Post on 18-Dec-2016
transcript
Fgf10 Expression Patterns in the Developing ChickInner Ear
Luis Oscar S�anchez-Guardado,1 Luis Puelles,2 and Matıas Hidalgo-S�anchez1*1Department of Cell Biology, School of Science, University of Extremadura, Badajoz E06071, Spain2Department of Human Anatomy and Psychobiology, School of Medicine, University of Murcia, Murcia E30100, Spain
ABSTRACTThe inner ear is a complex three-dimensional sensorial
structure with auditory and vestibular functions. It origi-
nates from the otic placode, which invaginates, forming
the otic vesicle; the latter gives rise to neurosensory
and nonsensory elements of the adult membranous
labyrinth. A hypothesis based on descriptive and
experimental evidence suggests that the acquisition of
discrete sensory patches during evolution of this pri-
mordium may be related to subdivision of an early pan-
sensory domain. In order to gain insight into this
developmental mechanism, we carried out a detailed
analysis of the spatial and temporal expression pattern
of the gene Fgf10, by comparing different markers of
otic patterning and hair cell differentiation. Fgf10
expression labels a sensory-competent domain included
in a Serrate-positive territory from which most of the
sensory epithelia arise. Our data show that Fgf10
transcripts are present initially in a narrow ventromedial
band of the rudimentary otocyst, extending between its
rostral and caudal poles. During development, this
Fgf10-expressing area splits repetitively into several
separate subareas, creating six of the eight sensory
organs present in birds. Only the lateral crista and the
macula neglecta were initially Fgf10 negative, although
they activated Fgf10 expression after their specification
as sensory elements. These results allowed us to deter-
mine a timetable of sensory specification in the devel-
oping chick inner ear. The comparison of the
expression pattern of Fgf10 with those of other markers
of sensory differentiation contributes to our understand-
ing of the mechanism by which vertebrate inner
ear prosensory domains have arisen during evolution.
J. Comp. Neurol. 521:1136–1164, 2013.
VC 2012 Wiley Periodicals, Inc.
INDEXING TERMS: fibroblastic growth factors; inner ear evolution; pansensory domain; otocyst; otic specification;
sensory patch; acoustic-vestibular ganglion; otic innervation
The vertebrate inner ear is an intricate and highly
asymmetric system of communicated cavities and con-
duits ultimately responsible for hearing and the analysis
of the head’s position and movement in 3D space. The
otic anlage arises from a portion of the cephalic ecto-
derm, named the otic placode, which invaginates and
then closes to form the otic vesicle or otocyst. During em-
bryonic development, the ovoid otic rudiment undergoes
important morphogenetic changes that lead to its trans-
formation into the highly complex 3D membranous laby-
rinth. A key aim of developmental studies in this field is to
understand the molecular and cellular mechanisms
involved in the early patterning of the otic primordium,
from the perspective of vertebrate evolution. In particular,
a central issue is how the diverse sensory and nonsen-
sory epithelia are generated (segregated) and distributed
in a specific spatial pattern (Abell�o and Alsina, 2007; Bok
et al., 2007; Schneider-Maunoury and Pujades, 2007;
Whitfield and Hammond, 2007). This process is thought
to be controlled by a network of diffusible signals (morph-
ogens) and a variously overlapping expression pattern of
transcription factors (Romand et al., 2006a; Abell�o and
Alsina, 2007; Bok et al., 2007; Fekete and Campero,
2007; Ohyama et al., 2007; S�anchez-Calder�on et al.,
2007a; Schimmang, 2007; Schneider-Maunoury and
Pujades, 2007; Whitfield and Hammond, 2007; Kelly and
Chen, 2009; Frenz et al., 2010; Ladher et al., 2010),
Grant sponsor: Spanish Ministery of Science; Grant numbers: BFU2010-19461 (to M.H.S.); BFU2005-09378-C02-01, BFU2008-04156; Grantsponsor: SENECA Fundation; Grant number: 04548/GERM/06-10891 (toL.P.); Grant sponsor: Junta-de-Extremadura predoctoral fellowship; Grantnumber: PRE/08031 (to L.-O.S.-G.).
*CORRESPONDENCE TO: Matıas Hidalgo-S�anchez, Department of CellBiology, University of Extremadura, Avda. de Elvas s/n, 06071 Badajoz,Spain. E-mail: mhidalgo@unex.es
VC 2012 Wiley Periodicals, Inc.
Received March 30, 2012; Revised June 22, 2012; Accepted September5, 2012
DOI 10.1002/cne.23224
Published online September 14, 2012 in Wiley Online Library(wileyonlinelibrary.com)
1136 The Journal of Comparative Neurology | Research in Systems Neuroscience 521:1136–1164 (2013)
RESEARCH ARTICLE
which establish boundaries and differential fates within
the developing otic epithelium (Fekete and Wu, 2002).
With respect to the specification of otic sensory epithe-
lia, an increasing body of evidence suggests that its even-
tual functional diversity (eight separate sensory organs in
the bird) results from refined subdivision of a primordial
single sensory patch over the course of vertebrate evolu-
tion (Fritzsch and Beisel, 2001; Fritzsch et al., 2002,
2010). Histological examination of the chick inner ear
have suggested that this primordial sensory area might
be located in the ventromedial wall of the otocyst, from
which all sensory organs seem to generate (Knowlton,
1967). At the molecular level, this sensory-competent do-
main is held to be regulated by components of the Notch
pathway, such as Serrate1 and Lunatic Fringe (LFng;
Adam et al., 1998; Cole et al., 2000), jointly with brain-
derived neurotrophic factor (BDNF; Fari~nas et al., 2001)
and cell adhesion molecules, such as BEN (Goodyear
et al., 2001). Further studies of the combined expression
patterns of additional regulatory genes should help us to
understand how such a remarkably complex origin of
individual sensory epithelia takes place with minimal vari-
ation within the otic rudiment of all vertebrates (e.g.,
Manley, 2000).
Fibroblastic growth factor (FGF) signaling molecules
have long been known to be involved in cell fate specifica-
tion in addition to cell proliferation, migration, and sur-
vival; they activate members of a family of tyrosine kinase
receptors, the FGF receptors (FGFRs; B€ottcher and
Niehrs, 2005; Dailey et al., 2005; Polanska et al., 2009;
Knights and Cook, 2010; Itoh and Ornitz, 2011). In the
developing nervous system, the diffusible FGFs are impli-
cated in the induction of early neural fate (for reviews,
see Wittler and Kessel, 2004; Heisenberg and Solnica-
Krezel, 2008), as well as in the regionalization of the cen-
tral nervous system (see Hidalgo-S�anchez et al., 2005;
Echevarrıa et al., 2005; and references therein). Given
their presence in the otic primordium (see below), FGFs
are attractive candidates for orchestrating differential
otic placode induction and morphogenesis of the otic
anlage. In particular, FGF signaling pathways might con-
trol, besides cell differentiation, the regional patterning of
the otic epithelium during vertebrate inner ear develop-
ment (reviewed by Frizsch et al., 2006; Bok et al., 2007;
Ohyama et al., 2007; Schimmang, 2007; Schneider-
Maunoury and Pujades, 2007; Streit, 2007; Whitfield and
Hammond, 2007; Kelly and Chen, 2009; for details on the
chick inner ear, see Hidalgo-S�anchez et al., 2000;
S�anchez-Calder�on et al., 2004, 2007b).
Among the FGFs, FGF10, which binds to FGFR2b with
high affinity (Igarashi et al., 1998; Ohuchi et al., 2000; Pir-
vola et al., 2000; Yu et al., 2000), has received increasing
interest as underlying a wide range of developmental
events regulating the formation of organs from all germ
layers (Ohuchi et al., 1997). Thus, Fgf10 gene activity has
been associated with several developmental events, such
as outgrowth of the limb bud, branching morphogenesis
of the lung, gastrointestinal tract formation, specification
of the ventral pancreas, tooth morphogenesis, and thy-
mus and parathyroid development (Colvin et al. 2001;
Tang et al., 2001; Zhang et al., 2005; Zakany et al., 2007;
Kobberup et al., 2010; Parsa et al., 2010; Neves et al.,
2011; see also references therein). It is also involved in
the differentiation of the nervous system (Umemori et al.,
2004; Hajihosseini et al., 2008; Potok et al., 2008; Parkin-
son et al., 2010; Sahara et al., 2009). Due to the dysgene-
sis or agenesis of a broad diversity of organs, Fgf10�/�
mutant mice die at birth (Ohuchi et al., 2000).
FGF signals have been shown to play a critical role in
inner ear induction in several species (Ohyama et al.,
2007). It is well known that FGF10 from the underlying
mesenchyme and FGF3 from the caudal hindbrain coop-
erate to control the expression of otic markers in the
cephalic ectoderm in both chick and mouse embryos
(Wright and Mansour, 2003; Ladher et al., 2005;
Zelarayan et al., 2007). Thus, Fgf3�/�/Fgf10�/� double-
mutant mice show microvesicles or no otic vesicles
(Alvarez et al., 2003; Wright and Mansour, 2003). Once
the otic placode is formed, low levels of Fgf10 expression
are detected in the mouse otic placode (Pirvola et al.,
2000), whereas Fgf10 expression is clearly observed in
the most anterior and medial portion of the chick otic pla-
code (Alsina et al., 2004). In the chick, this expression
domain subsequently evolves dynamically, forming an
anteromedial band placed at the equator of the otic vesi-
cle, and sharply excluding other parts of the otic anlage
Abbreviations
ac anterior cristaAG acoustic ganglionasc anterior semicircular canalAVG acoustic-vestibular ganglionbp basilar papillacc common cruscd cochlear ductect ectodermed endolymphatic ductes endolymphatic sacHB hindbrainhp horizontal pouchlc lateral cristalsc lateral semicircular canalml macula lagenamn macula neglectams macula sacculimu macula utriculip posterior Bmp4-positive patchpc posterior cristapsc posterior semicircular canals sacculetv tegmentum vasculosumu utricleVG vestibular ganglionvp vertical pouch
Fgf10 in the developing chick inner ear
The Journal of Comparative Neurology | Research in Systems Neuroscience 1137
(Alsina et al., 2004). Thus, Fgf10 is expressed in a well-
delimited region that largely coincides with the presump-
tive neural-sensory domain and anticipates the expres-
sion of several proneural and neurogenic genes such as
Ngn1, Delta1, Hes5, and Neuro D/M (Alsina et al., 2004).
As development proceeds, Fgf10 expression is
detected in the vestibular sensory patches of E12 mouse
embryos (Pauley et al., 2003; Lillev€ali et al., 2006). Fgf10
knockout mice show morphogenetic and innervation
alterations including complete loss of the posterior semi-
circular canal crista and all three semicircular canals
(Ohuchi et al., 2000, 2005; Pauley et al., 2003). It has
been proposed that Fgf10 activity may regulate cell
migration and cell death during semicircular canal forma-
tion (Chang et al., 2004; Ohuchi et al., 2005), probably in
direct or indirect cooperation with FGF3 signals (Zelar-
ayan et al., 2007), BMP2 (Chang et al., 2004), and Foxg1
(Pauley et al., 2006). In the auditory system, Fgf10 is like-
wise expressed in the developing mouse cochlea (Pauley
et al., 2003; Pan et al., 2011). Descriptive studies and
gain-of-function experiments suggest that Fgf10 can
induce the acoustic-vestibular ganglion (Pirvola et al.,
2000; Pauley et al., 2003; Alsina et al., 2004; Miyazaki
et al., 2006; Lillev€ali et al., 2006). Taken together, these
results at the otocyst stage strongly support the hypothe-
sis that Fgf10 is involved in initial otic neurogenesis and
specification of sensory patches in the developing inner
ear (Pirvola et al., 2000; Ohuchi et al., 2005; Pauley et al.,
2003; Alsina et al., 2004; Lillev€ali et al., 2006; Pujades
et al., 2006; Pan et al., 2011; see also Abell�o and Alsina,
2007).
In order to gain insight into the differential sensory
patch specification taking place in this sensory organ
complex of vertebrates, in particular in birds, we charac-
terized in detail the spatial and temporal expression pat-
tern of the Fgf10 gene, held to be a marker of the chick
panneural otic area, during embryonic development
(Alsina et al., 2004; Pujades et al., 2006). The Bmp4 and
Serrate1 expression patterns are also considered to pro-
vide key landmarks in morphological interpretation of the
otic vesicle, being markers of all sensory patches (Wu
and Oh, 1996; Adam et al., 1998; Cole et al., 2000). Com-
parison of the expression pattern of Fgf10 with that of
other sensory markers—such as Cath1, Delta1, Hes5,
LFng, and BEN, among others—helped us to better under-
stand their individual roles in a context of multiple, and
possibly partly redundant, functions.
Taken together, the results obtained corroborate a pos-
sible contribution of the Fgf10 gene to normal patterning
and specification of the chick otic sensory epithelia. The
topologic position and relative timing of the molecular
specification events for each sensory epithelium are
determined more precisely in the developing chick inner
ear, a well-documented experimental model. In addition,
our descriptive analysis presents discussions of the evo-
lution of the vertebrate inner ear, strongly supporting the
pansensory subdivision hypothesis previously proposed
by Fritzsch and colleagues (Fritzsch and Beisel, 2001;
Frizsch et al., 2002, 2010).
MATERIALS AND METHODS
Tissue processingChick embryos were obtained from fertilized White
Leghorn chick eggs incubated in a humidified atmosphere
at 38�C. All embryos were treated according to the rec-
ommendations of the European Union and the Spanish
government for laboratory animals. Embryos ranging
between stages HH14 and HH41 (Hamburger and Hamil-
ton, 1951), were fixed by immersion, or via intracardiac
perfusion, with 4% paraformaldehyde in 0.1 M phosphate-
buffered saline solution (PBS; pH 7.4), at 4�C overnight.
The fixed embryos were rinsed and cryoprotected in 10%
sucrose solution in PBS and embedded in the same
TABLE 1.
Gene Probes Used for ISH and Their Principal Characteristics
Gene
symbol
NCBI
accession no. Size (bp) Position
Antisense probe
enzyme/polymerase Reference/laboratory
Bmp4 NM 205237.1 806 1–807 Cla1/T7 S�anchez-Guardado et al., 2009Bmp7 XM_417496.3 975 706–1,680 XhoI/T3 Merino et al., 1999Cath1 CR 40596.1 899 1,843–2,743 Not1/T3 EST clone: ChEST686k4Delta1 NM_204973.1 588 1,596–2,183 Not1/T3 Adam et al., 1998Fgf10 NM_204696.1 686 214–900 NcoI/Sp6 Alsina et al., 2004Fgf19 NM_204674.1 795 366–1,160 Not1/T3 EST clone: ChEST230k6FGFR3 NM_205509.2 677 1–678 BamHI/T7 Bermingham-McDonogh et al., 2001Hes5 NM_001012695.1 1188 1–1,188 Not1/T3 Fior and Henrique, 2005LFNG NM_204948.1 473 2,190–2,663 Not1/T3 EST clone: ChEST378f3Msx1 NM_205488.2 721 588–1,309 Not1/T3 EST clone: ChEST325e24Serrate1 XM 415035.3 706 436–1,141 HindIII/T7 Adam et al., 1998Sox2 NM_205188.1 552 462–1,013 SalI/T7 Uchikawa et al., 1999
S�anchez-Guardado et al.
1138 The Journal of Comparative Neurology |Research in Systems Neuroscience
buffered sucrose solution with added 10% gelatin. The
blocks were frozen for 1 minute in isopentane cooled to
�70�C by dry ice, and then stored at �80�C. Cryostat se-
rial sections 20 lm thick were cut in the transverse and
horizontal planes, mounted as parallel sets on SuperFrost
slides, and stored at �80�C until use. Twenty embryos
were used per stage.
RNA probes for in situ hybridizationIn situ hybridization (ISH) was performed on cryosec-
tions according to S�anchez-Guardado et al. (2009). The
chick Fgf10 subclone was kindly provided by Fernando
Giraldez. Plasmid information is provided in Table 1. All
riboprobes were labeled with digoxigenin-11-UTP (Roche,
Mannheim, Germany) according to the manufacturer’s
instructions.
