Early Development of the Vertebrate

Inner Ear
MARTA MAGARIN

OS,
1,2,3
JULIO CONTRERAS,
1,2,4
MARI

A R. ABURTO,
1,2
AND ISABEL VARELA-NIETO
1,2,5
*
1
Instituto de Investigaciones Biomedicas Alberto Sols, CSIC-UAM, Madrid, Spain
2
Unit 761, Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER),
Instituto de Salud Carlos III, Madrid, Spain
3
Departamento de Biolog a, Universidad Autonoma de Madrid, Spain
4
Facultad de Veterinaria, Universidad Complutense de Madrid, Spain
5
Instituto de Investigacion Sanitaria Hospital, Universitario La Paz, IdiPAZ, Madrid, Spain
ABSTRACT
This is a review of the biological processes and the main signaling
pathways required to generate the different otic cell types, with particu-
lar emphasis on the actions of insulin-like growth factor I. The sensory
organs responsible of hearing and balance have a common embryonic ori-
gin in the otic placode. Lineages of neural, sensory, and support cells are
generated from common otic neuroepithelial progenitors. The sequential
generation of the cell types that will form the adult inner ear requires
the coordination of cell proliferation with cell differentiation programs,
the strict regulation of cell survival, and the metabolic homeostasis of otic
precursors. A network of intracellular signals operates to coordinate the
transcriptional response to the extracellular input. Understanding the
molecular clues that direct otic development is fundamental for the
design of novel treatments for the protection and repair of hearing loss
and balance disorders. Anat Rec, 295:17751790, 2012. VC
2012 Wiley
Periodicals, Inc.
Key words: apoptosis; autophagy; deafness; IGF-I; miRNA;
notch signaling; otic precursors; SOX2
AN INTRODUCTION TO THE ANATOMY OF
THE ADULT INNER EAR
The inner ear is a structurally complex sensory sys-
tem formed by the organs responsible for hearing and
balance. The inner ear is located inside the temporal
bone and it is formed by soft canals and cavities, named
the membranous labyrinth, filled by endolymph and
encased by the bony labyrinth. Between the membra-
nous labyrinth and the bony labyrinth there is a space
filled by a different fluid the perilymph.
In mammals, a coiled structure, the cochlea, is respon-
sible for hearing and contains the organ of Corti, the
sensory receptor, where the mechanosensory hair cells
transduce the sound stimuli and generate the electro-
chemical signal response that otic neurons will transmit
to the brain (Raphael and Altschuler, 2003; Fig. 1). The
cochlea is a complex and integrated system, the damage
of a specific cell type can lead to the damage of other
cochlear elements and to hearing loss. Genetic and envi-
ronmental factors can damage cochlear cells causing
deafness (Eisen and Ryugo, 2007; Liu and Yan, 2007;
Raviv et al., 2010; Fetoni et al., 2011). The cochlea has
three major functional parts: the lateral wall, the organ
of Corti, and the spiral ganglion (Fig. 1). The lateral
wall, with the spiral ligament and the stria vascularis,
is essential to the normal physiology of hearing (Jin
et al., 2007). The stria vascularis is responsible for endo-
lymph production (Takeuchi et al., 2000), a specialized
extracellular fluid with intracellular characteristics like
Grant sponsor: MICINN, Spain; Grant number: SAF2008 and
SAF2011; Grant sponsor: CSIC I3P
*Correspondence to: Prof. Isabel Varela-Nieto, Instituto de
Investigaciones Biomedicas Alberto Sols, Consejo Superior de
Investigaciones Cient ficas (CSIC), Universidad Autonoma de
Madrid (UAM), Arturo Duperier 4, 28029 Madrid, Spain. Fax:
34 915854401. E-mail: ivarela@iib.uam.es
Received 24 July 2012; Accepted 24 July 2012.
DOI 10.1002/ar.22575
Published online 8 October 2012 in Wiley Online Library
(wileyonlinelibrary.com).
THE ANATOMICAL RECORD 295:17751790 (2012)
V VC
2012 WILEY PERIODICALS, INC.
high K

and low Na

and Ca
2
concentrations (Wange-
mann, 2006; Jin et al., 2008; Patuzzi, 2011). The
melanocytes or intermediate cells of the stria vascularis
play an important role to preserve the cochlear function
in stress situations (Murillo-Cuesta et al., 2010), and
their alteration causes deafness (Cohen-Salmon et al.,
2007).
The organ of Corti is located within the cochlear duct
or scala media laying on the basilar membrane along its
entire length from the base to the apex of the cochlea.
This organ consists of two types of functional hair cells
and support cells (Kimura, 1975; Santi and Tsuprun,
2001). The hair cells are the receptors that transduce
the mechanical stimulus to an electrochemical signal by
means of stereocilia arranged in a V or W-shaped bun-
dle (Hudspeth, 2008). There are two types organized in
parallel rows, one row of inner hair cells (IHC) and three
rows of outer hair cells (OHC) separated by the Tunnel
Figure 1.
1776 MAGARIN

