Development and Evolution of the Vestibular Sensory Apparatus of the Mammalian Ear

Home , Helix (ear), Saccule, Sensory nervous system

Journal of Vestibular Research 15 (2005) 225–241 225 IOS Press

Development and evolution of the vestibular sensory apparatus of the mammalian ear

Kirk W. Beisel, Yesha Wang-Lundberg, Adel Maklad and Bernd Fritzsch ∗ Creighton University, Omaha, NE and BTNRH, Omaha, NE, USA

Received 21 June 2005 Accepted 3 November 2005

Abstract. Herein, we will review molecular aspects of vestibular ear development and present them in the context of evolutionary changes and hair cell regeneration. Several genes guide the development of anterior and posterior canals. Although some of these genes are also important for horizontal canal development, this canal strongly depends on a single gene, Otx1. Otx1 also governs the segregation of saccule and utricle. Several genes are essential for otoconia and cupula formation, but protein interactions necessary to form and maintain otoconia or a cupula are not yet understood. Nerve fiber guidance to specific vestibular endorgans is predominantly mediated by diffusible neurotrophic factors that work even in the absence of differentiated hair cells. Neurotrophins, in particular Bdnf, are the most crucial attractive factor released by hair cells. If Bdnf is misexpressed, fibers can be redirected away from hair cells. Hair cell differentiation is mediated by Atoh1. However, Atoh1 may not initiate hair cell precursor formation. Resolving the role of Atoh1 in postmitotic hair cell precursors is crucial for future attempts in hair cell regeneration. Additional analyses are needed before gene therapy can help regenerate hair cells, restore otoconia, and reconnect sensory epithelia to the brain.

Keywords: Ear, development, sensory epithelia, sensory neurons, otoconia, cupula

1. Introduction c) Sound reaches the cochlea where the basilar membrane motion and hair cell stimulation con- verts specific frequencies into tonotopic informa- The mammalian ear contains three sensory systems: tion that encodes place of cochlear stimulation a) Angular acceleration perception is accomplished and intensity. The spiral ganglion neurons re- by the semicircular canals to direct specific en- ceive this signal and then relay this information dolymphatic movement to crista organs where to the brainstem, but not the cerebellum. hair cells convert it into electric signals. These Excluding the problem of the origin and diversifi- signals are conducted by specific vestibular affer- cation of the cochlea [55], what makes a functional ent fibers to the brainstem and the cerebellum. vestibular system must be minimally understood at four b) Linear acceleration is detected by hair cells by interconnected levels requiring knowledge of both ear movement of a gelatinous mass that contains development and evolution. otoconia in relationship to the endorgans where i) We need to understand how the ear is patterned movement is converted into electric signals con- such that morphogenesis and histogenesis hap- ducted to brainstem and cerebellum. pens at the right place at the right time. For example, if a canal crista forms near the endolymphatic duct, this abnormal position would NOT ∗Corresponding author: B. Fritzsch, Creighton University Depart- ment of Biomedical Sciences Omaha, NE 68178, USA. Tel.: +1 402 permit the perception of angular acceleration 280 2915; Fax: +1 402 280 5556; E-mail: [email protected]. mediated by endolymphatic movement as angu-

lar accelerations would only cause limited fluid 2. Vertebrate vestibular ear evolution shows movements, much like in plugged canals [115]. changes in linear and angular acceleration ii) Hair cells have to develop at the right place with sensory systems the right polarity or a mix thereof to be able to extract information from the stimuli. Having The origin of the ear has sparked various hypotheses. ◦ The rather ubiquitous distribution of statocyts in all an- hair cells oriented 90 with respect to their nor- imal radiations suggests that bearing mechanosensory mal orientation in the canal cristae would result cells aggregated into statocysts are ancient structures in virtually no stimulation no matter how rapidly derived from single mechanosensory cells [17,49,90]. the head is turned. Gene expression supports such a notion as it shows iii) The extracellular matrix has to be appropriately that certain genes are involved in statocyst and sensory formed to permit detection of the stimulus. Hav- cell development across phyla [51,73]. The chordate ing a cupula-like gelatinous structure on the utri- most closely related to vertebrates, the lancelet, has cle would render this epithelium virtually in- neither an ear nor organs that resemble the lateral line capable to perceive linear acceleration as the organs [41,75]. All craniate vertebrates have an ear that cupula lacks the higher density to cause relative consists of two discrete sensory systems, angular and linear acceleration sensors. In cyclostome vertebrates movements in linear acceleration. Conversely, such as lampreys and hagfish, the gravistatic receptor attaching otoconia to the canal cupula causes a is a single macula covered with otoconia [79]. Evo- clinical syndrome known as cupulolithiasis as it lution has apparently turned this otoconia containing introduces linear acceleration sensation capacity organ, which resembles the invertebrate statocyst, into into an angular acceleration sensor. two or three different organs (utricle, saccule, lagena), iv) Last, but not least, the extracted information has each with a distinct orientation in space [79,119]. Such to be conducted in discrete and partial overlap- diversification in vertebrate ears implies, but does not ping fashion to the hindbrain, including the cere- prove, that vertebrate ear evolution is primarily the di- bellum, for appropriate information processing. versification of a statocyst. Interestingly, the evolution of the angular and linear acceleration system of the ear In this overview we will first briefly introduce major of cephalopods indicates a comparable diversification aspects of vestibular vertebrate ear evolution to high- from a simple moluscan statocyst [16,94] as proposed light how selection has shaped an unknown ancestral here for the evolution of the vertebrate ear from a hy- sensory organ into a system with three semicircular pothetical statocyst of an unknown vertebrate ancestor. canals and crista organs to extract angular acceleration In parallel to the segregation and diversification of otoconia baring organs is a diversification and morpho- in the three cardinal planes and evolved one (macula logical evolution of angular acceleration organs. Hag- communis), two (utricle and saccule) or three sensory fish have only a single canal and Lampreys have only organs (utricle, saccule, lagena) for perception of hori- two vertical canals. Both taxa have only two cristae zontal and vertical linear acceleration, including grav- organs, an anterior and a posterior crista. This suggests ity. Next, we will analyze, in order, 1) current molec- that the horizontal crista evolved in jawed vertebrates ular developmental insights into patterning events and together with the segregation of a single otoconia bear- morphogenesis of canals and utricular and saccular re- ing organ into two [45,97]. Interestingly, cristae or- cesses; 2) molecular biology of cupula and otoconia gans of hagfish lack cupulae. Instead, the entire am- composition; 3) development and molecular biology of pullary lumen is surrounded by hair cells which extent sensory neuron development, including known guiding their elongated kinocilium and stereocilia freely into the canals (Fig. 1). These data suggest that evolution of principles for connecting these neurons specifically to canal cristae in jawed vertebrates involved transforma- the vestibular sensory organs; and 4) molecular basis tions of sensory epithelia (from an organ surrounding of sensory epithelial development (including hair cells) the entire lumen to an organ restricted to a small sen- as revealed by recent analysis of mutant and transgenic sory patch), formation of a cupula to change an organ mice. We follow roughly the sequence of appearance that measures only angular velocity into an organ that of these events in ear development, with hair cell dif- measures pressure of endolymph at the cupula [63], ferentiation being one of the last events. and generation of a new canal (the horizontal canal) K.W. Beisel et al. / Development and evolution of the vestibular sensory apparatus 227

of the gravistatic organ by losing the capacity to incorporate otoconia. Molecular similarities also suggest that the cupula of cristae may have evolved out of extracellular matrix [54] of gravistatic organs in which otoconia were embedded. Developmental and evolutionary data support that the tectorial membrane of the basilar papilla (or cochlea in vertebrates) derives from gravistatic organs, suggesting that tectorial membrane formation was secondarily modiﬁed by eliminating the capacity to incorporate otoconia into the matrix [45, 54]. Development of the ear has to provide the molecular causality for such evolutionary changes (Fig. 1) and has to explain how three (instead of one) canals form, how three cristae form, and how two or more instead of one linear acceleration sensor form, including the necessary and associated changes in hair cell and sensory neurons.

