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doi:10.1111/cga.12072

Congenital Anomalies 2015; 55, 17–25

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REVIEW ARTICLE

Signaling regulating inner ear development: Cell fate determination, patterning, morphogenesis, and defects Yuji Nakajima Department of Anatomy and Cell Biology, Graduate School of Medicine, Osaka City University, Osaka, Japan

ABSTRACT

The membranous labyrinth of the inner ear is a highly complex organ that detects sound and balance. Developmental defects in the inner ear cause congenital hearing loss and balance disorders. The membranous labyrinth consists of three semicircular ducts, the utricle, saccule, and endolymphatic ducts, and the cochlear duct. These complex structures develop from the simple otic placode, which is established in the cranial ectoderm adjacent to the neural crest at the level of the hindbrain at the early neurula stage. During development, the otic placode invaginates to form the otic vesicle, which subsequently gives rise to neurons for the vestibulocochlear ganglion, the non-sensory and sensory epithelia of the membranous labyrinth that includes three ampullary crests, two maculae, and the organ of Corti. Combined paracrine and autocrine signals including fibroblast growth factor, Wnt, retinoic acid, hedgehog, and bone morphogenetic protein regulate fate determination, axis formation, and morphogenesis in the developing inner ear. Juxtacrine signals mediated by Notch pathways play a role in establishing the sensory epithelium, which consists of mechanosensory hair cells and supporting cells. The highly differentiated organ of Corti, which consists of uniformly oriented inner/outer hair cells and specific supporting cells, develops during fetal development. Developmental alterations/arrest causes congenital malformations in the inner ear in a spatiotemporal-restricted manner. A clearer understanding of the mechanisms underlying inner ear development is important not only for the management of patients with congenital inner ear malformations, but also for the development of regenerative therapy for impaired function. Key Words: development, growth factors, inner ear, malformations, Notch

INTRODUCTION The inner ear exists in the petrous part of the temporal bone and consists of a membranous labyrinth and bony labyrinth. The membranous labyrinth consists of at least six mechanosensory epithelia, which include the organ of Corti in the cochlea for sound, three ampullary crests in the base of three semicircular ducts for angular acceleration, and two maculae, macula utriculi and sacculi, for gravity/linear acceleration. Electrical signals, which are translated from the mechanical signals evoked in these sensory epithelia, are transmitted to the brain stem via the vestibulocochlear nerve Correspondence: Yuji Nakajima, MD, PhD, Department of Anatomy and Cell Biology, Graduate School of Medicine, Osaka City University, 1-4-3 Asahimachi, Abenoku, Osaka, 545-8585 Japan. Email: [email protected] Received April 5, 2014; revised and accepted June 7, 2014.

(cranial nerve VIII). During organogenesis, reciprocal signaling between the embryonic tissues successively occurs to establish highly organized functional tissues and organs in a spatiotemporalrestricted manner. Developmental arrest/alterations as well as acquired injuries in the inner ear cause functional disorders for hearing and balance. The aim of this review is to discuss signaling that regulates the key developmental events responsible for the establishment of the complicated membranous labyrinth. Several excellent reviews on inner ear development have been published (Torres and Giraldez 1998; Abelló and Alsina 2007; Bok et al. 2007a; Driver and Kelley 2009; Groves and Fekete 2012; Magarinos et al. 2012; Neves et al. 2013).

EMBRYOGENESIS OF THE INNER EAR AND ITS MALFORMATIONS The membranous labyrinth and vestibulocochlear neurons are derived from an ectodermal thickening named the otic placode (OP), which develops from the cranial ectoderm immediately lateral to the neural crest at the level of the hindbrain at the neurula stage. The OP invaginates and is then being pinched off from the ectoderm, which results in the formation of the otic vesicle (otocyst). The posterior rim and its anterior domain of the OP give rise to non-sensory semicircular and cochlear epithelia, respectively (Abelló et al. 2007). Epithelial cells in the anteromedial domain of the OP/otic vesicle further differentiate to the proneurosensory epithelium, from which neural progenitors delaminate and give rise to neuroblasts of the vestibulocochlear ganglion (Satoh and Fekete 2005). After neural cell migration, the resulting prosensory domain later develops into five vestibular sensory patches and the organ of Corti (basilar papilla in birds). The dorsal epithelium of the otic vesicle expands to form the vertical pouch; its opposing epithelia at the future anterior and posterior semicircular ducts later fuse to be absorbed resulting in the formation of two superior semicircular ducts and the common crus (Fig. 1, Martin and Swanson 1993; Chang et al. 2004). The lateral semicircular duct develops from the lateral pouch and the endolymphatic duct from the dorsomedial epithelium of the otic vesicle. The five sensory patches that develop in the vestibular labyrinth are three cristae in the anterior, posterior, and lateral ampulla; and two maculae in the utricle and saccule. These cristae detect head rotation while the maculae maintain static equilibrium. The organ of Corti, which develops specifically in the mammalian cochlea, is a highly differentiated sensory epithelium for sound. The sensory epithelium basically consists of sensory hair cells and supporting cells, while highly differentiated sensory and supporting cells develop in the organ of Corti, that is, three lateral rows of outer hair cells and a medial row of inner hair cells, with inner phalangeal cells (supporting inner hair cells), outer Deiters’ cells (supporting outer hair cells), and pillar cells (demarcating the inner and outer hair cells and making the tunnel of Corti). © 2014 Japanese Teratology Society

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Fig. 1 Inner ear development and corresponding malformations. Modified from Jackler et al. (1987) and Yasuda et al. (2007). an, anterior semicircular duct; c, cochlear duct; CS, Carnegie stage; SCC, semicircular canal (duct); e, endolymphatic duct; l, lateral SCC; p, posterior SCC; pl, primordium of the lateral semicircular duct; v, vestibular pouch; W, age in weeks. Note that the superior SCC includes the anterior SCC and the posterior SCC.

Table 1 Classification of inner ear malformations based on embryogenesis Complete labyrinth aplasia (Michel) Cochlear malformations Aplasia Common cavity Hypoplasia Incomplete partition (Mondini) Semicircular canal malformations Aplasia Small buds Superior and lateral SCC dysplasia Lateral SCC aplasia/dysplasia Enlarged vestibular aqueduct Note that the superior semicircular canal (SCC) includes the anterior SCC and the posterior SCC. The most common cochlear malformation is incomplete partition, and the most frequently diagnosed SCC malformation is lateral SCC dysplasia (Jackler et al. 1987; Brenski and Arjmand 2003).