OS ET AL.
of Corti. The stereocilia of OHC are embedded in the
Tectorial Membrane, an extracellular component that
covers the organ of Corti throughout the cochlea. Sup-
port cells, Deiters cells, Hensens cells, Claudiuss cells,
participate in regulating the ionic and nutrients homeo-
stasis (Forge and Wright, 2002; Chang et al., 2008).
Alterations in their functions are a frequent cause of
hearing impairment (Lefebvre and Van de Water, 2000).
The spiral ganglion is located within the cochlear
modiolus and it is formed by the cell bodies of bipolar
neurons that connect the hair cells of the organ of Corti
with the brain (Nayagam et al., 2011). The dendritic
ends of spiral neurons innervate the hair cells, type I
neurons are the most abundant subtype (95%) and in-
nervate the IHC. Type II neurons innervate several
OHC. The axons of spiral neurons leave the spiral gan-
glion and pass through the basis of the modiolus to form
the cochlear division of the cochleo-vestibular nerve
towards the cochlear nuclei at the brainstem. Sound infor-
mation progresses in a complex multisynaptic, parallel,
and ascendant pathway from the cochlea through the
brainstem nuclei to the auditory cortex (Webster et al.,
1992; Fig. 2). The cochlear nuclei, olivar complex, nucleus
of lateral lemniscus, inferior colliculus, and medial genic-
ulate complex are part of the rely nuclei that transmit the
information to the cortex, where the auditory information
is processed in multiple areas. The tonotopic organization
present at the cochlea is maintained along the pathway.
In addition, neurons from the brainstem (superior olivary
complex) also contact hair cells in a centrifugal control
mechanism of the auditory pathway.
The vestibular system contains the balance receptors
with highly specialized mechanoreceptor hair cells (Gold-
berg, 1991; Fig. 1). Three sensory organs called cristae
located at the base of the semicircular canals are respon-
sible of balance perception, whereas the two maculae of
the sacculus and utriculus detect linear and angular
acceleration (Highstein and Fay, 2004).
To sum up, the mammalian inner ear contains six sen-
sory patches: the coiled cochlear duct or organ of Corti,
which is the auditory receptor, the maculae, and the three
cristae corresponding to the three semicircular canals are
responsible of balance perception. Sensory patches are con-
nected to the brain nuclei by the fibers of the spiral and
vestibular ganglions that form the eighth cranial nerve.
DEVELOPMENT OF THE VERTEBRATE
INNER EAR
The sensory organs of the inner ear have a common
embryonic origin at the otic placode. The cells that consti-
tute the adult inner ear originate from the embryonic otic
placode with the exceptions of the melanocytes of the stria
vascularis and the ganglionar Schwann cells that are of
neural crest origin (DAmico-Martel and Noden, 1983;
Rubel and Fritzsch, 2002; Fritzsch et al., 2011). Cranial
placodes are regions of the ectoderm that generate a wide
variety of cell types, including elements of the sense
organs and most of the sensory neurons of the cranial
nervous system. The different placodes derive from a com-
mon preplacodal region, which surrounds the neural plate
(Streit, 2007). Before the otic placode becomes visible, the
ectodermal cells that will form it undergo a genetic pro-
gram to express preplacodal transcription factors as the
Dlx family, Sox9a and Foxi1 (Ekker et al., 1992; Groves
and Bronner-Fraser, 2000; Solomon and Fritz, 2002; Liu
et al., 2003). There is also induction from the underlying
mesenchyme that produces growth factors of the fibro-
blast growth factor family (FGF10, FGF19, or FGF15
depending on the species) and from the hindbrain, which
secretes FGF3 (Maroon et al., 2002; Leger and Brand,
2002; Wright and Mansour, 2003).
After induction, the otic placode invaginates beneath
the surface ectoderm to form the otic cup, which pinches
off in birds and mice, or cavitates in fish to produce the
otic vesicle or otocyst (Fig. 3; Haddon and Lewis, 1996;
Fig. 1. Anatomy of the inner ear. A: Schematic view of the inner ear
showing the cochlear and vestibular parts and the sensory areas,
crista, macula, and organ of Corti (shadowed). B: Whole mount prepa-
ration of the cochlea showing the basal turn, the apex, the round win-
dow (RW), and the oval window (OW). Note the pigmented stria
vascularis through the lateral wall (melanin granules at intermediate
cell layers). Scale bar 0.5 mm. C: Low magnification of a midmodiolar
section from a mouse cochlea. Note the scala vestibule (SV) limiting
by the Reissners membrane (RS) with the scala media (SM) where the
auditory receptor is located (red boxes), the scala tympani (ST) with
the basilar membrane (BM), and the spiral ganglion (SG, in the osse-
ous Rosenthals canal). The lateral wall (LW) is directly in contact with
the osseous otic capsule. Scale bar 0.5 mm. D: The organ of Corti
containing the neurosensorial cells (inner hair cell, IHC; outer hair cells,
OHC), the nonsensorial cells (DC: Deiter cells; HC: Hensen cells; CC:
Claudius cells; PC: pillar cells), the tectorial membrane (TM), the spiral
limbus (SL), the basilar membrane (BM), the tunnel of Corti (asterisk),
and the myelinated cochlear nerves fibers (CNF). Scale bar 50 lm (E
and F) Synaptophysin (Syn, in E) labeling of presynapses in the IHC
and efferent fibers arriving at the IHC and OHC, Neurofilament 200 kD
(NF, in F) labeling of the afferent fibers of the organ of Corti and the
synapse region. Scale bar 50 lm. (G) Phalloidin histochemistry of the
organ of Corti, labeling F-actin in viable sensory epithelium (stereocilia
and cuticular plate of hair cells, reticular lamina, and pillar cells). Scale
bar 50 lm. H: Semithin section showing the cytoarchitecture of the
spiral ganglion (SG). The inset shows electron microphotograph of the
ganglionar cells, surrounded by the Schwann cells (SC). The most
abundant neurons in the SG, the type I cells, present a myelin sheath,
with external compact myelin (CM) and internal loose myelin (LM).
Scale bar 50.1 lm (inset) and 30 lm. (I) Detail of the mouse lateral
wall showing the stria vascularis with the marginal cells (MC) close to
the scala media, the intermediate cells (IC), and the basal cells (BC).
The spiral ligament (SpL) is the most lateral part that is close to the
otic capsule of the cochlea. Scale bar 50 lm. J: Kir4.1 (KCNJ10, an
inwardly rectifying K

channel) expression in the stria vascularis of an

aging mouse. Note the relative loss of expression in some patches in
the stria (asterisks). This channel is related with the production of the
endocochlear potential, thus with an important role in auditory physiol-
ogy. Scale bar 50 lm. K: Na

