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doi:10.1111/cga.12072
REVIEW ARTICLE
Signaling regulating inner ear development: Cell fate determination,
patterning, morphogenesis, and defects
Yuji Nakajima
Department of Anatomy and Cell Biology, Graduate School of Medicine, Osaka City University, Osaka, Japan
ABSTRACT The membranous labyrinth of the inner ear
is a highly complex organ that detects sound and balance.
Developmental defects in the inner ear cause congenital hearing
loss and balance disorders. The membranous labyrinth consists
of three semicircular ducts, the utricle, saccule, and
endolymphatic ducts, and the cochlear duct. These complex
structures develop from the simple otic placode, which is estab-
lished in the cranial ectoderm adjacent to the neural crest at
the level of the hindbrain at the early neurula stage. During
development, the otic placode invaginates to form the otic
vesicle, which subsequently gives rise to neurons for the
vestibulocochlear ganglion, the non-sensory and sensory epithe-
lia of the membranous labyrinth that includes three ampullary
crests, two maculae, and the organ of Corti. Combined
paracrine and autocrine signals including fibroblast growth
factor, Wnt, retinoic acid, hedgehog, and bone morphogenetic
protein regulate fate determination, axis formation, and
morphogenesis in the developing inner ear. Juxtacrine signals
mediated by Notch pathways play a role in establishing the
sensory epithelium, which consists of mechanosensory hair cells
and supporting cells. The highly differentiated organ of Corti,
which consists of uniformly oriented inner/outer hair cells and
specific supporting cells, develops during fetal development.
Developmental alterations/arrest causes congenital malforma-
tions in the inner ear in a spatiotemporal-restricted manner. A
clearer understanding of the mechanisms underlying inner ear
development is important not only for the management of
patients with congenital inner ear malformations, but also for
the development of regenerative therapy for impaired function.
Key Words: development, growth factors, inner ear, malforma-
tions, Notch
INTRODUCTION
The inner ear exists in the petrous part of the temporal bone and
consists of a membranous labyrinth and bony labyrinth. The mem-
branous labyrinth consists of at least six mechanosensory epithelia,
which include the organ of Corti in the cochlea for sound, three
ampullary crests in the base of three semicircular ducts for angular
acceleration, and two maculae, macula utriculi and sacculi, for
gravity/linear acceleration. Electrical signals, which are translated
from the mechanical signals evoked in these sensory epithelia, are
transmitted to the brain stem via the vestibulocochlear nerve
Correspondence: Yuji Nakajima, MD, PhD, Department of Anatomy and
Cell Biology, Graduate School of Medicine, Osaka City University, 1-4-3
Asahimachi, Abenoku, Osaka, 545-8585 Japan. Email: yuji@med.osaka-
cu.ac.jp
Received April 5, 2014; revised and accepted June 7, 2014.
Congenital Anomalies 2015; 55, 17–25 17
(cranial nerve VIII). During organogenesis, reciprocal signaling
between the embryonic tissues successively occurs to establish
highly organized functional tissues and organs in a spatiotemporal-
restricted manner. Developmental arrest/alterations as well as
acquired injuries in the inner ear cause functional disorders for
hearing and balance. The aim of this review is to discuss signaling
that regulates the key developmental events responsible for the
establishment of the complicated membranous labyrinth. Several
excellent reviews on inner ear development have been published
(Torres and Giraldez 1998; Abelló and Alsina 2007; Bok et al.
2007a; Driver and Kelley 2009; Groves and Fekete 2012;
Magarinos et al. 2012; Neves et al. 2013).
EMBRYOGENESIS OF THE INNER EAR AND
ITS MALFORMATIONS
The membranous labyrinth and vestibulocochlear neurons are
derived from an ectodermal thickening named the otic placode
(OP), which develops from the cranial ectoderm immediately
lateral to the neural crest at the level of the hindbrain at the neurula
stage. The OP invaginates and is then being pinched off from the
ectoderm, which results in the formation of the otic vesicle
(otocyst). The posterior rim and its anterior domain of the OP give
rise to non-sensory semicircular and cochlear epithelia, respectively
(Abelló et al. 2007). Epithelial cells in the anteromedial domain of
the OP/otic vesicle further differentiate to the proneurosensory epi-
thelium, from which neural progenitors delaminate and give rise to
neuroblasts of the vestibulocochlear ganglion (Satoh and Fekete
2005). After neural cell migration, the resulting prosensory domain
later develops into five vestibular sensory patches and the organ of
Corti (basilar papilla in birds). The dorsal epithelium of the otic
vesicle expands to form the vertical pouch; its opposing epithelia at
the future anterior and posterior semicircular ducts later fuse to be
absorbed resulting in the formation of two superior semicircular
ducts and the common crus (Fig. 1, Martin and Swanson 1993;
Chang et al. 2004). The lateral semicircular duct develops from the
lateral pouch and the endolymphatic duct from the dorsomedial
epithelium of the otic vesicle. The five sensory patches that develop
in the vestibular labyrinth are three cristae in the anterior, posterior,
and lateral ampulla; and two maculae in the utricle and saccule.