Developmental arrest in the membranous labyrinth leads to various congenital inner ear defects in temporally and spatially restricted manners. These inner ear malformations include cochlear, semicircular canal, aqueduct, and combined malformations. Based on embryogenesis and radiological findings, cochlear malformations have been classified as aplasia, common cavity, hypoplasia, and incomplete partition, while semicircular canal malformations have been classified as aplasia, small buds, dysplasia of the three semicircular canals, and dysplasia of the lateral semicircular canal (Fig. 1, Table 1, Jackler et al. 1987; Sennaroglu and Saatci 2002; Brenski and Arjmand 2003). Developmental defects/arrest in the inner ear and its surrounding structures are often diagnosed in children with sensorineural hearing loss; therefore, an accurate morphological assessment of the inner ear by high-resolution CT (computed tomography) is important (Masuda et al. 2013). © 2014 Japanese Teratology Society

PREPLACODE REGION The sensory nervous system in the head is derived from both the neural crest and placodes, while that in the trunk is derived from the neural crest alone. At the late gastrula to early neurula stage, the developing ectoderm is subdivided into at least four distinct regions: the neural plate, neural crest, and epidermal ectoderm from the head to the trunk; and preplacode region (PPR) in only the head ectoderm. Therefore, the establishment of PPR may be precisely regulated by surrounding tissues in a spatiotemporal-restricted manner. Signals derived from the head and lateral mesoderm (including the heart-forming mesoderm) are important for defining and establishing the PPR adjacent to the neural crest at the cranial region (Fig. 2, Litsiou et al. 2005). Excess bone morphogenetic protein (BMP) and Wnt signals, which are secreted from the ectoderm and neural plate, respectively, act to suppress the expression of PPR marker genes, such as Six1, Six4, and Eya2, in the PPRforming ectoderm. Together with the BMP/Wnt signals, Cerberus (BMP/Wnt antagonist) and fibroblast growth factor (FGF), which are secreted from the mesoderm subjacent to the prospective PPR, upregulate the expression of PPR genes (Fig. 2, Litsiou et al. 2005). The anterior-posterior patterning of the PPR is regulated by mutual repression between Gbx2 and Otx2, that is, posteriorly localized Gbx2 is required for the formation of the OP, while anterior Otx2 is required for that of the olfactory, lens, and trigeminal placode in the anterior PPR (Steventon et al. 2012). Six, Dach, and Eya are expressed in developing organs including ear and eye (Li et al. 2003; Zou et al. 2004). The Six1 protein acts as both a repressor and activator in a context-dependent manner. The Six-Dach complex without Eya acts to repress target genes, while the Six-Dach-Eya complex, in which Eya exhibits phosphatase activity to suppress the co-repressor complex, recruits the co-activator to activate target genes for the promotion of organogenesis (Li et al. 2003). Development of the inner ear, nose, skeletal muscle, thymus, and kidney was shown to be severely affected in Six1-null mice, (Ozaki et al. 2003). In this mutant, the vestibular labyrinth is a single space with an expanded endolymphatic duct, and ventral structures (cochlea and vestibulocochlear ganglion) are absent (Ozaki et al. 2003). A previous study reported that the initial cell fate determination and migration of vestibulocochlear neurons were unaffected in Eya- and

Inner ear development

Fig. 2 Signals regulating the preplacode region (PPR). In the late gastrula to early neurula embryo, PPR (pink) is determined in the head ectoderm (Ect), which is immediately lateral to the neural crest (NCr, green) and dorsal to the heart/head mesoderm. Excess bone morphogenetic protein (BMP) and Wnt signals, which are derived from the ectoderm (yellow) and neural plate (NP, blue), respectively, suppress PPR genes. Fibroblast growth factor (FGF) and Cerberus (Cer, a BMP/Wnt antagonist), which are secreted from the heart/head mesoderm, can upregulate the expression of PPR genes.

Six1-null mutant mice; however, early neurogenesis was affected in the mutant inner ear (Zou et al. 2004). Genes expressed in the ventral otic vesicle of the Six1-null developing inner ear, including Otx1, Otx2, Lunatic fringe (Lfng), Fgf3, Fgf10, Bmp4, Gata3, and Nkx5.1, were found to be suppressed (Ozaki et al. 2003; Zou et al. 2004). In humans, an impaired EYA1-SIX1 regulatory network is one of the candidates involved in branchio-oto-renal syndrome, which is associated with the incomplete partition of the cochlear duct as well as semicircular canal dysplasia (Propst et al. 2005; Kochhar et al. 2007; Senel et al. 2009).

THE OTIC PLACODE The otic placode (OP) is induced by combined signaling that is mediated by the head mesoderm and hindbrain in vertebrates (Fig. 3). During the inner ear development, mesodermal tissue adjacent to the hindbrain expresses FGF, which defines the posterior PPR (precursor region of OP and epibranchial placode) and induces the expression of Wnt8a and FGF in the hindbrain. Signaling mediated by anterior hindbrain-derived Wnt8a and FGF was shown to involve the differentiation of OP (Ladher et al. 2000, 2010; Urness et al. 2010; Groves and Fekete 2012). An explantation experiment revealed that endoderm-derived FGF8 acts as an upstream signal of FGF in the chick mesoderm (Ladher et al. 2005). The expression of Fgf10 in the mesoderm subjacent to prospective OP ectoderm of Fgf3-null and hypomorphic Fgf8 mouse embryo was reduced, which resulted in the failed formation of the OP, suggesting that FGF8/3 acts as an upstream signal of Fgf10 in the head mesoderm during the induction of the OP. Taken together with these findings, pharyngeal endoderm-derived FGF8 plays an upstream role in the FGF signaling cascade to induce the OP in both the chick and mouse (Ladher et al. 2005). In addition, hindbrain-derived Wnt signaling was reported to suppress the expression of the epibranchial placode gene, Foxi2, which indicated that Wnt signaling also defined the area of the otic region (Freter et al. 2008) (Fig. 3).

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Fig. 3 Signals regulating otic placode (OP) formation. Head mesodermsecreted FGF, the expression of which is induced by the pharyngeal endoderm (Pha) FGF8, defines the posterior preplacode region (PPR, pink) and upregulates the expression of Wnt8a and FGF in the hindbrain (HB). Anterior hindbrain-derived FGF and Wnt8a specify the PPR to OP (green). Wnt8a suppresses the epibranchial placode (Epi, purple) gene, Foxi2, to demarcate the area of the OP.

Fgf3 is expressed in the hindbrain adjacent to the prospective OP at the level of rhombomere 4–6, as well as in the OP itself, at the onset of OP formation in the mouse. Fgf10 was shown to be expressed in the head mesenchyme underlying the prospective OP-forming ectoderm (Wright and Mansour 2003; Alvarez et al. 2003). Gain-of-function experiments demonstrated that the ectopic expression of Fgf10 in the hindbrain was capable of inducing an OP-like structure, whereas Fgf3 only exhibited faint inducible activity (Alvarez et al. 2003). The Fgf10- and Fgf3-null mutants displayed only mild defects during formation of the otic vesicle; however, double knockout mutant had a hypoplastic otic vesicle, in which the expression of some otic marker genes, such as Pax2, Dlx5, and Sox9, was absent (Wright and Mansour 2003; Alvarez et al. 2003). These findings indicated the redundant role of FGFs and also that head mesoderm-derived FGF10 acts as one of the inductive signals for OP formation in the mouse. Human LAMM (labyrinth aplasia, microtia, and microdontia) syndrome, which is caused by homoallelic mutations in FGF3, presents as a series of inner ear malformations including complete labyrinth aplasia as well as common cavity and incomplete partitions (Ramsebner et al. 2010).