In situ hybridizationThe frozen sections were postfixed for 10 minutes with
4% paraformaldehyde in PBS, and then washed with PBS
for 30 minutes. They were then acetylated in a well-mixed
solution containing 3.2 ml triethanolamine, 600 ll acetic
anhydride, 420 ll HCl (36%), and 234 ll H2O for 10
minutes at room temperature, permeabilized subse-
quently in 1% Triton X-100 for 30 minutes, and washed
twice with PBS for 15 minutes. Thereafter, prehybridiza-
tion was allowed to occur at room temperature for 2
hours in a solution containing 50% formamide, 10% dex-
tran sulfate, 5X Denhardt’s solution, 250 mg/ml yeast
tRNA, and salt solution. The salt solution contained 0.2 M
NaCl, 10 mM Tris-HCl, 1 mM Tris-Base, 5 mM NaH2PO4-
2H2O, 5 mM Na2HPO4, and 0.5 M EDTA. Digoxigenin-la-
beled RNA probes were diluted in the prehybridization so-
lution (200–300 ng/ml). Diluted probe solution was
heated for 5 minutes at 80�C, and then cooled on ice for
5 minutes before addition of 200 ll to each slide. The
slides were next covered with coverslips. Hybridization
was performed overnight in a humidified horizontal cham-
ber at 72�C. After hybridization, the sections were
washed with 0.2X standard saline citrate (SSC) at 72�C
for 1 hour and rinsed twice in 0.2X SSC at room tempera-
ture. Then the sections were washed with a solution con-
taining 100 mM NaCl, 0.1% Triton X-100, and 100 mM
Tris-HCl (pH 7.5). After treatment with 10% normal goat
serum (Sigma, St. Louis, MO) and 0.1 M lysine monohy-
drochloride in the same solution for 2 hours, to block the
tissue, the sections were incubated overnight with alka-
line phosphatase–conjugated antidigoxigenin Fab frag-
ments (1:3,500; Roche). The sections were washed twice
with the same buffer and then incubated in 100 mM
NaCl, 50 mM MgCl2, 0.1% Tween 20, and 100 mM Tris-
HCl (pH 9.5). For the coloring reaction, nitroblue tetrazo-
lium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate
(BCIP) were used as substrates (Roche). After ISH, the
sections were washed three times in PBS and 0.05% Tri-
ton X-100 (PBS-T) and coverslipped with Mowiol (Calbio-
chem, San Diego, CA). The Mowiol solution contains 30%
glycerol, 12% polyvinyl alcohol 40-88 (Fluka, Buchs, Swit-
zerland), and 120 mM Tris-HCl (pH 8.5). The correspond-
ing sense probes did not yield any signal.
Immunohistochemistry staining procedureThe immunoreactions were performed following the
indications of S�anchez-Guardado et al. (2009, 2011).
Briefly, cryosections were washed in PBS and incubated
with a solution containing 0.1 M lysine monohydrochlor-
ide, 1% normal goat serum (NGS), and 0.25% Triton X-100
in PBS, to reduce the nonspecific background. Sections
were incubated with 3A10 or anti-BEN monoclonal anti-
bodies. The primary antibody was reacted with biotinyl-
ated goat anti-mouse secondary antibody (1:100; Sigma),
and then with ExtrAvidin-biotin-horseradish peroxidase
complex (1:200; Sigma). All antibodies were diluted in a
solution containing 1% NGS and 0.25% Triton X-100 in
PBS. The incubations were performed without coverslips,
and were terminated by rinsing three times with PBS-T.
The histochemical detection of the peroxidase activity
was carried out by using 0.03% diaminobenzidine (DAB)
and 0.005% H2O2. After the immunoreactions, the sec-
tions were rinsed three times with PBS-T and then cover-
slipped with Mowiol.
Antibody characterizationAntibody information is provided in Table 2. The mono-
clonal antibody 3A10 was obtained by using as immuno-
gen ventral spinal cord/cyclophosphamide treatment/
assorted nervous tissue (from chick nervous tissue). It
has been used as a marker for differentiating neurons in
the developing chick nervous system (Yamada et al.,
1991; Storey et al., 1992; Hill et al., 1995; Perez et al.,
1999). In the developing inner ear, the 3A10 antibody is a
useful tool to label axons of the acoustic-vestibular gan-
glion (AVG) neurons and to aid in the identification of pre-
sumptive sensory epithelia (Adam et al. 1998; S�anchez-
Calder�on et al. 2004, 2005, 2007b; Battisti and Fekete,
2008; S�anchez-Guardado et al., 2009, 2011; see also
Sienknecht and Fekete, 2008, 2009). The detailed immu-
nization procedure used to generate the BEN Mab mono-
clonal antibodies from the bursa of Fabricius was previ-
ously described (Pourqui et al., 1990; Bardet et al.,
2006). The anti-BEN monoclonal antibodies recognized a
cell adhesion molecule of the Ig superfamily (also known
as SC-1 or DM-GRASP) at the cell surface of the peripher-
ally projecting neurons (Pourqui et al., 1990) and olivocer-
ebellar projections (Ch�edotal et al., 1996). BEN immuno-
reactivity was also detected in the otic epithelium and
Fgf10 in the developing chick inner ear
The Journal of Comparative Neurology | Research in Systems Neuroscience 1139
axons of the developing chick inner ear (Goodyear et al.,
2001).
ImagingAll preparations were photographed with a Zeiss Axio-
phot microscope equipped with a Zeiss AxioCam camera
(Carl Zeiss, Oberkochen, Germany) and AxioVision
2.0.5.3. software, and the images were saved in 4-MB
TIFF format. These were size-adjusted, cropped, contrast-
enhanced, and annotated with Adobe Photoshop version
7.0 software (Adobe Systems, San Jose, CA). All illustra-
tions were produced with Adobe Photoshop software.
RESULTS
Fgf10 expression pattern at the late oticvesicle stage
It is well known that Fgf10 is expressed in an antero-
medial domain of the otic placode, and that, at the otic
vesicle stage, it labels early on in the otic prosensory do-
main (Alsina et al., 2004; Pujades et al., 2006). To charac-
terize the distribution of Fgf10 transcripts in the chick
otic epithelium as development proceeds, we performed
a detailed study by using in situ hybridization on trans-
verse and horizontal serial sections through the develop-
ing inner ear between stages HH18 (2.5 days of incuba-
tion) and HH38 (12th day of incubation).
At stages HH18–21, serial transverse sections showed
an unbroken anteroposterior band of Fgf10 expression,
located in the ventromedial part of the otic anlage (Fig. 1A–
D). This continuous domain was best observed in a horizon-
tal section (Fig. 1E). The Fgf10 expression was stronger at
the rostral than at the caudal level (Fig. 1E). The Fgf10-la-
beled domain included at least the presumptive territories
of the anterior crista, which was already innervated by
3A10-immunoreactive otic dendrites (ac; Fig. 1A,E), and the
posterior crista (pc; Fig. 1C,D). It is well known that Ser-
rate1 (a ligand of Notch receptors) is also involved in the
patterning of the vertebrate otocyst (Adam et al., 1998;
Cole et al., 2000; Kiernan et al., 2001; Brooker et al.,
2006), defining the otic prosensory domain long before the
differentiation of hair cells occurs (Abell�o and Alsina, 2007).
A single domain of Serrate1 expression likewise foreshad-
ows the appearance of sensory patches in the chick inner
ear rudiment (Adam et al., 1998; Cole et al., 2000).
At stages HH18–20, comparison of the Fgf10 and Ser-
rate1 expression domains in horizontal sections showed
that Serrate1 labels a long anteroposterior band (Fig. 1G),
in which the Fgf10-positive band was included (compare
arrowheads in Fig. 1F,G). Sox2 appears to be involved in
the development of inner ear sensory domains (Uchikawa
et al., 1999; Kiernan et al., 2005, 2006; Daudet et al.,
2007; Hume et al., 2007; Neves et al., 2007, 2011, 2012;
TABLE2.
AntibodiesUsedin
This
Study
Antibody
Antigen
Immunogen
Manufacturer,species
antibodywasraisedin,
mono-vs.polyclonal,
cat.no.
Dilu
tion
Antibody
IDfrom
NIF
Antibodylin
kReference
3A
10
Neuro
fila
ment
ass
oci
ate
dN
am
e:
chic
knerv
ous
tiss
ue
Ori
gin
:ve
ntr
al
spin
al
cord
/cy
clop
hosp
ham
ide
treatm
ent/
ass
ort
ed
nerv
ous
tiss
ue
Deve
lop
menta
lS
tud
ies
Hyb
rid
om
aB
ank
(DS
HB
),m
ouse
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onocl
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#3
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31
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4htt
p:/
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bod
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org
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B_5
31
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BEN
Peri
phera
lp
roje
ctin
gneuro
ns,
clim
bin
gfib
ers
,hem
op
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tic
pre
curs
ors
,im
matu
reand
act
ivate
dT
lym
phocy
tes
Nam
e:
burs
aof
Fab
rici
us
Ori
gin
:ch
ick
em
bry
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icole
Le
Douari
nand
Oliv
ier
Pourq
ui� e
,D
SH
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mouse
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onocl
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#B
EN
1:1
00
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p:/
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shb
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wa.e
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2P
ourq
ui� e
et
al.,
19
90
;B
ard
et
et
al.,
20
06
.
Anti
-mouse
IgG
(whole
mole
cule
),b
ioti
nco
nju
gate
Mouse
IgG
Nam
e:
puri
fied
mouse
IgG
Ori
gin
:m
ouse
Sig
ma,
goat,
poly
clonal,
#B
72
64
1:1
00
N/
Ahtt
p:/
/w
ww
.sig
maald
rich
.com
/ca
talo
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pro
duct
/si
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4?l
ang¼
es&
regio
n¼
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Guard
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al.,
20
11
.
S�anchez-Guardado et al.
1140 The Journal of Comparative Neurology |Research in Systems Neuroscience
Dabdoub et al., 2008). At the HH18 stage, Sox2-express-
ing cells were detected in the anteromedial domain of the
otic rudiment (Fig. 1H). The rostral border of the Sox2-
positive domain was coincident with that of the Fgf10-
positive domain, but not its caudal border (compare
arrowheads in Fig. 1F,H). As development proceeded, the
Sox2 expressing domain extended caudally to include the
presumptive territory of the posterior crista at stage
HH21 (Fig. 1K), besides the anterior crista (Fig. 1L).
The Atoh1 gene, a close homologue of Atonal in Dro-
sophila, is a proneural bHLH gene expressed in sensory
patches in the developing vertebrate inner ear. Math1
and Cath1 genes are required for differentiation of sen-
sory hair cells in different phyla (Bermingham et al., 1999;
Zheng and Gao, 2000; Chen et al., 2002; Kawamoto et al.,
2003; Fritzsch et al., 2005; Matei et al., 2005; Pujades
et al., 2006; Millimaki et al., 2007, 2010; see also Fritzsch
et al., 2006, 2010). At stages HH20–21, horizontal sec-
tions through the developing avian inner ear showed scat-
tered Cath1-positive cells in the innervated anterior crista
(ac; Fig. 1I) and in the developing posterior sensory patch,
which contains the posterior crista (pc; Fig. 1I; Wu and
Oh, 1996). These Cath1-expressing cells were contained
within two separate Bmp4-expressing areas (Fig. 1J). At
stage HH21, the lateral borders of the territories express-
ing Fgf10, Cath1, Sox2, and Bmp4 were coincident (see
arrowheads in Fig. 1I–L; Fgf10, not shown). At this
moment, other sensory elements are not yet clearly speci-
fied (Oh et al., 1996; Wu and Oh, 1996; S�anchez-Calder�on
et al., 2004, 2005, 2007b; S�anchez-Guardado et al.,
2009, 2011). Figure 1M and N shows 3D diagrams of an-
terior and posterior views, respectively, of an otic vesicle
(HH21), summarizing all these results.
Fgf10, Cath1, and Bmp4 expressionpatterns at stage HH24
At stage HH24, the inner ear undergoes significant mor-
phogenetic changes. Morphologically, we can distinguish a
vertical part of the otic pouch, located dorsolaterally (vp;
Fig. 2A–D) from a horizontal part of the pouch, found later-
ally (hp; Fig. 2C,D). The endolymphatic duct and sac de-
velop in the dorsomedial portion of the otic anlage (ed and
es; Fig. 2B–D), whereas the cochlear duct can be clearly
observed in its ventral portion (cd; Fig. 2D,E). The differen-
tiation of some sensory epithelia starts to be more evident,
as revealed by Bmp4 expression (Fig. 2M–Q; Oh et al.,
1996; Wu and Oh, 1996). At this developmental stage, the
prospective territory of the macula utriculi can be barely or
inconstantly identified by a very low Bmp4 signal (mu; Fig.
2N), which appears adjacent to the lateral crista, as
observed in transverse sections (mu, lc; Fig. 2B,G,N). The
macula lagena and the macula neglecta cannot be easily
recognized at this stage by using this marker (Wu and Oh,
1996; see also S�anchez-Calder�on et al., 2004, 2005,
2007b; S�anchez-Guardado et al., 2009, 2011). However,
Bmp4 is not a specific marker of sensory patches, because
it is also expressed in nonsensory tissues, such as the
Figure 1. Fgf10 expression pattern at the otic vesicle stage.
A–D: Transverse sections through stage HH21 inner ear from ros-
tral (A) to caudal (D) levels, treated with the Fgf10 probes and
3A10 antibodies. Fgf10 expression showed a rostrocaudal band
located in the ventromedial portion of the otic anlage (A–D), better
observed in a corrected horizontal section at stage HH18
(E, indicated in M and N). E–L: Horizontal sections through stages
HH18 (E–H) and HH21 (I–L), treated with the indicated markers. At
stage HH18, the Fgf10-positive band was included within a Ser-
rate1-expressing domain (see arrowheads in F,G), whereas the
Sox2-expressing domain was included within the anteromedial por-
tion of the Fgf10-positive band (compare arrowheads in E,H). At
stage HH21, the Fgf10-expressing domain contained the anterior
and posterior cristae, Cath1-, Sox2-, and Bmp4-positive (I–L). M,N:
3D diagrams of Fgf10, Serrate1, and Cath1 expression patterns, as
well as weak and strong expression of Bmp4, at the otic vesicle
stage. The horizontal sections are indicated in M and N. For abbre-
viations, see list. Orientation: C, caudal; D, dorsal; M, medial; R, ros-
tral. Scale bar ¼ 14 lm in B (applies to A–D); 8 lm in H (applies to
E–H); 9 lm in K (applies to I–L).
Fgf10 in the developing chick inner ear
The Journal of Comparative Neurology | Research in Systems Neuroscience 1141
semicircular canals and an abneural portion of the coch-
lear duct (see below; large arrows in Fig. 3P; see also Oh
et al., 1996; Wu and Oh, 1996).
Cath1 expression identifies the prospective sensory
patches better in the developing inner ear. Cath1 was
expressed in the anterior and posterior cristae (ac and
pc; Fig. 2F,L), the first sensory epithelia to be differenti-
ated (Wu and Oh, 1996). Some scattered Cath1-positive
cells were detected in the lateral crista (lc; arrows in Fig.
2G,H) and in the macula sacculi (ms; arrows in Fig. 2I,J).
Neither the macula utriculi nor the basilar papilla, the lat-
ter located in the cochlear duct, showed significant Cath1
expression (mu and bp; Fig. 2G,K). However, the pres-
ence of a few cells expressing Cath1 weakly cannot be
completely disregarded.
As regards Fgf10, almost all sensory epithelia differen-
tiated at this stage showed Fgf10 expression (Fig. 2A–E).
The continuous anteroposterior band of strong Fgf10
expression described at the otic vesicle stage (Fig. 1)
apparently was still continuous across the transverse sec-
tion series at HH24 (Fig. 2A–E). Interestingly, the lateral
crista, identified by the presence of a reduced number of
Cath1-positive cells (arrows in Fig. 2G,H), seemed to lie
outside of the Fgf10-positive domain (lc; compare arrow-
heads in Fig. 2B,G,N).
Horizontal sections of the otic epithelium allowed us to
corroborate the Fgf10, Cath1, and Bmp4 expression pat-
terns described at stage HH24 (Fig. 3). Fgf10 transcripts
were observed in several, but not all, recognized sensory
elements (ac, mu, ms, bp, and pc; Fig. 3A–E); the macula
lagena and macula neglecta are not yet identified (Oh
et al., 1996; Wu and Oh, 1996). In the anterior portion of
the otic primordium, a small area of the Fgf10-expressing
domain showed weaker Fgf10 expression, possibly sug-
gesting that the anteroposterior Fgf10-positive band
starts to be subdivided, and the macula utriculi and mac-
ula sacculi begin to be separated, at this stage (arrows in
Fig. 3C,D).
As mentioned above, Cath1 expression was clearly
detected in the anterior and posterior cristae (ac and pc;
Fig. 3F,G), whereas scattered Cath1-expressing cells
were observed in the lateral crista and macula sacculi (lc
and ms; arrows in Fig. 3I–K). It is interesting to note that,
again, the presumptive lateral crista domain, which was
weakly Bmp4-positive (lc; Fig. 3P,Q), was excluded from
the Fgf10-expressing area (Fig. 3C,D). At this level, the
Fgf10-expressing area showed a sharp border just adja-
cent to the lateral crista (compare arrowheads in Fig.