These cristae detect head rotation while the maculae maintain static
equilibrium. The organ of Corti, which develops specifically in the
mammalian cochlea, is a highly differentiated sensory epithelium
for sound. The sensory epithelium basically consists of sensory hair
cells and supporting cells, while highly differentiated sensory and
supporting cells develop in the organ of Corti, that is, three lateral
rows of outer hair cells and a medial row of inner hair cells, with
inner phalangeal cells (supporting inner hair cells), outer Deiters’
cells (supporting outer hair cells), and pillar cells (demarcating the
inner and outer hair cells and making the tunnel of Corti).
© 2014 Japanese Teratology Society
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Fig. 1 Inner ear development and corresponding malformations. Modified from Jackler et al. (1987) and Yasuda et al. (2007). an, anterior semicircular duct;
c, cochlear duct; CS, Carnegie stage; SCC, semicircular canal (duct); e, endolymphatic duct; l, lateral SCC; p, posterior SCC; pl, primordium of the
lateral semicircular duct; v, vestibular pouch; W, age in weeks. Note that the superior SCC includes the anterior SCC and the posterior SCC.
Table 1 Classification of inner ear malformations based on
embryogenesis
Complete labyrinth aplasia (Michel)
Cochlear malformations
Aplasia
Common cavity
Hypoplasia
Incomplete partition (Mondini)
Semicircular canal malformations
Aplasia
Small buds
Superior and lateral SCC dysplasia
Lateral SCC aplasia/dysplasia
Enlarged vestibular aqueduct
Note that the superior semicircular canal (SCC) includes the
anterior SCC and the posterior SCC. The most common cochlear
malformation is incomplete partition, and the most frequently diag-
nosed SCC malformation is lateral SCC dysplasia (Jackler et al.
1987; Brenski and Arjmand 2003).
Developmental arrest in the membranous labyrinth leads to
various congenital inner ear defects in temporally and spatially
restricted manners. These inner ear malformations include cochlear,
semicircular canal, aqueduct, and combined malformations. Based
on embryogenesis and radiological findings, cochlear malforma-
tions have been classified as aplasia, common cavity, hypoplasia,
and incomplete partition, while semicircular canal malformations
have been classified as aplasia, small buds, dysplasia of the three
semicircular canals, and dysplasia of the lateral semicircular canal
(Fig. 1, Table 1, Jackler et al. 1987; Sennaroglu and Saatci 2002;
Brenski and Arjmand 2003). Developmental defects/arrest in the
inner ear and its surrounding structures are often diagnosed in
children with sensorineural hearing loss; therefore, an accurate
morphological assessment of the inner ear by high-resolution CT
(computed tomography) is important (Masuda et al. 2013).
© 2014 Japanese Teratology Society
Y. Nakajima
PREPLACODE REGION
The sensory nervous system in the head is derived from both the
neural crest and placodes, while that in the trunk is derived from
the neural crest alone. At the late gastrula to early neurula stage, the
developing ectoderm is subdivided into at least four distinct
regions: the neural plate, neural crest, and epidermal ectoderm from
the head to the trunk; and preplacode region (PPR) in only the head
ectoderm. Therefore, the establishment of PPR may be precisely
regulated by surrounding tissues in a spatiotemporal-restricted
manner. Signals derived from the head and lateral mesoderm
(including the heart-forming mesoderm) are important for defining
and establishing the PPR adjacent to the neural crest at the cranial
region (Fig. 2, Litsiou et al. 2005). Excess bone morphogenetic
protein (BMP) and Wnt signals, which are secreted from the ecto-
derm and neural plate, respectively, act to suppress the expression
of PPR marker genes, such as Six1, Six4, and Eya2, in the PPR-
forming ectoderm. Together with the BMP/Wnt signals, Cerberus
(BMP/Wnt antagonist) and fibroblast growth factor (FGF), which
are secreted from the mesoderm subjacent to the prospective PPR,
upregulate the expression of PPR genes (Fig. 2, Litsiou et al. 2005).
The anterior-posterior patterning of the PPR is regulated by mutual
repression between Gbx2 and Otx2, that is, posteriorly localized
Gbx2 is required for the formation of the OP, while anterior Otx2 is
required for that of the olfactory, lens, and trigeminal placode in the
anterior PPR (Steventon et al. 2012).
Six, Dach, and Eya are expressed in developing organs including
ear and eye (Li et al. 2003; Zou et al. 2004). The Six1 protein acts
as both a repressor and activator in a context-dependent manner.
The Six-Dach complex without Eya acts to repress target genes,
while the Six-Dach-Eya complex, in which Eya exhibits phos-
phatase activity to suppress the co-repressor complex, recruits
the co-activator to activate target genes for the promotion of
organogenesis (Li et al. 2003). Development of the inner ear, nose,
skeletal muscle, thymus, and kidney was shown to be severely
affected in Six1-null mice, (Ozaki et al. 2003). In this mutant, the
vestibular labyrinth is a single space with an expanded
endolymphatic duct, and ventral structures (cochlea and
vestibulocochlear ganglion) are absent (Ozaki et al. 2003). A pre-
vious study reported that the initial cell fate determination and
migration of vestibulocochlear neurons were unaffected in Eya- and
 Inner ear development
Fig. 2 Signals regulating the preplacode region (PPR). In the late gastrula
to early neurula embryo, PPR (pink) is determined in the head
ectoderm (Ect), which is immediately lateral to the neural crest
(NCr, green) and dorsal to the heart/head mesoderm. Excess bone
morphogenetic protein (BMP) and Wnt signals, which are derived
from the ectoderm (yellow) and neural plate (NP, blue), respec-
tively, suppress PPR genes. Fibroblast growth factor (FGF) and
Cerberus (Cer, a BMP/Wnt antagonist), which are secreted from the
heart/head mesoderm, can upregulate the expression of PPR genes.