-K

-ATPase expression in the stria vas-

cularis, another functional marker of striatal healthiness that are
related with ion homeostasis and auditory physiology (Patuzzi, 2011).
Scale bar 50 lm. L and M: The sensory epithelium of the vestibular
inner ear: the macula and the cristae gross anatomy. L: Cytoarchitec-
ture of a semithin section of the macula (L) and cristae ampullaris (M)
showing the morphological characteristics of these vestibular recep-
tors. Note the arrangement of the hair cells (HC) with the stereocilia
(asterisks), the supporting cells (SC), the basement membrane
(arrows), the otoconial membrane (OM) with otolites, and the vestibu-
lar nerve fibers (VNF). Scale bar 50 lm. N and O: Detail of the macula
(N) and cristae ampullaris (O) showing the myosin VIIa expression
(red, labeling sensory hair cells) and neurofilament 200 kD expression
(green, labeling macula nerve fibers). Arrows show the afferent calyx
of type I hair cells.
SENSORY AND NEURONAL CELL FATE SPECIFICATION 1777
Sanchez-Calderon et al., 2007). As development continues,
the otic vesicle undergoes reshaping processes that modify
the simple epithelial sac and transform it into a sophisti-
cated fluid-filled labyrinth composed by interconnected
chambers where the hair and supporting cells from the dif-
ferent sensory epithelia are located forming a complex cell
mosaic (Anniko, 1983; Kelly and Chen, 2009). Simultane-
ously to these changes, the inner ear recruits nearby
mesenchymal cells that will form the bony capsule that
surrounds the labyrinth (Chang et al., 2002).
Otic neuroblasts delaminate from the otic cup and otic
vesicle to form the acoustic-vestibular ganglion (AVG), which
contains the neural precursors of the spiral auditory and ves-
tibular ganglia that form a single ganglion at early
developmental stages. Otic neurons extend their processes
connecting the sensory epithelium to the brainstem nuclei
through the eighth cranial nerve (Hemond and Morest, 1991;
Adamet al., 1998; Rubel and Fritzsch, 2002; Figs. 2, 3A).
The general structure of the inner ear is comparable
in all vertebrates (Fig. 3B) but the rate at which the
inner ear develops is highly variable among species. The
mechanisms coordinating cell proliferation and survival
with differentiation remain poorly understood during
embryonic development and also in postnatal regenera-
tion. Here, we will review the biological processes and
the main signaling pathways required to generate the
different otic cell types. The contribution of cell survival,
apoptosis, proliferation, differentiation, and autophagy
will be discussed, with particular emphasis on the
actions of insulin-like growth factor I (IGF-I). There are
excellent recent reviews focused on complementary
aspects of otic sensory development (Kelley, 2007; Kelly
and Chen, 2009; Kwan et al., 2009; Chatterjee et al.,
2010; Fritzsch et al., 2011).
THE SPECIFICATION OF THE PROSENSORY
COMPETENT DOMAIN
Multipotent progenitor cells from the otic epithelia
will generate the three lineages of prosensory (the future
hair and supporting cells), proneural (future auditory
and vestibular neurons), and nonsensory (other otocyst-
derived cells) cells (Fig. 4). A common origin for neuro-
nal and sensory cells has also been proposed (Kelley,
2006; Raft et al., 2007). Neural specification starts with
the activation of a transcriptional program of expression
of proneural genes, being Neurog1 required for the spec-
ification of the neuronal fate. Gain of function studies
have demonstrated the formation of ectopic neurons in
regions where Neurog1 is over-expressed (Perron et al.,
1999; Kim et al., 2001), whilst Neurog1 inactivation
causes loss of sensory ganglia neurons and also of hair
cells (Ma et al., 2000), suggesting that at least in some
areas exist common progenitors for both cell lineages.
Accordingly, SOX2, a marker of the prosensory region is
also expressed in the proneural domain. A key common
feature of the SOX family of transcription factors is their
ability to maintain self-renewal state and pluripotency
of progenitors (Wegner and Stolt, 2005; Takahashi and
Yamanaka, 2006). In the zebrafish inner ear, however,
SOX2 is not required for the initial development of hair
cells (Millimaki et al., 2010). Lineage tracing in the
chicken otocyst showed that although some neuroblast
or sensory cells can be derived from a common progeni-
tor, this is not very frequent. Therefore, these data
indicate that most otic neurosensory cells have either a
proneural or a prosensory origin (Satoh and Fekete,
2005; Kelley, 2006). Further work is still required to
understand this very early stage of cell fate specification
that may vary among species.
The Notch Signaling Pathway
Several studies have shown that Notch signaling par-
ticipates in the earliest induction stages of the
prosensory domain. Members of the canonical Notch
pathway Notch1, Lunatic fringe (Lfng), Delta-like1
(Dll1), and Hes5 are differentially expressed in the otic
placode/cup (Adam et al., 1998; Cole et al., 2000; Groves
and Bronner-Fraser, 2000; Daudet and Lewis, 2005;
Jeon et al., 2011). The nonprosensory region expresses
Hes1 and the Notch ligand Jagged1 (Jag1), but not Lfng
and Dll1. In the chicken basilar papilla, ectopic activa-
tion of Notch signaling induced sensory patches in
nonsensory regions of the cochlea (Daudet and Lewis,
2005). Accordingly, the pharmacological inhibition of
Notch in vitro during prosensory specification blocks the
prosensory phase of development in the mammalian
cochlea (Hayashi et al., 2008). In addition, the
Fig. 2. The auditory pathway. A: Schematic view of the relay nuclei
of the multisynaptic and complex ascendant auditory pathway. B:
Example showing the ABR register of normal (/) and deaf mutant
(/) mice. The auditory brainstem response (ABR) reflects the
evoked potential response of auditory activity in the auditory nerve
and subsequent fiber tracts and nuclei within the auditory brainstem.
1778 MAGARIN