ANTERIOR-POSTERIOR PATTERNING At the early otic placode/vesicle stage, cells possessing both neurogenic and sensory lineages in the anteromedial domain express Fgf10 and Sox3 (Alsina et al. 2004; Abelló et al. 2007; 2010). Later these cells express Lim homeodomain transcription factor, Islet1 and homeobox transcription factor, Prox1 (Stone et al. 2003; Radde-Gallwitz et al. 2004). Neurogenin1 and NeuroD are later upregulated in neurogenic cells in this domain. The sensory marker gene Lfng was shown to be co-expressed in this neurogenic epithelium (Cole et al. 2000). Lineage tracing experiments with fluorescent dye in chick otic cup (16–18 somite stage) clarified that the anterior domain of the otic cup, posterior rim of the otic cup, and posteromedial ventral region of the otic cup gave rise to neurosensory domain, non-sensory epithelium of the semicircular ducts, and non-sensory epithelium of the cochlear duct, respectively (Abelló et al. 2007). A more detailed lineage analysis revealed that the prosensory region was located more peripheral to the © 2014 Japanese Teratology Society

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Y. Nakajima (Frenz et al. 1996). The most prominent abnormality in this model was the absence/hypoplasia of the vestibulocochlear ganglion, in which neural cells are developed from the anterior domain of the otic vesicle, which suggested that excess levels of RA may alter anterior positional information to posterior positional information, resulting in a reduction in neuronal differentiation.

DORSAL-VENTRAL PATTERNING

Fig. 4 Anterior-posterior (AP) axis formation in the otic placode. At the early otic placode/cup stage, the retinoic acid (RA)-synthesizing enzyme, Raldh2 (retinaldehyde dehydrogenese2), is expressed in the mesoderm posterior to the otic placode (OP), while RA-catabolizing enzyme (Cyp26) is expressed in the ectoderm, which is anterior to the OP. RA inhibits expression of the anterior genes of the OP, which suggests that the posterior-to-anterior gradient of RA regulates the AP axis of the OP. FGF8, which is expressed in the ectoderm anterior to the placode, upregulates the expression of anterior otic genes. Note that a dorsal view of the cranial half of the neurula embryo is shown.

neurogenic region, but also that some neurons and sensory cells developed from common progenitors (Satoh and Fekete 2005). At the early otic cup stage in the chick (11–15 somite stage), experiments demonstrated that AP (anterior–posterior) inversion of the otic cup (epithelium) alone did not affect the expression of anterior placode genes, while inversion with the associated preotic ectoderm altered the expression of anterior marker genes in the posterior otic cup, which suggested that signaling from tissue surrounding the otic cup defined the AP polarity of the otic cup (Bok et al. 2011). Retinoic acid (RA) is known to regulate the AP axis in the embryonic body and organs. At the early OP/cup stage in the chick embryo, the RA-synthesizing enzyme, retinaldehyde dehydrogenese2 (Raldh2) is expressed in the mesoderm just posterior to the otic region, while the P450-associated RA-catabolizing enzyme, Cyp26, is expressed in the ectoderm anterior to the OP, suggesting an posterior-to-anterior gradient of RA in the developing placode. Bead implantation experiments in chick embryos revealed that the posterior-to-anterior gradient of RA activity defined, at least partly, AP polarity in the inner ear (Fig. 4; Bok et al. 2011). Pax2 and Sox3 were found to be expressed in the otic and epibranchial ectoderm at the onset of otic development at the 5 somite stage. The expression of Sox3 subsequently becomes restricted to the anteromedial (neurosensory) domain of the otic region at the 10 somite stage by FGF8, which is expressed in the ectoderm anterior to the otic ectoderm (Abelló et al. 2010). Sox3 can induce anterior neurogenic markers, such as Sox2 and Delta1, and was shown to repress the non-neurogenic posterior gene, Lmx1 (Abelló et al. 2010). Therefore, in addition to RA, FGF8 plays a role in the establishment of AP polarity in the OP (Fig. 4). Once the neurosensory region is determined in the anterior domain, several Notch-related genes are expressed in a region-specific manner; that is, Lfng (a Notch modulator), Delta1 (ligand), and Hes5 (repressor) in the anterior region of the placode, and Serrate1 (ligand) and Hairy1 (Hey1, repressor) in the posterior domain. Notch1 (receptor) is expressed in the entire otic region. Various types of cochlear and vestibular malformations as well as a defective cartilaginous otic capsule were induced in mouse embryos exposed to maternally administered excess levels of RA © 2014 Japanese Teratology Society

After development of the otic vesicle is completed, establishing the dorsal-ventral (DV) axis is necessary to further generate the complex inner ear morphology consisting of the dorsal vestibular labyrinth and ventral cochlea. The early otic vesicle is located closely to the hindbrain (rhombomere 4–6) as well as the midline structure, the notochord. The ablation of the floor plate in the hindbrain and notochord, from which Shh (Sonic hedgehog) is secreted, caused a defective cochlea in chick embryos, and inhibiting Shh activity led to the same result (Bok et al. 2005). The experimental inversion of the DV axis in the hindbrain with the notochord resulted in an inverted DV axis in the otic vesicle, in which the expression of ventral marker genes including NeuroD, Lfng, and Six1 is shifted dorsally (Bok et al. 2005). These results indicated that Shh from the floor plate and notochord plays a critical role in establishing the DV axis in the inner ear. The ventral structures of the inner ear, such as the cochlea and vestibulocochlear ganglion, are absent in Shh-null mutant mice. Ventral markers including Otx1/2 and Pax2 were reported to be downregulated in this mutant, while the dorsal marker gene Dlx5 was upregulated, which suggested that the Shh signal may be required to specify the ventral structure of the otic vesicle (Riccomagno et al. 2002). The neurogenic markers, Neurogenin1 and NeuroD, were also downregulated; therefore, the vestibulocochlear ganglion was absent in the mutant inner ear. However, expression of the sensory markers, Bmp4, Lfng, and Fgf3 in the crests and maculae remained unaltered, which suggested that Shh is required for neurogenic specification, but not for sensory lineage during inner ear development (Riccomagno et al. 2002). Gli3 is one of the transcriptional mediators for the Shh signal and is thought to act not only as a transcriptional activator, but also as a transcriptional repressor that is dependent on Shh protein levels (Jacob and Briscoe 2003). Shh-dependent Gli3 functions are required for pattern formations during development, such as the DV axis in the spinal cord and neocortex in the brain (Komada 2012). Using several combinations of mutant alleles for Shh, Gli2, and Gli3, Bok et al. (2007b) reported that in addition to the Gli2/Gli3dependent activator signal, Gli3-mediated repression was required for the morphology of the semicircular ducts (Fig. 5). In addition, formation of the distal-most cochlear duct at the ventral region requires a strong Shh activator signal, while the proximal cochlear and saccule requires a weaker Shh signal controlled by Gli2mediated activator and Gli3-mediated repressor signals (Bok et al. 2007b).