3C,I,J,P).
To confirm whether the Fgf10-expressing domain
excludes the presumptive territory of the lateral crista at
stage HH24, we used several other markers of sensory
patches. Fgf19 expression was described in the macula
utriculi, with a stronger expression at its lateral border,
abutting the Fgf19-negative lateral crista (mu; Fig. 4B;
S�anchez-Calder�on et al., 2007b). Thus, Fgf19 expression
Figure 2. Fgf10, Cath1, and Bmp4 expression patterns at stage
HH24. Transverse sections through the inner ear. The probes used
are noted in each column. All sections were treated with 3A10
immunoreactions. A–E: Fgf10 expression labeled several sensory
epithelia: the anterior crista (ac; A), the macula utriculi (mu; B), the
macula sacculi (ms; C), the basilar papilla (bp; D), and the posterior
crista (pc; E). F–Q: The lateral crista (lc; B,G,N) was devoid of Fgf10
expression (lc; B). Some of these sensory epithelia were Bmp4-posi-
tive (M–Q). Cath1 transcripts were clearly observed in the anterior
and posterior cristae (ac and pc; F,L). Scattered Cath1-positive
cells were detected in the lateral crista and macula sacculi (lc and
ms; arrows in G–J). The macula utriculi and the basilar papilla did
not show any Cath1 expression (mu and bp; G,K). The arrowheads
in B and G point to the border between Fgf10 and Cath1 expres-
sion. The horizontal sections shown in Figure 3 are also indicated.
For abbreviations, see list. Orientation: D, dorsal; M, medial. Scale
bar ¼ 15 lm in J (applies to H,J); 28 lm in Q (applies to A–G,I,K–
Q). [Color figure can be viewed in the online issue, which is avail-
able at wileyonlinelibrary.com.]
S�anchez-Guardado et al.
1142 The Journal of Comparative Neurology |Research in Systems Neuroscience
is a good marker of the border between the Fgf19-nega-
tive lateral crista and the Fgf19-positive macula utriculi.
The lateral borders of both the Fgf10- and the Fgf19-posi-
tive domains were coincident (arrowheads in Fig. 4A,B),
thus corroborating that the lateral crista is Fgf10 nega-
tive. The Hes5 gene, belonging to the hairy and enhancer
of split family of genes, encodes a bHLH transcription fac-
tor, which is considered to be necessary for inner ear
development (Adam et al., 1998; Zine et al., 2001;
Kelly and Chen, 2007). In the developing chick inner ear,
Hes5 is expressed in cells within the proneural domain at
the otic cup stage (Abell�o et al., 2007). Also, the Notch
ligand Delta1 is clearly or strongly expressed in differenti-
ating sensory patches during chick inner ear development
(Adam et al., 1998; Bryant et al., 2002). In our chick stage
HH24 embryos, the lateral borders of both Hes5 and
Delta1 expression domains were also coincident with
those of Fgf10 and Fgf19 genes (arrowheads in Fig. 4A–
D,G). Interestingly, strong Serrate1 expression (Fig. 4F)
was detected in the Fgf10-, Fgf19-, Hes5-, and Delta1-
expressing domains (Fig. 4A–D,G). Moreover, the weakly
Serrate1-expressing territory extended far from the men-
tioned limit of those expression patterns (arrowhead in
Fig. 4F), similarly to the case of Bmp4 expression (large
arrow in Fig. 3P).
Other evidence confirming the conclusion that the lat-
eral crista is early on excluded from the Fgf10-expressing
domain derives from the expression patterns of Sox2,
Lunatic Fringe (LFng), and the cell adhesion molecule
BEN, three postulated markers of otic sensory epithelium
specification. At stage HH24, Sox2 expression was also
absent in the lateral crista (arrowhead in Fig. 4H). LFng, a
vertebrate homologue of Drosophila fringe, is implicated
in positioning boundaries during embryogenesis (Laufer
et al., 1997; Rodrıguez-Esteban et al., 1997). This gene is
also involved in inner ear patterning (Laufer et al., 1997;
Adam et al., 1998; Cole et al., 2000). It has been reported
that its expression in the chick lateral crista is still absent
at stages HH23/24, but is evident at stage HH25 (Cole
et al., 2000). BEN labels a band in which chick sensory
epithelia develop, but its direct implication in lateral
crista specification is not clearly demonstrated at stage
Figure 3
Figure 3. Fgf10, Cath1, and Bmp4 expression patterns at stage
HH24. Horizontal sections through the inner ear. A–E: Fgf10
expression was detected in some, but not all, sensory elements:
anterior crista (ac; B), posterior crista (pc; A), macula sacculi (ms;
C and D), macula utriculi (mu; C and D), and basilar papilla (bp; C–
E). The lateral crista (lc; C,D) and the macula neglecta (mn; B; for
its innervation, see G,H) were Fgf10-negative (B) and Bmp4-positive
(O). The short arrows in C and D point to the incipient weakly
Fgf10-expressing area between the macula utriculi and macula sac-
culi. F–R: Note the strong Cath1 expression observed in the ante-
rior and posterior cristae (ac and pc; F and G), whereas some
Cath1-expressing cells were detected in the lateral crista and mac-
ula sacculi (lc and ms; short arrows in I–K). The arrowheads in C–E
and P–R point to the border of the Fgf10-expressing territory. In P,
the large arrow points to the Bmp4 expressing area adjacent to
the lc. The horizontal sections shown in Figure 2 are also indi-
cated. For abbreviations, see list. Orientation: A, anterior; M,
medial. Scale bar ¼ 15 lm in K (applies to H,J,K); 33 lm in R
(applies to A–G,I,L–R). [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]
Fgf10 in the developing chick inner ear
The Journal of Comparative Neurology | Research in Systems Neuroscience 1143
HH24 (Goodyear et al., 2001). Our horizontal sections
across the stage HH24 otic rudiment showed that the
prospective lateral crista was clearly excluded from the BEN-
and LFng-positive domains (see arrowheads in Fig. 4J,L).
In the caudal part of the otic rudiment, the continuous
dorsoventral band of Bmp4 expression, which has been
proposed to develop the caudalmost otic sensory
elements in the chick (posterior crista, macula neglecta,
Figure 4. Specification of lateral crista, macula utriculi, and macula neglecta at stage HH24. Horizontal sections treated with the indi-
cated markers. A–L: The lateral border of Fgf10 (A,D), Fgf19 (B), Hes5 (C), Delta1 (G), Sox2 (H), BEN (J), and LFng (L) staining domains
were coincident, labeling the utriculi (mu)/lateral crista (lc) boundary (arrowheads). This boundary coincided with the border of strong Ser-
rate1 expression (arrowhead in F). The lateral crista expressed the Serrate1 gene weakly (F). The presumptive territory of the macula
neglecta (mn in D–L), innervated by 3A10- (arrowhead in E) and BEN-staining (arrowheads in K) axons, was Fgf10-negative and Serrate1-
positive (mn; D,F). However, a few chick embryos showed very low levels of Fgf10 and Sox2 expression in the future macula neglecta
(H,I). For abbreviations, see list. Orientation: M, medial; R, rostral. Scale bar ¼ 50 lm in G (applies to A–D,F,G); 15 lm in K (applies to
E,I,K); 40 lm in L (applies to H,J,L). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
S�anchez-Guardado et al.
1144 The Journal of Comparative Neurology |Research in Systems Neuroscience
basilar papilla, and macula lagena; Wu and Oh, 1996), did
not correspond exactly with the Fgf10-expressing domain
(compare Fig. 3A–E and N–R). There was an Fgf10-nega-
tive gap (Fig. 3B,O) between the posterior crista in the
vestibule (pc; Fig. 3A,N) and the Bmp4-positive zone in
the cochlear duct (Fig. 3C,P). This small area expresses
Bmp4 weakly (Fig. 3O), and is innervated by pioneering
3A10-positive fibers (Fig. 3G,H), although no Cath1-
expressing cells were observed within it (Fig. 3G,H). It is
tempting to assign the future macula neglecta to this
Bmp4-positive and Fgf10-negative domain (mn; Fig.
3B,O), because it is located always just adjacent and
slightly ventral and medial to the posterior crista (Wu and
Oh, 1996; see also S�anchez-Calder�on et al., 2004, 2005,
2007b; S�anchez-Guardado et al., 2009, 2011). Thus, the
macula neglecta, similarly to the lateral crista, seems to
be excluded from the derivatives of the continuous otic
anteroposterior band of Fgf10 expression observed at
stages HH18–21 (Fig. 1).
At stage HH24, a patch of Serrate1 expression possibly
corresponding to the macula neglecta anlage was
observed in the caudal part of the inner ear (mn; Fig. 4F).
At this developmental stage, most of the chick embryos
treated with the Fgf10 and Sox2 probes showed no
expression for Fgf10 and Sox2 genes (Fig. 4D,E; for Sox2,
not shown), although a few embryos started to express
very low levels of expressions for these genes (Fig. 4H,I).
BEN-immunoreactive otic dendrites have arrived at this
caudal Serrate1-positive domain (mn in Fig. 4F,J; arrow-
heads in K), located just ventral to the BEN-positive pos-
terior crista (not shown). This Serrate1-positive area
lacked expression of other sensory epithelial markers
(Hes5, Delta1, and LFng; Fig. 4C,G,L). Because this por-
tion of the otic epithelium receives otic fibers from the
acoustic-vestibular ganglion (Figs. 3G,H, 4D–L), and
Fgf10 and Sox2 expression has started to be observed,
one may consider it to be the presumptive territory of a
sensory element, although no differentiated hair cells
were detected within it at this developmental stage (see
Fig. 3G,H for Cath1 expression). Therefore, we tentatively
consider it to represent the future macula neglecta.
Figure 5 summarizes the Fgf10 expression pattern in
the inner ear at stage HH24. The Fgf10-expressing do-
main contains several sensory epithelia: the anterior
crista (ac), the macula utriculi (mu), the macula sacculi
(ms), the basilar papilla (bp), and the posterior crista (pc).
The area showing weak Fgf10 expression between the
macula utriculi and macula sacculi is also indicated
(arrowhead in Fig. 5A). The expression patterns of all the
studied genes strongly suggested that the boundary
between the macula utriculi and lateral crista was well
defined at stage HH24. The lateral crista lies outside the
superposed Fgf10-, Fgf19-, Hes5-, and Delta1-expressing
domains, but is Serrate1-, Bmp4-, and Cath1-positive. The
possible location of the macula neglecta, identified by
Serrate1 expression outside the Fgf10-positive domain,
was also suggested (mn; Fig. 5B). At this developmental
stage, none of the known markers identifies the prospec-
tive macula lagena (see below for subsequent develop-
mental stages).
Fgf10 expression and sensory organspecification at stage HH25–26
As development proceeds to 4.5–5 days of incubation
(stages HH24þ–26), changes in the expression patterns
of the genes considered above became apparent with
respect to the developing lateral crista domain (Fig. 6).
Fgf10 expression was now observed in the presumptive
territory of the lateral crista (lc; Fig. 6A,B, stage HH24þ;
Fig. 6G,H, stage HH25; Fig. 6M, stage HH26), with an evi-
dent beginning of expression in the stage HH24þembryos (see the low amount of Fgf10 transcripts in Fig.
6B,H; arrows). The lateral crista showed a greater number
of Cath1-expressing cells (Fig. 6C,D) than at the previous
stage (HH24; Figs. 2G, 3I,J). In contrast to the status
described at the previously analyzed stage (HH24; Fig. 4),
expression of both Hes5 and Delta1 now extended later-
ally into the macula utriculi/lateral crista boundary (small
arrowheads in Fig. 6E,F,K,P; see also Fig. 4C,G), this bor-
der still being clearly defined by the Fgf19 expression do-
main (not shown; S�anchez-Calder�on et al., 2007b). With
respect to the Sox2 gene, its expression was not yet
Figure 5. A,B: 3D diagrams of the chick otic anlage at stage
HH24 (A, anterior view; B, posterior view) summarizing the Fgf10
and Cath1 expression patterns. The weak and strong levels of
Bmp4 expression are also distinguished. The lateral crista (lc; A)
and the macula neglecta (mn; B) were Fgf10-negative. Note Fgf10
downregulation between the utricular and saccular maculae
(arrowhead in A). The horizontal sections shown in Figure 3 are
also indicated. For abbreviations, see list. Orientation: C, caudal;
D, dorsal; M, medial; R, rostral.
Fgf10 in the developing chick inner ear
The Journal of Comparative Neurology | Research in Systems Neuroscience 1145
Figure 6. Specification of the lateral crista, utricular and saccular maculae, and macula neglecta at stage HH24þ-26. Horizontal (A–P)
and transverse (Q–V) sections treated with the probes indicated in each picture and 3A10 immunoreactions. The lateral crista/macula
utriculi boundary is indicated (arrowheads in A–H, J–M, O,P). Fgf10 (A,B,G,H,M), Cath1 (C,D), Hes5 (E,F,P), and Delta1 (K) were observed in
the developing lateral crista (lc). Sox2 expression was also detected in the lateral crista at stage HH26 (O), but not at stage HH25 (L).
These expressions were included in a larger domain of Serrate1 expression (arrow in J). The separation between the macula utriculi and
macula sacculi was also evident (large arrowheads in G,J,L,M,O,P). A small portion of the Serrate1-positive macula neglecta (mn; J) showed
very low levels of Fgf10 (mn; G,I,M,N) and Sox2 (mn; L,O) expression, being devoid of Delta1 (not shown), and Hes5 (mn; P). In transverse
sections (Q–V), the macula lagena, Msx1-positive (ml; U), was included in an Fgf10- (Q), Serrate1- (R), and Bmp7- (S) expressing domain,
but was Sox2- and FGFR3- negative (T,V). The arrowheads in Q–V point to the basilar papilla/macula lagena border. For abbreviations, see
list. Orientation: D, dorsal; M, medial; R, rostral. Scale bar ¼ 15 lm in F (applies to B,D,F); 12 lm in H (applies to H,K,I,N); 40 lm in P
(applies to A,C,E,G,J,L,M,O,P); 60 lm in U (applies to Q–V).
Figure 7. Fgf10, Cath1, and Bmp4 expression patterns at stage HH27. Transverse sections. The probes used are noted in each column.
All sections were treated with 3A10 immunoreactions. A–D: Fgf10 expression was observed in all sensory elements, and was less evident
in the macula neglecta (mn; D). The arrowheads in B and C point to areas with low expression of Fgf10. E–G: Cath1-expressing cells were
detected in the cristae (ac, lc, and pc; E,G,I), the macula utriculi (mu; F,G), and the macula sacculi (ms; G). H,I: A few Cath1-positive cells
were observed in the basilar papilla (not shown), but none in the macula lagena (ml; H) or the macula neglecta (mn; I). J–M: Different lev-
els of Bmp4 expression were observed within most sensory patches. The horizontal sections shown in Figure 8 are also indicated. For
abbreviations, see list. Orientation: D, dorsal; M, medial. Scale bar ¼ 60 lm in M (applies to A–M). [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
Fgf10 in the developing chick inner ear
The Journal of Comparative Neurology | Research in Systems Neuroscience 1147
Figure 8. Fgf10, Cath1, and Bmp4 expression patterns at stage HH27. Horizontal sections. The probes used are noted in each column. All
sections were treated with 3A10 immunoreactions. The expression patterns of Fgf10 (A–G), Cath1 (H–N), and Bmp4 (O–U) genes are shown.
Note the gap of Fgf10 expression between the anterior crista (ac; A) and the ventrally placed lateral crista (lc; D) and macula utriculi (mu; D;
large arrowhead in B). The lateral crista started to be separate (small arrowhead in D). The separation between the macula utriculi (mu) and
the macula sacculi (ms) was evident (large arrowhead in D). Fgf10 expression was included in all sensory epithelia, the macula neglecta
showing very low levels of Fgf10 expression in its dorsalmost portion (mn; B,C) but not in the caudal portion (mn; D). Cath1 expression was
detected in all cristae, the utricular and saccular maculae, and the basilar papilla (H–M), the macula lagena being devoid of Cath1-expressing
cells (ml; N). The arrowheads in F, M, and T point to the border of Fgf10 expression. The arrow in S points to a Bmp4-positive domain in the
caudal otic epithelium, which was devoid of Fgf10 and Cath1 expression (see also arrows in E,L). The horizontal sections shown in Figure 7
are also indicated. For abbreviations, see list. Orientation: M, medial; R, rostral. Scale bar ¼ 30 lm in J (applies to C,J,Q); 63 lm in T (applies
to A,B,D–I,K–P,R–U). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
evident at stage HH25 (Fig. 6L), but it was so at stage
HH26 (Fig. 6O). Note that Serrate1 expression was main-
tained in the lateral aspect of the stage HH25 otic anlage
(short arrow in Fig. 6J).
