Fgf10 expression patterns in the developing chick inner ear

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: [email protected]

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



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.

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This

Study

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Manufacturer,species

antibodywasraisedin,

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cat.no.

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NIF

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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).



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.]



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.]



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.]



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.



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.]



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



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.



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.]



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.]



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.



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.]



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.



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



(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



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).



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



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.



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



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,



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.



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.



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Fgf10 expression patterns in the developing chick inner ear

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