OS ET AL.
conditional activation of the Notch pathway in otic
regions at early mouse developmental stages induces
prosensory markers in the whole otic epithelium (Hart-
man et al., 2010). These results support a model where
early activation of Notch promotes the prosensory char-
acter in specific regions of the developing otocyst.
Nonetheless, there is still some controversy. Basch et al.,
(2011) showed that ectopic activation of Notch signaling
did not induce ectopic sensory patches in nonsensory
regions of the cochlea suggesting that Notch signaling is
not sufficient for prosensory specification in the mouse
cochlea. In addition, they show that conditionally inacti-
vation of RBPjk, the mediator of Notch signaling, is not
followed by a reduction on the prosensory markers, but
instead by a shorter life of hair and supporting cells.
Bone Morphogenetic Protein 4 (BMP4)
BMP4 is a member of the TGFb superfamily of
secreted signaling molecules that has been proposed to
Fig. 3. Inner ear development of vertebrates. A: Scheme of the early
development of the vertebrate inner ear. The inner ear originates from
the otic placode, a thickening of the head ectoderm that invaginates to
form the otic cup and separates from the ectoderm to form the otocyst
or otic vesicle, an ellipsoid-shaped structure able to generate most of
the cell types of the adult inner ear. In parallel to otic vesicle shaping, the
neuronal precursors (yellow) delaminate from the otic cup ventral region,
migrate and differentiate to produce the postmitotic otic neurons (red)
that form the acoustic-vestibular ganglion. (Modified from Ref. Varela-
Nieto et al., 2004) B: Schematic drawing of the inner ear of adult verte-
brates. From left to right: zebrafish, birds, and mouse. The origin and
early developmental stages of the inner ear are very similar in verte-
brates, although ear development in mammals is delayed with respect to
that of birds and, in zebrafish, the otic cup cavitates to form the otocyst.
All vertebrates show the general vestibular (light purple) and auditory
sensory components (dark purple), but with considerable differences, of
which the most notable is that the auditory region is a coiled cochlea in
mammals that contain the organ of Corti, and a straight cochlear duct in
birds called the basilar papilla. Zebrafish auditory function is carried out
by the saccule and the lagena. Abbreviations: A: anterior; ac: anterior
crista; bp: basilar papilla; co: cochlea; D: dorsal; l: lagena; lc: lateral
crista; M: medial pc: posterior crista; s: saccule; u: utricle.
SENSORY AND NEURONAL CELL FATE SPECIFICATION 1779
play a key role in the specification of prosensory patches
(Oh et al., 1996; Wu and Oh, 1996; Cole et al., 2000).
Although BMP4 expression is in agreement with an in-
ductive sensory role, functional in vitro studies have
provided opposing results (Li et al., 2005; Pujades et al.,
2006). This controversy evidences the complexity of
BMP4 actions in different developmental scenarios.
The Fibroblast Growth Factor (FGF) Family
The FGF family is composed by a large number of
ligands and four receptors (FGFR) implicated in the reg-
ulation of cell differentiation, proliferation, growth,
motility, and survival (Wright and Mansour, 2003). FGF
signaling plays a key role during vertebrate inner ear
development and participates in the early induction of
the otocyst (Schimmang, 2007), in prosensory specifica-
tion (Pirvola et al., 2002; Sanchez-Calderon et al., 2007;
Millimaki et al., 2007), otic neurogenesis and neurito-
genesis (Nicholl et al., 2005; Wei et al., 2007), and in
pillar cell differentiation (Doetzlhofer et al., 2009; San-
chez-Calderon et al., 2010). FGF ligands present
redundant actions and complex interactions, therefore a
combination of experimental approaches and the study
of mouse, chicken and zebrafish animal models are being
used to further understand and unravel FGF actions in
inner ear development (Kelly and Chen, 2009).
The HMG-Box Transcription Factor SOX2
The high mobility group (HMG)-box transcription fac-
tor SOX2 is a marker of the prosensory region, as well
as of the otocyst proneural domain (Fig. 5). Studies in the
chicken embryo evidenced that inductive signals regulate
directly SOX2 expression in the neural tube and that
SOX2 is responsible for neural fate acquisition in prolifer-
ating precursors (Rex et al., 1997; Bylund et al., 2003;
Graham et al., 2003). Early in development during otic ves-
icle stages, SOX2 and other SOX proteins such as SOX3
are expressed by proliferating cells in the prosensory do-
main. SOX2 wide expression in the prosensory domain
becomes restricted to the supporting cells as development
continues (Kiernan et al., 2005; Neves et al., 2007). Indeed,
several genes that are initially expressed in the prosensory
domain are afterwards restricted to the supporting cells
(Jag1, Lfng, and p27
kip
). These spatiotemporal patterns
emphasize the different and even opposing functions that
certain factors may have at different developmental stages,
which greatly difficult the identification of specific prosen-
sory markers (Kelley, 2007).
Two mutant mice deficient for Sox2 have been
described, Light coat and circling (Lcc) and Yellow sub-
marine (Ysb). Both mutants show defects in hearing and
balance (Kiernan et al., 2005), the cochleae of Lcc mice
lack neurons in the spiral ganglion (Puligilla et al.,
2010) and neither hair cells nor supporting cells
Fig. 4. Cell specification in the otic vesicle. Three cell lineages are
generated from the otic vesicle: prosensory, neural, and nonsensory.
Auditory and vestibular neurons are generated from a common neuro-
nal progenitor. Hair and supporting cells derive from the prosensory
progenitors. The drawing summarizes some of the factors required to
produce cellular diversity. Among others to be identified, SOX2, Islet-
1, the Notch pathway, Eya1, BMP4, and the miR-200 miRNA family
are required to generate the pool of prosensory cells. The Notch sig-
naling pathway is also playing a role in the differentiation of hair cells.
Ids release the interacting partners of Atoh1, and the Ahoh1 positive
cells will express JAG2 and Dll1, which by activating the Notch signaling
targets Hes1 and Hes5 in the surrounding cells, direct the supporting
cell fate. The expression of Neurog1, Islet-1, and SOX2 lead the neural
cell fate. Tbx1 is required to delimit the neurogenic domain in the oto-
cyst. The sequential activation of the proneural genes NeuroM and Neu-
roD will promote the formation of the vestibular and auditory neurons.
IGF-I/IGF1R signaling modulates cell survival and proliferation, although
a direct role in cell fate specification has not been shown yet.
1780 MAGARIN