ONSET OF NEURAL AND SENSORY CELL DEVELOPMENT Neurosensory progenitors develop in the anterior domain of the otic cup at the onset of neural development in the inner ear. They express the HMG-box transcription factor Sox2, which is thought to maintain the multipotency of tissue-specific stem cells including neurons (Takahashi and Yamanaka 2006). A previous study demonstrated that the expression of Sox2 is regulated by a signaling cascade

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Fig. 5 Dorsal-ventral (DV) patterning of the inner ear. The otic vesicle, which is located adjacent to the hindbrain (HB), gives rise to the ventral cochlea duct (CD) and dorsal semicircular ducts. At this time, the floor plate (FP) and notochord (NCh) secrete Shh (Sonic hedgehog) to establish the DV axis of the inner ear and neural tube. The Shh signal is converted to Gli2/3 activator and Gli3 repressor activities in a Shh concentration-dependent manner. ES, endolymphatic duct; LSCD, lateral semicircular duct; SSCD, superior semicircular duct; S, saccule; U, utricle

including FGF, BMP, Wnt, and Tbx6 (Takemoto 2013). At the onset of neural development in the OP, prospective neuroblasts are specified in the neurosensory epithelium and then delaminate to generate neurons for the vestibulocochlear ganglion, from which bipolar neurons will connect hair cells to the brain via the vestibulocochlear nerve. Delaminated neurons lose their expression of Sox2 as well as proliferative activity. At the onset of neural determination, prospective neuroblasts express Neurogenin1, the expression of which is positively regulated by Sox2. Neurogenin1 has been reported to repress the expression of Sox2 by a negative feedback loop (Evsen et al. 2013). Therefore, negative feedback inhibition to Sox2 by Neurogenin1 (also by NeuroD) is required for progressive neurogenesis (Fig. 6). In cultured otic vesicles, FGF10 was found to reduce the proliferation of neurosensory progenitors, and, consequently, the expansion of NeuroD-expressing neural cells. On the other hand, SU5402 (FGF receptor inhibitor) stimulates the proliferation of neurosensory progenitors, thereby reducing the number of mature neurons. Therefore, the FGF signal acts on the progenitor cells, resulting in their exit from the cell cycle and commitment to a neural fate (Alsina et al. 2004). Neurogenin1 and/or Sox3 also upregulate the expression of Delta1 (Notch ligand) in neurogenic cells. This then activates Notch signaling in neighboring cells, which inhibits Neurogenin1 by Hes-mediated lateral inhibition, which suppresses neurogenesis and the maintenance of Sox2 expression, resulting in the preservation of undifferentiated sensory progenitors (Jeon et al. 2011, Fig. 6). Once the prosensory domains are established, Jagged1 (another Notch ligand) acts to maintain the expression of Sox2 probably by Hesr-mediated lateral induction (Hayashi et al. 2008a; Neves et al. 2011, Fig. 6). Prosensory progenitors later differentiate to Sox2-negative hair cells and Sox2positive supporting cells (Neves et al. 2007). Nascent hair cells express Delta1 during this process to accelerate hair cell fate in a cell-autonomous manner, while Delta1 activates Notch signaling in the neighboring cells to suppress Atoh1 via Hes1/5 to maintain supporting cells in a non-cell-autonomous manner (Chrysostomou et al. 2012). Sox2 has been shown to exhibit both positive and negative regulatory functions for Atoh1 in a stage-dependent

Fig. 6 Neurosensory development. In inner ear development, neural cells and sensory cells develop from the proneurosensory domain, which is located in the anteromedial region of the otic vesicle and expresses Sox2. At the onset of neural development, Sox2 initiates the expression of Neurogenin1 (Ngn1), which then suppresses Sox2 and upregulates the expression of NeuroD. Proneural cells also express the Notch ligand, Delta1 (Dl1), which activates Notch signaling in the neighboring cells to inhibit a neural fate, thereby maintaining Sox2-positive prosensory cells by lateral inhibition. Once the prosensory cells are determined, the Notch ligand, Jagged1 (Jag1), acts to maintain these prosensory cells probably by lateral induction. In some prosensory cells, Sox2 induces Atoh1 for hair cell differentiation. Prospective hair cells express Dl1, which binds to the Notch receptors of neighboring cells to maintain Sox2positive supporting cells by lateral inhibition.

fashion (Neves et al. 2012). Supporting cells still express Sox2 and proliferating characteristics, which suggests that Sox2 may be involved in the maintenance of stem cell characteristics as well as regenerative capacity.

SEMICIRCULAR DUCTS Three-dimensional morphogenesis has been examined in the inner ear by a paint-fill method (Bissonnette and Fekete 1996; Morsli et al. 1998; Mansour and Schoenwolf 2005). After completion of the pear-shaped otic vesicle (otocyst), two distinct bulges are formed, the pars superior (dorsal bulge) and pars inferior (ventral bulge), from which the semicircular ducts/utricle and cochlear duct/ saccule will later develop, respectively. The endolymphatic duct simultaneously emerges from the dorsal-medial surface of the otic vesicle. Two distinct pouches, the dorsal pouch and lateral pouch, subsequently develop from the pars superior. The dorsal pouch gives rise to two superior (vertical) ducts, the anterior and posterior semicircular ducts, while the lateral pouch gives rise to the lateral (horizontal) semicircular duct. At the onset of semicircular duct formation, the opposing epithelia in the center of each pouch fuse to form the epithelial fusion plate, which is thereafter resorbed by epithelial-mesenchymal transition and apoptosis, resulting in the formation of tube-like semicircular ducts (Kobayashi et al. 2008). © 2014 Japanese Teratology Society

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Fig. 7 Semicircular duct development. Wnt/β-catenin signaling stimulates the expression of BMP4/FGF10 in the cristae (yellow domain), which then induce the expression of Bmp2 in the prospective semicircular duct (SCD) epithelium. BMP2 induces the expression of Dlx5 in the perimeter rim of the pouch (future SCD) to inhibit Netrin1 expression, thereby maintaining the epithelial structure of the SCD. In the fusion plate of the pouch, Netrin1 is maintained to expand the surrounding mesenchyme, which then pushes the facing epithelia to fuse (fusion plate), resulting in the absorption of the fusion plate.

Each semicircular duct connects with the utricle, and the two superior (anterior and posterior) ducts possess a common duct, the crus membranous commune. The anterior and posterior ends of the vertical semicircular ducts (opposite sides of the common crus) and the anterior end of the lateral duct have their own ampulla connecting to the utricle. A mutant mouse with normal semicircular ducts and no sensory crista has not been yet generated, which suggests that the crista is involved in the formation of the semicircular ducts (Chang et al. 2004). In the developing crista (Fig. 7), Wnt/β-catenin signaling stimulates the expression of BMP4 and FGF10, which then induces the expression of Bmp2 in the prospective semicircular duct epithelium adjacent to the cristae (Chang et al. 1999, 2002, 2004; Gerlach et al. 2000). BMP2 has been shown to induce the expression of Dlx5 in the perimeter rim of the canal pouch to inhibit the expression of Netrin1, thereby maintaining the epithelial structure of the semicircular ducts (Chang et al. 2004, 2008; Rakowiecki and Epstein 2013). The expression of Netrin1 is maintained in the fusion plate of the canal pouch in order to expand the surrounding mesenchyme, which pushes the center of the epithelial pouch to generate the fusion plate. The fusion plate will then dissociate via apoptosis and epithelia-mesenchymal transition, leaving tube-like semicircular ducts (Salminen et al. 2000; Kobayashi et al. 2008).