3. Genes involved in patterning the ear

The ear starts as an epithelial thickening, the otic placode [147] that forms lateral to rhombomeres 5 and 6 of the hindbrain, except for lampreys and hagfish [42,102]. Placodes show specific genetic markers Fig. 1. A brief survey of major evolutionary changes in the ear is pre- prior to any morphological distinction [5,104]. Pre- sented. Hagfish, a jawless vertebrate, have a single torus that contains cisely how large and how long lasting such a prepla- two canal cristae and a single common macula (A). Mammals such as mice have three canal cristae and two organs for linear acceleration codal area is remains at the moment speculative [15, perception with otoconia as well as a cochlea (B). The canal cristae 104]. Interestingly, the data across vertebrates suggest show differences in their organization. Mammals have a crista that that even orthologues of genes expressed in these areas sits on a ridge separated by a cruciate eminence (CE) that is covered by a cupula (removed in D to show the hair cell bundles). In contrast, such as Foxi1 [104,134] and Fgf10 [15,103,109] may hagfish have no cupula and the sensory hair cells are arranged to form not be conserved in their function for placode induc- a ring around the largest diameter of ampullary enlargement (C). AC, tion. Such data may indicate that placodes are tran- anterior crista; C, cochlea; CE; cruciate eminence; HC, horizontal sient embryological adaptation and are modified to fit crista; MC, macula communis; PC, posterior crista; S, saccule; U, utricle. Bar indicates 100 μm. Modified after [45]. into the different developmental pathways of various vertebrates. Given that hair cells develop only com- together with the horizontal canal crista and the ap- paratively late out of placodes, these thickenings may propriate afferent fibers to connect this organ to the therefore present an embryological adaptation compa- brain [45]. rable to the placenta: necessary for development but Combined with the molecular developmental conser- uniquely evolved for such developmental purposes. vation of mechanosensory cells and the rather ubiqui- Like the nearby central nervous system, the otic pla- tous presence of statocysts in aquatic invertebrates, the code undergoes invagination to form the otic cup and limited changes in the linear acceleration system argues later the otocyst that closes off from the ectoderm. The that the evolutionary origin of the vertebrate ear was otocyst will form between the first somite and the more a statocyst like organ, with the canal system evolving rostral somitomeres. The developing otocyst will there- independently. Hagfish have only extracellular matrix fore be exposed to some of the same factors emanating containing otoconia covering the single common mac- from the notochord and spinal cord to pattern somites ula [79,87,119]. Neither the lateral line system [13] nor such as sonic hedgehog (Shh), which upregulate genes the canal cristae are covered with extracellular matrix. specific for muscle and tendon formation such as the ba- This suggests that those covering structures evolved af- sic helix-loop-helix transcription factor (bHLH), paired ter the organs had evolved, possibly out of the matrix box (Pax), fibroblast growth factor (Fgf) and forkhead 228 K.W. Beisel et al. / Development and evolution of the vestibular sensory apparatus box (Fox) genes [6,14]. In the ear, genes of these fami- lies are the bHLH genes, Neurog1, Atoh1 and Neurod1, the Pax family member, Pax2 [12,18,120], Fgf members Fgf3, Fgf10, and several Fox members [60,104]. Thus, the major players of early otic placode formation may be under molecular governance comparable to the nearby somites. One of the first morphogenetic events of the otocyst is the growth of a plate that will form the anterior and posterior vertical canal by embryonic day 12.5 [95, 100]. Through adhesion and cell death of the central parts, the remaining ducts are formed [19,22,39]. Par- tially overlapping with this is the growth of another plate that will eventually form the horizontal or lateral canal (Fig. 2). At this stage, a separation will divide the superior part and the inferior part, leaving the utriculo- saccular foramen [19]. In the mouse approximately 5 days after the first appearance of the placodal thickening, the vestibular portion of the ear is formed. An increasing number of genes have been identified through targeted mutations that play major roles in ear morphogenesis [23,83,103]. The genes we chose to present in some detail belong to several classes of factors that play major roles in morphogenesis: FGFs, EYA/SIX/DACH heteromeric complexes and zinc finger proteins. Fig. 2. Morphogenetic defects of three null mutant lines are shown. FGF’s are a large family of 22 predominantly se- Fgf10 null mutants lack formation of any canals (A,B) but form creted ligands (except for FGF11-14) that signal to 7 a single undifferentiated pouch that carries the reduced and mal- formed anterior and horizontal cristae. These mutants lack a pos- specific receptors [differently spliced out of four re- terior crista entirely (B). Gata3 null mutants show arrest of ear for- ceptor (Fgfr) genes] in a paracrine or autocrine fash- mation at the level of the otocyst with barely recognizable initia- ion. Upon ligand binding, FGFR’s homodimerize tion of sensory epithelia formation (D). Interestingly, the expression of Gata3 as revealed with the β-galactosidase marker shows disor- and activate various intracellular signaling pathways ganization suggestive of alterations of expression of Gata3 (C,D). that ultimately change gene expression in the targeted Otx1 null mutants lack formation of the horizontal canal and show cell [106]. The most important FGF’s for early ear de- a fusion of the utricular and saccular epithelium across the enlarged velopment are Fgf10, Fgf3 [152] and Fgf8 [76]. Fgf10 utriculo-saccular foramen (E,F). The loss of a horizontal canal and fusion of the utriculo-saccular epithelium in the absence of Otx1 sug- is expressed at the placodal stage in the mesoderm un- gests that Otx1 expression in the ear was a crucial step in vertebrate derlying the otic placode. As the placode thickens, ear evolution to segregate the common macula of cyclostomes into Fgf10 expression moves into the otic ectoderm [103, two gravistatic organs, the utricle and saccule. AC, anterior crista; 109,112,154]. Fgf3 is initially only found in the nearby HC, horizontal crista; PC, posterior crista; S, saccule; SN, sensory neurons; U, utricle. Bar indicates 100 μm. Modified after [44,68, rhombomeres 5 and 6 of the hindbrain. Eventually, 109]. Fgf3 is expressed in the antero-ventral aspect of the otocyst, overlapping with Fgf10 [154]. Fgf9 is only ex- released from inner hair cells may determine the cell pressed in the periotic mesenchyme [114]. Expression fate of pillar cells, which express FGFR3 [27,112]. of Fgfr1 and Fgfr2b, which is the receptor for FGF3 Fgf10 null mutant mice (Fig. 2) have no canal for- and FGF10 [152], is initially overlapping in the otic mation [103,109] and show reduction and incomplete placode. During morphogenesis of the ear, Fgfr2b is separation of canal sensory epithelia [109]. Double expressed in the dorsal half of the otocyst, expanding knockouts of both Fgf3 and Fgf10 show only limited into the cochlea caudally [112] suggesting its involve- formation of occasional micro-vesicles, if any vesi- ment in canal morphogenesis. cles form at all [4,153]. An ear forms in single Fgf3 FGFs are essential for mammalian ear formation null mutants but there is variable size reduction [4]. and histogenesis [27,109,113]. For example, FGF8 Fgf9 null mutants also have reduced canal formation K.W. Beisel et al. / Development and evolution of the vestibular sensory apparatus 229 somewhat similar to Fgf10 null mice [114], suggest- brates related to proliferation, morphogenesis and cell ing that mesenchymal-epithelial interactions are essen- fate assignment [108]. Most interesting for the ear is tial for canal morphogenesis. How FGF’s recruit other the known interaction of GATA factors with BMP,FGF, genes such as Bmp4 [20–22,53], Hmx2/3 (previously FOX, HMX and T-box signaling. Interaction between Nkx5) [11,57,149], Dlx [98,133], Gata3 [68], Otx1 [44, GATA and FGF’s to neuralize ectoderm may be ances- 101] and Sox2 [71] is not understood. The reduction tral to chordates [10]. GATA factors also play roles in or loss of semicircular canals is consistent with FGF’s sensory organ formation in insects [108,129], possibly established role in branching morphogenesis [109,112] through the activation of bHLH factors [9]. also apparent in lung formation [114]. Modeling func- Like some Fox genes [60,104], Gata3 is expressed at tional molecular interactions between FGF mediated the level of the otic placode [77]. Targeted disruption of FGFR intracellular signaling and other factors target- Gata3 leads to an arrest of ear development at the level ing similar promoter regions requires a much deeper of the otocyst [68] with little morphogenesis beyond knowledge of genes, their qualitative and quantitative a sac (Fig. 2). How GATA3 interacts with other tran- expression patterns, and their functional interactions in scription factors involved in ear morphogenesis [40, ear development than is currently available [23]. 156] is still uncertain. However, multiple binding sites Morphogenesis of the embryonic ear depends on for GATAfactors, Otx and E-box motifs in the promoter the EYA/SIX/DACH complex [34,156,157], an evolu- regions of Fox genes have been described [32] and an tionary conserved gene network [59]. This develop- interaction of GATA factors with FGF’s and OTX may mental module or cassette is used in a variety of or- be important for neuralization [10]. Given the early gans and across phyla to regulate genes that permit ear expression of Gata3 it is not at all surprising to see cell migration [136], proliferation, and cell death [157]. influences on transcription factors regulating morpho- Null mutants show that Eya1 and Six1 are crucial for genesis and histogenesis of the ear [77]. Both BMP any development of the ear past the otocyst stage as mediated and direct signaling by GATA3 [9] as well as only microvesicles form in Eya1/Six1 double null mu- GATA3 interaction with FOX and T-box proteins [60, tants [157,159]. Biological redundancy is suggested by 108] could effect the formation of sensory neurons. the formation of the ear in altered Pax2 gene expres- Our laboratory is currently exploring the expression of sion in spontaneous and null mutations [34,142,156, several of these genes in Gata3 null mutant mice. 157]. The haploid insufficiency observed in human and Other major factors in early ear morphogenesis have mouse Eya1 mutations suggest that alterations in gene been reviewed recently such as retinoids, Hox and other dosage can affect morphogenesis [1,2,67,146,157]. transcription factors [11,23,83,124] and will not be Six1 affects the expression of genes known to be im- dealt with here. It suffices to say that none of the genes portant in ear morphogenesis such as Bmp4, Fgf10 and and their effect on ear morphogenesis is any further Fgf3 [21,109,154], which are downregulated in Six1 along in dissecting the molecular causality toward de- null mutants. Six1 also alters the expression pattern of velopmental defects than those three examples given two genes known to be involved in ear morphogenesis, above. Overall, these gene interactions seem to directly Hmx3 and Gata3 [11,68] suggesting a direct involve- specify distinct areas to undergo morphogenesis and ment of Six1 in maintenance of their correct expres- histogenesis, possibly through the formation of polar sion pattern [157,159]. A related gene, Eya4, leads to coordinates by which the position of these future mor- late-onset deafness at the DFNA10 locus and endolym- phogenetic and histogenetic events are specified [40]. phatic hydrops [110,151]. Clearly, Eya1 and Six1 are Most interestingly for understanding the evolution of the best understood genes in terms of their effect on ear morphogenesis is that certain processes can be de- other important genes in ear morphogenesis and still velopmentally uncoupled. For example, formation of a explaining their function remains tentative in the ab- horizontal canal and segregated utricular and saccular sence of any information about most of their down- epithelia (Fig. 2) depend on the Otx1 gene [19],whereas stream genes [159]. Further dissection of their role in formation and positioning of a horizontal crista appears ear development must be approached by using condi- to be governed by other genes [44,45]. tional mutant mouse lines to understand the contextual role these regulatory genes are playing without com- promising the viability of these mutants owing to their 4. Molecular composition of otoconia and cupulae defect of other vital organs [159]. GATA factors participate directly in several signal- As already stated in the introduction, the ear of verte- transduction pathways in both invertebrates and vertebrates is a complex structure housing three sensory sys- 230 K.W. Beisel et al. / Development and evolution of the vestibular sensory apparatus tems which perceive linear acceleration (gravity), an- have incorporated several chains of sulfated polysac- gular acceleration, and sound [79,119]. Specificity of charides. Oc90 mRNA expression starts early in the stimulus acquisition in the ear is achieved by associat- ear of mice (around embryonic day 9.5) and expression ing particular sensory epithelia such as utricle, saccule, is restricted to non-sensory areas of the ear. This unex- canal cristae and cochlea with acellular matrix proteins pected expression pattern of OC90 changed the tradi- inside the inner ear space that mediate unique mechani- tional view that otoconia are formed from secreted vesi- cal stimuli. Otoconia provide the inertia mass to gener- cles from supporting cells. In essence, Oc90 expres- ate shearing forces for the utricle and saccule when sub- sion negatively defines sensory epithelia in developing jected to linear acceleration such as gravity. The otoco- and adult mice [141,150]. OC90 could thus present nia are typically composed of calcium carbonate crys- an early marker for non-sensory parts of the ear and tals partially embedded in a glycoprotein matrix that is could help elucidate changes in gene composition be- tethered by proteinaceous filaments to the underlying tween non-sensory and sensory parts of the ear using sensory epithelium (Fig. 3). The crystals form inside quantitative and global gene expression analyses. the endolymphatic space of the inner ear, which con- Another important protein that attaches the acellu- tains a fluid composed of high levels of potassium with lar matrix structures of the ear to the sensory epithe- very little calcium. Through unknown control mech- lia is otogelin (otog). In Otog mutant animals, otoco- anisms, the crystals are subject to disassembly under nia, cupulae and the tectorial membrane detach from certain conditions, including age- and sex-dependent the sensory epithelia [131] rendering the mutants unre- alterations [125,141]. In contrast, the cupulae of the sponsive to sound and vestibular stimuli. Like OC90, semicircular canal cristae and the tectorial membrane otogelin is expressed early (embryonic day 10.5) and of the cochlea do not contain any calcium carbonate is apparently specifically expressed in the differentiat- as this would likely change the stiffness of these struc- ing supporting cells [36]. This early expression thus tures and alter their physical properties in angular ac- suggests that some supporting cell commitment may celeration and sound perception. Such data are sup- predate proliferation of hair cells [126]. ported by the human condition of cupulolithiasis where A crucial protein for otoconia formation is dislocated otoconia are associated with the posterior otopetrin [65]. If mutated, as in the tilt mutation, no crista cupula and modify its physical responses such otoconia will form [65,105]. In situ hybridization pro- that the patients suffer from dizziness known as benign files of this gene demonstrated expression only in the paroxysmal positional vertigo, BPPV [128,135]. otoconia bearing sensory maculae, presenting a distin- In recent years the biochemical composition of oto- guishing feature from other sensory organs of the ear. conia, tectorial membrane and cupulae has been ana- More recently, the gene responsible for another otoco- lyzed and several genes essential for their formation and nia mutant mouse has been identified: head tilt mice, attachment to the underlying sensory epithelia of the carry a mutation in Nox3 [74,107]. As with other mu- ear have been characterized [54,65,78,105,131,141]. tations, it remains unclear how absence of this protein These data suggest specific compositional differences relates to loss of otoconia [132]. between all three acellular structures of the ear. Thus Unequal distribution profiles have been reported for far, seven major proteins of the extracellular matrices the two proteins common to all acellular structures, have been identified in the mammalian ear [54,141]. α-tectorin and β-tectorin, using immunocytochem- Additional data on another protein, otoancorin, that istry [54]. Interestingly, β-tectorin is only found in the links the stereocilia of the hair cells to the otoconia are inner layers of mammalian otoconia and is restricted emerging [160]. to the striola region in birds thus indicating a possible Seven proteins have been identified to be related to role in the specification of striola. Despite that specu- otoconia formation (Fig. 3). The predominant protein lation, its function remains unclear as residual capacity in otoconia of mammals is otoconin 90/95 (OC90) [145, to sense linear acceleration has not been tested in either 150]. OC90 has conserved the structural features of the Tecta or Tectb null mutant mice [54,78,144]. Expres- secreted phospholipase A2 (PLA2) except for the cat- sion data [118] show that in the saccule and utricle, alytic activity of the enzyme. Its highest structural sim- Tecta mRNA is detected at E12.5, but Tectb mRNA ilarity is to the calcium-dependent group X PLA2. The is not observed until E14.5. Expression of β-tectorin PLA2 like domains in OC90 have a higher content of mRNA ceases after P15, whereas β-tectorin mRNA ex- anionic residues than their non-otic homologues, gen- pression continues within the striolar region of the utri- erating a highly acidic pH. In addition, these proteins cle until at least P150. The results suggest that Tecta K.W. Beisel et al. / Development and evolution of the vestibular sensory apparatus 231