OS ET AL.
differentiate (Kiernan et al., 2005). In addition, muta-
tions in human SOX2 cause sensorineural deafness
(Hagstrom et al., 2005). Due to the missing expression of
Sox2 in Jag1 mouse mutants, an interesting hypothesis
is that the Notch pathway element Jag1 could induce
the expression of Sox2 (Dabdoub et al., 2008).
Insulin-Like Growth Factor I
IGF-I belongs to the family of polypeptides of insulin
that plays a central role in embryonic development and
adult nervous system homeostasis by endocrine, auto-
crine, and paracrine mechanisms (Murillo-Cuesta et al.,
2011, 2012). IGF-I is secreted by the developing chicken
otocysts and it is expressed in the mouse inner ear
throughout development (Camarero et al., 2002; San-
chez-Calderon et al., 2010). Igf1r is expressed in the
sensory patches of otocysts from HH19 chicken (Aburto
et al., 2012) and E15.5 mouse (Sanchez-Calderon et al.,
2010) embryos. One of the earliest cell fate determina-
tion steps in the specification of the proneurosensory
field is the expression of the bHLH proneural gene
Fig. 5. Spatiotemporal expression patterns of SOX2 and B-RAF
during vertebrate inner ear development. In early development, SOX2
expression (red throughout the figure) marks, in chicken and mouse,
the prosensory and the sensory epithelia. SOX2 is initially expressed
in all prosensory and sensory regions (AD, A
0
and B
0
), later the
expression in hair cells diminishes (C
0
and D
0
) and is restricted to sup-
porting cells (boxed area in E
0
). B-RAF (green throughout the figure)
although shows a basal expression at very early developmental stages
in most tissues, is expressed in abundance in sensory regions of
chicken and mouse (AD, A
0
and B
0
). B-RAF is also highly expressed
in hair cells (E and F, C
0
and D
0
asterisk and arrows) although the
expression in the mouse OHC diminishes at the postnatal P30 stage
(boxed area in E
0
, asterisk and arrows). While B-RAF is intensively
expressed in otic neurons of the AVG and the SG, SOX2 is expressed
in nonoverlapping regions of glial cells in chicken (B and D) and
mouse (F
0
, arrows in the boxed area). Both SOX2 and B-RAF are also
expressed in the vestibular component in nonoverlapping regions (B-
RAF higher expression is found in hair cells, while SOX2 is abundantly
expressed in supporting cells. The boxed areas show higher magnifi-
cations of the selected regions. The bracket in B
0
indicates the sen-
sory region. The bracket in G
0
J
0
indicates the differential expression
of B-RAF and SOX2 in the macula. Abbreviations: AVG: acoustic-ves-
tibular ganglion; bp: basilar papilla; IHC: inner hair cells; OHC: outer
hair cells; ms, macula sacculi; mu: macula utriculi; sc: supporting
cells; sg: spiral ganglion. Orientation: D, dorsal; M, media. Scale bars:
200 lm applies to A
0
and C
0
; 100 lm applies to AE, E
0
, and G
0
I
0
: 50
lm applies to B
0
and F
0
; 25 lm applies to D
0
and to E
0
inset. (Partially
reproduced from Ref. Magari~ nos et al., 2010).
SENSORY AND NEURONAL CELL FATE SPECIFICATION 1781
Neurog1 (Adam et al., 1998; Fritzsch et al., 2010). Neu-
rog1 activates the proneural genes NeuroD and NeuroM,
all have the potential to activate the expression of Igf1
and Igf1r genes. An appealing hypothesis is that upon
prosensory specification by other growth factors and
morphogenetic proteins, Igf1 expression is upregulated
by proneural genes to master early neurogenesis. IGF-I
actions and IGF1R expression are mediated by an intra-
cellular signaling network (Murillo-Cuesta et al., 2011,
2012; Fig. 6), and precedes that of neurotrophins and
TrkA expression in neurons (Li et al., 2009). Indeed, otic
neurons gradually reduce their expression of the Igf1r,
while they increase the neurotrophin receptor TrkC
(Aburto et al., 2012), suggesting that IGF-I is a key
trophic factor during the otic neuronal progenitor phase
of early inner ear development.
BIRTH AND DIFFERENTIATION OF HAIR AND
SUPPORTING CELLS
Hair and supporting cells are originated after the
specification of the prosensory domain (Driver and Kel-
ley, 2009). During early development, it seems to be a
default program by which the first cells specified are the
hair cells that in turn, by lateral inhibition, block their
fate to neighbor cells. Atoh1, a basic helixloophelix
(bHLH) transcription factor is a key player in hair cell
production and development. Although its expression
pattern is somehow controversial (Bermingham et al.,
1999; Lanford et al., 2000; Chen et al., 2002), loss and
gain of function studies have proven that Atoh1 is neces-
sary and sufficient to induce hair cell formation in
sensory and nonsensory cells (Bermingham et al., 1999;
Zheng and Gao, 2000; Kawamoto et al., 2003; Woods
et al., 2004; Jones et al., 2006). It is interesting to note
that the solely expression of Atoh1 is able to generate
hair cells in nonsensory patches, suggesting that the
sensorial competence is not restricted to certain regions
of the otic epithelium (Kelley, 2006).
Before hair cell formation starts there is a broad
expression of a family of proteins, the Ids (Jones et al.,
2006) that prevent hair cell differentiation by the inacti-
vation of Atoh1. Ids are inhibitors of differentiation that
contain a HLH domain that competes with Atoh1 for its
dimerization partners E47 and E2A. The Ids do not have
a DNA binding domain, but they can sequester the fac-
tors required by Atoh1 to form a functional heterodimer
(Norton, 2000). The expression of the Id family is
reduced in hair cells as development continues, conse-
quently releasing the factors necessary for Atoh1
activity, and thus allowing the differentiation of hair
cells (Jones et al., 2006). Once hair cells have differenti-
ated, the surrounding cells become supporting cells by a
lateral inhibition mechanism. The Notch pathway that
had had a previous role in prosensory specification is
also implicated in individual cell differentiation. The
Atoh1 positive cells begin to express two Notch ligands
Jagged2 (Jag2), and Dll1 (Lanford et al., 1999; Morrison
et al., 1999; Hartman et al., 2007). This leads to Notch1
activation and to intracellular expression of Notch tar-
gets such as Hes1, Hes5, Hey1, and Hey2 in the future
supporting cells (Lanford et al., 2000; Zheng et al., 2000;
Zine et al., 2001; Murata et al., 2006; Hayashi et al.,
2008; Li et al., 2008; Doetzlhofer et al., 2009). There are
also other factors that act as Atoh1 antagonist such as
SOX2 and Prox1 (Zheng et al., 2000; Dabdoub et al.,
2008; Doetzlhofer et al., 2009).
In summary, Notch activity through evolution is
required for sensory development to specify the prosen-
sory regions in the otic cup and otocyst, and later to
promote cell type differentiation to hair and supporting
cells (Eddison et al., 2000; Daudet and Lewis, 2005).
The Role of MicroRNAs in Inner Ear
Development
MicroRNAs (miRNA) are small noncoding RNA, which
bind to the three untranslated region of target mRNA to
suppress their translation (Kloosterman and Plasterk,
2006). miRNA have been implicated in multiple biologi-
cal processes across phyla including development and
disease (Bartel, 2009; Lewis and Steel, 2010). miRNA
started to be an inner ear topic when the expression of
the miR-183 family (composed by miR-182, miR-96, and
miR-183) was reported in the inner ear of zebrafish
embryos (Wienholds et al., 2005). This family is highly
conserved throughout evolution and it is expressed in
ciliated neurosensorial organs (Pierce et al., 2008). These
miRNA are expressed in hair cells in chicken, and
mouse, and in lower levels in mouse sensory neurons of
acoustic and vestibular structures (Darnell et al., 2006;
Weston et al., 2006; Li and Fekete, 2010). Indeed, muta-
tions in miR-96 cause hearing loss in mice and men
(Lewis et al., 2009; Menc a et al., 2009). The dynamic
expression of the miR-183 family in the mouse begins at
the otocyst stage and has the higher expression in differ-
entiating hair cells, suggesting that this family is
involved in hair cell differentiation and maturation
(Sacheli et al., 2009). Another miRNA family, the miR-
200 (miR-200a, miR-200b, miR-200c, miR-141, and miR-
429) has been associated to inner ear prosensory specifi-
cation. The miR-200 family is expressed in the inner ear
epithelia of zebrafish, chicken, and mouse (Wienholds
et al., 2005; Darnell et al., 2006; Weston et al., 2006).
Because of their known targets and interacting path-
ways, it has been suggested that it plays a key role in
establishing the prosensory epithelial domains (Soukup,
2009).
The importance of miRNAs in the inner ear is further
evidenced by the conditional deletion of the enzyme
Dicer specifically in the otic placode. These mutants
show a severe phenotype similar to that found in the
Sox2 null mouse, with profound defects in inner ear neu-
rogenesis and sensory epithelial histogenesis (Kiernan
et al., 2005; Friedman et al., 2009; Soukup et al., 2009).
The emerging role of miRNAs in inner ear development
suggests that they may be of use in novel therapeutic
strategies aimed to hair cell regeneration (Beisel et al.,
2008).
DEVELOPMENTAL OTIC NEUROGENESIS
AND INNERVATION
The AVG contains cells derived from the otic epithelia
that will transit through different stages to become post-
mitotic neurons finally. Otic neurogenesis can therefore
be separated into different cellular stages that are char-
acterized by the distinct expression of a combination of
molecular markers. After the regionalization of the otic
cup/vesicle by the expression of a defined set of genes
1782 MAGARIN

