Identification of the Otopetrin Domain,a conserved domain in vertebrate otopetrins and invertebrate otopetrin-like family members

Background  

Otopetrin 1 (Otop1) encodes a multi-transmembrane domain protein with no homology to known transporters, channels, exchangers, or receptors. Otop1 is necessary for the formation of otoconia and otoliths, calcium carbonate biominerals within the inner ear of mammals and teleost fish that are required for the detection of linear acceleration and gravity. Vertebrate Otop1 and its paralogues Otop2 and Otop3 define a new gene family with homology to the invertebrate Domain of Unknown Function 270 genes (DUF270; pfam03189).     

Otolith matrix proteins OMP-1 and Otolin-1 are necessary for normal otolith growth and their correct anchoring onto the sensory maculae

Fish otoliths are highly calcified concretions deposited in the inner ear and serve as a part of the hearing and balance systems. They consist mainly of calcium carbonate and a small amount of organic matrix. The latter component is considered to play important roles in otolith formation. Previously, we identified two major otolith matrix proteins, OMP-1 (otolith matrix protein-1) and Otolin-1, from salmonid species. To assess the function of these proteins in otolith formation, we performed antisense morpholino oligonucleotide (MO)-mediated knockdown of omp-1 and otolin-1 in zebrafish embryos. We first identified zebrafish cDNA homologs of omp-1 (zomp-1) and otolin-1 (zotolin-1). Whole-mount in situ hybridization then revealed that the expression of both zomp-1 and zotolin-1 mRNAs is restricted to the otic vesicles. zomp-1 mRNA was expressed from the 14-somite stage in the otic placode, but the zOMP-1 protein was detected only from 26-somite stage onwards. In contrast, zotolin-1 mRNA expression became clear around 72 hpf. MOs designed to inhibit zomp-1 and zotolin-1 mRNA translation, respectively, were injected into 1-2 cell stage embryos. zomp-1 MO caused a reduction in otolith size and an absence of zOtolin-1 deposition, while zotolin-1 MO caused a fusion of the two otoliths, and an increased instability of otoliths after fixation. We conclude that zOMP-1 is required for normal otolith growth and deposition of zOtolin-1 in the otolith, while zOtolin-1, a collagenous protein, is involved in the correct arrangement of the otoliths onto the sensory epithelium of the inner ear and probably in stabilization of the otolith matrix.     

Immunohistochemical localization of two otolith matrix proteins in the otolith and inner ear of the rainbow trout,<Emphasis Type="Italic"> Oncorhynchus mykiss</Emphasis>: comparative aspects between the adult inner ear and embryonic otocysts

The fish otolith consists mainly of calcium carbonate and organic matrices, the latter of which may play important roles in the process of otolith formation. We previously identified two otolith matrix proteins, named otolith matrix protein-1 (OMP-1) and otolin-1, from the rainbow trout, Oncorhynchus mykiss, and the chum salmon, O. keta. In this study, recombinant proteins corresponding to OMP-1 and otolin-1 were synthesized using yeast and bacterial expression systems, respectively, to produce specific antibodies against each protein. Immunohistochemical analysis using these antisera revealed that in the otoliths of adult fish, OMP-1 and otolin-1 were colocalized along the daily rings possibly formed by alternate deposition of calcium carbonate and organic matrices. In the adult inner ear, OMP-1 was produced at most of the saccular epithelium, while otolin-1 was produced at a limited part of cylindrical cells located at the marginal zone of the sensory epithelium. In the embryonic inner ear, these proteins had already existed in the otolith primordia when calcification had commenced. In addition, otolin-1 was localized in the fibrous materials connecting otolith primordia and sensory epithelium at this stage. These results indicate that these proteins are required as essential components for otolith formation and calcification.     

First steps of otolith formation of the zebrafish: role of glycogen?