COCHLEA At the onset of cochlear formation, cells in the medioanterior region of the otic vesicle proliferate and the cochlear bud elongates to form the cochlear duct, in which the specific sensory epithelium, the organ of Corti, develops. Once the fate of hair cells is determined, future hair cells exit the cell cycle; therefore, elongation/growth of the cochlear duct is thought to occur via convergent extension movements of cochlear cells, which is regulated by Wnt-planar cell © 2014 Japanese Teratology Society

Fig. 8 Epithelial differentiation of the organ of Corti. At early mammalian cochlear development, the medial cochlea epithelium is subdivided into three distinct regions from the medial (neural) to the lateral (abneural) site, i.e., Kölliker’s organ (neural side of the cochlear duct), the prosensory auditory domain, and the outer sulcus. Bmp4 is expressed in the outer epithelial region, and, thus, the establishment of a BMP signaling gradient, which is required to form three distinct epithelial regions. FGF20, the expression of which is directly regulated by Notch signals, is required for differentiation of the auditory sensory epithelium, which consists of hair cells and supporting cells. Inner hair cell-secreted FGF8 locally controls the differentiation of pillar cells from FGFR3-positive supporting cells. The Wnt-PCP pathway regulates not only the planar cell polarity of the hair cells, but also extension of the cochlear duct.

polarity (PCP) pathways including Rho-associated coiled-coil kinase (ROCK) and non-muscle myosin II activity (Yamamoto et al. 2009; Fritzsch et al. 2011). The cochlear duct did not grow in mutant mice in which Shh pathways related to fate determination/ cell proliferation were affected, which resulted in a common cavity or hypoplastic cochlea with or without lateral semicircular duct malformation (Riccomagno et al. 2002; Bok et al. 2007b). A shortened cochlea with a larger number of hair cell rows and other inner ear malformations occur in the Foxg1 mutant, in which embryonic morphogenesis including cell fate determination and proliferation is affected (Pauley et al. 2006). Mutant mice, in which the Wnt-PCP pathways are affected, were found to have a shortened cochlea with a larger number of disorganized hair cell rows (Yamamoto et al. 2009). A shortened cochlea reflects the incomplete partition of cochlea (Mondini) in human inner ear malformations (Table 1). Mice in which the Wnt-PCP pathways were deleted also displayed other congenital defects, such as conotruncal heart defects, skeletal defects, and social abnormalities (Etheridge et al. 2008). Cochlear elongation and hair cell development are coordinated in a contextdependent manner because hair cell development was shown to be affected in the Aho1 conditional mutant while almost normal growth was observed in the cochlear duct (Pan et al. 2011). A shortened cochlea with a smaller number of turns (Mondini) represents a common inner ear malformation diagnosed in children with congenital sensorineural hearing loss, and syndromes associated with this type of defect include trisomy 21, DiGeorge, CHARGE, Pendred, congenital cytomegalovirus infection, and congenital rubella (Brenski and Arjmand 2003). During early mammalian cochlear development, the medial thickened cochlea epithelium is subdivided into three distinct regions from the medial (neural) to lateral (abneural) site along the short axis of the cochlear duct, that is, Kölliker’s organ (neural side of the cochlear duct), the prosensory auditory domain, and the outer sulcus. At this time, Bmp4 is expressed in the outer sulcus region; thus, a BMP signal gradient is established in the developing sensory domain and is high at the lateral site and low at the neural site (Fig. 8, Ohyama et al. 2010). The prosensory domain and outer sulcus region did not form in the Alk3-CKO;Alk6+/− mutant, in

Inner ear development which BMP signals were conditionally deleted in the developing inner ear. In the cultured cochlea, a high dose of the BMP4 protein was found to upregulate the expression of outer sulcus markers and downregulate the Kölliker’s organ’s genes, while an intermediate dose induced a large number of prosensory cells (Ohyama et al. 2010). These findings suggested that BMP signaling with a lateralto-medial gradient is required for the patterning of the cochlear sensory epithelium along the short axis. Previous studies reported defects in hair cells and supporting cells in the developing cochlear duct of mutant mice with the tissue-specific deletion of Fgfr1 or Jagged1-Notch signaling (Kiernan et al. 2001; Pirvola et al. 2002). The expression of Fgf20 and its receptor Fgfr1 precedes the differentiation of hair cells and supporting cells in the developing cochlea duct. In a cultured murine developing inner ear, SU5402 (FGF receptor inhibitor) or the anti-FGF20 neutralizing antibody were found to effectively inhibit the formation of the sensory epithelium, including both hair cells and supporting cells, when added to the medium before/during the formation of the sensory epithelium. These findings suggested that signaling mediated by FGF20/FGFR1 may play a role in generating the sensory domain of the developing cochlear duct (Fig. 8, Hayashi et al. 2008b). Recent experiments showed that the FGFR1mediated MAP kinase activation through FGFR substrate (Frs) 2/3 is necessary for the maintenance of Sox2-positive sensory progenitors and their subsequent commitment to the sensory cell differentiation (Ono et al. 2014). The Notch ligand Jagged1 is expressed earlier than that of Fgf20 in the developing inner ear. The expression of Fgf20 was absent in Jagged1-conditional mutant mice, and the Fgf20 promoter region possessed an Rbpj-binding domain, which indicated that the expression of Fgf20 is directly regulated by Notch signaling (Munnamalai et al. 2012). Not only the expression of Fgf20, but also the generation of hair cells and supporting cells was inhibited in a cultured cochlea duct treated with DAPT (Notch inhibitor) before the onset of hair cell specification, and this inhibitory effect was reversed by adding the FGF20 protein to the medium (Munnamalai et al. 2012). These findings suggested that FGF20 may be involved in the specification and/or maintenance of the prosensory domain under the control of Notch signaling pathways The sensitivity of the prosensory epithelium to DAPT is known to be stage-dependent; treating the early cochlea (ED12.5 in mouse embryo) with DAPT led to a reduction in the number of hair cells and supporting cells because of the blockade of lateral induction, whereas, conversely, the treatment with DAPT at a later stage (ED13.5) increased the number of hair cells because of the blockade of lateral inhibition. At the earlier stage, Notch signalingdependent Sox2 maintains the prosensory region; therefore, Notch-induced FGF20 may amplify/maintain Sox2 to specify the prosensory domain, from which hair cells and supporting cells will develop (Dabdoub et al. 2008; Munnamalai et al. 2012). In the organ of Corti, hair cell differentiation progresses in a medial-tolateral direction by an, as yet, unidentified mechanism. Fgf20-null mutant mice lack not only outer hair cells, but also differentiated supporting cells, such as Deiters’ cells (Huh et al. 2012). Therefore, a fine-tuned balance between FGF and BMP signaling may be a prerequisite for establishing the proper number of hair cell rows. Phospho-Erk, a downstream signal of FGF, was shown to phosphorylate the linker region of Smad1 to disrupt BMP-mediated Smad1 signaling (Pera et al. 2003). After the formation of hair cells in the cochlea, inner hair cells expressed Fgf8 and outer hair cells and supporting cells expressed Fgfr3 (Jacques et al. 2007). Mutant mice without FGF8/FGFR3 signaling have defective pillar cells and deafness, which indicated that inner hair cell-derived FGF8 locally controls the differentiation of supporting cells into pillar cells,