Concerning specification of the utricular and saccular
maculae, the downregulation of Fgf10 between their pre-
sumptive domains was more evident now (large arrow-
heads in Fig. 6G, stage HH25; Fig. 6M, stage HH26) than
before (stage HH24; arrows in Fig. 3C,D). The advanced
specification of these two maculae was also confirmed by
the restricted expression of Serrate1, Sox2, and Hes5 in
two separate patches (see large arrowheads in Fig.
6J,L,O,P).
Regarding the prospective macula neglecta in the cau-
dal aspect of the stage HH25/26 inner ear (Fig. 6G,I,J,M–
P), this sensory element showed some Fgf10- and Sox2-
positive cells in a small portion of its presumptive domain
(mn; Fig. 6G,I,L,N,O), being always located within a Ser-
rate1-positive territory (mn; Fig. 6J). At these stages, this
sensory patch was devoid of Delta1 (not shown) and
Hes5 expression (mn; Fig. 6P).
Concerning the developing macula lagena, we checked
whether its presumptive domain was included within an
Fgf10-expressing domain at stages HH25–26, using addi-
tional molecular markers. BMP7, another member of the
bone morphogenetic protein family, is involved in specifi-
cation of the vestibular and auditory sensory patches,
and its expression has been reported to label the macula
lagena and basilar papilla jointly (Oh et al., 1996; see Fig.
6S). Msx1, coding a homeobox-containing transcription
factor, is considered to be a marker of the macula lagena
(Wu and Oh, 1996). Msx1 transcripts were exclusively
detected in this sensory patch, present at the end of the
cochlear duct, but not in the basilar papilla (Fig. 6U).
Comparing Bmp7 and Msx1 expression with that of Fgf10,
we concluded that, at stages HH25–26, the presumptive
macula lagena develops within continuous Fgf10-positive
and Serrate1-positive domains (Fig. 6Q,R). At this devel-
opmental stage, the macula lagena was, however, devoid
of Sox2 and FGFR3 expression (Fig. 6T,V), the latter being
a restricted marker of the basilar papilla (Bermingham-
McDonogh et al., 2001). This may also be the case for
earlier stages.
Fgf10, Cath1, and Bmp4 expression patternsat stage HH27
At stage HH27, the morphogenetic progress was more
evident, and all the sensory epithelia could be easily rec-
ognized by conventional markers and/or sensory patch
innervation. Transverse sections through the stage HH27
otic anlage showed that Fgf10 expression was strongly
present in all sensory elements (Fig. 7A–D). The
innervated macula neglecta (mn; Fig. 7D), which was in
close proximity to the Fgf10-positive posterior crista (pc;
Fig. 7D), showed Fgf10 expression in its dorsal part (not
shown; see Fig. 8B,C) but not in its ventral part (Fig. 7D;
see also Fig. 8D). These sections also showed that the an-
terior crista was quite separated from the nearest vestib-
ular sensory patches (not shown), a feature that was
more noticeable in horizontal sections (see below; Fig. 8).
Nevertheless, the lateral crista and the macula lagena
have not yet finished separating from their closest sen-
sory elements (the macula utriculi and the basilar papilla,
respectively) at this developmental stage, although a
decrease of Fgf10 expression in the intervening territories
can be observed (arrowheads in Fig. 7B,C). Interestingly,
the presumptive domains of the macula sacculi and the
basilar papilla were included in a continuous dorsoventral
band with homogenous Fgf10 expression (Fig. 7B).
With respect to Bmp4 expression, high levels of Bmp4
transcripts were observed in all cristae and the macula
sacculi (ac, lc, pc, ms; Fig. 7J,K,M), whereas only weak
expression was detected in the macula utriculi, basilar
papilla, and the macula lagena (mu, bp, ml; Fig. 7K,L).
Fgf10 expression was, accordingly, a better marker of
developing sensory elements than Bmp4 expression.
Cath1-expressing cells were present in all cristae (Fig.
7E,G,I), in a small area of the macula utriculi (mu; Fig.
7F,G), and in the entire macula sacculi (ms; Fig. 7G). The
basilar papilla showed very few Cath1-labeling cells (not
shown in transverse sections; see Fig. 8M). Cath1 expres-
sion was absent at the macula lagena (ml; Fig. 7H) and
the macula neglecta (mn; Fig. 7I). The restricted location
of Cath1-positive cells in some, but not all, developing
sensory elements contrasts with the arrival of 3A10-im-
munoreactive otic dendrites at each of the sensory epi-
thelium patches (for innervation patterns, see also
S�anchez-Calder�on et al., 2004, 2005, 2007b; S�anchez-
Guardado et al., 2009, 2011).
Serial horizontal sections through the inner ear at
stage HH27 (Fig. 8A–U) confirmed the Fgf10, Cath1, and
Bmp4 expression patterns described above for transverse
sections (Fig. 7). Regarding Fgf10 expression in the ros-
tral part of the otic anlage, a clear-cut nonexpressing gap
was evident (large arrowhead in Fig. 8B) between the an-
terior crista (ac; Fig. 8A) and the neighboring ventral sen-
sory epithelia: the lateral crista and the macula utriculi (lc
and mu; Fig. 8D). Concerning the mutual delimitation of
these sensory elements, Fgf10 expression has started to
decrease in the epithelial territory separating them (small
arrowhead in Fig. 8D). A portion of otic epithelium wholly
devoid of Fgf10 expression was also detected between
the developing macula utriculi and macula sacculi (large
arrowhead in Fig. 8D), making the separation between
these sensory patches more evident (see arrows in
Fgf10 in the developing chick inner ear
The Journal of Comparative Neurology | Research in Systems Neuroscience 1149
Fig. 3C,D). In the caudal part of the otic anlage, the dorsal
Fgf10-expressing posterior crista (Fig. 8A) was observed
in close proximity to the adjoining macula neglecta, which
was very weakly Fgf10-positive in its dorsalmost portion
(Fig. 8B,C) and Fgf10-negative in its ventralmost portion
(Fig. 8D), and very weakly Bmp4-positive in this particular
embryo (Fig. 8P–R).
After examining the Bmp4-expressing domains at stage
HH27 in more detail, we observed a Bmp4-expressing
area in the posterior part of the inner ear (arrow in Fig.
8S), which was not labeled by Fgf10 (arrow in Fig. 8E).
This Bmp4-expressing area corresponds to an abneural
portion of the cochlear duct (Oh et al., 1996; Wu and Oh,
1996; Cole et al., 2000). As noted in transversal sections,
the macula sacculi and the basilar papilla were included
in a continuous dorsoventral band of Fgf10 expression
(Fig. 8D–F), which was weakly labeled by Bmp4 expres-
sion (Fig. 8R–T).
With respect to Cath1 expression, it was easy to note
the presence of abundant Cath1-expressing cells in all
cristae (Fig. 8H–K). Scattered Cath1-positive cells were
also detected in the entire macula sacculi (Fig. 8K,L) and
in the lateral portion of the macula utriculi (Fig. 8K). The
proximal portion of the basilar papilla, but not its distal
portion, showed a reduced number of Cath1-expressing
cells (bp; Fig. 8M). Cath1 expression was absent in the
macula neglecta (mn; Fig. 8I–K) and the macula lagena
(ml; Fig. 8N), suggesting that hair cell specification is not
yet defined in these sensory elements at this develop-
mental stage.
Figure 9 summarizes the Fgf10 expression pattern in
the inner ear at stage HH27. All sensory epithelia, except
the macula neglecta (Fig. 9B), were Fgf10-positive (Fig. 9).
The incipient separation between the lateral crista and the
macula utriculi, on the one hand, and the basilar papilla
and the macula lagena, on the other hand, was indicated
by weaker Fgf10 expression (arrowheads in Fig. 9A). The
fact that the macula sacculi and the basilar papilla were
still included in a continuous dorsoventral band of Fgf10
expression was also patent (Fig. 9A). Interestingly, the dor-
soventral Bmp4-expressing band located in the caudal por-
tion of the inner ear at stage HH27, which extends from
the macula neglecta to the macula lagena (arrow in Fig.
9B), was devoid of Fgf10 expression and any other
markers of differentiated hair cells, in particular Cath1.
At stage HH28, expression of Fgf10 (Fig. 10A), Ser-
rate1 (Fig. 10B), Delta1 (Fig. 10C), and Hes5 (not shown)
strongly labeled the entire extent of the developing lat-
eral crista. An obvious gap of Fgf10 expression was
observed between the developing lateral crista and the
macula utriculi (small arrowhead in Fig. 10A). The ab-
sence of Fgf10 expression was also detected between
the utricular and saccular maculae (large arrowhead in
Fig. 10A), as previously noted for stages HH24 (Fig.
3C,D) and HH25–26 (Fig. 6G,M). Collectively, these
results strongly suggest a complete specification and
delimitation of these three sensory epithelia of the devel-
oping avian vestibule at stage HH28. Moreover, Serrate1
expression also labeled an Fgf10-negative portion of the
otic epithelium corresponding to a part of the horizontal
pouch caudal to both the lateral crista and the macula
utriculi (arrow in Fig. 10B). This suggests that Serrate1
expression does not mark exclusively sensory elements
in the developing chick inner ear. At stage HH29, the
Fgf10-positive lateral crista was clearly BEN-immunore-
active and expressed LFng (not shown), a feature not
observed at stage HH24 (Fig. 4J,L).
As mentioned above, the presumptive Fgf10-express-
ing macula lagena starts to be separated from the nearby
basilar papilla at stage HH27 (Fig. 7C). At stage HH30
(6.5 days of incubation), ISH performed on horizontal sec-
tions showed the strongly Fgf10-expressing macula
lagena to be a clear-cut sensory patch fully separated
from the basilar papilla (arrowhead in Fig. 10D). The pres-
ence of isolated transcripts of Serrate1 (Fig. 10E), Cath1
(Fig. 10F), Sox2 (Fig. 10G), Hes5 (not shown), and Delta1
(not shown) was also observed in the macula lagena, all
these signals being included within a Bmp4-expressing
area (not shown).
Figure 9. A,B: 3D diagrams of the chick otic anlage at stage
HH27 (A, anterior view; B, posterior view) summarizing the Fgf10
and Cath1 expression patterns. The weak and strong levels of
Bmp4 expression are also distinguished. The weak Fgf10 expres-
sion is indicated (arrowheads in A). A part of the macula neglecta
showed a weak Fgf10 expression (B). In the cochlear duct, the
abneural Bmp4-expressing domain was devoid of Fgf10 and Cath1
expression (arrow in B). The horizontal sections shown in Figure 8
are also indicated. Orientation: C, caudal; D, dorsal; M, medial; R,
rostral. Diagrams adapted from S�anchez-Guardado et al., 2009
and 2011.
S�anchez-Guardado et al.
1150 The Journal of Comparative Neurology |Research in Systems Neuroscience
Figure 10. Specification of the lateral crista, macula lagena, and macula neglecta at stage HH28-31. Horizontal sections treated with the
indicated probes and 3A10 immunoreactions. A–C: Strong expression of Fgf10 (A), Serrate1 (B), and Delta1 (C) in the lateral crista (lc)
was observed. The lateral crista was situated a long way from the macula utriculi (small arrowheads). The utricular and saccular maculae
were clearly separated (large arrowheads). The arrow in B points to a nonsensory portion of the otic epithelium, Serrate1 positive. D–G:
The macula lagena (ml) was identified by the strong expression of Fgf10 (D), Serrate1 (E), and Cath1 (F), and by a weak expression of
Sox2 (G). This macula was separated from the nearby basilar papilla (bp); the arrowheads point to the space between these two sensory
elements. H–M: At stage HH29, the innervated macula neglecta (mn), separated from the posterior crista (pc; arrowheads), expressed
Fgf10 (H,I) and Bmp4 (L,M). Very low expression of Sox2 (mn; K) and no Cath1 (mn; J) expression was detected. O–Q: At stage HH31, the
Fgf10-positive macula neglecta (mn; O) showed Cath1- and Hes5-positive cells (P,Q). For abbreviations, see list. Orientation: M, medial; R,
rostral. Scale bar ¼ 70 lm in C (applies to A–C); 30 lm in G (applies to D–G); 60 lm in L (applies to H,J,L); 45 lm in M (applies to
I,K,M); 70 lm in Q (applies to O–Q). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Fgf10 in the developing chick inner ear
The Journal of Comparative Neurology | Research in Systems Neuroscience 1151
The macula neglecta is one of the last sensory patches
to be specified, as indicated by the expression of Fgf10 in
the developing inner ear. In horizontal sections across the
dorsal aspect of the otic anlage at stage HH29 (6 days of
incubation; Fig. 10H–M), Fgf10-expressing cells were
more evident under the 3A10-positive otic dendrites that
already contacted the presumptive macula neglecta (mn;
Fig. 10H,I). At this developmental stage, the macula
Figure 11. Fgf10 expression pattern at stage HH34. Horizontal sections treated with the noted probes and the 3A10 antibodies. A–K: Fgf10
expression was detected in all sensory epithelia (A–E), which were clearly defined by the 3A10 immunoreaction and labeled by the expression of
Serrate1 (F,J), Cath1 (G,H), and Hes5 (I). Interestingly, an additional nonsensory Fgf10-expressing area was observed between the macula sacculi
and the basilar papilla (ms and bp; arrows in A,C), being Serrate1-positive (arrow in F) and Cath1-negative (arrow in G). In the cochlear duct, the
Fgf10-positive domain was larger than the innervated basilar papilla (bp; arrow in D; see also arrows in H–J), abutting the Otx2-positive tegmen-
tum vasculosum (tv; arrowheads in D,K). For abbreviations, see list. Orientation: M, medial; R, rostral. Scale bar ¼ 31 lm in A (applies to
A,B,C,F,G); 22 lm in E (applies to D,E,H–K). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
S�anchez-Guardado et al.
1152 The Journal of Comparative Neurology |Research in Systems Neuroscience
neglecta still showed low levels of Bmp4 and Sox2 expres-
sion (Fig. 10K-L), and was clearly labeled by Serrate1
expression (not shown). Although the macula neglecta
was clearly separated from the neighboring posterior
crista (arrowheads in Fig. 10H–M), we did not detect any
expression of Cath1 (Fig. 10J), Hes5 (not shown), or
Delta1 (not shown) in this developing sensory patch. In
contrast, the macula neglecta was completely specified
at stage HH31 (7 days of incubation; Fig. 10O–P). The
entire extent of the macula neglecta expressed Fgf10
(Fig. 10O), and the separation relative to the posterior
crista was larger (arrowheads in Fig. 10O–Q). Also, this
sensory element showed expression of Cath1 and Hes5
(Fig. 10P,Q), indicating differentiation of hair and support-
ing cells.
Fgf10 expression pattern at stage HH34At 8 days of incubation (stage HH34), the chick membra-
nous labyrinth had attained its mature shape and differen-
tial regionalization. All the sensory epithelia could be identi-
fied histologically and morphologically. Fgf10 expression
was strongly detected in all eight sensory epithelia of the
chick inner ear (Fig. 11A–E), and these were all innervated
by 3A10-immunoreactive otic dendrites (Fig. 11A–E). Simi-
lar results were obtained with others markers, such as Ser-
rate1, Cath1, and Hes5 (Fig. 11F–J for mu, ms, and bp).
Interestingly, a small area in the saccular-cochlear junction,
contiguous to the macula sacculi, expressed Fgf10 weakly
(arrows in Fig. 11A and C) and Serrate1 strongly (arrow in
Fig. 11F), but was Cath1 negative (Fig. 11G). This weakly
Fgf10-labeled area extended ventrally into the epithelium of
the proximal cochlear duct (cd; arrow in Fig. 11A), where it
contacted the strongly Fgf10-expressing basilar papilla (see
arrow in Fig. 11D). Within the cochlear duct, the weak
expression domain also continued as a strip of nonsensory
Fgf10-positive epithelium along the neural edge of the basi-
lar papilla. This region, which is presumed to correspond to
the future homogene cell region, was clearly devoid of
Cath1 expression (Fig. 11H), as well as Hes5 (Fig. 11I), and
Serrate1 (Fig. 11J). This weakly Fgf10-expressing area
extended toward the presumptive domain of the tegmen-
tum vasculosum (tv; Fig. 11D), which could be labeled spe-
cifically by Otx2 expression (tv; Fig. 11K; S�anchez-Calder�on
et al., 2004), and ended abutting it (compare arrowheads in
Fig. 11D,K). Therefore, at stage HH34, all the sensory otic
epithelia were defined by strong Fgf10 expression and
3A10-positive otic fibers. However, the macula sacculi and
Figure 12. A,B: 3D diagrams of a stage HH34 inner ear (A, anterior view; B, posterior view) summarizing the Fgf10 expression pattern.