Fig. 3. The regional differences of otoconia related gene expression in the utricle and structural and protein differences in the utricular otoconia as revealed by immunocytochemistry are shown for a guinea pig. Note the differences in the size of otoconia crystals in striola (S) and extrastriolar regions (see insert). Oc90/95 is expressed in the non-sensory epithelium of the ear (red), otopetrin is expressed throughout the sensory epithelium (yellow); osteopontin is in hair cells (dark blue); otogelin is in supporting cells (cyan); α-tectorin is in supporting cells (orange); β-tectorin is in the striola (green). All proteins are found throughout the otoconia (lilac) except for otogelin (cyan) and β-tectorin (green) which are in the lower layers. No information on concentration differences of proteins within the otoconia matrix are available. Modified after [84]. may play a developmental role prior to or concomitant epithelia [65,118]. How all these different proteins re- with hair-cell differentiation, but before the appearance late to the distinct fine structure observed in acellular of hair bundles, whereas, Tectb may play a maintenance matrices of the ear [54,141] as well as to the differ- or functional role. Although Tecta is only expressed ent crystal structure of otoconia [58,85] and the differ- transiently during otoconia development, Tectb is con- ent striolar/extrastriolar organization of otoconia [79] tinuously expressed within the striolar region of the remains unclear. utricle. Most important here is that the three different acellu- The last protein related to otoconia is osteopontin. lar structures of the mammalian ear, the cupulae of the Osteopontin is a non-collagenous bone matrix pro- cristae, the otoconia of the linear acceleration system tein that is involved in ossification processes. mRNA and the tectorial membrane represent variations of a has been identified in marginal cells of the stria vas- general molecular theme with a number of shared pro- cularis, spiral ganglion cells, vestibular sensory hair teins being present in all three of these structures [54]. cells and vestibular dark cells [127,137]. In contrast More information about the developmental expression to most other acellular matrix proteins, the mRNA of of these proteins is needed to reveal the ontogenetic dif- osteopontin is expressed exclusively in vestibular hair ferences between the different epithelia to understand cells [143]. In the utricle and saccule, osteopontin the developmental distinctions of these acellular cov- was detected by immunocytochemistry in the otoconia, ering structures. Likewise, a correlation is needed be- suggesting that this protein is a component protein of tween the stereocilia maturation of hair cells and the mammalian otoconia. development of the extracellular matrices. While knowledge of the compositions of the inner ear acellular structures has thus been advanced, basic questions related to the development and mainte- 5. Making sensory neurons and connecting them nance of otoconia, cupula and tectorial membrane re- to the epithelia main open. For example, how the proteins that form the otoconia assemble in the calcium poor endolym- Sensorineural development requires spatial and tem- phatic fluid and how this extracellular protein assembly poral regulation of many genes for the precise local- guides otoconia crystallization, but not calcification of ization of the delaminating sensory neurons, ultimately the other acellular matrices is unknown. What is in- upregulating the genes for sensory neuron and hair cell triguing is that certain otoconia proteins are expressed formation. For example, the reduction in general oto- outside the sensory epithelia [150]. In contrast, other cyst expression in Fgf10 might and the change in ex- proteins known to participate in otoconia formation, in pression of Tbx1 [109,116] might alter the topology particular otopetrin, α-tectorin and β-tectorin, are ap- of proneuronal gene expression. Also, other proteins parently strongly expressed in otoconia bearing sensory like GATA3, EYA1, SIX1 and FGF’s may be important 232 K.W. Beisel et al. / Development and evolution of the vestibular sensory apparatus for maintenance of the neurogenic capacity. Recent work on EYA1and SIX1 show reduction and even complete loss of sensory neuron formation after an initial start [157,159]. Likewise, Gata3 null mutants [68] and sonic hedgehog (Shh) null mutants [120] show reduced formation of sensory neurons. Loss-of-function (targeted null mutations of the re- spective genes) experiments have clarified some of the proneural genes crucial for inner ear primary sensory neuron development (Fig. 4). The work of Ma et al. [88] showed that inner ear primary sensory neuron formation requires the vertebrate bHLH gene, neurogenin 1 (Neurog1). As a consequence of the absence of primary sensory neuron formation in Neurog1 null mutants, the ear develops without brainstem connections as afferents do not form and neither efferents nor autonomic fibers appear to reach the ear in these animals [89]. Nevertheless, such ears develop fairly normally in their overall histology, including hair cells. This suggests that ear formation and development even of many hair cells is largely autonomous of innervation. While those hair cells that do form develop morphologically normal in the absence of innervation (except for some minor disorientation), hair cell numbers are reduced to various degrees in Neurog1 null mutant mice [96]. Most interestingly, the cochlea is shortened and the saccule is almost completely lost in these null mutants. In ad- Fig. 4. The effect of loss of hair cells (B,D) loss of a neurotrophin dition, extra rows of hair cells form in the shortened (C,E,F) or misexpression of a neurotrophin (F) on the pattern of cochlea. These data suggest a significant interaction vestibular innervation is shown. Late embryonic loss of hair cells between progenitor cells that form primary neurons and in Pou4f3 null mice (B) and absence of differentiated hair cells in Atoh1 null mice (D) are both compatible with a targeted projection progenitor cells that give rise to hair cells, supporting to vestibular sensory epithelia. Note, however, that the projection and other inner ear epithelial cells. The simplest expla- to the utricle is more profoundly affected (A,B,D). Loss of Bdnf nation would be a clonal relationship of primary sen- results in complete loss of crista innervation and reduced innervation of the utricle (E). Some projection to cristae organs can be rescued sory clones with hair cell/supporting cell clones [40, in mutants in which sensory neuron degeneration is blocked in the 43]. However, other possible interactions can not be absence of Bax (C). More fibers can be rescued to the canal cristae excluded [45] such as alteration of cell fate [96]. organs in Ntf3tgBDNF misexpressors (F) suggesting that a limited A gene that is immediately downstream of and expression of Ntf3 is able to guide fibers to the canal cristae (C) but is not sufficient to rescue survival of the neurons (E). Note that mostly regulated by Neurog1 is Neurod1, another misexpression of Bdnf under Ntf3 promoter control can also guide bHLH gene [88]. Null mutations of this gene have been fibers to areas of the ear that have minor Ntf3 expression but no analyzed and show severe reduction [72] to complete hair cells (arrow in F). AC, anterior crista; HC, horizontal crista; U, μ loss of sensory neurons [86]. Surviving vestibular sen- utricle. Bar indicates 100 m. Modified after [50,61,139,155]. sory neurons may be in an unusual position and project in an aberrant pattern to only parts of the cochlea and of sensory neurons in Neurod1 null mutants are in part only some vestibular sensory epithelia [47,72]. Judg- mediated by the reduced expression of a Pou domain ing from these data, Neurod1 appears to play a role in factor, Pou4f1 [64]. As with Neurod1, Pou4f1 affects neuronal differentiation and survival, migration to ap- upregulation of certain neurotrophin receptors and thus propriate areas, and target selection of peripheral neu- might be only indirectly achieving its effect. rites. Consistent with the effect on survival is the re- Recent molecular data suggest some candidate genes duction and/or absence of certain neurotrophinreceptor that are involved in regulating peripheral neuronal pro- genes known to be essential for neuronal survival [72, cess development. Those candidate genes belong to 86]. Moreover, it is possible that the effects on survival the family of ephrin receptor and ligand complex and K.W. Beisel et al. / Development and evolution of the vestibular sensory apparatus 233 semaphorin ligand and receptor system known to be ear, it also remains unclear why mammals have two. important in other developing systems [140]. Most In addition, the expression patterns described show a simplistically, one might assume that hair cells simply highly dynamic change in the vestibular system [38, attract growing neurites. If so, the molecular principle 111]. Data on transgenic knockin animals showed that of neurite attraction should be abolished with hair cell each neurotrophin can be functionally replaced by the ablation. A recent study on Pou4f3 null mutant mice other neurotrophin. This suggests that the topologi- that do not develop differentiated hair cells past early cal difference in expression matter more than any spe- neonatal stages, reported growth of afferents to absent cific molecular effect [3,28]. This apparent capacity or disappearing hair cells (Fig. 4) as well as expression of functional replacement of each neurotrophin by the of neurotrophic factors in these mutants [155]. Interest- other reinvigorated the question for the functional sig- ingly, Atoh1 null mutant mice that never even form dif- nificance of the presence of two neurotrophins as well ferentiated hair cells also display rather normal initial as their differential expression. fiber growth (Fig. 4) but retention of fibers to the end An answer for this puzzle was recently provided us- of gestation is largely in areas of expression of the Bdnf ing the transgenic knockin animals in which Bdnf has neurotrophin [50]. Moreover, even if entire sensory replaced Ntf3 (Ntf3tghBdnf ). Selective tracer injection epithelia formation is abrogated in specific null mutant in these animals showed reorganization of the periph- mice, initial growth of afferents is rather targeted [109], eral projection pattern of vestibular neurons into the suggesting that at least some of the pathfinding prop- basal turn of the cochlea [139] as well as rescue of erties must reside in the interaction between growing projection to canal cristae in Ntf3tghBdnf mice com- neurites and the otocyst soluble factors. bined with Bdnf null mutation (Fig. 4). Indeed, ab- Null mutants for the ephrin receptor B2, Ephb2, sence of Bdnf can result in some innervation of canal show circling behavior and altered axonal guidance of sensory epithelia (Fig. 4) in animals in which a gene midline crossing efferents, but no data on alteration of important for cell death (Bax) has been eliminated [61]. peripheral innervation have been provided [29]. Other These projections developed at the time the neurons are ephrin mutants exist but their phenotype has not yet known to become susceptible to the neurotrophins for been characterized for the ear and likely requires more their survival suggesting that they become both neu- detailed analysis than simple uniform fiber labeling as rotrophically as well as neurotropically dependent on detailed errors might be missed using non-specific tech- neurotrophinsat the same time. These data also suggest niques. Recently, a role of one semaphorin, Sema3a, that the differential expression of neurotrophins in the in providing a stop signal for growing afferents has ear plays a significant role in patterning the ear inner- been presented. Specifically, in the null mutant grow- vation. Interestingly, the central projection remained ing neurites do not stop at the vestibular sensory epithe- unchanged in these animals suggesting little if any in- lia but rather continue to grow and may extend outside fluence of neurotrophins on the patterning of the central the ear as far as the skin above the ear [56]. While projections. Overall, these data suggest that the guid- these data indicate some progress in the molecular basis ance to molecular basis processes that guide peripheral of peripheral neurite guidance, it also highlights how and central projection are distinct. rudimentary this insight is [46,50]. Most interesting are recent data on topographic seg- In contrast to this apparent paucity of data on molecu- regation of afferent fibers from gravistatic organs that lar guidance, extensive knowledge exists on the molec- have hair cells with different polarities [91,92]. These ular basis of sensory neuron survival [48]. Briefly, two data suggest that hair cells with one polarity receive neurotrophins (Bdnf, Ntf3) and their receptors (Ntrk2, an input different from those with an opposite polar- Ntrk3) as well as the so-called low affinity receptor ity, with the striola presenting a dividing line (Fig. 5a). p75 have been identified in the developing ear using These data also show that no topographic segregation various techniques. Double null mutations of either of afferent cells projecting to specific organs exists in the two neurotrophins or their two neurotrophin re- the mouse ear (Fig. 5b). How such sharp boundaries ceptors have shown complete loss of all sensory neu- form during development remains unclear. rons, demonstrating that those ligands and receptors In summary, certain molecular aspects of sensory are crucial for inner ear sensory neuron survival. Re- neuron formation, guidance and survival have been maining issues center on the differential function of clarified in recent years. Still, numerous open issues each neurotrophin receptor combination. Since most remain in this fast moving field and virtually no molec- other vertebrates express only one neurotrophin in the ular data exist for the basic patterning of central projec- 234 K.W. Beisel et al. / Development and evolution of the vestibular sensory apparatus