OS ET AL.
Fig. 6. Biological programmes that generate otic cell diversity. AC:
Programmed cell death in the developing inner ear. Apoptosis occurs in
vivo in selected regions (arrows) of the developing otic vesicle (C). Cryo-
stat section of a HH18 chicken embryo visualized by TUNEL (green
throughout the figure). Blocking apoptosis in organotypic cultures of
otic vesicles with BOC caused a reduction in the AVG size (arrow) and
the otic epithelium appeared thickened (arrowhead, A and B). Otic
vesicles were isolated from HH18 chicken embryos, made quiescent,
and cultured for 20 hr in serum-free culture medium without additions
(0S, A) or in the presence of BOC (50 lM; B). (DF) Growth factors are
required to induce the survival of specific cell populations in the otic
vesicle. Otic vesicles were culture without additions 0S (D), IGF-I (10
nM; E) or in the presence of LY (25 lM; F). TUNEL-levels decreased
markedly in the presence of IGF-I and increased dramatically with the
PI3K/AKT inhibitor. GI: Cell proliferation of otic progenitors is required
for correct morphogenesis and AVG neurogenesis. Proliferation was
measured by the incorporation of BrdU (red) during 1 hr. Cultured otic
vesicles in 0S medium (G), with IGF-I (10 nM; H), or in the presence of
Sor (5 lM; I) showed that IGF-I promoted otic proliferation, whilest Sor
impaired BrdU incorporation. Consequently, the size of Sor-treated otic
vesicles was severely reduced. Reproduced from Ref. Magari~ nos et al.,
2010. JL: IGF-I drives otic neurogenesis by adjusting the neuroblast/
neurons ratio. Otic vesicles cultured in 0S medium (J), containing IGF-I
(10 nM; K) or LY (25 lM; L) were double-immunostained for TuJ-1 (red)
and Islet-1 (green). Islet-1-positive population increases in the presence
of IGF-I (JK, dashed areas). In the presence of LY, there are fewer Islet-
1-positive neuroblasts in the AVG, while the remaining AVG presents
generalized TuJ-1 expression (L) Reproduced from Ref. Aburto et al.,
2012. MO: Autophagy is involved in inner ear development. HH18 cul-
tured otic vesicles were incubated 20 hr in the 0S condition (M) or with
the 3-MA (10 mM; N) and then cultured with An-V (red). Apoptotic cell
death was visualized by TUNEL (green) and An-V, an eat me flag for
the non-professional macrophages. When autophagy is inhibited (N),
TUNEL labeling increases and An-V is reduced, suggesting that apopto-
tic cell clearance is impaired. The otocysts present aberrant features
evidenced by TuJ-1 labeling (red; O). Representative images are shown
that were obtained from compiled confocal microscopy projections of
the otic vesicles. Orientation: AN, anterior; DO, dorsal. Scale bar, 150
lm. Abbreviations: An-V, Annexin-V; B AVG, Acoustic-vestibular gan-
glion; BOC, Boc-D-FMK (pan-caspase inhibitor); LY, LY294002 (PI3K/
AKT inhibitor); Sor, Sorafenib (RAF-MEK-ERK inhibitor); 3-MA, (3-
methyladenine).
including among others, Neurog1, LFng, Dl1, Sox2, and
Sox3 that specify the neural fate, neuroblasts migrate
from the proneural region of the otic cup toward the
closely mesenchymal space. The expression of the pro-
neural genes NeuroD and NeuroM define the following
stage in neural differentiation: the epithelial neuroblast
population (Chae et al., 2004; Sanchez-Calderon et al.,
2007). This population will migrate in a process that has
been intensely studied in chicken and mouse embryos
(DAmico-Martel and Noden, 1983; Alvarez et al., 1989;
Hemond and Morest 1991; Davies, 2007, 2011). Migra-
tion begins at the otic cup stage and reaches its
maximal rate in the early otic vesicle stage (Figs. 3A,
7A). The new formed ganglionic neuroblast population
can be identified by their location, round shape, and by
the expression of a defined set of transcription factors,
neurofilaments, and neurotrophins receptors. Including
the transcription factor Islet-1 (Hobert and Westphal,
2000), which begins to be expressed before migration by
epithelial neuroblasts (Adam et al., 1998; Camarero
et al., 2003; Li et al., 2004). Ganglionic neuroblasts
retain the capacity to undergo cell division to expand
their population and, accordingly, express receptors for
IGF-I and cell cycle activators as FoxM1. Once neuro-
blasts exit from the cell cycle, they differentiate into
postmitotic neurons that begin to project their processes
toward their peripheral and central targets (Whitehead
and Morest, 1985; Fekete and Campero, 2007). The imma-
ture post-mitotic neurons show a reduced expression of
early neural markers, and start to gradually express
another set of genes related to neuritogenesis and cell
cycle exit, as type III tubulin (TuJ1), G4, and the cyclins
inhibitor p27
Kip
(Sanchez-Calderon et al., 2007, 2010).
Finally, the mature otic neurons generate action poten-
tials, express synaptic receptors and neurotransmitters,
and reach the most differentiated and mature state
(Raphael and Altschuler, 2003). Mature neurons express
a whole set of genes related to neuronal trophic support
as the neurotrophins receptors TrkB and TrkC (Pirvola
et al., 1997; Brumwell et al., 2000; Kim et al., 2001).
Autophagy in the Developing Inner Ear
Autophagy is a self-degradative process of the cellular
cytosolic constituents that is crucial for balancing sour-
ces of energy in response to different extracellular
stimuli and for preventing the accumulation of misfolded
proteins or damaged organelles (Qu et al., 2007; Glick
et al., 2010; Ravikumar et al., 2010). Autophagy in ver-
tebrates has been shown to have key roles during
development (Levine and Klionsky 2004; Mizushima and
Levine 2010; Montero and Hurle 2010). In the inner ear,
autophagy genes are essential for the vestibular function
in mice (Mari~ no et al., 2010) and dead cells with auto-
phagic features have been observed in the damaged
cochlea (Taylor et al., 2008). Autophagy plays a role dur-
ing normal inner ear development (Aburto et al., in
press) and its inhibition by treatment with 3-methylade-
nine (3-MA; Rubinsztein et al., 2007) dramatically
impairs neurogenesis (Fig. 6). An appealing hypothesis
is that the degradation by autophagy of the otic cells
owns components provide the energy required for the
migration of the neuronal precursors. In this context, it
would be very interesting to examine early inner ear de-
velopment in Atg4b
/
and Atg5
/
deficient mice,
which show different degrees of vestibular defects (Mar-
i~ no et al., 2010).
Vestibular Versus Auditory Neuronal Cell Fate
Otic neuroblasts are the common progenitors of audi-
tory and vestibular neurons that will have defined traits
and innervate different brain centers and establish pre-
cise connections with their peripheral sensory targets.
There are no precise molecular markers of this specifica-
tion process. The sequential expression of NeuroM and
NeuroD by migrating neuroblasts could determine spe-
cific stages of maturation; in the chicken embryo,
evidence suggests that these genes do not identify differ-
ent neuron identities but rather a temporal appearance
of vestibular and auditory neurons (Bell et al., 2008).
Studies in mouse null mutants have also shown the tem-
porally regulated expression of Neurog1 to first generate
vestibular neurons and secondly cochlear neurons
(Koundakjian et al., 2007).
Neuritogenesis and Axonal Pathfinding
The innervation of specific sensory structures by otic
neurons is built in a stereotyped fashion (Fekete and
Campero, 2007; Koundakjian et al., 2007; Appler and
Goodrich, 2011). It has been suggested that otic neurons
send their projections by retracing back to the original
delamination place (Carney and Silver, 1983; Bruce
et al., 1997). However, there are some long distance con-
nections that would require the release of molecular
cues. Though the mechanisms and factors affecting axon
guidance in the inner ear are still under study and dis-
cussion, its basis are probably similar to those behind
the wiring of the central nervous system. Indeed, most
of the molecules that have been reported to play a role
in ear innervation are well-known chemoattractants, as
brain-derived neurotrophic factor, neurotrophin-3 (NT-3),
Shh, and the FGF family (Fari~ nas et al., 2001a,b;
Fritzsch et al., 2004; Fantetti and Fekete, 2011), or else
chemorepellent cues such as Semaphorin3/Npn1, Eph/
Ephrins, and Slit/Robo (Webber and Raz, 2006; Fekete
and Campero, 2007; Battisti and Fekete, 2008). It is also
interesting to underline that molecules that reorganize
the cytoskeleton such as the integrins (Davies, 2007,
2011), autophagy and pathways traditionally implicated
in axon outgrowth such as the RAF-MEK-ERK pathway,
have been also reported to act in early otic neuritogene-
sis (Zhong et al., 2007; Magari~ nos et al., 2010; Fig. 7).
Autophagy is emerging as a new player in neurite gener-
ation and degeneration (Yang et al., 2007; Koike et al.,
2008; Plowey et al., 2008). Unc-51, the homolog for Atg1
in C. elegans, is required for axonal guidance (Ogura
and Goshima, 2006) and it has been recently demon-
strated that neural soma survival is required for
adequate axonal maintenance and regeneration (Rodr -
guez-Muela et al., 2011).
Currently, the best available therapy for treating
hearing loss is the electrical stimulation of the spiral
ganglion neurons with cochlear implants. Therefore,
there is a need to improve our knowledge on how audi-
tory neurons survive, differentiate, and interact with
their targets to provide new avenues for the therapy of
inner ear disorders.
1784 MAGARIN