The sensory patches are represented by the hatched area. All sensory epithelia were Fgf10-labeled. Note a weak Fgf10 expression
between the macula sacculi (ms) and the basilar papilla (bp) (arrows). The weak Fgf10 expression near the basilar papilla is also shown
(arrowhead in A). The horizontal sections shown in Figure 11 are also indicated. For abbreviations, see list. Orientation: D, dorsal; M,
medial; P, posterior; R, rostral.
Fgf10 in the developing chick inner ear
The Journal of Comparative Neurology | Research in Systems Neuroscience 1153
the basilar papilla were included in a large dorsoventral
Fgf10-expressing band. Figure 12 summarizes Fgf10
expression in the inner ear at stage HH34.
Fgf10 expression pattern at stage HH36At stage HH36 (10 days of incubation), Fgf10 expres-
sion was completely confined to the differentiating sen-
sory epithelia. The weak Fgf10 expression detected at
stage HH34 between the macula sacculi and the basilar
papilla had finally disappeared (arrow in Fig. 13A),
although Serrate1 expression was still detected at this
level (arrow in Fig. 13B). In the rostral aspect of the coch-
lear duct, the nonsensory Fgf10 expression still observed
at stage HH34 was significantly reduced or not detected
at HH36. The border of Fgf10/Serrate1 expression was
coincident with the border of the innervated basilar pa-
pilla (bp; arrowheads in Fig. 13C,D). At stage HH38 (12
days of incubation), Fgf10 and Serrate1 expression pat-
terns were completely restricted to all the sensory epithe-
lia, as confirmed by the expression of the hair and sup-
porting cell markers Cath1 and Hes5 (not shown).
DISCUSSION
The sensory/neuronal fate assignments emerging
within the otic epithelium, including some new, function-
ally adapted sensorial formations, are key developmental
events in the evolutionary history of the vertebrate inner
ear. A current hypothesis based on descriptive and exper-
imental evidence proposes that the acquisition of new
discrete sensory epithelial patches during inner ear evolu-
tion was generated by the subdivision of previously exist-
ing sensory primordia, all of them deriving from a single
original domain. Thus, the progressive splitting up of sen-
sory patches, together with associated evolutionary mor-
phogenetic changes, has originated in vertebrates an
intricate 3D structure that shows up to nine separate
organs, held to derive from an ancestral inner ear (like
jawless vertebrates) with simpler sensory organs, namely,
two cristae and a single macula (Fritzsch and Beisel,
2001; Fritzsch et al., 2002). This developmental course
may be compared to that which occurs in the develop-
ment of the cranial placodes and lateral line organs
(Baker and Bronner-Fraser, 2001; Streit, 2007; Baker
et al., 2008; Ma and Raible, 2009; Ladher et al., 2010).
However, an alternative evolutionary path might involve
the appearance of new sensory epithelia by a de novo
creation (Bang et al., 2001; Bever and Fekete, 2002;
Hammond and Whitfield, 2006). This second hypothesis
was proposed on the basis of observations of the Bmp4
expression pattern (Oh et al., 1996; Wu and Oh, 1996).
Thus, both segregation and de novo creation of sensory
epithelia are considered as potential drivers of inner ear
evolution. In the last decade, a key aim of developmental
Figure 13. Fgf10 expression pattern at stage HH36. A–D: Horizontal section through stage HH36 inner ear treated with the indicated
probes and 3A10 antibodies. In the saccule (s; A,B), Fgf10 transcripts were restricted to the macula sacculi (ms; A), whereas Serrate1
mRNA was detected in a caudal adjacent area (compare arrows in A and B). In the cochlear duct (cd), Fgf10 and Serrate1 expression out-
side the basilar papilla (bp) were absent (compare arrowheads in C,D and small arrows in Fig. 11D,H–J). For abbreviations, see list. Orien-
tation: M, medial; R, rostral. Scale bar ¼ 25 lm in A (applies to A–D). [Color figure can be viewed in the online issue, which is available
at wileyonlinelibrary.com.]
S�anchez-Guardado et al.
1154 The Journal of Comparative Neurology |Research in Systems Neuroscience
studies has been to better understand the molecular and
cellular mechanisms involved in the early patterning
(regionalization) of the otic epithelium and the subse-
quent specification and delimitation of sensory epithelial
patches (Abell�o and Alsina, 2007; Bok et al., 2007;
Schneider-Maunoury and Pujades, 2007; Whitfield and
Hammond, 2007), seeking further understanding of inner
ear evolution (Fritzsch and Beisel, 2001; Fritzsch, 2003;
Fritzsch et al., 2002, 2006, 2010).
Pioneering histological evidence has suggested that
the different sensory organs of the developing chick inner
ear all derive from a thickened ventromedial portion of
the otic anlage wall, detectable at about the otic vesicle
stage (Knowlton, 1967). In fish embryos, the vestibular
maculae and cristae seem to originate from one continu-
ous primordial patch, termed the macula communis, also
located ventromedially in the developing inner ear (Had-
don and Lewis, 1996; Millimaki et al., 2007; Sweet et al.,
2011). The existence of a single common prosensory
area in the developing otic vesicle was also strongly sup-
ported by the studies mapping BEN protein, a cell adhe-
sion molecule (Goodyear et al., 2001). Goodyear and co-
workers showed that BEN immunostaining clearly labels a
common ventromedial area, in addition to other otic
regions. This BEN-expressing band subsequently splits
into all the sensory organs of the developing chick inner
ear from stage HH22 onward. The boundaries defined by
the combined expression patterns of Serrate1 and LFng,
two components of the Notch signaling pathway, have
allowed at least partial molecular characterization of the
sensory-competent domain in the chick rudimentary oto-
cyst (Adam et al., 1998; Cole et al., 2000). In this sense,
Fekete (1996) predicted that all of the sensory primordia
arise from a common sensory-competent annulus located
around the equator of the otocyst, and that this sensory-
competent domain would presage the appearance of dis-
crete BMP4-positive sensory patches (at compartment
boundaries). Thus, the sensory organs in the developing
chick inner ear do not arise independently of each other,
as was concluded from Bmp4 expression studies (Wu and
Oh, 1996).
Fgf10 expression in the developingvertebrate inner ear
It has been proposed that Fgf10 expression may be
involved in promoting neurogenesis during inner ear de-
velopment both in birds and mammals (Pirvola et al.,
2000; Pauley et al., 2003; Alsina et al., 2004; Ohuchi
et al., 2005). In the chick otic placode, Fgf10 expres-
sion is detected in its most anterior and medial
portion. This domain of Fgf10 expression evolves as
development proceeds, forming first a well-defined
anteromedial band located at the equator of the otic
vesicle (Alsina et al., 2004; present study). It has al-
ready been reported that this Fgf10-expressing domain
coincides with the presumptive neural-sensory domain,
and anticipates the expression of several proneural and
neurogenic genes such as Ngn1, Delta1, Hes5, and
NeuroD/M (Alsina et al., 2004). Hence, Fgf10 expres-
sion might define the proneural domain in the chick
inner ear. In the mammalian otic vesicle, Fgf10 expres-
sion starts to be observed in its lower half (Pauley
et al., 2003). A little later, Fgf10 expression appears re-
stricted to the anterior pole of the otic anlage and the
delaminating acoustic-vestibular neurons. As develop-
ment proceeds, all mammalian cristae are clearly Fgf10
positive, suggesting upregulation of expression in all
sensory elements but with variable individual patterns
(Pirvola et al., 2000; Pauley et al., 2003). When all
available data are taken together, Fgf10 is thought to
be involved, directly or indirectly, in vertebrate inner
ear morphogenesis and in the specification of otic vesi-
cle cells toward a sensory/neuronal lineage (Pauley
et al., 2003; Alsina et al., 2004; Ohuchi et al., 2005;
for review, see Abell�o and Alsina, 2007).
Our results strongly support the hypothesis of a poten-
tial involvement of Fgf10 in the specification of sensory
epithelia during chick inner ear development. The early
anteroposterior Fgf10-expressing domain in the ventro-
medial side of the rudimentary otocyst apparently demar-
cates a specific sensory-competent compartment,
entirely included within an ampler Serrate1-expressing
territory. However, this Fgf10-expressing domain does
not include all prospective sensory patches. The anterior
and posterior cristae, the utricular and saccular maculae,
the basilar papilla, and the macula lagena differentiate
within it, whereas the lateral crista and the macula
neglecta are excluded from this common domain, irre-
spective that they later independently come to express
Fgf10 as they differentiate. One may tentatively suggest
that these two sensory elements are generated de novo
in areas adjacent to the Fgf10-positive domain, and that
their specification is determined by some local combina-
tion of signaling pathways (see below), possibly including
the activity of Serrate1 and Bmp4, among other regulatory
proteins. Our comparative analysis of the correlative
expression of all known otic sensory patch markers, in
particular Cath1, helped us to determine the precise time-
table of sensory patch specification from either Fgf10-
positive or Fgf10-negative territories. The specification of
the prosensory domains in chick appears to involve both
segregation of a broad Fgf10-expression domain and sub-
sequent de novo specification of lateral crista and macula
neglecta (also requiring Fgf10); thus a combination of two
different mechanisms are at work.
Fgf10 in the developing chick inner ear
The Journal of Comparative Neurology | Research in Systems Neuroscience 1155
Specification of the semicircular canals andtheir associated cristae
The cristae of the vertebrate inner ear contribute to the
codification of the angular movement in all three cardinal
planes for the reflex control of eye and postural move-
ments (Fritzsch and Wake, 1988; Fritzsch et al., 2002).
The molecular mechanisms that control the development
of the anterior and posterior cristae seem to be different
from those regulating the specification of the lateral
crista, thought to be a later evolutionary acquisition
(absent in agnatha; Lewis, 1985). The quantitative differ-
ences in some gene expression patterns, as well as the
asymmetrical expression of some genes, lend support to
this assumption. In the developing chick inner ear, SOHo-
1, a homeobox-containing gene, initially labels weakly the
anterior and posterior cristae, whereas the lateral crista
only shows a robust SOHo-1 expression at stage HH24
(Kiernan et al., 1997). At the same developmental stage,
the Cath1-positive presumptive lateral crista clearly lies
outside the initial Fgf10 expressing domain, and is devoid
of other sensory organ markers such as BEN and LFng,
which do label the anterior and posterior cristae. In agree-
ment with this hypothesis, it has been reported that
FGF10 is essential for the correct morphogenesis of the
anterior and posterior semicircular canals and their asso-
ciated cristae, but not for the lateral crista. In the Fgf10
mutant the anterior and posterior cristae are much
smaller and barely segregated from the utricle, and their
semicircular canals are severely disorganized (Ohuchi
et al., 2000, 2005; Pauley et al., 2003).
The horizontal canal and the lateral crista are consid-
ered new acquisitions in jawed vertebrates (Lewis, 1985).
Numerous works have explored the molecular events that
may have been significant in this evolutionary scenario
(reviewed by Fritzsch et al., 2006). Mixynoids have a sin-
gle semicircular canal, which apparently duplicates spec-
ularly into the anterior and posterior canals already pres-
ent in lampreys. The lateral crista of gnathostomes might
have segregated via symmetric duplication from the dor-
sal sensory patch corresponding to the anterior papilla
(as suggested by their similar spatial orientation, at the
anterior end of the respective canals; Fritzsch and Wake,
1988), or from the macula utriculi. Interestingly, the po-
larity orientation of the hair cells of the lateral crista is
similar to that observed in a portion of the macula utriculi
(Lewis, 1985), suggesting a common field effect
(reviewed by Fritzsch et al., 2002, 2006). In our HH24
chick embryos, the Fgf10-expressing area clearly
excluded the prospective lateral crista. The nearest sen-
sory element, the Fgf10-positive macula utriculi, has a
clear-cut lateral border delimited likewise by Fgf19
expression (S�anchez-Calder�on et al., 2007b). In this
regard, a possible role of Fgf19/Fgf10 cooperation may
be speculated in the similarly polarized specification of
these two sensory patches, the lateral crista and the mac-
ula utricle, probably by setting up analogous diffusion
gradients that regulate positional cell identities.
However, based on BMP4 and LFng expression pat-
terns, Morsli and co-workers (1998) proposed that the
anterior and lateral cristae share a common origin in the
developing mammalian inner ear. Our descriptive results
do not exclude a possible origin of the lateral crista from
a domain near the anterior crista, matching observations
from Serrate1 expression studies (Adam et al., 1998). In
this sense, it is interesting to note that, although the
mouse Lmx1a expression is restricted to nonsensory ele-
ments of the mouse inner ear, the anterior and lateral
cristae are fused in Lmx1a null mutant mice, suggesting
that the presumptive territory of the lateral crista might
be initially in close proximity to that of the anterior crista
(Nichols et al., 2008).
Although the genetic mechanisms involved in the lat-
eral crista require further research, it is well known that
the Otx1 gene could control the emergence and complete
specification of the whole horizontal canal and lateral
crista system (Morsli et al., 1998; Acampora et al., 1996;
Tomsa and Langeland, 1999; Cantos et al., 2000; Mazan
et al., 2000). In mouse embryos, Otx1 is expressed in the
presumptive lateral crista, and Otx1 null mutants lack at
least a significant part of both the lateral crista and its
semicircular canal (Morsli et al., 1998; Fritzsch, 2001;
reviewed by Fritzsch et al., 2002), confirming the hypoth-
esis that the Otx1 gene is primarily responsible for the dif-
ference between the agnathan and gnathostome inner
ears (Hammond and Whitfield, 2006). In this molecular
context, it is plausible to accept a possible interaction
between Otx family members, in particular Otx1, and
Fgf10 (Miyazaki et al., 2006).
Specification of the utricular and saccularmaculae
The utricular and saccular maculae in the inner ear ves-
tibule play a key role in the balance system, sensing lin-
ear acceleration and gravity. In all vertebrates, these
maculae are close one to another, suggesting a possible
common origin from a continuous primordial patch (Adam
et al., 1998; Morsli et al., 1998; Fari~nas et al., 2001; Ham-
mond and Whitfield, 2006). Our descriptive results in the
developing chick inner ear show that both maculae
emerge from an Fgf10-expressing domain that starts to
divide at stage HH24 to generate the macula utriculi and
a saccular/basilar/lagenar domain. These findings are
strongly consistent with the evidence derived from the
Serrate1 expression pattern in chick (Adam et al., 1998),
and the Bmp4 and LFng expression patterns in mouse
S�anchez-Guardado et al.
1156 The Journal of Comparative Neurology |Research in Systems Neuroscience
(Morsli et al., 1998). Some differences were nevertheless
observed between the two maculae regarding Bmp4
expression, and the saccular macula was the first to
begin to differentiate, as indicated by the presence of
Cath1-positive hair cells. A possible relationship between
Fgf10 and genes known to be involved in the specification
of the utricule and saccule, such as Otx1/2, needs to be
considered (Morsli et al., 1998; S�anchez-Calder�on et al.,
2002, 2004; Beisel et al., 2005; Hammond and Whitfield,
2006; for a different developing system, see Hidalgo-
S�anchez et al., 2005).
Specification of the macula lagenaThe macula lagena is a variable sensory organ in the
gnathostome group (Fritzsch, 1992; Buran et al., 2005). It
has been indicated that the macula communis in lamprey
contains three clearly distinct macular areas, which could
correspond to the saccular, utricular, and lagenar macu-
lae (Avallone et al., 2005), suggesting an origin of these
three maculae from a single elongated epithelium. Fur-
thermore, it has been proposed that the lagenar macula
could originate from the saccular macula (Baird, 1974) or
from the utricular macula (Platt et al., 2004). According
to comparative morphological studies and consideration
of ciliary bundle orientation, the macula lagena may have
evolved three times independently in the evolution of the
vertebrate inner ear, always splitting off from the saccule
(Fritzsch, 1992). In contrast, the zebrafish macula lagena
could have arisen de novo, but not from other maculae
(Bang et al., 2001; Bever and Fekete, 2002). In the chick,
the presumptive territory of the macula lagena, clearly
identified by the expression of Msx1, is included within
the overlapping Fgf10- and Bmp7-positive domains at
stage HH24, and starts to be separated from the macula
sacculi/basilar papilla domain at stage HH27. Thus, both
lagenar macula and basilar papilla possibly share a com-
mon molecular program of development. However, new
descriptive and experimental studies are necessary to
shed more light on this question.