Fig. 5. The distribution of afferent fibers (A) and sensory neurons (B) after injections of two different lipophilic tracers into the cerebellum (red) and the superior vestibular nucleus (green) is shown in this 7 day old mouse. Note that red and green fibers are mixed to the canal cristae but are segregated along the striola in the utricle (u in A). These data imply that hair cell polarity reversal is accompanied by an almost complete segregation of fibers across the area of hair cell polarity reversal. Despite this extraordinary degree of segregation in their target organs, sensory neurons in the superior and inferior vestribular ganglion (SVG, IVG) are almost randomly mixed (B). AC, anterior crista; HC, horizontal crista; U, utricle. Bar indicates 200 μm. tions. Interesting future work will have to sort out how Unknown factors initiate the proliferation of hair the apparent relationship between sensory neurons and cell precursor inside the growing and dividing sensory hair cells work at a molecular level. patches, such as Sox2 [71], and initiate upregulation of the retinoblastoma gene to terminate hair cell mitosis [93]. Interestingly, at least the formation of some 6. Generating sensory epithelia and specifying hair cells is linked to the sensory neuron formation as position and polarity of hair cells Neurog1 null mutants are deficient in the number of hair cells and do never fully grow certain sensory patches, like the saccule [89]. It seems reasonable to assume Hair cell development requires precise spatial and that growth of epithelia depends on the proliferation of temporal regulation of many genes [45,71]. There is hair cell precursors, which may be set aside as early as inadequate molecular data to specify areas of sensory E10.5 in the mouse [45,96]. How all these early pat- patch formation but it is clear that the topology of these terning processes relate ultimately to the upregulation patches is dependent on the proper polarity acquisition of genes relevant for hair cell differentiation remains of the developing otocyst [40]. A candidate for the es- unclear. tablishment of dorso-ventral and possibly anterior pos- Hair cell differentiation requires a single bHLH gene, terior axis formation is Shh [120] in interaction with atonal homologue 1 (Atoh1), both during develop- Gbx2 [12,83], Wnt1/3a [121] and Gli3 [23]. Longitu- ment [8] and during regeneration [52,66,69]. Recent dinal studies suggest that sensory epithelia form as a data show that the gene relevant for hair cell precursor single patch that divides as the otocyst enlarges and be- formation is still unknown [25] as undifferentiated hair comes compartmentalized [38,100,109]. Null mutants cell precursors form in Atoh1 null mice [50]. Confirm- strongly support the involvement of Fgf10 and Sox2 in ing and extending this findings (Fig. 6) are recent data sensory patch and hair cell formation [71,103], but the on organ of Corti tissue culture showing that Atoh1 role of Bmp4 and the DELTA/NOTCH lateral inhibition is not required for the initial expression of MyoVIIa, process in this context is not completely clarified [6,26, MyoVI, nicotinic aceytcholine receptor α9, fimbrin, 31]. Most importantly, recent data suggest formation of POU4F3 and other hair cell markers [123]. This con- hair cell precursors in the absence of Atoh1, implying clusion is also supported by the precocious expression that Atoh1 can not activate the lateral inhibition system of the neurotrophin Bdnf in the cochlea, prior to the up- of delta and notch to sort out supporting cells from hair regulation of Atoh1, in what appears to be postmitotic cells [50]. hair cells [25,38,50]. Nevertheless, a possible involve- K.W. Beisel et al. / Development and evolution of the vestibular sensory apparatus 235 ment of Atoh1 in the selection of proliferating hair cell hair cell polarity require a more direct link with known precursors can not be ruled out for vestibular sensory molecules that are differentially distributed in various epithelia [96]. hair cells. Several crucial genes for hair cell differentiation and In summary, while our molecular understanding of maintenance are now characterized: Atoh1, Pou4f3, hair cell formation and differentiation is by far the most Gfi1 and Barhl1 [8,37,82,148,155]. More recent work advanced in all ear morphogenetic and histogenetic indicates that the Pou domain factor (Pou4f3) regulates processes, it also shows gaps that require much further the zinc finger protein, GFI1 [62]. How those genes in work to be connected in a logical and causal sequence turn tie into Barhl1 regulation as well as hair cell dif- in order to be used in a controlled fashion to remedy ferentiation remains unclear. It appears, however, that one aspect of vestibular loss of function: to replace lost major aspects of hair cell differentiation can be accomplished in the absence of either Pou4f3 or Gfi1 [62]. hair cells. Recent attempts to tie the functional matu- Thus Atoh1 is certainly able to promote hair cell dif- ration of hair cells [35] to the onset of transmitter re- ferentiation to the extent of stereocilia formation even lease mechanisms [130] remain to be completed using without those genes, but identification of hair cells ap- appropriate mutational analysis of conditional mutants pears to be even possible in the absence of Atoh1 [50] with conditional deletion of relevant genes. and those cells express the BDNF neurotrophin(Fig. 6). At age 70 in humans the decline in vestibular and Molecules that specify the hair cell competent area cochlear hair cell numbers may have depleted the ca- are unknown. Transfection experiments using Atoh1 pacity of the system to compensate and hearing and gene loaded viruses have shown that cells in the greater vestibular disorders become apparent. While devices epithelial ridge can transform into hair cells in neonates to restore hearing have been designed, such as cochlear and adults [52,66,69]. How Atoh1 is initially upregu- and cochlear nucleus implants, electronic devices for lated in the cells that will become hair cells is not fully vestibular information delivery are in their infancy. Us- established and may vary between different sensory ep- ing the insights into the molecular basis of hair cell ithelia, the species used, as well as the technical limita- formation to regenerate hair cells and neurons and to tions of the specific techniques employed [25,96,122]. connect them properly so that vestibular sensation is While the role of p27 in terminal mitosis of hair cell precursors is now clear [24], other genes relevant for fully restored in the elderly would be a desired outcome mitosis need to be studied are needed as well such as of these studies summarized above. retinoblastoma [93]. Atoh1 drives related HLH genes Significant progress toward these goals show that (Hes1 and Hes5) that regulate the mosaic of hair cell gene therapy using Atoh1 loaded adenoviruses can and supporting cell formation [31,52,158]. Mutation transform supporting cells into hair cells [66,69]. Re- in the Hes genes result in extra hair cell formation, generating hair cells by forcing them to divide in supporting the notion that these genes regulate the hair retinoblastoma null mutants seem to be an alternative cell/supporting cell mosaic and have limited effected on way that requires, however, further studies are needed the overall topology and extent of hair cell progenitor to avoid the possibility of cancer formation [93]. Other formation that is regulated by as yet unknown genes. avenues to achieve functional recovery are the regener- How all those cell fate determination process are ation of hair cells and sensory neurons from embryonic molecularly integrated into the process of cell contact or adult stem cells [80,81] and isolation of proliferating formation that uses a variety of different cadherins and ganglion cell precursors appears to be possible from related molecules [70] that appear to play crucial roles the human cochlea [117]. Implanting such cells into in hair cell polarity formation [6,30,33,99] remains un- ears depleted of either hair cells or sensory neurons or known. There is no direct connection with the differen- both might be a future direction to restore hearing and tial distribution of actin and espin to form the stereociliary staircase and the mechanism of stereociliary bun- vestibular signal sensing. Transfering those data from dle polarity is yet to be shown [35]. It is conceivable, the cochlea to the vestibular system is another chal- but unknown, that differential distribution of espin that lenge for the field [7,138]. Overall, these data support regulates the assembly of actin in a quantitative fashion the notion that curing hair cell loss in the cochlea and maybe at the basis of the differential growth of stere- the vestibular sensory organs might become a possibil- ocilia. Thus the effects described for various trans- ity although more work is needed before any of these membrane and secreted signals in the establishment of techniques can be used for clinical trials. 236 K.W. Beisel et al. / Development and evolution of the vestibular sensory apparatus