Six1-null mutant mice; however, early neurogenesis was affected in
the mutant inner ear (Zou et al. 2004). Genes expressed in the
ventral otic vesicle of the Six1-null developing inner ear, including
Otx1, Otx2, Lunatic fringe (Lfng), Fgf3, Fgf10, Bmp4, Gata3, and
Nkx5.1, were found to be suppressed (Ozaki et al. 2003; Zou et al.
2004). In humans, an impaired EYA1-SIX1 regulatory network is
one of the candidates involved in branchio-oto-renal syndrome,
which is associated with the incomplete partition of the cochlear
duct as well as semicircular canal dysplasia (Propst et al. 2005;
Kochhar et al. 2007; Senel et al. 2009).
THE OTIC PLACODE
The otic placode (OP) is induced by combined signaling that is
mediated by the head mesoderm and hindbrain in vertebrates
(Fig. 3). During the inner ear development, mesodermal tissue adja-
cent to the hindbrain expresses FGF, which defines the posterior
PPR (precursor region of OP and epibranchial placode) and induces
the expression of Wnt8a and FGF in the hindbrain. Signaling medi-
ated by anterior hindbrain-derived Wnt8a and FGF was shown to
involve the differentiation of OP (Ladher et al. 2000, 2010; Urness
et al. 2010; Groves and Fekete 2012). An explantation experiment
revealed that endoderm-derived FGF8 acts as an upstream signal of
FGF in the chick mesoderm (Ladher et al. 2005).
The expression of Fgf10 in the mesoderm subjacent to prospective
OP ectoderm of Fgf3-null and hypomorphic Fgf8 mouse embryo was
reduced, which resulted in the failed formation of the OP, suggesting
that FGF8/3 acts as an upstream signal of Fgf10 in the head meso-
derm during the induction of the OP. Taken together with these
findings, pharyngeal endoderm-derived FGF8 plays an upstream
role in the FGF signaling cascade to induce the OP in both the chick
and mouse (Ladher et al. 2005). In addition, hindbrain-derived Wnt
signaling was reported to suppress the expression of the epibranchial
placode gene, Foxi2, which indicated that Wnt signaling also defined
the area of the otic region (Freter et al. 2008) (Fig. 3).
19
Fig. 3 Signals regulating otic placode (OP) formation. Head mesoderm-
secreted FGF, the expression of which is induced by the pharyngeal
endoderm (Pha) FGF8, defines the posterior preplacode region
(PPR, pink) and upregulates the expression of Wnt8a and FGF in
the hindbrain (HB). Anterior hindbrain-derived FGF and Wnt8a
specify the PPR to OP (green). Wnt8a suppresses the epibranchial
placode (Epi, purple) gene, Foxi2, to demarcate the area of the OP.
Fgf3 is expressed in the hindbrain adjacent to the prospective OP
at the level of rhombomere 4–6, as well as in the OP itself, at the
onset of OP formation in the mouse. Fgf10 was shown to be
expressed in the head mesenchyme underlying the prospective
OP-forming ectoderm (Wright and Mansour 2003; Alvarez et al.
2003). Gain-of-function experiments demonstrated that the ectopic
expression of Fgf10 in the hindbrain was capable of inducing an
OP-like structure, whereas Fgf3 only exhibited faint inducible
activity (Alvarez et al. 2003). The Fgf10- and Fgf3-null mutants
displayed only mild defects during formation of the otic vesicle;
however, double knockout mutant had a hypoplastic otic vesicle, in
which the expression of some otic marker genes, such as Pax2,
Dlx5, and Sox9, was absent (Wright and Mansour 2003; Alvarez
et al. 2003). These findings indicated the redundant role of FGFs
and also that head mesoderm-derived FGF10 acts as one of the
inductive signals for OP formation in the mouse. Human LAMM
(labyrinth aplasia, microtia, and microdontia) syndrome, which is
caused by homoallelic mutations in FGF3, presents as a series of
inner ear malformations including complete labyrinth aplasia as
well as common cavity and incomplete partitions (Ramsebner et al.
2010).
