. Thus, mice whose
EphB4 gene has been mutated display abnormal migration of neural crest cells
arising from rhombomere 4 (Golding et al. 2000). Since this early work, many
experiments have highlighted diverse chemorepellent and chemoattracting factors
(Théveneau and Mayor 2012). However, these data are quite difficult to extrapolate
to humans because these molecules have a divergent function in mice, birds, and
amphibians (Mellott and Burke 2008).
Tissue Interactions
Branchial arches are made of tissues derived from the three germ layers and are the
site of multiple interactions. The endoderm plays an important role in these pro-
cesses (Couly et al. 2002); the ectoderm overlying the mesenchymal core also has a
major action (Shigetani et al. 2000), as is the case for the obturator membrane
(Edlund et al. 2014).
Blocking or genetic invalidation of SHH induces cell death affecting the branchial
arches (Ahlgren and Bronner-Fraser 1999; Yamagishi et al. 2006). SHH is produced
by the endoderm (Brito et al. 2006; Haworth et al. 2007) and induces FGF8
expression by surface ectoderm (Yamagishi et al. 2006; Haworth et al. 2007).
Endothelins are small peptides (21 amino acids) (ET-1, ET-2, and ET-3) that bind
to G protein-coupled receptors (ETA and ETB). The endothelins are produced as
preproendothelins, which are processed to form inactive intermediates called big
endothelins. The latter are proteolytically activated by endothelin-converting
enzymes (ECE). ET-1 and ET-2 can bind to ETA and ETB, respectively, whereas
ET-3 can only bind to ETB.
ET-1 is secreted by both ecto- and endodermal cells of the branchial arches 1, 2,
and 3 (Kurihara et al. 1994; Clouthier et al. 1998), by the endothelium of the arch
vessels, and by the cephalic mesoderm of branchial arches 1 and 2 (Clouthier
et al. 1998; Fig. 28). Furthermore, ET-1 / homozygous mice display abnormalities
of the branchial arch-derived tissues (Kurihara et al. 1994), showing that ET-1 plays
a major regulative role from the ectoderm to the underlying tissues. ETA is expressed
by neural crest cells as soon as they arise from the neural tube and by neural crest-
derived ectomesenchyme of the branchial arches 1, 2, and 3 (Clouthier et al. 1998)
(Fig. 28). ETA / mice display the same craniofacial phenotype as ET-1 / mice
(Clouthier et al. 1998), showing that ET-1 mediates its biological effects during
development via the ETA receptor. At last, ECE 1 / mice exhibit craniofacial
abnormalities that are virtually identical to the defects observed in the previously
described knockout (Yanagisawa et al. 1998).
FGF8 is expressed in the branchial arches of the mouse with a very dynamic
pattern. At the beginning, the expression is weak and homogeneous. Then, FGF8 is
expressed by the rostral ectoderm of the first branchial arch and finally by the oral
ectoderm (Trumpp et al. 1999). A mouse line in which Fgf8 has been invalidated in
36 M. Catala
the first branchial arch shows massive apoptosis of neural crest cells populating the
first arch and then a defect of all the derivatives of this arch (Trumpp et al. 1999). It is
interesting to note that the distal region of the first arch develops normally, contrary
to the proximal region. This finding indicates that there are two distinct domains in
the first branchial arch: a proximal domain whose development is dependent of
FGF8 and a distal domain that is not.
Foxi is expressed by the cells of the obturator membrane, and its genetic ablation
leads to a defect in FGF secreting centers in the branchial arches (Edlund et al. 2014).
Hox a2 as a Selector Gene for the Second Arch Identity