Fig. 6. The formation of Atoh1 and Bdnf expressing cells is shown in the absence of Atoh1, a gene necessary for hair cell differentiation. Atoh1 and Bdnf genes were replaced with the LacZ reporter gene and the distribution of β-galactosidase, the enzyme generated by the LacZ gene, was revealed with a histochemical reaction that generates a blue reaction product. Note that either Atoh1-LacZ or Bdnf-LacZ reveals all hair cells of vestibular epithelia (A,C,E) in mice heterozygotic for these genes. Interestingly, in mice null for Atoh1 and that show no differentiated hair cells, expression of Atoh1-LacZ (B) and Bdnf-LacZ (D) shows a distribution of cells consistent with the formation of sensory epithelia. In addition, those sensory epithelia receive an innervation that is somewhat targeted toward those cells (E,F). Note that Bdnf expression in the utricle depends on the expression of Atoh1 (D). AC, anterior crista; HC, horizontal crista; PC, posterior crista; S, saccule; SN, sensory neurons; U, utricle. Bar indicates 100 μm. Modiﬁed after [50].

7. Conclusion and outlook ear evolution such as specific fiber projection to distinct endorgans and their central targets are less likely Molecular progress has revealed some aspects of ear to depend on single genes. morphogenesis, sensory epithelia and extracellular matrix formation and formation and specification of sen- Acknowledgments sory neuron connections. Nevertheless, these insights provide only a beginning and numerous open ques- This work was supported by a grant from NIH (RO1 tions remain, requiring more sophisticated molecular DC005590; BF, KB) and NASA (NAG 2-1611; BF, analysis combined with physiological experiments in KB), COBRE (1P20RR018788-01; YWL, BF, KB) and viable mutant mice that carry ear directed mutations. LB692 (BF, KB). This investigation was conducted in Progress already indicates significant clinical potential a facility constructed with support from Research Fa- that may relate to treatments of vestibular sensation cilities Improvement Program Grant Number 1 C06 loss, by mediating hair cell and sensory neuron regen- RR17417-01 from the National Center for Research eration. Some aspects of vestibular ear evolution ap- Resources, National Institutes of Health. We acknowl- pear to relate to the recruitment of specific genes into edge the use of the confocal microscope facility of the existing network of gene expression that mediates the NCCB, supported by EPSCoR EPS-0346476 (CFD ear development. However, other aspects of vestibular 47.076). K.W. Beisel et al. / Development and evolution of the vestibular sensory apparatus 237

References [17] P. Burighel, N.J. Lane, G. Fabio, T. Stefano, G. Zaniolo, M.D. Carnevali and L. Manni, Novel, secondary sensory cell organ in ascidians: in search of the ancestor of the vertebrate [1] S. Abdelhak, V. Kalatzis, R. Heilig, S. Compain, D. Samson, lateral line, J Comp Neurol 461 (2003), 236–249. C. Vincent, F. Levi-Acobas, C. Cruaud, M. Le Merrer, M. [18] Q. Burton, L.K. Cole, M. Mulheisen, W. Chang and D.K. Mathieu, R. Konig, J. Vigneron, J. Weissenbach, C. Petit and Wu, The role of Pax2 in mouse inner ear development, Dev D. Weil, Clustering of mutations responsible for branchio- Biol 272 (2004), 161–175. oto-renal (BOR) syndrome in the eyes absent homologous [19] R. Cantos, L.K. Cole, D. Acampora, A. Simeone and D.K. region (eyaHR) of EYA1, Hum Mol Genet 6 (1997), 2247– Wu, Patterning of the mammalian cochlea, Proc Natl Acad 2255. Sci USA 97 (2000), 11707–11713. [2] S. Abdelhak, V. Kalatzis, R. Heilig, S. Compain, D. Sam- [20] W. Chang, F.D. Nunes, J.M. De Jesus-Escobar, R. Harland son, C. Vincent, D. Weil, C. Cruaud, I. Sahly, M. Leibovici, and D.K. Wu, Ectopic noggin blocks sensory and nonsensory M. Bitner-Glindzicz, M. Francis, D. Lacombe, J. Vigneron, organ morphogenesis in the chicken inner ear, Dev Biol 216 R. Charachon, K. Boven, P. Bedbeder, N. Van Regemorter, (1999), 369–381. J. Weissenbach and C. Petit, A human homologue of the [21] W. Chang, P. ten Dijke and D.K. Wu, BMP pathways are in- Drosophila eyes absent gene underlies branchio-oto-renal volved in otic capsule formation and epithelial-mesenchymal (BOR) syndrome and identifies a novel gene family, Nat signaling in the developing chicken inner ear, Dev Biol 251 Genet 15 (1997), 157–164. (2002), 380–394. [3] K. Agerman, J. Hjerling-Leffler, M.P. Blanchard, E. Scar- [22] W. Chang, J.V. Brigande, D.M. Fekete and D.K. Wu, The fone, B. Canlon, C. Nosrat and P. Ernfors, BDNF gene re- development of semicircular canals in the inner ear: role placement reveals multiple mechanisms for establishing neu- of FGFs in sensory cristae, Development 131 (2004), 4201– rotrophin specificity during sensory nervous system devel- 4211. opment, Development 130 (2003), 1479–1491. [23] W. Chang, L.K. Cole, R. Cantos and D.K. Wu, Molecular [4] Y. Alvarez, M.T. Alonso, V. Vendrell, L.C. Zelarayan, P. genetics of vestibular organ development, in: The Vestibular Chamero, T. Theil, M.R. Bosl, S. Kato, M. Maconochie, D. System, Vol. 19, S.M. Highstein, R.R. Fay and A.N. Popper, Riethmacher and T. Schimmang, Requirements for FGF3 and eds, New York: Springer Verlag, 2004, pp. 11–56. FGF10 during inner ear formation, Development 130 (2003), [24] P. Chen and N. Segil, p27(Kip1) links cell proliferation to 6329–6338. morphogenesis in the developing organ of Corti, Develop- [5] C.V. Baker and M. Bronner-Fraser, Vertebrate cranial pla- ment 126 (1999), 1581–1590. codes I. Embryonic induction, Dev Biol 232 (2001), 1–61. [25] P. Chen, J.E. Johnson, H.Y. Zoghbi and N. Segil, The role of [6] K.F. Barald and M.W. Kelley, From placode to polariza- Math1 in inner ear development: Uncoupling the establish- tion: new tunes in inner ear development, Development 131 ment of the sensory primordium from hair cell fate determi- (2004), 4119–4130. nation, Development 129 (2002), 2495–2505. [7] O. Bermingham-McDonogh and E.W. Rubel, Hair cell re- [26] L.K. Cole, I. Le Roux, F. Nunes, E. Laufer, J. Lewis and generation: winging our way towards a sound future, Curr D.K. Wu, Sensory organ generation in the chicken inner ear: Opin Neurobiol 13 (2003), 119–126. contributions of bone morphogenetic protein 4, serrate1, and [8] N.A. Bermingham, B.A. Hassan, S.D. Price, M.A. Vollrath, lunatic fringe, J Comp Neurol 424 (2000), 509–520. N. Ben-Arie, R.A. Eatock, H.J. Bellen, A. Lysakowski and [27] J.S. Colvin, B.A. Bohne, G.W. Harding, D.G. McEwen and H.Y. Zoghbi, Math1: an essential gene for the generation of D.M. Ornitz, Skeletal overgrowth and deafness in mice lack- inner ear hair cells, Science 284 (1999), 1837–1841. ing fibroblast growth factor receptor 3, Nat Genet 12 (1996), [9] N. Bertrand, D.S. Castro and F. Guillemot, Proneural genes 390–397. and the specification of neural cell types, Nat Rev Neurosci [28] V. Coppola, J. Kucera, M.E. Palko, J. Martinez-De Velasco, 3 (2002), 517–530. W.E. Lyons, B. Fritzsch and L. Tessarollo, Dissection of NT3 [10] V. Bertrand, C. Hudson, D. Caillol, C. Popovici and P. functions in vivo by gene replacement strategy, Development Lemaire, Neural tissue in ascidian embryos is induced by 128 (2001), 4315–4327. FGF9/16/20, acting via a combination of maternal GATA and [29] C.A. Cowan, N. Yokoyama, L.M. Bianchi, M. Henkemeyer Ets transcription factors, Cell 115 (2003), 615–627. and B. Fritzsch, EphB2 guides axons at the midline and is [11] E. Bober, S. Rinkwitz and H. Herbrand, Molecular ba- necessary for normal vestibular function, Neuron 26 (2000), sis of otic commitment and morphogenesis: a role for 417–430. homeodomain-containing transcription factors and signaling [30] A. Dabdoub, M.J. Donohue, A. Brennan, V. Wolf, M. Mont- molecules, Curr Top Dev Biol 57 (2003), 151–175. couquiol, D.A. Sassoon, J.C. Hseih, J.S. Rubin, P.C. Sali- [12] J. Bok, M. Bronner-Fraser and D.K. Wu, Role of the hind- nas and M.W. Kelley, Wnt signaling mediates reorientation brain in dorsoventral but not anteroposterior axial specifica- of outer hair cell stereociliary bundles in the mammalian tion of the inner ear, Development 132 (2005), 2115–2124. cochlea, Development 130 (2003), 2375–2384. [13] C.B. Braun, Schreiner organs: a new craniate chemosensory [31] N. Daudet and J. Lewis, Two contrasting roles for Notch ac- modality in hagfishes, J Comp Neurol 392 (1998), 135–163. tivity in chick inner ear development: specification of prosen- [14] A.E. Brent, R. Schweitzer and C.J. Tabin, A somitic com- sory patches and lateral inhibition of hair-cell differentiation, partment of tendon progenitors, Cell 113 (2003), 235–248. Development 132 (2005), 541–551. [15] S.T. Brown, K. Martin and A.K. Groves, Molecular basis of [32] E.S. David, N.H. Luke and B.T. Livingston, Characterization inner ear induction, Curr Top Dev Biol 57 (2003), 115–149. of a gene encoding a developmentally regulated winged he- [16] B. Budelmann, Hearing in Nonarthropod Invertebrates, in: lix transcription factor of the sea urchin Strongylocentrotus The Evolutionary Biology of Hearing, D.B. Webster, R.R. purpuratus, Gene 236 (1999), 97–105. Fay and A.N. Popper, eds. New York, NY: Springer Verlag; [33] A. Davies, C. Formstone, I. Mason and J. Lewis, Planar 1992, pp. 141–155. polarity of hair cells in the chick inner ear is correlated with 238 K.W. Beisel et al. / Development and evolution of the vestibular sensory apparatus