ANTERIOR-POSTERIOR PATTERNING
At the early otic placode/vesicle stage, cells possessing both neu-
rogenic and sensory lineages in the anteromedial domain express
Fgf10 and Sox3 (Alsina et al. 2004; Abelló et al. 2007; 2010). Later
these cells express Lim homeodomain transcription factor, Islet1
and homeobox transcription factor, Prox1 (Stone et al. 2003;
Radde-Gallwitz et al. 2004). Neurogenin1 and NeuroD are later
upregulated in neurogenic cells in this domain. The sensory marker
gene Lfng was shown to be co-expressed in this neurogenic epithe-
lium (Cole et al. 2000). Lineage tracing experiments with fluores-
cent dye in chick otic cup (16–18 somite stage) clarified that the
anterior domain of the otic cup, posterior rim of the otic cup, and
posteromedial ventral region of the otic cup gave rise to
neurosensory domain, non-sensory epithelium of the semicircular
ducts, and non-sensory epithelium of the cochlear duct, respectively
(Abelló et al. 2007). A more detailed lineage analysis revealed
that the prosensory region was located more peripheral to the
© 2014 Japanese Teratology Society
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Fig. 4 Anterior-posterior (AP) axis formation in the otic placode. At the
early otic placode/cup stage, the retinoic acid (RA)-synthesizing
enzyme, Raldh2 (retinaldehyde dehydrogenese2), is expressed
in the mesoderm posterior to the otic placode (OP), while
RA-catabolizing enzyme (Cyp26) is expressed in the ectoderm,
which is anterior to the OP. RA inhibits expression of the anterior
genes of the OP, which suggests that the posterior-to-anterior gra-
dient of RA regulates the AP axis of the OP. FGF8, which is
expressed in the ectoderm anterior to the placode, upregulates the
expression of anterior otic genes. Note that a dorsal view of the
cranial half of the neurula embryo is shown.
neurogenic region, but also that some neurons and sensory cells
developed from common progenitors (Satoh and Fekete 2005).
At the early otic cup stage in the chick (11–15 somite stage),
experiments demonstrated that AP (anterior–posterior) inversion of
the otic cup (epithelium) alone did not affect the expression of
anterior placode genes, while inversion with the associated preotic
ectoderm altered the expression of anterior marker genes in the
posterior otic cup, which suggested that signaling from tissue sur-
rounding the otic cup defined the AP polarity of the otic cup (Bok
et al. 2011). Retinoic acid (RA) is known to regulate the AP axis
in the embryonic body and organs. At the early OP/cup stage in
the chick embryo, the RA-synthesizing enzyme, retinaldehyde
dehydrogenese2 (Raldh2) is expressed in the mesoderm just poste-
rior to the otic region, while the P450-associated RA-catabolizing
enzyme, Cyp26, is expressed in the ectoderm anterior to the OP,
suggesting an posterior-to-anterior gradient of RA in the developing
placode. Bead implantation experiments in chick embryos revealed
that the posterior-to-anterior gradient of RA activity defined, at least
partly, AP polarity in the inner ear (Fig. 4; Bok et al. 2011).
Pax2 and Sox3 were found to be expressed in the otic and
epibranchial ectoderm at the onset of otic development at the 5
somite stage. The expression of Sox3 subsequently becomes
restricted to the anteromedial (neurosensory) domain of the otic
region at the 10 somite stage by FGF8, which is expressed in the
ectoderm anterior to the otic ectoderm (Abelló et al. 2010). Sox3
can induce anterior neurogenic markers, such as Sox2 and Delta1,
and was shown to repress the non-neurogenic posterior gene, Lmx1
(Abelló et al. 2010). Therefore, in addition to RA, FGF8 plays a
role in the establishment of AP polarity in the OP (Fig. 4). Once the
neurosensory region is determined in the anterior domain, several
Notch-related genes are expressed in a region-specific manner; that
is, Lfng (a Notch modulator), Delta1 (ligand), and Hes5 (repressor)
in the anterior region of the placode, and Serrate1 (ligand) and
Hairy1 (Hey1, repressor) in the posterior domain. Notch1 (receptor)
is expressed in the entire otic region.
Various types of cochlear and vestibular malformations as well as
a defective cartilaginous otic capsule were induced in mouse
embryos exposed to maternally administered excess levels of RA
© 2014 Japanese Teratology Society
Y. Nakajima
(Frenz et al. 1996). The most prominent abnormality in this model
was the absence/hypoplasia of the vestibulocochlear ganglion, in
which neural cells are developed from the anterior domain of the
otic vesicle, which suggested that excess levels of RA may alter
anterior positional information to posterior positional information,
resulting in a reduction in neuronal differentiation.
DORSAL-VENTRAL PATTERNING
After development of the otic vesicle is completed, establishing the
dorsal-ventral (DV) axis is necessary to further generate the
complex inner ear morphology consisting of the dorsal vestibular
labyrinth and ventral cochlea. The early otic vesicle is located
closely to the hindbrain (rhombomere 4–6) as well as the midline
structure, the notochord. The ablation of the floor plate in the
hindbrain and notochord, from which Shh (Sonic hedgehog) is
secreted, caused a defective cochlea in chick embryos, and inhibit-
ing Shh activity led to the same result (Bok et al. 2005). The
experimental inversion of the DV axis in the hindbrain with the
notochord resulted in an inverted DV axis in the otic vesicle, in
which the expression of ventral marker genes including NeuroD,
Lfng, and Six1 is shifted dorsally (Bok et al. 2005). These results
indicated that Shh from the floor plate and notochord plays a critical
role in establishing the DV axis in the inner ear.
The ventral structures of the inner ear, such as the cochlea and
vestibulocochlear ganglion, are absent in Shh-null mutant mice.
Ventral markers including Otx1/2 and Pax2 were reported to be
downregulated in this mutant, while the dorsal marker gene Dlx5
was upregulated, which suggested that the Shh signal may be
required to specify the ventral structure of the otic vesicle
(Riccomagno et al. 2002). The neurogenic markers, Neurogenin1
and NeuroD, were also downregulated; therefore, the
vestibulocochlear ganglion was absent in the mutant inner ear.