OS ET AL.
Fig. 7. Early otic neurogenesis. A: Neurogenic markers of otic neu-
rogenesis. Schematic representation of stages of development of otic
neurons. Postmitotic neurons depend on neurotrophins, but the fac-
tors expanding neuroblast populations are still to be fully defined.
Abbreviations: Del, delaminating; IN, immature neuron; MP, multipotent
progenitor; NBe epithelial neuroblast; NBg, ganglionar neuroblast; ON,
otic neuron. Adapted from Sanchez-Calderon et al., 2007. B: In vivo
early otic neurogenesis. (a) Cryostat section of a HH19 chicken
embryo inmunostained for the transcription factor Islet-1 (green) and
the neuron marker G4 (magenta). Panel b corresponds to the boxed
area in a, showing a higher magnification of the AVG. C: Schematic
representation of the AVG ex vivo culture. The AVG can be explanted
from the embryo at HH19. The figure shows a schematic drawing of
a HH19 chicken embryo showing the otic vesicle and the AVG loca-
tion and of an AVG immediately after dissection (0 hr) and after 24 hr
in culture. Factors and drugs can be added to the serum-free culture
medium to study their effects on AVG neuritogenesis. D: Signaling dur-
ing early otic neuritogenesis. (a and b) Inhibition of both IGF-I signaling
pathways, RAF-MERK-ERK and PI3K/AKT impaired otic neuritogene-
sis. (a) AVG explants were cultured in the 0S medium, or with Sor (2.5
lM) and immunostained for G4 (red) and Islet-1 (green). Sor-treated
AVG have shorter processes without affecting the size of the AVG
soma. Reproduced from Ref. Magari~ nos et al., 2010. (b) AVG cultured
in 0S or in the presence of LY (25 lM) and immunostained for TuJ-1
(magenta) and Islet-1 (green) showed that both the neuronal soma
area and the length of the neurites of the LY culture are smaller.
Reproduced from Ref. Aburto et al., 2010. Representative images are
shown from at least six otic vesicles per condition obtained in at least
three independent experiments. Compiled confocal microscopy projec-
tions of AVG are shown. (c) Inhibition of autophagy alters AVG neuro-
genesis. AVG explants cultured in the 0S condition or with 3-MA were
immunostained for G4 (green). Scale bar: 300 lm. Fluorescence images
were obtained from the compiled projections of confocal images of otic
vesicles and acoustic-vestibular ganglia. Abbreviations: B AVG: Acous-
tic-vestibular ganglion; LY: LY294002 (PI3K/AKT inhibitor); Sor: Sorafe-
nib (RAF-MEK-ERK inhibitor); 3-MA: (3-methyladenine).
SENSORY AND NEURONAL CELL FATE SPECIFICATION 1785
IGF-I SIGNALING IN INNER EAR
DEVELOPMENT
IGF-I is fundamental for the regulation of cochlear de-
velopment, growth, and differentiation and its mutations
are associated with hearing loss in mice and men
(Walenkamp and Wit, 2006; Murillo-Cuesta et al., 2011,
2012). Early hearing loss in Igf1 null mice is due to neu-
ronal loss and poor innervation of the sensory hair cells
a phenotype associated to sensorineural deafness (Cediel
et al., 2006). With ageing the stria vascularis degener-
ates and cellular traits of metabolic deafness, appear
earlier than in the wild type littermates (Riquelme
et al., 2010). Recent studies have also shown that low
levels of IGF-I correlate with different human syn-
dromes showing hearing loss (Murillo-Cuesta et al.,
2011, 2012; Rodriguez de la Rosa et al., 2011) and with
presbycusis (Riquelme et al., 2010).
IGF-I actions are mediated by a complex network of
intracellular molecules. IGF-I binding to IGF1R results
in its autophosphorylation in tyrosine residues (LeRoith
et al., 1995; Laviola et al., 2008), recruitment of insulin
receptor substrates leading to the activation of two main
downstream pathways: the PI3K-AKT and the RAF-
MEK-ERK phosphorylation cascade. During inner ear
development, the generation of cellular diversity
requires the strict regulation of biological processes that
are coordinated by the concerted action of extrinsic and
intrinsic factors. Otic epithelial cells have to escape apo-
ptosis, survive, nurture themselves by autophagy,
proliferate, migrate, and differentiate to generate the di-
versity of cell types of the adult inner ear. To this end,
IGF-I acts differentially over its signaling pathways: the
PI3K-AKT and the RAF-MEK-ERK cascade (Fig. 6).
Apoptosis Versus Survival
Programmed cell death plays an important role during
the earliest morphogenetic events of inner ear develop-
ment (Lang et al., 2000; Leon et al., 2004). Apoptosis
contributes to the regulation of cell number in the epi-
thelial and ganglionar neuroblast populations, and it
serves to remove aberrant cells. Total blockage of apopto-
sis with the caspase inhibitor Boc-D-FMK increases the
size of otic vesicles, caused an abnormal thickening in
all the otic epithelium and also caused a reduction in
the AVG size (Fig. 6AC) (Aburto et al., 2012). These
data suggest that apoptosis is required for morphogene-