polarized distribution of c-flamingo-1 protein, Dev Dyn 233 [52] W.Q. Gao, Hair cell development in higher vertebrates, Curr (2005), 998–1005. Top Dev Biol 57 (2003), 293–319. [34] R.J. Davis, W. Shen, Y.I. Sandler, T.A. Heanue and G. [53] L.M. Gerlach, M.R. Hutson, J.A. Germiller, D. Nguyen- Mardon, Characterization of mouse Dach2, a homologue of Luu, J.C. Victor and K.F. Barald, Addition of the BMP4 Drosophila dachshund, Mech Dev 102 (2001), 169–179. antagonist, noggin, disrupts avian inner ear development, [35] R.A. Eatock and K.M. Hurley, Functional development of Development 127 (2000), 45–54. hair cells, Curr Top Dev Biol 57 (2003), 389–448. [54] R.J. Goodyear and G.P. Richardson, Extracellular matrices [36] A. El-Amraoui, M. Cohen-Salmon, C. Petit and M.C. Simm- associated with the apical surfaces of sensory epithelia in the ler, Spatiotemporal expression of otogelin in the developing inner ear: molecular and structural diversity, J Neurobiol 53 and adult mouse inner ear, Hear Res 158 (2001), 151–159. (2002), 212–227. [37] L. Erkman, R.J. McEvilly, L. Luo, A.K. Ryan, F. Hoosh- [55] B. Grothe, E.C. Carr, J.H. Casseday, B. Fritzsch and C. mand, S.M. O’Connell, E.M. Keithley, D.H. Rapaport, A.F. Koppl,¨ The Evolution of Central Pathways and Their Neural Ryan and M.G. Rosenfeld, Role of transcription factors Brn- Processing Patterns, in: Springer Handbook of Auditory Re- 3.1 and Brn-3.2 in auditory and visual system development, search: Evolution of the Vertebrate Auditory System, G.A. Nature 381 (1996), 603–606. Manley, A.N. Popper and R.R. Fay, eds, New York: Spinger- [38] I. Farinas, K.R. Jones, L. Tessarollo, A.J. Vigers, E. Huang, Verlag, 2004, pp. 289–359. M. Kirstein, D.C. de Caprona, V. Coppola, C. Backus, L.F. [56] C. Gu, E.R. Rodriguez, D.V. Reimert, T. Shu, B. Fritzsch, Reichardt and B. Fritzsch, Spatial shaping of cochlear in- L.J. Richards, A.L. Kolodkin and D.D. Ginty, Neuropilin- nervation by temporally regulated neurotrophin expression, 1 Conveys Semaphorin and VEGF Signaling during Neural J Neurosci 21 (2001), 6170–6180. and Cardiovascular Development, Dev Cell 5 (2003), 45–57. [39] D.M. Fekete, S.A. Homburger, M.T. Waring, A.E. Riedl [57] T. Hadrys, T. Braun, S. Rinkwitz-Brandt, H.H. Arnold and and L.F. Garcia, Involvement of programmed cell death in E. Bober, Nkx5-1 controls semicircular canal formation in morphogenesis of the vertebrate inner ear, Development 124 the mouse inner ear, Development 125 (1998), 33–39. (1997), 2451–2461. [58] R. Hallworth, M.L. Wiederhold, J.B. Campbell and P.S. [40] D.M. Fekete and D.K. Wu, Revisiting cell fate specification Steyger, Atomic force microscope observations of otoconia in the inner ear, Curr Opin Neurobiol 12 (2002), 35–42. in the newt, Hear Res 85 (1995), 115–121. [41] B. Fritzsch, Similarities and differences in lancelet and cra- [59] I.M. Hanson, Mammalian homologues of the Drosophila eye niate nervous systems, Isr. J. Zool. 42 (1996), 147–160. specification genes, Semin Cell Dev Biol 12 (2001), 475–484. [42] B. Fritzsch, K. Barald and M. Lomax, Early embryology of [60] V. Hatini, X. Ye, G. Balas and E. Lai, Dynamics of placodal the vertebrate ear, in: Springer Handbook of Auditory Re- lineage development revealed by targeted transgene expres- search, Vol. XII, E.W. Rubel, A.N. Popper and R.R. Fay, eds, sion, Dev Dyn 215 (1999), 332–343. Development of the Auditory System., New York: Springer [61] D. Hellard, T. Brosenitsch, B. Fritzsch and D.M. Katz, Cra- Verlag, 1998, pp. 80–145. nial sensory neuron development in the absence of brain- [43] B. Fritzsch and K.W. Beisel, Evolution and development of derived neurotrophic factor in BDNF/Bax double null mice, the vertebrate ear, Brain Res Bull 55 (2001), 711–721. Dev Biol 275 (2004), 34–43. [44] B. Fritzsch, M. Signore and A. Simeone, Otx1 null mutant [62] R. Hertzano, M. Montcouquiol, S. Rashi-Elkeles, R. Elkon, mice show partial segregation of sensory epithelia compara- R. Yucel, W.N. Frankel, G. Rechavi, T. Moroy, T.B. Fried- ble to lamprey ears, Dev Genes Evol 211 (2001), 388–396. man, M.W. Kelley and K.B. Avraham, Transcription profil- [45] B. Fritzsch, K.W. Beisel, K. Jones, I. Farinas, A. Maklad, J. ing of inner ears from Pou4f3ddl/ddl identifies Gfi1 as a tar- Lee and L.F. Reichardt, Development and evolution of inner get of the Pou4f3 deafness gene, Hum Mol Genet 13 (2004), ear sensory epithelia and their innervation, J Neurobiol 53 2143–2153. (2002), 143–156. [63] S.M. Highstein, R.D. Rabbitt, G.R. Holstein and R.D. Boyle, [46] B. Fritzsch, Development of inner ear afferent connections: Determinants of spatial and temporal coding by semicircular forming primary neurons and connecting them to the devel- canal afferents, J Neurophysiol 93 (2005), 2359–2370. oping sensory epithelia, Brain Res Bull 60 (2003), 423–433. [64] E.J. Huang, W. Liu, B. Fritzsch, L.M. Bianchi, L.F. Reichardt [47] B. Fritzsch and K.W. Beisel, Molecular conservation and and M. Xiang, Brn3a is a transcriptional regulator of soma novelties in vertebrate ear development, Curr Top Dev Biol size, target field innervation and axon pathfinding of inner 57 (2003), 1–44. ear sensory neurons, Development 128 (2001), 2421–2432. [48] B. Fritzsch, V. Coppola, L. Tessarollo and L.F. Reichardt, [65] B. Hurle, E. Ignatova, S.M. Massironi, T. Mashimo, X. Neurotrophins in the ear: their roles in sensory neuron sur- Rios, I. Thalmann, R. Thalmann and D.M. Ornitz, Non- vival and fiber guidance, Progress in Brain Research 146 syndromic vestibular disorder with otoconial agenesis in (2003), 265–278. tilted/mergulhador mice caused by mutations in otopetrin 1, [49] B. Fritzsch and K.W.Beisel, Keeping sensory cells and evolv- Hum Mol Genet 12 (2003), 777–789. ing neurons to connect them to the brain: molecular con- [66] M. Izumikawa, R. Minoda, K. Kawamoto, K.A. Abrashkin, servation and novelties in vertebrate ear development, Brain D.L. Swiderski, D.F. Dolan, D.E. Brough and Y. Raphael, Behav Evol 64 (2004), 182–197. Auditory hair cell replacement and hearing improvement by [50] B. Fritzsch, V.A. Matei, D.H. Nichols, N. Bermingham, K. Atoh1 gene therapy in deaf mammals, Nat Med 11 (2005), Jones, K.W. Beisel and V.Y. Wang, Atoh1 null mice show 271–276. directed afferent fiber growth to undifferentiated ear sensory [67] K.R. Johnson, S.A. Cook, L.C. Erway, A.N. Matthews, L.P. epithelia followed by incomplete fiber retention, Dev Dyn Sanford, N.E. Paradies and R.A. Friedman, Inner ear and 233 (2005), 570–583. kidney anomalies caused by IAP insertion in an intron of the [51] B. Fritzsch and J. Piatigorsky, Ancestry of photic and me- Eya1 gene in a mouse model of BOR syndrome, Hum Mol chanic sensation? Science 308 (2005), 1113–1114. Genet 8 (1999), 645–653. K.W. Beisel et al. / Development and evolution of the vestibular sensory apparatus 239