However, expression of the sensory markers, Bmp4, Lfng, and Fgf3
in the crests and maculae remained unaltered, which suggested that
Shh is required for neurogenic specification, but not for sensory
lineage during inner ear development (Riccomagno et al. 2002).
Gli3 is one of the transcriptional mediators for the Shh signal and
is thought to act not only as a transcriptional activator, but also as a
transcriptional repressor that is dependent on Shh protein levels
(Jacob and Briscoe 2003). Shh-dependent Gli3 functions are
required for pattern formations during development, such as the DV
axis in the spinal cord and neocortex in the brain (Komada 2012).
Using several combinations of mutant alleles for Shh, Gli2, and
Gli3, Bok et al. (2007b) reported that in addition to the Gli2/Gli3-
dependent activator signal, Gli3-mediated repression was required
for the morphology of the semicircular ducts (Fig. 5). In addition,
formation of the distal-most cochlear duct at the ventral region
requires a strong Shh activator signal, while the proximal cochlear
and saccule requires a weaker Shh signal controlled by Gli2-
mediated activator and Gli3-mediated repressor signals (Bok et al.
2007b).
ONSET OF NEURAL AND SENSORY CELL
DEVELOPMENT
Neurosensory progenitors develop in the anterior domain of the otic
cup at the onset of neural development in the inner ear. They express
the HMG-box transcription factor Sox2, which is thought to main-
tain the multipotency of tissue-specific stem cells including neurons
(Takahashi and Yamanaka 2006). A previous study demonstrated
that the expression of Sox2 is regulated by a signaling cascade
 Inner ear development
Fig. 5 Dorsal-ventral (DV) patterning of the inner ear. The otic vesicle,
which is located adjacent to the hindbrain (HB), gives rise to the
ventral cochlea duct (CD) and dorsal semicircular ducts. At this
time, the floor plate (FP) and notochord (NCh) secrete Shh (Sonic
hedgehog) to establish the DV axis of the inner ear and neural tube.
The Shh signal is converted to Gli2/3 activator and Gli3 repressor
activities in a Shh concentration-dependent manner. ES,
endolymphatic duct; LSCD, lateral semicircular duct; SSCD, supe-
rior semicircular duct; S, saccule; U, utricle
including FGF, BMP, Wnt, and Tbx6 (Takemoto 2013). At the onset
of neural development in the OP, prospective neuroblasts are speci-
fied in the neurosensory epithelium and then delaminate to generate
neurons for the vestibulocochlear ganglion, from which bipolar
neurons will connect hair cells to the brain via the vestibulocochlear
nerve. Delaminated neurons lose their expression of Sox2 as well as
proliferative activity. At the onset of neural determination, prospec-
tive neuroblasts express Neurogenin1, the expression of which is
positively regulated by Sox2. Neurogenin1 has been reported to
repress the expression of Sox2 by a negative feedback loop (Evsen
et al. 2013). Therefore, negative feedback inhibition to Sox2
by Neurogenin1 (also by NeuroD) is required for progressive
neurogenesis (Fig. 6). In cultured otic vesicles, FGF10 was found to
reduce the proliferation of neurosensory progenitors, and, conse-
quently, the expansion of NeuroD-expressing neural cells. On the
other hand, SU5402 (FGF receptor inhibitor) stimulates the prolif-
eration of neurosensory progenitors, thereby reducing the number
of mature neurons. Therefore, the FGF signal acts on the progenitor
cells, resulting in their exit from the cell cycle and commitment to
a neural fate (Alsina et al. 2004). Neurogenin1 and/or Sox3 also
upregulate the expression of Delta1 (Notch ligand) in neurogenic
cells. This then activates Notch signaling in neighboring cells,
which inhibits Neurogenin1 by Hes-mediated lateral inhibition,
which suppresses neurogenesis and the maintenance of Sox2
expression, resulting in the preservation of undifferentiated sensory
progenitors (Jeon et al. 2011, Fig. 6). Once the prosensory domains
are established, Jagged1 (another Notch ligand) acts to maintain the
expression of Sox2 probably by Hesr-mediated lateral induction
(Hayashi et al. 2008a; Neves et al. 2011, Fig. 6). Prosensory pro-
genitors later differentiate to Sox2-negative hair cells and Sox2-
positive supporting cells (Neves et al. 2007). Nascent hair cells
express Delta1 during this process to accelerate hair cell fate in a
cell-autonomous manner, while Delta1 activates Notch signaling in
the neighboring cells to suppress Atoh1 via Hes1/5 to maintain
supporting cells in a non-cell-autonomous manner (Chrysostomou
et al. 2012). Sox2 has been shown to exhibit both positive and
negative regulatory functions for Atoh1 in a stage-dependent
21
Fig. 6 Neurosensory development. In inner ear development, neural cells
and sensory cells develop from the proneurosensory domain, which
is located in the anteromedial region of the otic vesicle and
expresses Sox2. At the onset of neural development, Sox2 initiates
the expression of Neurogenin1 (Ngn1), which then suppresses Sox2
and upregulates the expression of NeuroD. Proneural cells also
express the Notch ligand, Delta1 (Dl1), which activates Notch
signaling in the neighboring cells to inhibit a neural fate, thereby
maintaining Sox2-positive prosensory cells by lateral inhibition.