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which are unique to the mammalian cochlea (Fig. 8, Colvin et al. 1996; Puligilla et al. 2007). Low-frequency sensorineural hearing loss has been reported in a mouse model of human Muenke syndrome carrying Fgfr3P244R, and the organ of Corti in this mutant has not only extra pillar cells and fewer Deiters’ cells, but also extra hair cells in the apical cochlear duct (Mansour et al. 2009). A genetic reduction in the expression of FGF10, which normally activates FGFR2b or FGFR1b, can reverse the cochlear disorders associated with this model (Mansour et al. 2013). In the chick developing basilar papilla (homolog of the organ of Corti), FGF signaling mediated by FGFR3 was shown to be required for maintaining supporting cells, which are known to act as a stem cell pool for the regeneration of injured hair cells (Jacques et al. 2012). A previous study demonstrated that mammalian cochlear supporting cells were capable of proliferating and differentiating into hair cells when cultured. This finding suggests that the regenerative mechanisms observed in adult avians may be conserved in the developing mammalian inner ear (White et al. 2006). After the specification of hair cell development in the organ of Corti, hair cells begin to be oriented as a set of three rows of outer hair cells and a single row of inner hair cells. The hair cells then generate a V-shaped bundle of stereocilia on their apical surface, on which the vertex points to the lateral site. Cells specified to the organ of Corti no longer proliferate; thus, a defined number of postmitotic cells undergo convergent extension movement to elongate the cochlear duct in a manner basal to apical direction. The planar cell polarity of hair cells as well as polarized cochlear extension are known to be regulated by the Wnt-PCP pathway (Fig. 8, Curtin et al. 2003; Montcouquiol et al. 2003; Wang et al. 2005). In the V-shaped hair cell bundle, which possesses uniform mediolateral polarity on the apical surface of hair cells, a polarity/mitotic spindle-associated protein complex consisting of mInsc (mammalian Inscuteable), LGN (vertebrate partner of Insc [Pins]), and Gαi (heteromeric G protein) is localized in a lateral microvilli-free region of the apical surface of each hair cell. Loss-of-function experiments on the mInsc/LGN/Gαi complex revealed irregular bundles of stereocilia, which suggested that the mInsc/LGN/Gαi complex plays a role in the establishment of uniform bundles of stereocilia on the hair cell surface (Tarchini et al. 2013). Therefore, at least two pathways involving planer cell polarity are required for the establishment of a mature sensory epithelium in the organ of Corti.

PERSPECTIVES One of the most intriguing issues related to inner ear developmental biology is a clearer understanding of the mechanisms regulating the regeneration of hair cells. Adult mammalian hair cells do not regenerate after injury; therefore, hair cell injury after birth causes irreversible deafness. The loss of regenerative activity in the adult sensory epithelium has been attributed to an age-dependent reduction in the number of multi-potent progenitor/stem cells due to a loss of multipotency and proliferative activity. Therefore, elucidating the genetic and epigenetic mechanisms underlying such quiescence in the adult sensory epithelium may facilitate the development of strategies for inner ear regeneration. At the onset of sensory epithelium development, the prosensory domain exits the cell cycle to commit to a lineage for mechanosensory hair cells. However, the cochlear epithelium in the neonatal rodent possesses potent regenerative activity, which gradually decreases in an agedependent manner. In the adult chick inner ear, the sensory epithelium maintains regenerative activity throughout life; therefore, the © 2014 Japanese Teratology Society

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Y. Nakajima

sensory epithelium in adult birds can proliferate and differentiate to hair cells after injury. Further investigations are necessary to understand the structural, cellular, and molecular mechanisms responsible for the regenerative activity observed in the sensory epithelium in neonatal mammals and avians.

ACKNOWLEDGMENTS The author thanks S Uoya for technical assistance. This work was supported by a JSPS Grant-in-Aid for Scientific Research (C) 25460273.

DISCLOSURE None.

REFERENCES Abelló G, Alsina B. 2007. Establishment of a proneural field in the inner ear. Int J Dev Biol 51:483–493. Abelló G, Khatri S, Giraldez F, Alsina B. 2007. Early regionalization of the otic placode and its regulation by the Notch signaling pathway. Mech Dev 124:631–645. Abelló G, Khatri S, Radosevic M, Scotting PJ, Giraldez F, Alsina B. 2010. Independent regulation of Sox3 and Lmx1b by FGF and BMP signaling influences the neurogenic and non-neurogenic domains in the chick otic placode. Dev Biol 339:166–178. Alsina B, Abelló G, Ulloa E, Henrique D, Pujades C, Giraldez F. 2004. FGF signaling is required for determination of otic neuroblasts in the chick embryo. Dev Biol 267:119–134. Alvarez Y, Alonso MT, Vendrell V et al. 2003. Requirements for FGF3 and FGF10 during inner ear formation. Development 130:6329–6338. Bissonnette JP, Fekete DM. 1996. Standard atlas of the gross anatomy of the developing inner ear of the chicken. J Comp Neurol 368:620–630. Bok J, Bronner-Fraser M, Wu DK. 2005. Role of the hindbrain in dorsoventral but not anteroposterior axial specification of the inner ear. Development 132:2115–2124. Bok J, Chang W, Wu DK. 2007a. Patterning and morphogenesis of the vertebrate inner ear. Int J Dev Biol 51:521–533. Bok J, Dolson DK, Hill P, Ruther U, Epstein DJ, Wu DK. 2007b. Opposing gradients of Gli repressor and activators mediate Shh signaling along the dorsoventral axis of the inner ear. Development 134:1713–1722. Bok J, Raft S, Kong KA, Koo SK, Drager UC, Wu DK. 2011. Transient retinoic acid signaling confers anterior-posterior polarity to the inner ear. Proc Natl Acad Sci U S A 108:161–166. Brenski AC, Arjmand EM. 2003. Congenital Inner Ear Anomalies. In: Bluestone CD, Stool SE, Casselbrant ML et al., editors. Pediatric otolaryngology, Vol. 1. Philadelphia: SAUNDERS. p 441–455. Chang W, Brigande JV, Fekete DM, Wu DK. 2004. The development of semicircular canals in the inner ear: role of FGFs 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 vestibular apparatus that detects angular head movements. Plos Genet 4:e1000050. Chang W, Nunes FD, De Jesus-Escobar JM, Harland R, Wu DK. 1999. Ectopic noggin blocks sensory and nonsensory organ morphogenesis in the chicken inner ear. Dev Biol 216:369–381. Chang W, ten Dijke P, Wu DK. 2002. BMP pathways are involved in otic capsule formation and epithelial-mesenchymal signaling in the developing chicken inner ear. Dev Biol 251:380–394. Chrysostomou E, Gale JE, Daudet N. 2012. Delta-like 1 and lateral inhibition during hair cell formation in the chicken inner ear: evidence against cis-inhibition. Development 139:3764–3774. Cole LK, Le Roux I, Nunes F, Laufer E, Lewis J, Wu DK. 2000. Sensory organ generation in the chicken inner ear: contributions of bone morphogenetic protein 4, serrate1, and lunatic fringe. J Comp Neurol 424:509–520.