The first steps of otolith formation were studied by electron microscopy in zebrafish embryos at different postfertilization (PF) time intervals. Between 19 and 22 h PF, the otic cavity contains glycogen particles derived by an apocrine process from the apical portions of the epithelial cells of the inner ear. The particles are arranged in parallel arrays, then in pseudocrystalloid structures, and finally in concentric arrays to form dense clusters referred to as "spherules". At 23 h PF, a group of "globules", consisting of modified aggregated "spherules" surrounded by several free "spherules", forms the nascent otolith. At 30 h PF, fused globules form a roughly spherical otolith. Spherules undergoing their process of modification and aggregation, are located in its central part, and constitute the so-called "nucleus". At 50 h PF, the otolith is a flattened hemisphere. It is made up of fused globules surrounded by two concentric layers whose organization is similar to that observed in the otolith of the adult fish. At this stage, calcium may be detected in the otolith except in its nucleus. We suggest that glycogen molecules found in the nascent otolith might allow the insertion of molecules such as glycoproteins (collagens) which are known to fix calcium. As a result, glycogen might play a key role in initiating the formation of otoliths and possibly that of other calcified tissues.     

Mutated <Emphasis Type="Italic">otopetrin 1</Emphasis> affects the genesis of otoliths and the localization of Starmaker in zebrafish

Otoliths in bony fishes and otoconia in mammals are composite crystals consisting of calcium carbonate and proteins. These biominerals are part of the gravity and linear acceleration detection system of the inner ear. Mutations in otopetrin 1 have been shown to result in lack of otoconia in tilted and mergulhador mutant mice. The molecular function of Otopetrin 1, a novel protein that contains ten predicted transmembrane domains, however, has remained elusive. Here we show that a mutation in the orthologous gene in zebrafish is responsible for the complete absence of otoliths in backstroke mutants. We examined the localization of Starmaker, a secreted protein that is highly abundant in otoliths in backstroke mutants. Starmaker protein accumulated within cells of the otic epithelium, indicating a possible defect in secretion. Our data suggest that Otopetrin 1 in zebrafish may be involved in the protein trafficking of components required for formation of biominerals in the ear.     

Separate Na,K-ATPase genes are required for otolith formation and semicircular canal development in zebrafish

We have investigated the role of Na,K-ATPase genes in zebrafish ear development. Six Na,K-ATPase genes are differentially expressed in the developing zebrafish inner ear. Antisense morpholino knockdown of Na,K-ATPase alpha1a.1 expression blocked formation of otoliths. This effect was phenocopied by treatment of embryos with ouabain, an inhibitor of Na,K-ATPase activity. The otolith defect produced by morpholinos was rescued by microinjection of zebrafish alpha1a.1 or rat alpha1 mRNA, while the ouabain-induced defect was rescued by expression of ouabain-resistant zebrafish alpha1a.1 or rat alpha1 mRNA. Knockdown of a second zebrafish alpha subunit, alpha1a.2, disrupted development of the semicircular canals. Knockdown of Na,K-ATPase beta2b expression also caused an otolith defect, suggesting that the beta2b subunit partners with the alpha1a.1 subunit to form a Na,K-ATPase required for otolith formation. These results reveal novel roles for Na,K-ATPase genes in vestibular system development and indicate that different isoforms play distinct functional roles in formation of inner ear structures. Our results highlight zebrafish gene knockdown-mRNA rescue as an approach that can be used to dissect the functional properties of zebrafish and mammalian Na,K-ATPase genes.     

Susceptibility to motion sickness in fish: a parabolic aircraft flight study

Juvenile swordtail fish and larval cichlids were subjected to parabolic aircraft flights (PAFs) and individually observed. After the PAFs, inner ear otoliths and sensory epithelia were examined on the light microscopical level. Otolith asymmetry (differences in otolith size between the left and the right side) was especially pronounced in those fish, who exhibited a kinetotic behaviour (e.g., spinning movements) during microgravity. This speaks in favour of a theoretical concept according to which susceptibility to space motion sickness in humans may be based on asymmetric inner ear stones. The cell density of sensory epithelia was lower in kinetotic animals as compared to normally swimming fish. Thus, asymmetric otoliths can cause kinetosis in fish during PAFs, but susceptibility to kinetosis may also be based on an aberrative inner ear morphology.     