[68] A. Karis, I. Pata, J.H. van Doorninck, F. Grosveld, C.I. de [86] M. Liu, F.A. Pereira, S.D. Price, M.J. Chu, C. Shope, D. Zeeuw, D. de Caprona and B. Fritzsch, Transcription factor Himes, R.A. Eatock, W.E.Brownell, A. Lysakowski and M.J. GATA-3 alters pathway selection of olivocochlear neurons Tsai, Essential role of BETA2/NeuroD1 in development of and affects morphogenesis of the ear, J Comp Neurol 429 the vestibular and auditory systems, Genes Dev 14 (2000), (2001), 615–630. 2839–2854. [69] K. Kawamoto, S. Ishimoto, R. Minoda, D.E. Brough and Y. [87] A. Lysakowski and J.M. Goldberg, Morphophysiology of the Raphael, Math1 gene transfer generates new cochlear hair Vestibular Periphery, in: The Vestibular System, Vol.19, S.M. cells in mature guinea pigs in vivo, J Neurosci 23 (2003), Highstein, R.R. Fay, A.N. Popper, eds, New York: Springer 4395–4400. Verlag, 2004, pp. 57–152. [70] M.W. Kelley, Cell adhesion molecules during inner ear and [88] Q. Ma, Z. Chen, I. del Barco Barrantes, J.L de la Pompa and hair cell development, including notch and its ligands, Curr D.J. Anderson, neurogenin1 is essential for the determination Top Dev Biol 57 (2003), 321–356. of neuronal precursors for proximal cranial sensory ganglia, [71] A.E. Kiernan, A.L. Pelling, K.K. Leung, A.S. Tang, D.M. Neuron 20 (1998), 469–482. Bell, C. Tease, R. Lovell-Badge, K.P. Steel and K.S. Cheah, [89] Q. Ma, D.J. Anderson and B. Fritzsch, Neurogenin 1 null Sox2 is required for sensory organ development in the mam- mutant ears develop fewer, morphologically normal hair cells malian inner ear, Nature 434 (2005), 1031–1035. in smaller sensory epithelia devoid of innervation, J Assoc [72] W.Y.Kim, B. Fritzsch, A. Serls, L.A. Bakel, E.J. Huang, L.F. Res Otolaryngol 1 (2000), 129–143. Reichardt, D.S. Barth and J.E. Lee, NeuroD-null mice are [90] G.O. Mackie and C.L. Singla, Cupular organs in two species deaf due to a severe loss of the inner ear sensory neurons of Corella (Tunicata: Ascidiacea), Invert. Biol. 123 (2004), during development, Development 128 (2001), 417–426. 269–281. [73] Z. Kozmik, M. Daube, E. Frei, B. Norman, L. Kos, L.J. [91] A. Maklad and B. Fritzsch, The developmental segregation of Dishaw, M. Noll and J. Piatigorsky, Role of Pax Genes in posterior crista and saccular vestibular fibers in mice: A car- Eye Evolution. A Cnidarian PaxB Gene Uniting Pax2 and bocyanine tracer study using confocal microscopy, Develop. Pax6 Functions, Dev Cell 5 (2003), 773–785. Brain Research. 135 (2002). [74] K.H. Krause, Tissue distribution and putative physiological [92] A. Maklad and B. Fritzsch, Development of vestibular affer- function of NOX family NADPH oxidases, Jpn J Infect Dis ent projections into the hindbrain and their central targets, 57 (2004), S28–29. Brain Res Bull 60 (2003), 497–510. [75] T.C. Lacalli, Sensory systems in amphioxus: a window on the [93] J. Mantela, Z. Jiang, J. Ylikoski, B. Fritzsch, E. Zacksenhaus ancestral chordate condition, Brain Behav Evol 64 (2004), and U. Pirvola, The retinoblastoma gene pathway regulates 148–162. the postmitotic state of hair cells of the mouse inner ear, [76] R.K. Ladher, T.J. Wright, A.M. Moon, S.L. Mansour and Development 132 (2005), 2377–2388. G.C. Schoenwolf, FGF8 initiates inner ear induction in chick [94] H. Markl, The perception of gravity and of angular acceler- and mouse, Genes Dev 19 (2005), 603–613. ation in invertebrates, in: Handbook of Sensory Physiology, [77] G. Lawoko-Kerali, M.N. Rivolta and M. Holley, Expression Vol. VI/1 Vestibular System, H.H. Kornhuber, ed., Berlin: of the transcription factors GATA3 and Pax2 during develop- Springer Verlag, 1974, pp. 17–74. ment of the mammalian inner ear, J Comp Neurol 442 (2002), [95] P. Martin and G.J. Swanson, Descriptive and experimental 378–391. analysis of the epithelial remodellings that control semicir- [78] P.K. Legan, V.A. Lukashkina, R.J. Goodyear, M. Kossi, I.J. cular canal formation in the developing mouse inner ear, Dev Russell and G.P. Richardson, A targeted deletion in alpha- Biol 159 (1993), 549–558. tectorin reveals that the tectorial membrane is required for [96] V. Matei, S. Pauley, S. Kaing, D. Rowitch, K.W. Beisel, K. the gain and timing of cochlear feedback, Neuron 28 (2000), Morris, F. Feng, K. Jones, J. Lee and B. Fritzsch, Smaller 273–285. inner ear sensory epithelia in Neurog1 null mice are related [79] E.R. Lewis, E.L. Leverenz and W.S. Bialek, The vertebrate to earlier hair cell cycle exit, Dev Dyn 234 (2005), 633–650. inner ear, Boca Raton (1985), CRC Press, 248. [97] S. Mazan, D. Jaillard, B. Baratte and P. Janvier, Otx1 [80] H. Li, H. Liu and S. Heller, Pluripotent stem cells from the gene-controlled morphogenesis of the horizontal semicircu- adult mouse inner ear, Nat Med 9 (2003), 1293–1299. lar canal and the origin of the gnathostome characteristics, [81] H. Li, G. Roblin, H. Liu and S. Heller, Generation of hair Evol Dev 2 (2000), 186–193. cells by stepwise differentiation of embryonic stem cells, [98] G.R. Merlo, L. Paleari, S. Mantero, B. Zerega, M. Adamska, Proc Natl Acad Sci USA 100 (2003), 13495–13500. S. Rinkwitz, E. Bober and G. Levi, The Dlx5 homeobox [82] S. Li, S.M. Price, H. Cahill, D.K. Ryugo, M.M. Shen and M. gene is essential for vestibular morphogenesis in the mouse Xiang, Hearing loss caused by progressive degeneration of embryo through a BMP4-mediated pathway, Dev Biol 248 cochlear hair cells in mice deficient for the Barhl1 homeobox (2002), 157–169. gene, Development 129 (2002), 3523–3532. [99] M. Montcouquiol and M.W. Kelley, Planar and vertical sig- [83] Z. Lin, R. Cantos, M. Patente and D.K. Wu, Gbx2 is required nals control cellular differentiation and patterning in the for the morphogenesis of the mouse inner ear: a downstream mammalian cochlea, J Neurosci 23 (2003), 9469–9478. candidate of hindbrain signaling, Development 132 (2005), [100] H. Morsli, D. Choo, A. Ryan, R. Johnson and D.K. Wu, 2309–2318. Development of the mouse inner ear and origin of its sensory [84] H.H. Lindeman, Studies on the morphology of the sensory organs, J Neurosci 18 (1998), 3327–3335. regions o fht evestibular apparatus, Advances in Anatomy, [101] H. Morsli, F. Tuorto, D. Choo, M.P. Postiglione, A. Simeone Embryology and Cell Biology 42 (1969), 7–113. and D.K. Wu, Otx1 and Otx2 activities are required for the [85] U. Lins, M. Farina, M. Kurc, G. Riordan, R. Thalmann, I. normal development of the mouse inner ear, Development Thalmann and B. Kachar, The otoconia of the guinea pig 126 (1999), 2335–2343. utricle: internal structure, surface exposure, and interactions [102] Y. Murakami, M. Pasqualetti, Y. Takio, S. Hirano, F.M. Ri- with the filament matrix, J Struct Biol 131 (2000), 67–78. jli and S. Kuratani, Segmental development of reticulospinal 240 K.W. Beisel et al. / Development and evolution of the vestibular sensory apparatus