© 2014 Japanese Teratology Society

Colvin JS, Bohne BA, Harding GW, McEwen DG, Ornitz DM. 1996. Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3. Nat Genet 12:390–397. Curtin JA, Quint E, Tsipouri V et al. 2003. Mutation of Celsr1 disrupts planar polarity of inner ear hair cells and causes severe neural tube defects in the mouse. Curr Biol 13:1129–1133. Dabdoub A, Puligilla C, Jones JM et al. 2008. Sox2 signaling in prosensory domain specification and subsequent hair cell differentiation in the developing cochlea. Proc Natl Acad Sci U S A 105:18396–18401. Driver EC, Kelley MW. 2009. Specification of cell fate in the mammalian cochlea. Birth Defects Res C Embryo Today 87:212–221. Etheridge SL, Ray S, Li S et al. 2008. Murine dishevelled 3 functions in redundant pathways with dishevelled 1 and 2 in normal cardiac outflow tract, cochlea, and neural tube development. Plos Genet 4:e1000259. Evsen L, Sugahara S, Uchikawa M, Kondoh H, Wu DK. 2013. Progression of neurogenesis in the inner ear requires inhibition of Sox2 transcription by neurogenin1 and neurod1. J Neurosci 33:3879–3890. Frenz DA, Liu W, Galinovic-Schwartz V, Van De Water TR. 1996. Retinoic acid-induced embryopathy of the mouse inner ear. Teratology 53:292–303. Freter S, Muta Y, Mak SS, Rinkwitz S, Ladher RK. 2008. Progressive restriction of otic fate: the role of FGF and Wnt in resolving inner ear potential. Development 135:3415–3424. Fritzsch B, Jahan I, Pan N, Kersigo J, Duncan J, Kopecky B. 2011. Dissecting the molecular basis of organ of Corti development: where are we now? Hear Res 276:16–26. Gerlach LM, Hutson MR, Germiller JA, Nguyen-Luu D, Victor JC, Barald KF. 2000. Addition of the BMP4 antagonist, noggin, disrupts avian inner ear development. Development 127:45–54. Groves AK, Fekete DM. 2012. Shaping sound in space: the regulation of inner ear patterning. Development 139:245–257. Hayashi T, Kokubo H, Hartman BH, Ray CA, Reh TA, Bermingham-McDonogh O. 2008a. Hesr1 and Hesr2 may act as early effectors of Notch signaling in the developing cochlea. Dev Biol 316:87– 99. Hayashi T, Ray CA, Bermingham-McDonogh O. 2008b. Fgf20 is required for sensory epithelial specification in the developing cochlea. J Neurosci 28:5991–5999. Huh SH, Jones J, Warchol ME, Ornitz DM. 2012. Differentiation of the lateral compartment of the cochlea requires a temporally restricted FGF20 signal. Plos Biol 10:e1001231. Jackler RK, Luxford WM, House WF. 1987. Congenital malformations of the inner ear: a classification based on embryogenesis. Laryngoscope 97:2–14. Jacob J, Briscoe J. 2003. Gli proteins and the control of spinal-cord patterning. EMBO Rep 4:761–765. Jacques BE, Dabdoub A, Kelley MW. 2012. Fgf signaling regulates development and transdifferentiation of hair cells and supporting cells in the basilar papilla. Hear Res 289:27–39. Jacques BE, Montcouquiol ME, Layman EM, Lewandoski M, Kelley MW. 2007. Fgf8 induces pillar cell fate and regulates cellular patterning in the mammalian cochlea. Development 134:3021–3029. Jeon SJ, Fujioka M, Kim SC, Edge AS. 2011. Notch signaling alters sensory or neuronal cell fate specification of inner ear stem cells. J Neurosci 31:8351–8358. Kiernan AE, Ahituv N, Fuchs H et al. 2001. The Notch ligand Jagged1 is required for inner ear sensory development. Proc Natl Acad Sci U S A 98:3873–3878. Kobayashi Y, Nakamura H, Funahashi J. 2008. Epithelial-mesenchymal transition as a possible mechanism of semicircular canal morphogenesis in chick inner ear. Tohoku J Exp Med 215:207–217. Kochhar A, Fischer SM, Kimberling WJ, Smith RJ. 2007. Branchio-otorenal syndrome. Am J Med Genet A 143a:1671–1678. Komada M. 2012. Sonic hedgehog signaling coordinates the proliferation and differentiation of neural stem/progenitor cells by regulating cell cycle kinetics during development of the neocortex. Congenit Anom Kyoto 52:72–77. Ladher RK, Anakwe KU, Gurney AL, Schoenwolf GC, Francis-West PH. 2000. Identification of synergistic signals initiating inner ear development. Science 290:1965–1967.