Once the prosensory cells are determined, the Notch ligand,
Jagged1 (Jag1), acts to maintain these prosensory cells probably by
lateral induction. In some prosensory cells, Sox2 induces Atoh1 for
hair cell differentiation. Prospective hair cells express Dl1, which
binds to the Notch receptors of neighboring cells to maintain Sox2-
positive supporting cells by lateral inhibition.
fashion (Neves et al. 2012). Supporting cells still express Sox2 and
proliferating characteristics, which suggests that Sox2 may be
involved in the maintenance of stem cell characteristics as well as
regenerative capacity.
SEMICIRCULAR DUCTS
Three-dimensional morphogenesis has been examined in the inner
ear by a paint-fill method (Bissonnette and Fekete 1996; Morsli
et al. 1998; Mansour and Schoenwolf 2005). After completion of
the pear-shaped otic vesicle (otocyst), two distinct bulges are
formed, the pars superior (dorsal bulge) and pars inferior (ventral
bulge), from which the semicircular ducts/utricle and cochlear duct/
saccule will later develop, respectively. The endolymphatic duct
simultaneously emerges from the dorsal-medial surface of the otic
vesicle. Two distinct pouches, the dorsal pouch and lateral pouch,
subsequently develop from the pars superior. The dorsal pouch
gives rise to two superior (vertical) ducts, the anterior and posterior
semicircular ducts, while the lateral pouch gives rise to the lateral
(horizontal) semicircular duct. At the onset of semicircular duct
formation, the opposing epithelia in the center of each pouch fuse to
form the epithelial fusion plate, which is thereafter resorbed by
epithelial-mesenchymal transition and apoptosis, resulting in the
formation of tube-like semicircular ducts (Kobayashi et al. 2008).
© 2014 Japanese Teratology Society
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Fig. 7 Semicircular duct development. Wnt/β-catenin signaling stimulates
the expression of BMP4/FGF10 in the cristae (yellow domain),
which then induce the expression of Bmp2 in the prospective semi-
circular duct (SCD) epithelium. BMP2 induces the expression of
Dlx5 in the perimeter rim of the pouch (future SCD) to inhibit
Netrin1 expression, thereby maintaining the epithelial structure of
the SCD. In the fusion plate of the pouch, Netrin1 is maintained to
expand the surrounding mesenchyme, which then pushes the facing
epithelia to fuse (fusion plate), resulting in the absorption of the
fusion plate.
Each semicircular duct connects with the utricle, and the two supe-
rior (anterior and posterior) ducts possess a common duct, the crus
membranous commune. The anterior and posterior ends of the
vertical semicircular ducts (opposite sides of the common crus) and
the anterior end of the lateral duct have their own ampulla connect-
ing to the utricle. A mutant mouse with normal semicircular ducts
and no sensory crista has not been yet generated, which suggests
that the crista is involved in the formation of the semicircular ducts
(Chang et al. 2004). In the developing crista (Fig. 7), Wnt/β-catenin
signaling stimulates the expression of BMP4 and FGF10, which
then induces the expression of Bmp2 in the prospective semicircular
duct epithelium adjacent to the cristae (Chang et al. 1999, 2002,
2004; Gerlach et al. 2000). BMP2 has been shown to induce the
expression of Dlx5 in the perimeter rim of the canal pouch to inhibit
the expression of Netrin1, thereby maintaining the epithelial struc-
ture of the semicircular ducts (Chang et al. 2004, 2008; Rakowiecki
and Epstein 2013). The expression of Netrin1 is maintained in the
fusion plate of the canal pouch in order to expand the surrounding
mesenchyme, which pushes the center of the epithelial pouch to
generate the fusion plate. The fusion plate will then dissociate via
apoptosis and epithelia-mesenchymal transition, leaving tube-like
semicircular ducts (Salminen et al. 2000; Kobayashi et al. 2008).
COCHLEA
At the onset of cochlear formation, cells in the medioanterior region
of the otic vesicle proliferate and the cochlear bud elongates to form
the cochlear duct, in which the specific sensory epithelium, the
organ of Corti, develops. Once the fate of hair cells is determined,
future hair cells exit the cell cycle; therefore, elongation/growth of
the cochlear duct is thought to occur via convergent extension
movements of cochlear cells, which is regulated by Wnt-planar cell
© 2014 Japanese Teratology Society
Y. Nakajima
Fig. 8 Epithelial differentiation of the organ of Corti. At early mammalian
cochlear development, the medial cochlea epithelium is subdivided
into three distinct regions from the medial (neural) to the lateral
(abneural) site, i.e., Kölliker’s organ (neural side of the cochlear
duct), the prosensory auditory domain, and the outer sulcus. Bmp4
is expressed in the outer epithelial region, and, thus, the establish-
ment of a BMP signaling gradient, which is required to form three
distinct epithelial regions. FGF20, the expression of which is
directly regulated by Notch signals, is required for differentiation of
the auditory sensory epithelium, which consists of hair cells and
supporting cells. Inner hair cell-secreted FGF8 locally controls the
differentiation of pillar cells from FGFR3-positive supporting cells.