and branchiomotor neurons in lamprey: insights into the evo- [119] G. Retzius, Das Gehororgan¨ der Wirbeltiere: II. Das lution of the vertebrate hindbrain, Development 131 (2004), Gehororgan¨ der Amnioten, Stockholm: Samson und Wallin 983–995. (1884), 345. [103] H. Ohuchi, A. Yasue, K. Ono, S. Sasaoka, S. Tomonari, A. [120] M.M. Riccomagno, L. Martinu, M. Mulheisen, D.K. Wu Takagi, M. Itakura, K. Moriyama, S. Noji and T. Nohno, Iden- and D.J. Epstein, Specification of the mammalian cochlea is tification of cis-element regulating expression of the mouse dependent on Sonic hedgehog, Genes Dev 16 (2002), 2365– Fgf10 gene during inner ear development, Dev Dyn 233 2378. (2005), 177–187. [121] M.M. Riccomagno, S. Takada and D.J. Epstein, Wnt- [104] T. Ohyama and A.K. Groves, Expression of mouse Foxi class dependent regulation of inner ear morphogenesis is balanced genes in early craniofacial development, Dev Dyn 231 (2004), by the opposing and supporting roles of Shh, Genes Dev 19 640–646. (2005), 1612–1623. [105] D.M. Ornitz, B.A. Bohne, I. Thalmann, G.W. Harding and [122] B.B. Riley, Genes controlling the development of the ze- R. Thalmann, Otoconial agenesis in tilted mutant mice, Hear brafish inner ear and hair cells, Curr Top Dev Biol 57 (2003), Res 122 (1998), 60–70. 357–388. [106] D.M. Ornitz and P.J. Marie, FGF signalling pathways in en- [123] M.N. Rivolta, A. Halsall, C.M. Johnson, M.A. Tones and chondral and intramembranous bone development and hu- M.C. Holley, Transcript profiling of functionally related man genetic disease, Genes Dev 16 (2002), 1446–1465. groups of genes during conditional differentiation of a Mam- [107] R. Paffenholz, R.A. Bergstrom, F. Pasutto, P. Wabnitz, R.J. malian cochlear hair cell line, Genome Res 12 (2002), 1091– Munroe, W. Jagla, U. Heinzmann, A. Marquardt, A. Bareiss, 1099. J. Laufs, A. Russ, G. Stumm, J.C. Schimenti and D.E. [124] R. Romand and I. Varela-Nieto, Development of Auditory Bergstrom, Vestibular defects in head-tilt mice result from and Vestibular Systems-3. Molecular Development of the mutations in Nox3, encoding an NADPH oxidase, Genes Dev Inner Ear, Curr Top Dev Biol 57 (2003), 1–481. 18 (2004), 486–491. [125] M.D. Ross, L.-G. Johnsson and D. Peacor, Observations [108] R.K. Patient and J.D. McGhee, The GATA family (verte- on normal and degenerating human otoconia, Ann Otol 85 brates and invertebrates), Curr Opin Genet Dev 12 (2002), (1976), 310–326. 416–422. [126] R.J. Ruben, Development of the inner ear of the mouse: a ra- [109] S. Pauley, T.J. Wright, U. Pirvola, D. Ornitz, K. Beisel and B. dioautographic study of terminal mitoses, Acta Otolaryngol: Fritzsch, Expression and function of FGF10 in mammalian Suppl 220 (1967), 221–244. inner ear development, Dev Dyn 227 (2003), 203–215. [127] M. Sakagami, Role of osteopontin in the rodent inner ear as [110] M. Pfister, T. Toth, H. Thiele, B. Haack, N. Blin, H.P.Zenner, revealed by in situ hybridization, Med Electron Microsc 33 I. Sziklai, P. Murnberg and S. Kupka, A 4-bp Insertion in the (2000), 3–10. eya-Homologous Region (eyaHR) of EYA4 Causes Hearing [128] F. Salvinelli, L. Firrisi, M. Casale, M. Trivelli, L. D’Ascanio, Impairment in a Hungarian Family Linked to DFNA10, Mol F. Lamanna, F. Greco and S. Costantino, Benign paroxysmal Med 8 (2002), 607–611. positional vertigo: diagnosis and treatment, Clin Ter 155 [111] U. Pirvola, J. Ylikoski, J. Palgi, E. Lehtonen, U. Arumae (2004), 395–400. and M. Saarma, Brain-derived neurotrophic factor and neu- [129] M. Sato and K. Saigo, Involvement of pannier and u-shaped rotrophin 3 mRNAs in the peripheral target fields of devel- in regulation of decapentaplegic-dependent wingless expres- oping inner ear ganglia, Proc Natl Acad Sci USA 89 (1992), sion in developing Drosophila notum, Mech Dev 93 (2000), 9915–9919. 127–138. [112] U. Pirvola, B. Spencer-Dene, L. Xing-Qun, P. Kettunen, [130] T. Schimmang, J. Tan, M. Muller, U. Zimmermann, K. Ro- I. Thesleff, B. Fritzsch, C. Dickson and J. Ylikoski, hbock, I. Kopschall, A. Limberger, L. Minichiello and M. FGF/FGFR-2(IIIb) signaling is essential for inner ear mor- Knipper, Lack of Bdnf and TrkB signalling in the postnatal phogenesis, J Neurosci 20 (2000), 6125–6134. cochlea leads to a spatial reshaping of innervation along the [113] U. Pirvola, J. Ylikoski, R. Trokovic, J. Hebert, S. McConnell tonotopic axis and hearing loss, Development 130 (2003), and J. Partanen, FGFR1 Is Required for the Development of 4741–4750. the Auditory Sensory Epithelium, Neuron 35 (2002), 671. [131] M.C. Simmler, M. Cohen-Salmon, A. El-Amraoui, L. Guil- [114] U. Pirvola, X. Zhang, J. Mantela, D.M. Ornitz and J. Ylikoski, laud, J.C. Benichou, C. Petit and J.J. Panthier, Targeted dis- Fgf9 signaling regulates inner ear morphogenesis through ruption of otog results in deafness and severe imbalance, Nat epithelial-mesenchymal interactions, Dev Biol 273 (2004), Genet 24 (2000), 139–143. 350–360. [132] C. Sollner, H. Schwarz, R. Geisler and T. Nicolson, Mutated [115] R.D. Rabbitt, R. Boyle and S.M. Highstein, Physiology of otopetrin 1 affects the genesis of otoliths and the localization the semicircular canals after surgical plugging, Ann N Y Acad of Starmaker in zebrafish, Dev Genes Evol 214 (2004), 582– Sci 942 (2001), 274–286. 590. [116] S. Raft, S. Nowotschin, J. Liao and B.E. Morrow, Suppres- [133] K.S. Solomon and A. Fritz, Concerted action of two dlx par- sion of neural fate and control of inner ear morphogenesis by alogs in sensory placode formation, Development 129 (2002), Tbx1, Development 131 (2004), 1801–1812. 3127–3136. [117] H. Rask-Andersen, M. Bostrom, B. Gerdin, A. Kinnefors, G. [134] K.S. Solomon, T. Kudoh, I.B. Dawid and A. Fritz, Zebrafish Nyberg, T. Engstrand, J.M. Miller and D. Lindholm, Regen- foxi1 mediates otic placode formation and jaw development, eration of human auditory nerve. In vitro/in video demon- Development 130 (2003), 929–940. stration of neural progenitor cells in adult human and guinea [135] T.M. Squires, M.S. Weidman, T.C. Hain and H.A. Stone, A pig spiral ganglion, Hear Res 203 (2005), 180–191. mathematical model for top-shelf vertigo: the role of sedi- [118] A. Rau, P.K.Legan and G.P.Richardson, Tectorin mRNA ex- menting otoconia in BPPV, J Biomech 37 (2004), 1137–1146. pression is spatially and temporally restricted during mouse [136] A. Streit, Extensive cell movements accompany formation of inner ear development, J Comp Neurol 405 (1999), 271–280. the otic placode, Dev Biol 249 (2002), 237–254. K.W. Beisel et al. / Development and evolution of the vestibular sensory apparatus 241

[137] T. Takemura, M. Sakagami, T. Nakase, T. Kubo, Y.Kitamura [150] Y. Wang, P.E. Kowalski, I. Thalmann, D.M. Ornitz, D.L. and S. Nomura, Localization of osteopontin in the otoconial Mager and R. Thalmann, Otoconin-90, the mammalian oto- organs of adult rats, Hear Res 79 (1994), 99–104. conial matrix protein, contains two domains of homology [138] R. Taylor and A. Forge, Developmental biology. Life after to secretory phospholipase A2, Proc Natl Acad Sci USA 95 deaf for hair cells? Science 307 (2005), 1056–1058. (1998), 15345–15350. [139] L. Tessarollo, V. Coppola and B. Fritzsch, NT-3 replacement [151] S. Wayne, N.G. Robertson, F. DeClau, N. Chen, K. Verho- with brain-derived neurotrophic factor redirects vestibular even, S. Prasad, L. Tranebjarg, C.C. Morton, A.F. Ryan, G. nerve fibers to the cochlea, J Neurosci 24 (2004), 2575–2584. Van and R.J. Smith, Mutations in the transcriptional activator [140] M. Tessier-Lavigne and C.S. Goodman, The molecular biol- EYA4 cause late-onset deafness at the DFNA10 locus, Hum ogy of axon guidance, Science 274 (1996), 1123–1133. Mol Genet 10 (2001), 195–200. [141] R. Thalmann, E. Ignatova, B. Kachar, D.M. Ornitz and I. [152] T.J. Wright, E.P. Hatch, H. Karabagli, P. Karabagli, G.C. Thalmann, Development and maintenance of otoconia: bio- Schoenwolf and S.L. Mansour, Expression of mouse fibrob- chemical considerations, Ann N Y Acad Sci 942 (2001), 162– last growth factor and fibroblast growth factor receptor genes 178. during early inner ear development, Dev Dyn 228 (2003), [142] M. Torres and F. Giraldez, The development of the vertebrate 267–272. inner ear, Mech Dev 71 (1998), 5–21. [153] T.J. Wright and S.L. Mansour, Fgf3 and Fgf10 are required [143] Y. Uno, A. Horii, M. Umemoto, T. Hasegawa, K. Doi, A. for mouse otic placode induction, Development 130 (2003), Uno, T. Takemura and T. Kubo, Effects of hypergravity on 3379–3390. morphology and osteopontin expression in the rat otolith [154] T.J. Wright and S.L. Mansour, FGF signaling in ear devel- organs, J Vestib Res 10 (2000), 283–289. opment and innervation, Curr Top Dev Biol 57 (2003), 225– [144] K. Verhoeven, L. Van Laer, K. Kirschhofer, P.K. Legan, D.C. 259. Hughes, I. Schatteman, M. Verstreken, P. Van Hauwe, P. [155] M. Xiang, A. Maklad, U. Pirvola and B. Fritzsch, Brn3c null Coucke, A. Chen, R.J. Smith, T. Somers, F.E. Offeciers, P. mutant mice show long-term, incomplete retention of some Van de Heyning, G.P. Richardson, F. Wachtler, W.J. Kimber- afferent inner ear innervation, BMC Neurosci 4 (2003), 2. ling, P.J. Willems, P.J.Govaerts and G. Van Camp, Mutations [156] P.X. Xu, J. Adams, H. Peters, M.C. Brown, S Heaney and R. in the human alpha-tectorin gene cause autosomal dominant Maas, Eya1-deficient mice lack ears and kidneys and show non-syndromic hearing impairment, Nat Genet 19 (1998), abnormal apoptosis of organ primordia, Nat Genet 23 (1999), 60–62. 113–117. [145] E. Verpy, M. Leibovici and C. Petit, Characterization of [157] W. Zheng, L. Huang, Z.B. Wei, D. Silvius, B. Tang and otoconin-95, the major protein of murine otoconia, provides P.X. Xu, The role of Six1 in mammalian auditory system insights into the formation of these inner ear biominerals, development, Development 130 (2003), 3989–4000. Proc Natl Acad Sci USA 96 (1999), 529–534. [158] A. Zine, A. Aubert, J. Qiu, S. Therianos, F. Guillemot, R. [146] C. Vincent, V. Kalatzis, S. Abdelhak, H. Chaib, S. Com- Kageyama and F. de Ribaupierre, Hes1 and Hes5 activities pain, J. Helias, F.M. Vaneecloo and C. Petit, BOR and BO are required for the normal development of the hair cells in syndromes are allelic defects of EYA1, Eur J Hum Genet 5 the mammalian inner ear, J Neurosci 21 (2001), 4712–4720. (1997), 242–246. [159] D. Zou, D. Silvius, B. Fritzsch and P.X. Xu, Eya1 and Six1 [147] C. von Kupffer, The development of the cranial nerves of are essential for early steps of sensory neurogenesis in mam- vertebrates, J Comp Neurol 1 (1891), 246–264, 315–332. malian cranial placodes, Development 131 (2004), 5561– [148] D. Wallis, M. Hamblen, Y. Zhou, K.J. Venken, A. Schu- 5572. macher, H.L. Grimes, H.Y. Zoghbi, S.H. Orkin and H.J. [160] I. Zwaenepoel, M. Mustapha, M. Leibovici, E. Verpy, R. Bellen, The zinc finger transcription factor Gfi1, implicated Goodyear, X.Z. Liu, S. Nouaille, W.E. Nance, M. Kanaan, in lymphomagenesis, is required for inner ear hair cell differ- K.B. Avraham, F. Tekaia, J. Loiselet, M. Lathrop, G. Richard- entiation and survival, Development 130 (2003), 221–232. son and C. Petit, Otoancorin, an inner ear protein restricted to [149] W. Wang, J.F. Grimmer, T.R. Van De Water and T. Lufkin, the interface between the apical surface of sensory epithelia Hmx2 and Hmx3 homeobox genes direct development of the and their overlying acellular gels, is defective in autosomal murine inner ear and hypothalamus and can be functionally recessive deafness DFNB22, Proc Natl Acad Sci USA 99 replaced by Drosophila Hmx, Dev Cell 7 (2004), 439–453. (2002), 6240–6245.