It is well known from classic experiments performed in avian embryos that the
neural crest populating the first branchial arch contains its own identity and can
develop as a mandible when transplanted into the second arch (Noden 1983).
The difference in term of Hox gene expression between the first and the second
arches is that no Hox genes are expressed by the cells of the first arch, whereas cells
of the second arch express Hox a2. If one performs a knockout of the Hox a2 gene,
the cells of the two arches will be identical as to Hox expression. This experiment
could test the role of Hox a2 in the control of positional identity. In these cases, the
bones of the second arch are not formed and are replaced by duplicated bones of the
first arch (Rijli et al. 1993; Gendron-Maguire et al. 1993). The reverse experiment, i.
e., overexpression of Hox a2 in the first branchial arch, leads to a homeotic
transformation of the first branchial arch into a second one (Grammatopoulos
et al. 2000).
These two experiments prove that Hox a2 is responsible for the positional
identity of the cells of the second branchial arch. However, the precise timing of
the acquisition of this identity remains to be established. By performing hetero-
topic transplantations, Prince and Lumsden (1994) showed that regulation of Hox
a2 expression is independent in the neural tube and in the neural crest. It can thus
be concluded that the positional identity of neural crest cells is acquired after their
emigration from the neural tube. However, is the acquisition an early event during
neural crest migration or is it a late one? To test this problem, Rijli et al. used a very
elegant strategy (Pasqualetti et al. 2000): they used an inducible system in Xenopus
embryos and found that overexpression of Hox a2 in the first branchial arch after
completion of migration is still capable of inducing a homeotic transformation.
This shows that neural crest cells acquire a positional identity late during their
development after they have reached their final destination. Such a conclusion is
also established in mice using an inducible system of knockout experiments
(Santagali et al. 2005), showing that plasticity is still operating after neural crest
cells have migrated. Invalidation of all the Hoxa genes expressed in branchial
arches leads to transformation of arches 2, 3, and 4 into arch 1 (Minoux
et al. 2009). This shows that the Hox code is responsible for anteroposterior
patterning of the branchial arches.
Dlx Genes Play a Role in the Development of the Second Branchial Arch
Dlx-2 / mice display abnormal development of the proximal derivatives of both the
first and the second branchial arches (Qiu et al. 1995). Dlx-1 / mice display an
inconstant defect of the development of the proximal derivatives of the second arch
(Qiu et al. 1997). Dlx-5 / mice are not affected by the development of the
derivatives of branchial arches 2–6, in spite of its expression in these tissues
(Acampora et al. 1999; Depew et al. 1999).
Conclusion
The development of the head and neck involves several tissues originating from the
three germ layers. Their development is complex, and their regulation involves
numerous tissue interactions. Many genes controlling these processes have been
identified, allowing one to understand certain human genetic defects or serving as
candidate genes for the molecular explanation of some syndromes. A good under-
standing of these processes is an important prerequisite for understanding the defects
affecting the cephalic pole of the human being.
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Embryology of The Head and Neck: Martin Catala

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Embryology of the Head and Neck

# Springer-Verlag Berlin Heidelberg 2016 1

Fig. 1 Head of a human

Fig. 2 Head of a human

Fig. 3 Head of a human

Fig. 4 Aspect of the parietal

Fig. 5 The vault of the skull

Fig. 6 Histological section

The Vault of the Skull

Fig. 7 Dorsal view of a chick

Embryonic Origin and Subsequent Development

Fig. 8 The embryonic origin

Fig. 9 Mesencephalic neural

Fig. 10 Section through the

Migration of the Cells

integrins. This system allows a general ﬂow of migration. It is likely that

The second tissue involved in skull mesenchyme development is the superﬁcial

Control at the Genetic Level

The Genetic Control of the Osteoblastic Lineage

The Genes Involved in the Development of the Vault

Msx Genes and Their Role in the Maintenance of Undifferentiated Cells

The Receptors of FGF Are Involved in the Control of Suture Development

Fig. 11 The base of the

Twist Is a Transcription Factor Preventing the Differentiation of Sutural Cells

Fig. 12 Sagittal section of a

Fig. 13 Sagittal section of

Fig. 14 Sagittal section of

Fig. 15 The two ossiﬁcation

The Base of the Skull

Fig. 16 The embryonic

Embryonic Origin and Subsequent Development

Migration of the Cells

Fig. 17 The aspect of the

Fig. 18 In situ hybridization

Control at the Genetic Level

Sonic Hedgehog and the Basicranium

Fig. 19 Dorsal view of a

The Genes Involved in the Development of the Supraoccipital Bone

Homeotic Genes in Drosophila

Fig. 20 The pathways of

Homeotic Genes in Vertebrates

Fig. 21 The different cell

Hox d4, a Key Gene for Vertebral Identity

Fig. 22 Face view of a

Fig. 23 Fusion of two

Face and Neck

Fig. 25 Histological aspect

Embryonic Origin and Subsequent Development

Interactions Between the Facial Buds

Neural Tube, Surface Ectoderm, and Frontonasal Mesenchyme: A Ménage à

The Early Role of Endoderm in Cephalic Patterning

development of cephalic mesenchyme (Couly et al. 2002). In ﬁsh, FGFs appear to

The Orbit Is Formed Through Interactions with the Eyeball

Control at the Genetic Level

Embryonic Origin and Subsequent Development

Migration of the Cells

Control at the Genetic Level

Hox a2 as a Selector Gene for the Second Arch Identity

Schowing J. Inﬂuence inductrice de l’encéphale embryonnaire sur le développement du cr^ane chez

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