The Wnt-PCP pathway regulates not only the planar cell polarity of
the hair cells, but also extension of the cochlear duct.
polarity (PCP) pathways including Rho-associated coiled-coil
kinase (ROCK) and non-muscle myosin II activity (Yamamoto
et al. 2009; Fritzsch et al. 2011). The cochlear duct did not grow in
mutant mice in which Shh pathways related to fate determination/
cell proliferation were affected, which resulted in a common cavity
or hypoplastic cochlea with or without lateral semicircular duct
malformation (Riccomagno et al. 2002; Bok et al. 2007b). A short-
ened cochlea with a larger number of hair cell rows and other inner
ear malformations occur in the Foxg1 mutant, in which embryonic
morphogenesis including cell fate determination and proliferation is
affected (Pauley et al. 2006). Mutant mice, in which the Wnt-PCP
pathways are affected, were found to have a shortened cochlea with
a larger number of disorganized hair cell rows (Yamamoto et al.
2009). A shortened cochlea reflects the incomplete partition of
cochlea (Mondini) in human inner ear malformations (Table 1).
Mice in which the Wnt-PCP pathways were deleted also displayed
other congenital defects, such as conotruncal heart defects, skeletal
defects, and social abnormalities (Etheridge et al. 2008). Cochlear
elongation and hair cell development are coordinated in a context-
dependent manner because hair cell development was shown to be
affected in the Aho1 conditional mutant while almost normal
growth was observed in the cochlear duct (Pan et al. 2011). A
shortened cochlea with a smaller number of turns (Mondini) repre-
sents a common inner ear malformation diagnosed in children with
congenital sensorineural hearing loss, and syndromes associated
with this type of defect include trisomy 21, DiGeorge, CHARGE,
Pendred, congenital cytomegalovirus infection, and congenital
rubella (Brenski and Arjmand 2003).
During early mammalian cochlear development, the medial
thickened cochlea epithelium is subdivided into three distinct
regions from the medial (neural) to lateral (abneural) site along the
short axis of the cochlear duct, that is, Kölliker’s organ (neural side
of the cochlear duct), the prosensory auditory domain, and the outer
sulcus. At this time, Bmp4 is expressed in the outer sulcus region;
thus, a BMP signal gradient is established in the developing sensory
domain and is high at the lateral site and low at the neural site
(Fig. 8, Ohyama et al. 2010). The prosensory domain and outer
sulcus region did not form in the Alk3-CKO;Alk6+/− mutant, in
 Inner ear development
which BMP signals were conditionally deleted in the developing
inner ear. In the cultured cochlea, a high dose of the BMP4 protein
was found to upregulate the expression of outer sulcus markers and
downregulate the Kölliker’s organ’s genes, while an intermediate
dose induced a large number of prosensory cells (Ohyama et al.
2010). These findings suggested that BMP signaling with a lateral-
to-medial gradient is required for the patterning of the cochlear
sensory epithelium along the short axis.
Previous studies reported defects in hair cells and supporting
cells in the developing cochlear duct of mutant mice with the
tissue-specific deletion of Fgfr1 or Jagged1-Notch signaling
(Kiernan et al. 2001; Pirvola et al. 2002). The expression of Fgf20
and its receptor Fgfr1 precedes the differentiation of hair cells and
supporting cells in the developing cochlea duct. In a cultured
murine developing inner ear, SU5402 (FGF receptor inhibitor) or
the anti-FGF20 neutralizing antibody were found to effectively
inhibit the formation of the sensory epithelium, including both hair
cells and supporting cells, when added to the medium before/during
the formation of the sensory epithelium. These findings suggested
that signaling mediated by FGF20/FGFR1 may play a role in gen-
erating the sensory domain of the developing cochlear duct (Fig. 8,
Hayashi et al. 2008b). Recent experiments showed that the FGFR1-
mediated MAP kinase activation through FGFR substrate (Frs) 2/3
is necessary for the maintenance of Sox2-positive sensory progeni-
tors and their subsequent commitment to the sensory cell differen-
tiation (Ono et al. 2014). The Notch ligand Jagged1 is expressed
earlier than that of Fgf20 in the developing inner ear. The expres-
sion of Fgf20 was absent in Jagged1-conditional mutant mice, and
the Fgf20 promoter region possessed an Rbpj-binding domain,
which indicated that the expression of Fgf20 is directly regulated by
Notch signaling (Munnamalai et al. 2012). Not only the expression
of Fgf20, but also the generation of hair cells and supporting cells
was inhibited in a cultured cochlea duct treated with DAPT (Notch
inhibitor) before the onset of hair cell specification, and this inhibi-
tory effect was reversed by adding the FGF20 protein to the
medium (Munnamalai et al. 2012). These findings suggested that
FGF20 may be involved in the specification and/or maintenance of
the prosensory domain under the control of Notch signaling path-
ways The sensitivity of the prosensory epithelium to DAPT is
known to be stage-dependent; treating the early cochlea (ED12.5 in
mouse embryo) with DAPT led to a reduction in the number of hair
cells and supporting cells because of the blockade of lateral induc-
tion, whereas, conversely, the treatment with DAPT at a later stage
(ED13.5) increased the number of hair cells because of the block-
ade of lateral inhibition. At the earlier stage, Notch signaling-
dependent Sox2 maintains the prosensory region; therefore,
Notch-induced FGF20 may amplify/maintain Sox2 to specify the
prosensory domain, from which hair cells and supporting cells will
develop (Dabdoub et al. 2008; Munnamalai et al. 2012). In the
organ of Corti, hair cell differentiation progresses in a medial-to-
lateral direction by an, as yet, unidentified mechanism. Fgf20-null
mutant mice lack not only outer hair cells, but also differentiated
supporting cells, such as Deiters’ cells (Huh et al. 2012). Therefore,
a fine-tuned balance between FGF and BMP signaling may be a
prerequisite for establishing the proper number of hair cell rows.