Specification of the auditory sensoryepithelium
It has been proposed that the hearing system segre-
gated from a pre-existing sensory epithelium, either the
saccule or the utricle, developing in sarcopterygians by
additional changes into a specific organ, together with
the acquisition of a tectorial membrane instead of an oto-
lith-laden gelatinous cupula, and the secondary associa-
tion with a perilymphatic space (Fritzsch et al., 1997). In
this evolutionary context, it has been proposed that the
utricle may have been generated in parallel as an addi-
tional receiver of sound pressure in some fishes (Lewis,
1985; Fritzsch, 2001; see also Fritzsch et al., 2006). How-
ever, it is also widely held that the saccule probably gave
rise to the basilar papilla of land vertebrates in the basal
tetrapod ancestors (Fritzsch, 1992). The dynamic expres-
sion patterns of LFng, BDNF, and NT-3 support the hy-
pothesis of a segregation of the basilar papilla from the
saccule (Morsli et al., 1998; Fari~nas et al., 2001). In
mouse, the Bmp4 and LFng expression patterns similarly
corroborate that the macula sacculi and basilar papilla
remain connected until late in development (Morsli et al.,
1998). The cochleo-saccular dysplasia defects described
in humans corroborate this suggestion (Sampaio et al.,
2004; Kariya et al., 2005).
Our descriptive results in chick also showed a clear
relationship between the macula sacculi and the basilar
papilla. Both sensory elements were included in a contin-
uous Fgf10-positive band extending dorsoventrally, which
exclusively contained these elements by stage HH30.
Their segregation was noted late in the development of
the chick inner ear (HH36). It is tentative to speculate
that, over the course of inner ear evolution, variation in
the regulation of the FGF signaling pathway, in particular
FGF10, within this common dorsoventral domain may
have been responsible for the appearance of an auditory
organ, the basilar papilla. In this sense, the FGFR3 gene,
whose expression is restricted to the chick basilar papilla
(Bermingham-McDonogh et al., 2001), interestingly was
never detected in the contiguous macula sacculi and
macula lagena (M.H.S., manuscript in preparation), and
may have played a key role in the appearance of this sen-
sory element during inner ear evolution in vertebrates.
Nevertheless, more detailed fate maps and functional
studies of the relevant signaling pathways are necessary
to check this hypothesis.
Specification of the macula neglectaThe macula neglecta is a small oval-shaped sensory ep-
ithelium normally located between the posterior crista
and the utriculo-saccular foramen, which receives inner-
vation from a branch of the posterior crista nerve. This
sensory element, present in most vertebrates (fish, rep-
tiles, birds, and some mammals) but not easily recog-
nized, is suspected to be involved in perception of vibra-
tory and auditory stimuli, although its function remains to
be definitively determined (Hoshino and Kodama, 1976;
Corwin, 1978, 1981; Lewis, 1985; Fritzsch and Wake,
1988).
In some amphibian species, the macula neglecta and
the amphibian papilla were thought to share a common
origin, because they are at first joined, but separate histo-
logically late in development (Fritzsch and Wake, 1988).
It has alternatively also been postulated that the macula
neglecta and the posterior crista may have a common
Fgf10 in the developing chick inner ear
The Journal of Comparative Neurology | Research in Systems Neuroscience 1157
origin (Montandon et al., 1970; Nichols et al., 2008).
Mice mutants null for Limx1a show dysmorphic inner
ears, in which the posterior crista and the papilla neglecta
are fused (Nichols et al., 2008), a result that is consistent
with this second hypothesis.
According to our results in the developing chick inner
ear, the macula neglecta starts to show Fgf10 expression
at stage HH24–25. This sensory element was clearly
identified later, at stage HH31, by the expression of vari-
ous hair cell markers, such as Cath1 and Hes5. However,
this sensory patch already receives 3A10- and BEN-
immunostaning fibers from the vestibular ganglion by
stage HH24. Together, these findings strongly suggest
that, in the developing chick inner ear, the macula
neglecta was initially excluded from the Fgf10-expressing
domain, similar to the case of the lateral crista, and was
the last sensory element to acquire Cath1 expression.
One may tentatively hypothesize that the macula
neglecta represents a relatively late acquisition in verte-
brate inner ear evolution, and probably results from de
novo acquisition (patterning) in gnathostomes, as in the
case of the lateral crista. The rest of the cristae and mac-
ulae, and also the basilar papilla, may be represented as
a common sensory patch in the ancestral plan of the ver-
tebrate inner ear. They apparently segregated subse-
quently one from another during otic vesicle morphogene-
sis and evolution. Further descriptive and experimental
studies will have to be performed in different groups of
vertebrates to confirm these ideas.
Fgf10 expression pattern related to Sox2,Serrate1, and Cath1 expression
Several members of the transcription factor Sox gene
family are linked to the acquisition of competences and
later commitment of cells to a neural fate (Pevny and
Plackek, 2005). Sox2 appears to be involved in the devel-
opment of inner ear sensory epithelia (Uchikawa et al.,
1999; Kiernan et al., 2005, 2006; Daudet et al., 2007;
Hume et al., 2007; Neves et al., 2007, 2011, 2012; Chang
et al., 2008; Dabdoub et al., 2008; Oesterle et al., 2008;
Ahmed et al., 2012a; Mak et al., 2009; Millimaki et al.,
2010). Thus, Sox2 mutant mice show disorders in the de-
velopment of sensory patches and hair cells or even their
absence (Kiernan et al., 2005).
In the developing avian inner ear, Sox2 is detected in
proliferating cells of neurogenic and prosensory regions
from the otic cup to the early otic vesicle (Neves et al.,
2007). Before prosensory specification (stage HH12–14),
the Sox2-positive region is very broad in the otic anlage
(Neves et al., 2011). At the HH16 stage, the Sox2-labeling
domain becomes more restricted, with Sox2-positive cells
being exclusively detected in the anteromedial domain of
the otic vesicle (Neves et al., 2007). This territory is con-
sidered to be the proneural domain (Alsina et al., 2004),
being complementary to the posterior HNK1-positive
non-neural region (Neves et al., 2007). Our comparative
study of Fgf10 and Sox2 expression patterns using ISH at
the HH18 stage showed that the Sox2-expressing domain
formed a rostrocaudal gradient of expression included
within the Fgf10-positive band. At the HH20 stage, Sox2
expression extended caudally, with the Sox2 and Fgf10
expression patterns being coincident and labeling the an-
terior and posterior cristae. As development proceeded,
the Fgf10 and Sox2 expression patterns evolved in paral-
lel until the HH24 stage, when these domains excluded
the developing lateral crista and macula neglecta. Soon
after, these sensory patches acquired first Fgf10 and
then Sox2 expression, more evidently for the lateral
crista.
Our data therefore strongly support the hypothesis
that the lateral crista and the macula neglecta emerge as
independent patches outside the Fgf10- and Sox2-posi-
tive territory. Regarding the macula lagena, this sensory
epithelium was included within the Fgf10-expressing do-
main at stage HH24. Although the macula lagena was
Sox2-negative at this developmental stage, it started to
express the Sox2 gene by stage HH28–30. Taken to-
gether, our results show that Fgf10 and Sox2 are directly
involved in the specification of all sensory patches of the
developing chick inner ear. Because Sox2 expression fol-
lowed that of Fgf10 in the developing sensory patches,
we would propose Fgf10 expression as a better sensory
marker than Sox2 expression.
Regarding the molecular mechanisms involved in the
specification of all the sensory epithelia, it is interesting
to note that the expression of SOX3, another member of
the SoxB1 transcription factor family, as is SOX2 (Uchi-
kawa et al., 1999), depends on FGF8 and BMP signaling
pathways at the chick otic stage (Abell�o et al., 2010).
Also, the FGF signaling pathway activates Sox2 expres-
sion through the enhancer N-1c in the genesis of the pos-
terior neural plate (Takemoto et al., 2006). In this molecu-
lar context, Sox2 could be involved directly in the
proliferative activity restricted to the proneural domain
(Neves et al., 2007), probably promoted by the FGF activ-
ity in cell proliferation, survival, and differentiation (Yun
et al., 2010). More experimental studies should be per-
formed to better understand whether Fgf10 could induce
or maintain Sox2 expression in the developing sensory
epithelia of vertebrate inner ears.
Serrate1, a ligand of Notch receptors, could define a
common requirement for sensory commitment, probably
conferring a prosensory potential on the otic epithelium
(Adam et al., 1998; Cole et al., 2000; Kiernan et al., 2001;
Brooker et al., 2006; see also Daudet and Lewis, 2005).
S�anchez-Guardado et al.
1158 The Journal of Comparative Neurology |Research in Systems Neuroscience
We showed that, by at least the HH18 stage, Fgf10
expression, and therefore Sox2 expression, was included
within a larger Serrate1 domain. From these descriptive
results, it can be tentatively considered that Serrate1
might be necessary to induce and/or maintain Fgf10
expression, and also, directly or indirectly, to regulate
Sox2 expression. It has been reported that Serrate1 is
necessary to preserve Sox2 expression in the chick and
mouse inner ear (Kiernan et al., 2006; Daudet et al.,
2007; Neves et al., 2011), although it is not sufficient to
induce Sox2 expression de novo (Neves et al., 2011).
Atoh1 (Math1 and Cath1) is a bHLH transcription factor
necessary for differentiation of sensory hair cells in the
otic sensory epithelia (Chen et al., 2002; Kawamoto et al.,
2003; Matei et al., 2005; Pujades et al., 2006; Millimaki
et al., 2007, 2010; Pan et al., 2011; Sweet et al., 2011).
Our results showed a direct relationship between Sox2 and
Cath1 expression patterns after stage HH24, with the
exception of the early specification of the Sox2-negative
and Cath1-positive lateral crista. It is well known that Sox2
can cooperate with Eya1 and Six1 to control Atoh1 expres-
sion and thus to specify hair cell/neuron fate in the devel-
oping inner ear (Ahmed et al., 2012a,b). In the mouse,
Sox2 disturbance leads to failure to express the Math1
gene and to lack of hair cell formation, suggesting that
Sox2 acts upstream of Math1 (Kiernan et al., 2005). Also,
Atoh1a and Sox2 control sensory development in all
regions of the zebrafish otocyst (Sweet et al., 2011). How-
ever, it has been reported that Sox2 is required for mainte-
nance and regeneration of zebrafish hair cells, but not for
their initial formation; knockdown of Sox2 does not prevent
hair cell production (Millimaki et al., 2010). Even though
Sox2 directly activates Atoh1, it could also promote Atoh1
inhibition following unknown mechanisms mediated by
Hes5, Hey1, and Idl-3 (Neves et al., 2012). New experimen-
tal studies should be carried out to resolve the Sox2/
Atoh1 dual interaction, particularly when specification of
all the sensory epithelia is taking place (for the developing
cochlea, see Dabdoub et al., 2008).
Subdivision of the Fgf10-expressing band bysignaling pathways
Retinoic acid (RA), a biologically active metabolite of
vitamin A, is necessary for the appropriate development
of the vertebrate inner ear (Romand et al., 2001, 2002,
2004, 2006a; S�anchez-Guardado et al., 2009), probably
by means of its modulation of other signaling pathways in
a dose-dependent action (Ross et al., 2000; Mark and
Chambon, 2003; Romand et al., 2006a). In chick inner
ear development, it has been suggested that RA, gener-
ated in the dorsomedial epithelium of the otic anlage,
might diffuse ventrally and regulate the specification of
sensory patches (S�anchez-Guardado et al., 2009).
Remarkably, the vestibular cristae, the utricular macula,
and the saccular macula, were all bordered by the
Raldh3-expressing domain by stage HH24, whereas the
basilar papilla, macula lagena, and macula neglecta were
delimited later by stage HH27 (S�anchez-Guardado et al.,
2009). Therefore, RA could fix the final position of the
Raldh3-Gbx2/Bmp4-Fgf8–positive border located in the
vestibule, just dorsal to the Bmp4-expressing macula sac-
culi at HH24 (S�anchez-Calder�on et al., 2004; S�anchez-
Guardado et al., 2009). Mutual regulation among long-
range diffusible RA, FGF, and BMP signals in the develop-
ing chick inner ear might determine the exact location
and extension of each sensory patch within the Serrate1-
and Fgf10-positive sensory-competent domains. Further
refinement of sensory organ specification may be con-
trolled by short-range signals from the otic epithelia, pio-
neering sensory ganglionic fibers, or surrounding tissues.
Notably, an excess or deficiency of RA in the otic anlage
produced by application of agonists or antagonists per-
turbs inner ear morphogenesis and organogenesis by
altering the FGF10 signaling pathway (Liu et al., 2008;
Frenz et al., 2010). In addition, it is now known that RA
inhibits the FGF signaling pathway in the developing neu-
ral tube (Diez-del-Corral et al., 2003). Given that the
Fgf10-positive domain clearly abuts the Raldh3-express-
ing area (present study; S�anchez-Guardado et al., 2009),
we suggest that an RA–FGF10 interaction may be opera-
tive in subdividing the initial proneural Fgf10-expressing
domain observed in the chick inner ear at the otic vesicle
stage.
CONCLUSIONS
A number of issues need to be resolved to better
understand the molecular mechanisms governing the
changes of a simple sac-like otocyst through various mor-
phogenetic events and the formation of the sensory epi-
thelia during vertebrate inner ear evolution. The sensory/
nonsensory fate, and even the functionally appropriate
sensorial acquisitions, may be regulated by RA–FGF re-
ciprocal interactions jointly with other signaling pathways
such as BMPs and SHH derived from the otic epithelium
and/or from surrounding tissues. Such molecular effects
play a key role in early patterning, subsequent differential
sculpting of the membranous labyrinth, and consequent
segregation of sensory epithelia. The well-defined Fgf10
expression pattern reported here in the developing chick
inner ear may be included in the boundary model of otic
patterning proposed by Fekete and Wu (2002). The early
borders of Fgf10 expression would define a nearly pan-
neural compartment in which the subsequent progressive
segregation of six out of eight sensory epithelia could
Fgf10 in the developing chick inner ear
The Journal of Comparative Neurology | Research in Systems Neuroscience 1159
take place (Fritzsch et al., 2002). Two other sensory
patches—the lateral crista and the macula neglecta—appa-
rently result, induced de novo in adjacent areas, probably
by singular patterning events emerging relatively later in
evolution. However, our descriptive results merely add
weight to some of the previously considered explanatory
options, without providing a definitive demonstration.
More detailed descriptive studies should be performed
regarding signaling pathways and regulatory genes in order
to elucidate the mechanisms responsible for the genera-
tion de novo of these ‘‘recent’’ domains, the lateral crista
and macula neglecta. In addition, studies aimed at obtain-
ing a fate map of the otic placode should also be consid-
ered to resolve hypotheses about the exact origin of all the
sensory epithelia in the developing chick inner ear.
ACKNOWLEDGMENTS
We thank the members of our scientific group for help-
ful discussions and Rub�en Corral-San-Miguel for expert
technical assistance. We also express our gratitude to Dr.
Fernando Giraldez for providing us with the chick Fgf10
probe.
CONFLICT OF INTEREST STATEMENT
The authors declare no actual or potential conflict of
interest.
ROLE OF AUTHORS
All authors had full access to all the data in the study
and take responsibility for the integrity of the data and
the accuracy of the data analysis. Study concept and
design: L.-O.S.-G., M.H.S., L.P.. Acquisition of data:
L.-O.S.-G.. Analysis and interpretation of data: L.-O.S.-G.,
M.H.S., L.P. Drafting of the manuscript: M.H.S. Critical
revision of the manuscript for important intellectual
content: L.P. Obtained funding: M.H.S., L.P.
LITERATURE CITEDAbell�o G, Alsina B. 2007. Establishment of a proneural field in
the inner ear. Int J Dev Biol 51:483–493.Abell�o G, Khatri S, Giraldez F, Alsina B. 2007. Early regionali-
zation of the otic placode and its regulation by the Notchsignaling pathway. Mech Dev 124:631–645.
Abell�o G, Khatri S, Radosevic M, Scotting PJ, Giraldez F,Alsina B. 2010. Independent regulation of Sox3 and Lmx1bby FGF and BMP signaling influences the neurogenic andnon-neurogenic domains in the chick otic placode. DevBiol 339:166–178.
Acampora D, Mazan S, Avantaggiato V, Barone P, Tuorto F,Lallemand Y, Brulet P, Simeone A. 1996. Epilepsy andbrain abnormalities in mice lacking the Otx1 gene. NatGenet 14:218–222.