Inner ear development Ladher RK, O’Neill P, Begbie J. 2010. From shared lineage to distinct functions: the development of the inner ear and epibranchial placodes. Development 137:1777–1785. Ladher RK, Wright TJ, Moon AM, Mansour SL, Schoenwolf GC. 2005. FGF8 initiates inner ear induction in chick and mouse. Genes Dev 19:603–613. Li X, Oghi KA, Zhang J et al. 2003. Eya protein phosphatase activity regulates Six1-Dach-Eya transcriptional effects in mammalian organogenesis. Nature 426:247–254. Litsiou A, Hanson S, Streit A. 2005. A balance of FGF, BMP and WNT signalling positions the future placode territory in the head. Development 132:4051–4062. Magarinos M, Contreras J, Aburto MR, Varela-Nieto I. 2012. Early development of the vertebrate inner ear. Anat Rec (Hoboken) 295:1775– 1790. Mansour SL, Li C, Urness LD. 2013. Genetic rescue of Muenke syndrome model hearing loss reveals prolonged FGF-dependent plasticity in cochlear supporting cell fates. Genes Dev 27:2320–2331. Mansour SL, Schoenwolf GC. 2005. Morphogenesis of the inner ear. In: Kelly M, Wu DK, Popper AN, Fay RR, editors. Development of the Inner Ear. New York: Springer. p 43–84. Mansour SL, Twigg SRF, Freeland RM, Wall SA, Li C, Wilkie AOM. 2009. Hearing loss in a mouse model of Muenke syndrome. Hum Mol Genet 18:43–50. Martin P, Swanson GJ. 1993. Descriptive and experimental analysis of the epithelial remodellings that control semicircular canal formation in the developing mouse inner ear. Dev Biol 159:549–558. Masuda S, Usui S, Matsunaga T. 2013. High prevalence of inner-ear and/or internal auditory canal malformations in children with unilateral sensorineural hearing loss. Int J Pediatr Otorhinolaryngol 77:228– 232. Montcouquiol M, Rachel RA, Lanford PJ, Copeland NG, Jenkins NA, Kelley MW. 2003. Identification of Vangl2 and Scrb1 as planar polarity genes in mammals. Nature 423:173–177. Morsli H, Choo D, Ryan A, Johnson R, Wu DK. 1998. Development of the mouse inner ear and origin of its sensory organs. J Neurosci 18:3327– 3335. Munnamalai V, Hayashi T, Bermingham-McDonogh O. 2012. Notch prosensory effects in the Mammalian cochlea are partially mediated by Fgf20. J Neurosci 32:12876–12884. Neves J, Abello G, Petrovic J, Giraldez F. 2013. Patterning and cell fate in the inner ear: a case for Notch in the chicken embryo. Dev Growth Differ 55:96–112. Neves J, Kamaid A, Alsina B, Giraldez F. 2007. Differential expression of Sox2 and Sox3 in neuronal and sensory progenitors of the developing inner ear of the chick. J Comp Neurol 503:487–500. Neves J, Parada C, Chamizo M, Giraldez F. 2011. Jagged 1 regulates the restriction of Sox2 expression in the developing chicken inner ear: a mechanism for sensory organ specification. Development 138:735–744. Neves J, Uchikawa M, Bigas A, Giraldez F. 2012. The prosensory function of Sox2 in the chicken inner ear relies on the direct regulation of Atoh1. PLoS ONE 7:e30871. Ohyama T, Basch ML, Mishina Y, Lyons KM, Segil N, Groves AK. 2010. BMP signaling is necessary for patterning the sensory and nonsensory regions of the developing mammalian cochlea. J Neurosci 30:15044– 15051. Ono K, Kita T, Sato S et al. 2014. FGFR1-Frs2/3 signalling maintains sensory progenitors during inner ear hair cell formation. Plos Genet:e1004118. Ozaki H, Nakamura K, Funahashi J et al. 2003. Six1 controls patterning of the mouse otic vesicle. Development 131:551–562. Pan N, Jahan I, Kersigo J et al. 2011. Conditional deletion of Atoh1 using Pax2-Cre results in viable mice without differentiated cochlear hair cells that have lost most of the organ of Corti. Hear Res 275:66–80. Pauley S, Lai E, Fritzsch B. 2006. Foxg1 is required for morphogenesis and histogenesis of the mammalian inner ear. Dev Dyn 235:2470–2482. Pera EM, Ikeda A, Eivers E, De Robertis EM. 2003. Integration of IGF, FGF, and anti-BMP signals via Smad1 phosphorylation in neural induction. Genes Dev 17:3023–3028.

25

Pirvola U, Ylikoski J, Trokovic R, Hebert JM, McConnell SK, Partanen J. 2002. FGFR1 is required for the development of the auditory sensory epithelium. Neuron 35:671–680. Propst EJ, Blaser S, Gordon KA, Harrison RV, Papsin BC. 2005. Temporal bone findings on computed tomography imaging in branchio-oto-renal syndrome. Laryngoscope 115:1855–1862. Puligilla C, Feng F, Ishikawa K et al. 2007. Disruption of fibroblast growth factor receptor 3 signaling results in defects in cellular differentiation, neuronal patterning, and hearing impairment. Dev Dyn 236:1905–1917. Radde-Gallwitz K, Pan L, Gan L, Lin X, Segil N, Chen P. 2004. Expression of Islet1 marks the sensory and neuronal lineages in the mammalian inner ear. J Comp Neurol 477:412–421. Rakowiecki S, Epstein DJ. 2013. Divergent roles for Wnt/beta-catenin signaling in epithelial maintenance and breakdown during semicircular canal formation. Development 140:1730–1739. Ramsebner R, Ludwig M, Parzefall T et al. 2010. A FGF3 mutation associated with differential inner ear malformation, microtia, and microdontia. Laryngoscope 120:359–364. Riccomagno MM, Martinu L, Mulheisen M, Wu DK, Epstein DJ. 2002. Specification of the mammalian cochlea is dependent on Sonic hedgehog. Genes Dev 16:2365–2378. Salminen M, Meyer BI, Bober E, Gruss P. 2000. Netrin 1 is required for semicircular canal formation in the mouse inner ear. Development 127:13–22. Satoh T, Fekete DM. 2005. Clonal analysis of the relationships between mechanosensory cells and the neurons that innervate them in the chicken ear. Development 132:1687–1697. Senel E, Gulleroglu BN, Senel S. 2009. Additional temporal bone findings on computed tomography imaging in branchio-oto-renal syndrome. Laryngoscope 119:832. Sennaroglu L, Saatci I. 2002. A new classification for cochleovestibular malformations. Laryngoscope 112:2230–2241. Steventon B, Mayor R, Streit A. 2012. Mutual repression between Gbx2 and Otx2 in sensory placodes reveals a general mechanism for ectodermal patterning. Dev Biol 367:55–65. Stone JS, Shang JL, Tomarev S. 2003. Expression of Prox1 defines regions of the avian otocyst that give rise to sensory or neural cells. J Comp Neurol 460:487–502. Takahashi K, Yamanaka S. 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676. Takemoto T. 2013. Mechanism of cell fate choice between neural and mesodermal development during early embryogenesis. Congenit Anom Kyoto 53:61–66. Tarchini B, Jolicoeur C, Cayouette M. 2013. A molecular blueprint at the apical surface establishes planar asymmetry in cochlear hair cells. Dev Cell 27:88–102. Torres M, Giraldez F. 1998. The development of the vertebrate inner ear. Mech Dev 71:5–21. Urness LD, Paxton CN, Wang X, Schoenwolf GC, Mansour SL. 2010. FGF signaling regulates otic placode induction and refinement by controlling both ectodermal target genes and hindbrain Wnt8a. Dev Biol 340:595–604. Wang J, Mark S, Zhang X et al. 2005. Regulation of polarized extension and planar cell polarity in the cochlea by the vertebrate PCP pathway. Nat Genet 37:980–985. White PM, Doetzlhofer A, Lee YS, Groves AK, Segil N. 2006. Mammalian cochlear supporting cells can divide and trans-differentiate into hair cells. Nature 441:984–987. Wright TJ, Mansour SL. 2003. Fgf3 and Fgf10 are required for mouse otic placode induction. Development 130:3379–3390. Yamamoto N, Okano T, Ma X, Adelstein RS, Kelley MW. 2009. Myosin II regulates extension, growth and patterning in the mammalian cochlear duct. Development 136:1977–1986. Yasuda M, Yamada S, Uwabe C, Shiota K, Yasuda Y. 2007. Threedimensional analysis of inner ear development in human embryos. Anat Sci Int 82:156–163. Zou D, Silvius D, Fritzsch B, Xu PX. 2004. Eya1 and Six1 are essential for early steps of sensory neurogenesis in mammalian cranial placodes. Development 131:5561–5572.

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