sis and neurogenesis fulfilling a complementary role in
the epithelial neurogenic area to allow the neuroblasts
to physically detach from the epithelium. Otic vesicle
cells require the actions of growth factors and neurotro-
phins to survive. For example, blockade of endogenous
IGF-I activity in otic vesicle explants, increases cell
death and inhibits the formation of the AVG, which
shows that IGF-I is essential to promote survival in the
otic precursors (Fig. 6D,E; Camarero et al., 2003).
Accordingly, IGF-I reduces caspase-3 activation and
TUNEL staining. However, the associated cell survival
is spatiotemporally regulated so that it results in the
persistence of cell death of specific populations. Cell
death mainly occurs in postmitotic neurons that down-
regulate the expression of high affinity IGF-I receptors.
To promote the survival of neural progenitors, IGF-I acts
through the PI3K-AKT pathway as indicated by the loss
of epithelial and ganglionic neuroblast after blockade of
either PI3K or AKT activity with the inhibitor LY294002
(Kong and Yamori, 2008; LY; Fig. 6F,JL). Therefore,
IGF-I/AKT signaling is fundamental for survival of pro-
liferative otic neuroblasts and for the maintenance of
the undifferentiated state. This suggests a crucial role of
this pathway in establishing the final number of neu-
rons and the timing at which neuron generation
proceeds during otic development.
Cell Proliferation
There is previous evidence that IGF-I can promote
proliferation of neural progenitors (Aberg et al., 2003;
Mairet-Coello et al., 2009), and of neural stem cells in
culture (Arsenijevic et al., 2001). Depending on the cell
type and context, these actions of IGF-I are mediated by
distinct signaling pathways (Varela-Nieto et al., 2003; Ye
and DErcole, 2006; Magari~ nos et al., 2010; Murillo-
Cuesta et al., 2011). The basic mechanism underlying
these actions is the capacity of IGF-I to promote G1/S
cell cycle progression by regulating cyclin kinase activa-
tion via the activation of specific signaling pathways
(Hodge et al., 2004; Mairet-Coello et al., 2009). In
chicken otocyst cultures, IGF-I promotes otic prolifera-
tion as assessed by complementary cellular and
molecular approaches (Frago et al., 1998, 2003; Sanz
et al., 1999; Magari~ nos et al., 2010). The activation of
the RAF-MEK-ERK cascade is essential for cellular pro-
liferation and it plays a fundamental role in the G1/S
transition (Schreck and Rapp, 2006; Chambard et al.,
2007), accordingly, blockade of the RAF pathway with
Sorafenib (Sor; Schreck et al., 2006) produces a dramatic
decrease in otocyst cell proliferation (Fig. 6GI; Mag-
ari~ nos et al., 2010) and increases the level of apoptotic
cells. Nevertheless, RAF activity is essential for the pro-
gression of otocyst cell proliferation but not for cell
survival because IGF-I rescues otic progenitors by acti-
vating the PI3K-AKT pathway even in the presence of
the RAF kinase inhibitor Sor.
Therefore, IGF-I orchestrates cell proliferation and
survival in the otic vesicle through distinct pathways,
although cross talk between signaling pathways also
occurs, as reported in other cell contexts (Moelling et al.,
2002; Magari~ nos et al., 2010). The Igf1 deficient mouse
showed a significant reduction of the activated forms of
the proteins ERK1/2 and AKT in the cochlea in prenatal
stages and a great increase of the activated p38a MAP
kinase. In summary, IGF-I deficiency in the cochlea
decreases the activity of the signaling pathways that
regulate cell survival and proliferation, and increases
those involved in the cellular response to stress (San-
chez-Calderon et al., 2010).
CONCLUSIONS
Hearing and balance sensory organs have a common
embryonic origin. Otic progenitor cells will transit
trough stages of commitment and differentiation to gen-
erate sensory cells, support cells, and neurons for the
vestibular and spiral ganglions. This transit is exqui-
sitely regulated by extracellular signals. Extracellular
signals as growth factors and morphogenetic molecules
will elicit a network of intracellular signals leading to
the expression of master transcription factors that will
1786 MAGARIN

OS ET AL.
generate gene expression spatiotemporal patterns.
Future therapies for hearing loss and balance disorders
will benefit from the increasing knowledge on the molec-
ular clues that direct otic development.
ACKNOWLEDGEMENTS
The authors thank the Image Unit (IIBM, Madrid) for
their technical support. The anti-Islet-1 monoclonal anti-
body was developed by Drs T.M. Jessel and J. Dodd and
it was obtained from the Developmental Studies Hybrid-
oma Bank, maintained at the Iowa University, Depart-
ment of Biological Sciences. They warmly thank the
critical comments and generous sharing of results of our
colleagues at the Neurobiology of Hearing group.
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