Adam J, Myat A, Le Roux I, Eddison M, Henrique D, Ish-Horo-wicz D, Lewis J. 1998. Cell fate choices and the expressionof Notch, Delta and Serrate homologues in the chick inner
ear: parallels with Drosophila sense-organ development.Development 125:4645–4654.
Ahmed M, Wong EY, Sun J, Xu J, Wang F, Xu PX. 2012a.Eya1-Six1 interaction is sufficient to induce hair cell fate inthe cochlea by activating Atoh1 expression in cooperationwith Sox2. Dev Cell 22:377–390.
Ahmed M, Xu J, Xu PX. 2012b. EYA1 and SIX1 drive the neu-ronal developmental program in cooperation with the SWI/SNF chromatin-remodeling complex and SOX2 in the mam-malian inner ear. Development 139:1965–1977.
Alsina B, Abello G, Ulloa E, Henrique D, Pujades C, Giraldez F.2004. FGF signaling is required for determination of oticneuroblasts in the chick embryo. Dev Biol 267:119–134.
Alvarez Y, Alonso MT, Vendrell V, Zelarayan LC, Chamero P,Theil T, Bosl MR, Kato S, Maconochie M, Riethmacher D,Schimmang T. 2003. Requirements for FGF3 and FGF10during inner ear formation. Development 130:6329–6338.
Avallone B, Fascio U, Senatore A, Balsamo G, Bianco PG,Marmo F. 2005. The membranous labyrinth during larvaldevelopment in lamprey (Lampetra planeri, Bloch, 1784).Hear Res 201:37–43.
Baird IL. 1974. Some aspects of the comparative anatomyand evolution of the inner ear in submammalian verte-brates. Brain Behav Evol 10:11–36.
Baker CV, Bronner-Fraser M. 2001. Vertebrate cranial placo-des I. Embryonic induction. Dev Biol 232:1–61.
Baker CV, O’Neill P, McCole RB. 2008. Lateral line, otic andepibranchial placodes: developmental and evolutionarylinks? J Exp Zool B Mol Dev Evol 310:370–383.
Bang PI, Sewell WF, Malicki JJ. 2001. Morphology and celltype heterogeneities of the inner ear epithelia in adult andjuvenile zebrafish (Danio rerio). J Comp Neurol 438:173–190.
Bardet SM, Cobos I, Puelles E, Martınez-De-La-Torre M,Puelles L. 2006. Chicken lateral septal organ and other cir-cumventricular organs form in a striatal subdomain abut-ting the molecular striatopallidal border. J Comp Neurol.499:745–67.
Battisti AC, Fekete DM. 2008. Slits and Robos in the develop-ing chicken inner ear. Dev Dyn 237:476–484.
Beisel KW, Wang-Lundberg Y, Maklad A, Fritzsch B. 2005. De-velopment and evolution of the vestibular sensory appara-tus of the mammalian ear. J Vestib Res 15:225–241.
Bermingham NA, Hassan BA, Price SD, Vollrath MA, Ben-ArieN, Eatock RA, Bellen HJ, Lysakowski A, Zoghbi HY. 1999.Math1: an essential gene for the generation of inner earhair cells. Science 284:1837–1841.
Bermingham-McDonogh O, Stone JS, Reh TA, Rubel EW. 2001.FGFR3 expression during development and regeneration ofthe chick inner ear sensory epithelia. Dev Biol 238:247–259.
Bever MM, Fekete DM. 2002. Atlas of the developing innerear in zebrafish. Dev Dyn 223:536–543.
Bok J, Chang W, Wu DK. 2007. Patterning and morphogenesisof the vertebrate inner ear. Int J Dev Biol 51:521–533.
B€ottcher RT, Niehrs C. 2005. Fibroblast growth factor signalingduring early vertebrate development. Endocr Rev 26:63–77.
Brooker R, Hozumi K, Lewis J. 2006. Notch ligands with con-trasting functions: Jagged1 and Delta1 in the mouse innerear. Development 133:1277–1286.
Bryant J, Goodyear RJ, Richardson GP. 2002. Sensory organdevelopment in the inner ear: molecular and cellular mech-anisms. Br Med Bull 63:39–57.
Buran BN, Deng X, Popper AN. 2005. Structural variation inthe inner ears of four deep-sea elopomorph fishes. J Mor-phol 265:215–225.
Cantos R, Cole LK, Acampora D, Simeone A, Wu DK. 2000.Patterning of the mammalian cochlea. Proc Natl Acad SciU S A 97:11707–11713.
S�anchez-Guardado et al.
1160 The Journal of Comparative Neurology |Research in Systems Neuroscience
Chang W, Brigande JV, Fekete DM, Wu DK. 2004. The devel-opment of semicircular canals in the inner ear: role ofFGFs in sensory cristae. Development 131:4201–4211.
Chang W, Lin Z, Kulessa H, Hebert J, Hogan BL, Wu DK.2008. Bmp4 is essential for the formation of the vestibularapparatus that detects angular head movements. PLoSGenet 4:e1000050.
Ch�edotal A, Pourqui O, Ezan F, San Clemente H, Sotelo C.1996. BEN as a presumptive target recognition moleculeduring the development of the olivocerebellar system. JNeurosci 16:3296–310.
Chen P, Johnson JE, Zoghbi HY, Segil N. 2002. The role ofMath1 in inner ear development: uncoupling the establish-ment of the sensory primordium from hair cell fate deter-mination. Development 129:2495–2505.
Cole LK, Le Roux I, Nunes F, Laufer E, Lewis J, Wu DK. 2000.Sensory organ generation in the chicken inner ear: contri-butions of bone morphogenetic protein 4, serrate1, andlunatic fringe. J Comp Neurol 424:509–520.
Colvin JS, White AC, Pratt SJ, Ornitz DM. 2001. Lung hypopla-sia and neonatal death in Fgf9-null mice identify this geneas an essential regulator of lung mesenchyme. Develop-ment 128:2095–2106.
Corwin J. 1981. Peripheral auditory physiology in the lemonshark: evidence of parallel otolithic and nonotolithic sounddetection J Comp Physiol 142:379–390.
Corwin JT. 1978. The relation of inner ear structure to the feedingbehavior in sharks and rays. In: Becker RP, Johari O, eds. Scan-ning electron microscopy. O’Hare, IL: SEM. pp 1105–1112.
Dabdoub A, Puligilla C, Jones JM, Fritzsch B, Cheah KS, PevnyLH, Kelley MW. 2008. Sox2 signaling in prosensory domainspecification and subsequent hair cell differentiation in thedeveloping cochlea. Proc Natl Acad Sci U S A 105:18396–18401.
Dailey L, Ambrosetti D, Mansukhani A, Basilico C. 2005.Mechanisms underlying differential responses to FGF sig-naling. Cytokine Growth Factor Rev 16:233–247.
Daudet N, Lewis J. 2005. Two contrasting roles for Notch ac-tivity in chick inner ear development: specification of pros-ensory patches and lateral inhibition of hair-celldifferentiation. Development 132:541–551.
Daudet N, Ariza-McNaughton L, Lewis J. 2007. Notch signal-ling is needed to maintain, but not to initiate, the forma-tion of prosensory patches in the chick inner ear.Development 134:2369–2378.
Diez del Corral R, Olivera-Martinez I, Goriely A, Gale E, MadenM, Storey K. 2003. Opposing FGF and retinoid pathways con-trol ventral neural pattern, neuronal differentiation, and seg-mentation during body axis extension. Neuron 40:65–79.
Echevarria D, Belo JA, Martinez S. 2005. Modulation of Fgf8activity during vertebrate brain development. Brain ResBrain Res Rev 49:150–157.
Fari~nas I, Jones KR, Tessarollo L, Vigers AJ, Huang E, KirsteinM, de Caprona DC, Coppola V, Backus C, Reichardt LF,Fritzsch B. 2001. Spatial shaping of cochlear innervationby temporally regulated neurotrophin expression. J Neuro-sci 21:6170–6180.
Fekete DM. 1996. Cell fate specification in the inner ear. CurrOpin Neurobiol 6:533–541.
Fekete DM, Campero AM. 2007. Axon guidance in the innerear. Int J Dev Biol 51:549–556.
Fekete DM, Wu DK. 2002. Revisiting cell fate specification inthe inner ear. Curr Opin Neurobiol 12:35–42.
Fior R, Henrique D. 2005. A novel hes5/hes6 circuitry of neg-ative regulation controls Notch activity during neurogene-sis. Dev Biol 281:318–333.
Frenz DA, Liu W, Cvekl A, Xie Q, Wassef L, Quadro L, Nieder-reither K, Maconochie M, Shanske A. 2010. Retinoid
signaling in inner ear development: a ‘‘Goldilocks’’ phenom-enon. Am J Med Genet A 152A:2947–2961.
Fritzsch B. 1992. The water-to-land transition: evolution of thetetrapod basilar papilla, middle ear and auditory nuclei. In:Webster DB, Fay RR, Popper AN, eds. The evolutionarybiology of hearing. Berlin: Springer-Verlag. pp 351–375.
Fritzsch B. 2001. The morphology and function of fish ears.In: Ostrander GK, ed. The laboratory fish. New York: Elsev-ier. pp 250–259.
Fritzsch B. 2003. Development of inner ear afferent connec-tions: forming primary neurons and connecting them to thedeveloping sensory epithelia. Brain Res Bull 60:423–433.
Fritzsch B, Beisel KW. 2001. Evolution and development ofthe vertebrate ear. Brain Res Bull 55:711–721.
Fritzsch B, Wake MH. 1988. The inner ear of gymnophineamphibians and its nerve supply: a comparative study ofregressive events in a complex sensory system (Amphibia,Gymnophina). Zoomorphology 108:201–217.
Fritzsch B, Silos-Santiago I, Bianchi LM, Fari~nas I. 1997. Therole of neurotrophic factors in regulating the developmentof inner ear innervation. Trends Neurosci 20:159–164.
Fritzsch B, Beisel KW, Jones K, Fari~nas I, Maklad A, Lee J,Reichardt LF. 2002. Development and evolution of innerear sensory epithelia and their innervation. J Neurobiol 53:143–156.
Fritzsch B, Matei VA, Nichols DH, Bermingham N, Jones K, BeiselKW, Wang VY. 2005. Atoh1 null mice show directed afferentfiber growth to undifferentiated ear sensory epithelia fol-lowed by incomplete fiber retention. Dev Dyn 233:570–583.
Fritzsch B, Beisel KW, Hansen LA. 2006. The molecular basisof neurosensory cell formation in ear development: a blue-print for hair cell and sensory neuron regeneration? Bioes-says 28:1181–1193.
Fritzsch B, Eberl DF, Beisel KW. 2010. The role of bHLH genesin ear development and evolution: revisiting a 10-year-oldhypothesis. Cell Mol Life Sci 67:3089–3099.
Goodyear RJ, Kwan T, Oh SH, Raphael Y, Richardson GP.2001. The cell adhesion molecule BEN defines a prosen-sory patch in the developing avian otocyst. J Comp Neurol434:275–288.
Haddon C, Lewis J. 1996. Early ear development in theembryo of the zebrafish, Danio rerio. J Comp Neurol 365:113–128.
Hajihosseini MK, De Langhe S, Lana-Elola E, Morrison H, Spar-shott N, Kelly R, Sharpe J, Rice D, Bellusci S. 2008. Local-ization and fate of Fgf10-expressing cells in the adultmouse brain implicate Fgf10 in control of neurogenesis.Mol Cell Neurosci 37:857–868.
Hamburger V, Hamilton HL. 1951. A series of normal stagesin the development of the chick embryo. Dev Dyn 195:231–272.
Hammond KL, Whitfield TT. 2006. The developing lamprey earclosely resembles the zebrafish otic vesicle: otx1 expres-sion can account for all major patterning differences.Development 133:1347–1357.
Heisenberg CP, Solnica-Krezel L. 2008. Back and forthbetween cell fate specification and movement during verte-brate gastrulation. Curr Opin Genet Dev 18:311–316.
Hidalgo-S�anchez M, Alvarado-Mallart R, Alvarez IS. 2000.Pax2, Otx2, Gbx2 and Fgf8 expression in early otic vesicledevelopment. Mech Dev 95:225–229.
Hidalgo-S�anchez M, Millet S, Bloch-Gallego E, Alvarado-MallartRM. 2005. Specification of the meso-isthmo-cerebellarregion: the Otx2/Gbx2 boundary. Brain Res Brain Res Rev49:134–149.
Hill J, Clarke JD, Vargesson N, Jowett T, Holder N. 1995. Exog-enous retinoic acid causes specific alterations in the devel-opment of the midbrain and hindbrain of the zebrafish
Fgf10 in the developing chick inner ear
The Journal of Comparative Neurology | Research in Systems Neuroscience 1161
embryo including positional respecification of the Mauthnerneuron. Mech Dev 50:3–16.
Hoshino T, Kodama A. 1976. Scanning electron microscopicstudy of the cat papilla neglecta. Arch Otorhinolaryngol212:141–146.
Hume CR, Bratt DL, Oesterle EC. 2007. Expression of LHX3and SOX2 during mouse inner ear development. Gene ExprPatterns 7:798–807.
Igarashi M, Finch PW, Aaronson SA. 1998. Characterization ofrecombinant human fibroblast growth factor (FGF)-10reveals functional similarities with keratinocyte growth fac-tor (FGF-7). J Biol Chem 273:13230–13235.
Itoh N, Ornitz DM. 2011. Fibroblast growth factors: from mo-lecular evolution to roles in development, metabolism anddisease. J Biochem 149:121–130.
Kariya S, Cureoglu S, Schachern PA, Sampaio AL, PaparellaMM, Kusunoki T, Oktay MF, Nishizaki K. 2005. Quantitativestudy of the vestibular sensory epithelium in cochleosaccu-lar dysplasia. Otol Neurotol 26:495–499.
Kawamoto K, Ishimoto S, Minoda R, Brough DE, Raphael Y.2003. Math1 gene transfer generates new cochlear haircells in mature guinea pigs in vivo. J Neurosci 23:4395–4400.
Kelly M, Chen P. 2007. Shaping the mammalian auditory sen-sory organ by the planar cell polarity pathway. Int J DevBiol 51:535–547.
Kelly MC, Chen P. 2009. Development of form and function inthe mammalian cochlea. Curr Opin Neurobiol 19:395–401.
Kiernan AE, Nunes F, Wu DK, Fekete DM. 1997. The expres-sion domain of two related homeobox genes defines a com-partment in the chicken inner ear that may be involved insemicircular canal formation. Dev Biol 191:215–229.
Kiernan AE, Ahituv N, Fuchs H, Balling R, Avraham KB, SteelKP, Hrabe de Angelis M. 2001. The Notch ligand Jagged1is required for inner ear sensory development. Proc NatlAcad Sci U S A 98:3873–3878.
Kiernan AE, Pelling AL, Leung KK, Tang AS, Bell DM, Tease C,Lovell-Badge R, Steel KP, Cheah KS. 2005. 2005. Sox2 isrequired for sensory organ development in the mammalianinner ear. Nature 434:1031–1035.
Kiernan AE, Xu J, Gridley T. 2006. The Notch ligand JAG1 isrequired for sensory progenitor development in the mam-malian inner ear. PLoS Genet 2:e4.
Knights V, Cook SJ. De-regulated FGF receptors as therapeu-tic targets in cancer. Pharmacol Ther 125:105–117.
Knowlton VY. 1967. Effects of extraembryonic membrane defi-ciency on differentiation of the embryonic avian brain andsense organs. Acta Anat (Basel) 66:420–445.
Kobberup S, Schmerr M, Dang ML, Nyeng P, Jensen JN,MacDonald RJ, Jensen J. 2010. Conditional control of thedifferentiation competence of pancreatic endocrine andductal cells by Fgf10. Mech Dev 127:220–234.
Ladher RK, Wright TJ, Moon AM, Mansour SL, Schoenwolf GC.2005. FGF8 initiates inner ear induction in chick andmouse. Genes Dev 19:603–613.
Ladher RK, O’Neill P, Begbie J. 2010. From shared lineage todistinct functions: the development of the inner ear andepibranchial placodes. Development 137:1777–1785.
Laufer E, Dahn R, Orozco OE, Yeo CY, Pisenti J, Henrique D,Abbott UK, Fallon JF, Tabin C. 1997. Expression of Radicalfringe in limb-bud ectoderm regulates apical ectodermalridge formation. Nature 386:366–373.
Lewis CM. 1985. The vertebrate inner ear. Boca Raton, FL:CRC Press.