Phospho-Erk, a downstream signal of FGF, was shown to phospho-
rylate the linker region of Smad1 to disrupt BMP-mediated Smad1
signaling (Pera et al. 2003). After the formation of hair cells in the
cochlea, inner hair cells expressed Fgf8 and outer hair cells and
supporting cells expressed Fgfr3 (Jacques et al. 2007). Mutant mice
without FGF8/FGFR3 signaling have defective pillar cells and
deafness, which indicated that inner hair cell-derived FGF8 locally
controls the differentiation of supporting cells into pillar cells,
23
which are unique to the mammalian cochlea (Fig. 8, Colvin et al.
1996; Puligilla et al. 2007). Low-frequency sensorineural hearing
loss has been reported in a mouse model of human Muenke syn-
drome carrying Fgfr3P244R, and the organ of Corti in this mutant has
not only extra pillar cells and fewer Deiters’ cells, but also extra hair
cells in the apical cochlear duct (Mansour et al. 2009). A genetic
reduction in the expression of FGF10, which normally activates
FGFR2b or FGFR1b, can reverse the cochlear disorders associated
with this model (Mansour et al. 2013). In the chick developing
basilar papilla (homolog of the organ of Corti), FGF signaling
mediated by FGFR3 was shown to be required for maintaining
supporting cells, which are known to act as a stem cell pool for the
regeneration of injured hair cells (Jacques et al. 2012). A previous
study demonstrated that mammalian cochlear supporting cells were
capable of proliferating and differentiating into hair cells when
cultured. This finding suggests that the regenerative mechanisms
observed in adult avians may be conserved in the developing mam-
malian inner ear (White et al. 2006).
After the specification of hair cell development in the organ of
Corti, hair cells begin to be oriented as a set of three rows of outer
hair cells and a single row of inner hair cells. The hair cells then
generate a V-shaped bundle of stereocilia on their apical surface, on
which the vertex points to the lateral site. Cells specified to the
organ of Corti no longer proliferate; thus, a defined number of
postmitotic cells undergo convergent extension movement to elon-
gate the cochlear duct in a manner basal to apical direction. The
planar cell polarity of hair cells as well as polarized cochlear exten-
sion are known to be regulated by the Wnt-PCP pathway (Fig. 8,
Curtin et al. 2003; Montcouquiol et al. 2003; Wang et al. 2005). In
the V-shaped hair cell bundle, which possesses uniform medio-
lateral polarity on the apical surface of hair cells, a polarity/mitotic
spindle-associated protein complex consisting of mInsc (mamma-
lian Inscuteable), LGN (vertebrate partner of Insc [Pins]), and Gαi
(heteromeric G protein) is localized in a lateral microvilli-free
region of the apical surface of each hair cell. Loss-of-function
experiments on the mInsc/LGN/Gαi complex revealed irregular
bundles of stereocilia, which suggested that the mInsc/LGN/Gαi
complex plays a role in the establishment of uniform bundles of
stereocilia on the hair cell surface (Tarchini et al. 2013). Therefore,
at least two pathways involving planer cell polarity are required for
the establishment of a mature sensory epithelium in the organ of
Corti.
PERSPECTIVES
One of the most intriguing issues related to inner ear developmental
biology is a clearer understanding of the mechanisms regulating the
regeneration of hair cells. Adult mammalian hair cells do not regen-
erate after injury; therefore, hair cell injury after birth causes irre-
versible deafness. The loss of regenerative activity in the adult
sensory epithelium has been attributed to an age-dependent reduc-
tion in the number of multi-potent progenitor/stem cells due to a
loss of multipotency and proliferative activity. Therefore, elucidat-
ing the genetic and epigenetic mechanisms underlying such
quiescence in the adult sensory epithelium may facilitate the devel-
opment of strategies for inner ear regeneration. At the onset of
sensory epithelium development, the prosensory domain exits the
cell cycle to commit to a lineage for mechanosensory hair cells.
However, the cochlear epithelium in the neonatal rodent possesses
potent regenerative activity, which gradually decreases in an age-
dependent manner. In the adult chick inner ear, the sensory epithe-
lium maintains regenerative activity throughout life; therefore, the
© 2014 Japanese Teratology Society
24
sensory epithelium in adult birds can proliferate and differentiate to
hair cells after injury. Further investigations are necessary to under-
stand the structural, cellular, and molecular mechanisms responsi-
ble for the regenerative activity observed in the sensory epithelium
in neonatal mammals and avians.
ACKNOWLEDGMENTS
The author thanks S Uoya for technical assistance. This work was
supported by a JSPS Grant-in-Aid for Scientific Research (C)
25460273.
DISCLOSURE
None.
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