Lillev€ali K, Haugas M, Matilainen T, Pussinen C, Karis A, Salmi-nen M. 2006. Gata3 is required for early morphogenesisand Fgf10 expression during otic development. Mech Dev123:415–429.
Liu W, Levi G, Shanske A, Frenz DA. 2008. Retinoic acid-induced inner ear teratogenesis caused by defective Fgf3/Fgf10-dependent Dlx5 signaling. Birth Defects Res B DevReprod Toxicol 83:134–144.
Ma EY, Raible DW. 2009. Signaling pathways regulating zebra-fish lateral line development. Curr Biol 19:R381–386.
Mahoney RAA, Zhang J, Shim K. 2011. Sprouty1 and Sprouty2limit both the size of the otic placode and hindbrain Wnt8aby antagonizing FGF signaling. Dev Biol 353:94–104.
Mak AC, Szeto IY, Fritzsch B, Cheah KS. 2009. Differentialand overlapping expression pattern of SOX2 and SOX9 ininner ear development. Gene Expr Patterns 9:444–453.
Manley GA. 2000. Cochlear mechanisms from a phylogeneticviewpoint. Proc Natl Acad Sci U S A 97:11736–11743.
Mark M, Chambon P. 2003. Functions of RARs and RXRs invivo: genetic dissection of the retinoid signaling pathway.Pure Appl Chem 75:1709–1732.
Matei V, Pauley S, Kaing S, Rowitch D, Beisel KW, Morris K,Feng F, Jones K, Lee J, Fritzsch B. 2005. Smaller inner earsensory epithelia in Neurog 1 null mice are related to ear-lier hair cell cycle exit. Dev Dyn 234:633–650.
Mazan S, Jaillard D, Baratte B, Janvier P. 2000. Otx1 gene-controlled morphogenesis of the horizontal semicircularcanal and the origin of the gnathostome characteristics.Evol Dev 2:186–193.
Merino R, Rodriguez-Leon J, Macias D, Ganan Y, EconomidesAN, Hurle JM. 1999. The BMP antagonist Gremlin regulatesoutgrowth, chondrogenesis and programmed cell death inthe developing limb. Development 126:5515–5522.
Millimaki BB, Sweet EM, Dhason MS, Riley BB. 2007. Zebrafishatoh1 genes: classic proneural activity in the inner ear andregulation by Fgf and Notch. Development 134:295–305.
Millimaki BB, Sweet EM, Riley BB. 2010. Sox2 is required formaintenance and regeneration, but not initial development, ofhair cells in the zebrafish inner ear. Dev Biol 338:262–269.
Miyazaki H, Kobayashi T, Nakamura H, Funahashi J. 2006.Role of Gbx2 and Otx2 in the formation of cochlear gan-glion and endolymphatic duct. Dev Growth Differ 48:429–438.
Montandon P, Gacek RR, Kimura RS. 1970. Crista neglecta inthe cat and human. Ann Otol Rhinol Laryngol 79:105–112.
Morsli H, Choo D, Ryan A, Johnson R, Wu DK. 1998. Develop-ment of the mouse inner ear and origin of its sensoryorgans. J Neurosci 18:3327–3335.
Neves J, Kamaid A, Alsina B, Giraldez F. 2007. Differentialexpression of Sox2 and Sox3 in neuronal and sensory pro-genitors of the developing inner ear of the chick. J CompNeurol 503:487–500.
Neves J, Parada C, Chamizo M, Giraldez F. 2011. Jagged 1regulates the restriction of Sox2 expression in the develop-ing chicken inner ear: a mechanism for sensory organspecification. Development 138:735–744.
Neves J, Uchikawa M, Bigas A, Giraldez F. 2012. The prosen-sory function of Sox2 in the chicken inner ear relies onthe direct regulation of Atoh1. PLoS One 7:e30871.
Nichols DH, Pauley S, Jahan I, Beisel KW, Millen KJ, FritzschB. 2008. Lmx1a is required for segregation of sensory epi-thelia and normal ear histogenesis and morphogenesis.Cell Tissue Res 334:339–358.
Oesterle EC, Campbell S, Taylor RR, Forge A, Hume CR.2008. Sox2 and JAGGED1 expression in normal and drug-damaged adult mouse inner ear. J Assoc Res Otolaryngol9:65–89.
Oh SH, Johnson R, Wu DK. 1996. Differential expression ofbone morphogenetic proteins in the developing vestibularand auditory sensory organs. J Neurosci 16:6463–6475.
Ohuchi H, Nakagawa T, Yamamoto A, Araga A, Ohata T,Ishimaru Y, Yoshioka H, Kuwana T, Nohno T, Yamasaki M,
S�anchez-Guardado et al.
1162 The Journal of Comparative Neurology |Research in Systems Neuroscience
Itoh N, Noji S. 1997. The mesenchymal factor, FGF10, ini-tiates and maintains the outgrowth of the chick limb budthrough interaction with FGF8, an apical ectodermal factor.Development 124:2235–2244.
Ohuchi H, Hori Y, Yamasaki M, Harada H, Sekine K, Kato S,Itoh N. 2000. FGF10 acts as a major ligand for FGF recep-tor 2 IIIb in mouse multi-organ development. Biochem Bio-phys Res Commun 277:643–649.
Ohuchi H, Yasue A, Ono K, Sasaoka S, Tomonari S, Takagi A,Itakura M, Moriyama K, Noji S, Nohno T. 2005. Identifica-tion of cis-element regulating expression of the mouseFgf10 gene during inner ear development. Dev Dyn 233:177–187.
Ohyama T, Groves AK, Martin K. 2007. The first steps towardshearing: mechanisms of otic placode induction. Int J DevBiol 51:463–472.
Pan N, Jahan I, Kersigo J, Kopecky B, Santi P, Johnson S,Schmitz H, Fritzsch B. 2011. Conditional deletion of Atoh1using Pax2-Cre results in viable mice without differentiatedcochlear hair cells that have lost most of the organ ofCorti. Hear Res 275:66–80.
Parkinson N, Collins MM, Dufresne L, Ryan AK. 2010. Expres-sion patterns of hormones, signaling molecules, and tran-scription factors during adenohypophysis development inthe chick embryo. Dev Dyn 239:1197–1210.
Parsa S, Kuremoto K, Seidel K, Tabatabai R, Mackenzie B,Yamaza T, Akiyama K, Branch J, Koh CJ, Al Alam D, KleinOD, Bellusci S. 2010. Signaling by FGFR2b controls the re-generative capacity of adult mouse incisors. Development137:3743–3752.
Pauley S, Wright TJ, Pirvola U, Ornitz D, Beisel K, Fritzsch B.2003. Expression and function of FGF10 in mammalianinner ear development. Dev Dyn 227:203–215.
Pauley S, Lai E, Fritzsch B. 2006. Foxg1 is required for mor-phogenesis and histogenesis of the mammalian inner ear.Dev Dyn 235:2470–2482.
Perez SE, Rebelo S, Anderson DJ. 1999. Early specification ofsensory neuron fate revealed by expression and functionof neurogenins in the chick embryo. Development 126:1715–1728.
Pevny L, Placzek M. 2005. SOX genes and neural progenitoridentity. Curr Opin Neurobiol 15:7–13.
Pirvola U, Spencer-Dene B, Xing-Qun L, Kettunen P, Thesleff I,Fritzsch B, Dickson C, Ylikoski J. 2000. FGF/FGFR-2(IIIb)signaling is essential for inner ear morphogenesis. J Neuro-sci 20:6125–6134.
Platt C, Jorgensen JM, Popper AN. 2004. The inner ear of thelungfish Protopterus. J Comp Neurol 471:277–288.
Polanska UM, Fernig DG, Kinnunen T. 2009. Extracellularinteractome of the FGF receptor-ligand system: complex-ities and the relative simplicity of the worm. Dev Dyn 238:277–293.
Potok MA, Cha KB, Hunt A, Brinkmeier ML, Leitges M, KispertA, Camper SA. 2008. WNT signaling affects gene expres-sion in the ventral diencephalon and pituitary glandgrowth. Dev Dyn 237:1006–1020.
Pourqui O, Coltey M, Thomas JL, Le Douarin NM. 1990. Awidely distributed antigen developmentally regulated in thenervous system. Development 109:743–752.
Pujades C, Kamaid A, Alsina B, Giraldez F. 2006. BMP-signal-ing regulates the generation of hair-cells. Dev Biol 292:55–67.
Rodriguez-Esteban C, Schwabe JW, De La Pena J, Foys B,Eshelman B, Izpisua Belmonte JC. 1997. Radical fringepositions the apical ectodermal ridge at the dorsoventralboundary of the vertebrate limb. Nature 386:360–366.
Romand R, Albuisson E, Niederreither K, Fraulob V, ChambonP, Dolle P. 2001. Specific expression of the retinoic acid-
synthesizing enzyme RALDH2 during mouse inner ear de-velopment. Mech Dev 106:185–189.
Romand R, Hashino E, Dolle P, Vonesch JL, Chambon P, Ghy-selinck NB. 2002. The retinoic acid receptors RARalphaand RARgamma are required for inner ear development.Mech Dev 119:213–223.
Romand R, Niederreither K, Abu-Abed S, Petkovich M, FraulobV, Hashino E, Dolle P. 2004. Complementary expressionpatterns of retinoid acid-synthesizing and -metabolizingenzymes in pre-natal mouse inner ear structures. GeneExpr Patterns 4:123–133.
Romand R, Dolle P, Hashino E. 2006a. Retinoid signaling ininner ear development. J Neurobiol 66:687–704.
Romand R, Kondo T, Fraulob V, Petkovich M, Dolle P, HashinoE. 2006b. Dynamic expression of retinoic acid-synthesizingand -metabolizing enzymes in the developing mouse innerear. J Comp Neurol 496:643–654.
Ross SA, McCaffery PJ, Drager UC, De Luca LM. 2000. Retinoidsin embryonal development. Physiol Rev 80:1021–1054.
Sahara S, O’Leary DD. 2009. Fgf10 regulates transition period ofcortical stem cell differentiation to radial glia controlling gen-eration of neurons and basal progenitors. Neuron 63:48–62.
Sampaio AL, Cureoglu S, Schachern PA, Kusunoki T, PaparellaMM, Oliveira CA. 2004. Cochleosaccular dysplasia: a mor-phometric and histopathologic study in a series of tempo-ral bones. Otol Neurotol 25:530–535.
S�anchez-Calderon H, Martin-Partido G, Hidalgo-S�anchez M.2002. Differential expression of Otx2, Gbx2, Pax2, andFgf8 in the developing vestibular and auditory sensoryorgans. Brain Res Bull 57:321–323.
S�anchez-Calderon H, Martin-Partido G, Hidalgo-S�anchez M.2004. Otx2, Gbx2, and Fgf8 expression patterns in thechick developing inner ear and their possible roles in oticspecification and early innervation. Gene Expr Patterns 4:659–669.
S�anchez-Calderon H, Martin-Partido G, Hidalgo-S�anchez M.2005. Pax2 expression patterns in the developing chickinner ear. Gene Expr Patterns 5:763–773.
S�anchez-Calderon H, Francisco-Morcillo J, Martin-Partido G,Hidalgo-S�anchez M. 2007a. Fgf19 expression patterns inthe developing chick inner ear. Gene Expr Patterns 7:30–38.
S�anchez-Calderon H, Milo M, Leon Y, Varela-Nieto I. 2007b. Anetwork of growth and transcription factors controls neuro-nal differentation and survival in the developing ear. Int JDev Biol 51:557–570.
S�anchez-Guardado LO, Ferran JL, Mijares J, Puelles L, Rodri-guez-Gallardo L, Hidalgo-S�anchez M. 2009. Raldh3 geneexpression pattern in the developing chicken inner ear. JComp Neurol 514:49–65.
S�anchez-Guardado LO, Ferran JL, Rodriguez-Gallardo L,Puelles L, Hidalgo-S�anchez M. 2011. Meis gene expressionpatterns in the developing chicken inner ear. J Comp Neu-rol 519:125–147.
Schimmang T. 2007. Expression and functions of FGF ligandsduring early otic development. Int J Dev Biol 51:473–481.
Schneider-Maunoury S, Pujades C. 2007. Hindbrain signals inotic regionalization: walk on the wild side. Int J Dev Biol51:495–506.
Sienknecht UJ, Fekete DM. 2008. Comprehensive Wnt-relatedgene expression during cochlear duct development inchicken. J Comp Neurol 510:378–395.
Sienknecht UJ, Fekete DM. 2009. Mapping of Wnt, frizzled,and Wnt inhibitor gene expression domains in the avianotic primordium. J Comp Neurol 517:751–764.
Storey KG, Crossley JM, De Robertis EM, Norris WE, SternCD. 1992. Neural induction and regionalisation in the chickembryo. Development 114:729–741.
Fgf10 in the developing chick inner ear
The Journal of Comparative Neurology | Research in Systems Neuroscience 1163
Streit A. 2007. The preplacodal region: an ectodermal domainwith multipotential progenitors that contribute to senseorgans and cranial sensory ganglia. Int J Dev Biol 51:447–461.
Sweet EM, Vemaraju S, Riley BB. 2011. Sox2 and Fgf interactwith Atoh1 to promote sensory competence throughoutthe zebrafish inner ear. Dev Biol 358:113–121.
Takemoto T, Uchikawa M, Kamachi Y, Kondoh H. 2006. Con-vergence of Wnt and FGF signals in the genesis of poste-rior neural plate through activation of the Sox2 enhancerN-1. Development 133:297–306.
Tang N, Marshall WF, McMahon M, Metzger RJ, Martin GR.2001. Control of mitotic spindle angle by the RAS-regu-lated ERK1/2 pathway determines lung tube shape. Sci-ence 333:342–345.
Tomsa JM, Langeland JA. 1999. Otx expression during lampreyembryogenesis provides insights into the evolution of thevertebrate head and jaw. Dev Biol 207:26–37.
Uchikawa M, Kamachi Y, Kondoh H. 1999. Two distinct sub-groups of Group B Sox genes for transcriptional activatorsand repressors: their expression during embryonic organo-genesis of the chicken. Mech Dev 84:103–120.
Umemori H, Linhoff MW, Ornitz DM, Sanes JR. 2004. FGF22and its close relatives are presynaptic organizing mole-cules in the mammalian brain. Cell 118:257–270.
Whitfield TT, Hammond KL. 2007. Axial patterning in the devel-oping vertebrate inner ear. Int J Dev Biol 51:507–520.
Wittler L, Kessel M. 2004. The acquisition of neural fate inthe chick. Mech Dev 121:1031–1042.
Wright TJ, Mansour SL. 2003. Fgf3 and Fgf10 are required formouse otic placode induction. Development 130:3379–3390.
Wu DK, Oh SH. 1996. Sensory organ generation in the chickinner ear. J Neurosci 16:6454–6462.
Yamada T, Placzek M, Tanaka H, Dodd J, Jessell TM. 1991.Control of cell pattern in the developing nervous system:polarizing activity of the floor plate and notochord. Cell 64:635–647.
Yu K, Herr AB, Waksman G, Ornitz DM. 2000. Loss of fibro-blast growth factor receptor 2 ligand-binding specificity inApert syndrome. Proc Natl Acad Sci U S A 97:14536–14541.
Yun YR, Won JE, Jeon E, Lee S, Kang W, Jo H, Jang JH, ShinUS, Kim HW. 2010. Fibroblast growth factors: biology,function, and application for tissue regeneration. J TissueEng 2010:218142.
Zakany J, Zacchetti G, Duboule D. 2007. Interactions betweenHOXD and Gli3 genes control the limb apical ectodermalridge via Fgf10. Dev Biol 306:883–893.
Zelarayan LC, Vendrell V, Alvarez Y, Dominguez-Frutos E, TheilT, Alonso MT, Maconochie M, Schimmang T. 2007. Differ-ential requirements for FGF3, FGF8 and FGF10 duringinner ear development. Dev Biol 308:379–391.
Zhang X, Stappenbeck TS, White AC, Lavine KJ, Gordon JI,Ornitz DM. 2006. Reciprocal epithelial-mesenchymal FGFsignaling is required for cecal development. Development133:173–180.
Zheng JL, Gao WQ. 2000. Overexpression of Math1 inducesrobust production of extra hair cells in postnatal rat innerears. Nat Neurosci 3:580–586.
Zine A, Aubert A, Qiu J, Therianos S, Guillemot F, KageyamaR, de Ribaupierre F. 2001. Hes1 and Hes5 activities arerequired for the normal development of the hair cells inthe mammalian inner ear. J Neurosci 21:4712–4720.
S�anchez-Guardado et al.
1164 The Journal of Comparative Neurology |Research in Systems Neuroscience