The role of hair cells, cilia and ciliary motility in otolith formation in the zebrafish otic vesicle

Otoliths are biomineralised structures required for the sensation of gravity, linear acceleration and sound in the zebrafish ear. Otolith precursor particles, initially distributed throughout the otic vesicle lumen, become tethered to the tips of hair cell kinocilia (tether cilia) at the otic vesicle poles, forming two otoliths. We have used high-speed video microscopy to investigate the role of cilia and ciliary motility in otolith formation. In wild-type ears, groups of motile cilia are present at the otic vesicle poles, surrounding the immotile tether cilia. A few motile cilia are also found on the medial wall, but most cilia (92-98%) in the otic vesicle are immotile. In mutants with defective cilia (iguana) or ciliary motility (lrrc50), otoliths are frequently ectopic, untethered or fused. Nevertheless, neither cilia nor ciliary motility are absolutely required for otolith tethering: a mutant that lacks cilia completely (MZovl) is still capable of tethering otoliths at the otic vesicle poles. In embryos with attenuated Notch signalling [mindbomb mutant or Su(H) morphant], supernumerary hair cells develop and otolith precursor particles bind to the tips of all kinocilia, or bind directly to the hair cells' apical surface if cilia are absent [MZovl injected with a Su(H)1+2 morpholino]. However, if the first hair cells are missing (atoh1b morphant), otolith formation is severely disrupted and delayed. Our data support a model in which hair cells produce an otolith precursor-binding factor, normally localised to tether cell kinocilia. We also show that embryonic movement plays a minor role in the formation of normal otoliths.     

Cilia-driven fluid flow as an epigenetic cue for otolith biomineralization on sensory hair cells of the inner ear

Ciliary motility is necessary for many developmental and physiological processes in animals. In zebrafish, motile cilia are thought to be required for the deposition of otoliths, which comprise crystals of protein and calcium carbonate, on hair cells of the inner ear. The identity of the motile cilia and their role in otolith biogenesis, however, remain controversial. Here, we show that the ear vesicle differentiates numerous motile cilia, the spatial distribution of which changes as a function of the expression pattern of the ciliogenic gene foxj1b. By contrast, the hair cells develop immotile kinocilia that serve as static tethers for otolith crystallization. In ears devoid of all cilia, otoliths can form but they are of irregular shapes and sizes and appear to attach instead to the hair cell apical membranes. Moreover, overproduction of motile cilia also disrupts otolith deposition through sustained agitation of the precursor particles. Therefore, the correct spatial and temporal distribution of the motile cilia is crucial for proper otolith formation. Our findings support the view that the hair cells express a binding factor for the otolith precursors, while the motile cilia ensure that the precursors do not sediment prematurely and are efficiently directed towards the hair cells. We also provide evidence that the kinocilia are modified motile cilia that depend on Foxj1b for their differentiation. We propose that in hair cells, a Foxj1b-dependent motile ciliogenic program is altered by the proneural Atoh proteins to promote the differentiation of immotile kinocilia.     

On the role of the central nervous system in regulating the mineralisation of inner-ear otoliths of fish

Summary. Stato- or otoliths are calcified structures in the organ of balance and equilibrium of vertebrates, the inner ear, where they enhance its sensitivity to gravity. The compact otoliths of fish are composed of the calcium carbonate polymorph aragonite and a small fraction of organic molecules. The latter form a protein skeleton which determines the morphology of an otolith as well as its crystal lattice structure. This short review addresses findings according to which the brain obviously plays a prominent role in regulating the mineralisation of fish otoliths and depends on the gravity vector. Overall, otolith mineralisation has thus been identified to be a unique, neuronally guided biomineralisation process. The following is a hypothetical model for regulation of calcification by efferent vestibular neurons: (1) release of calcium at tight junctions in the macular epithelia, (2) macular carbonic anhydrase activity (which in turn is responsible for carbonate deposition), (3) chemical composition of matrix proteins. The rationale and evidence that support this model are discussed. Correspondence and reprints: Zoological Institute, University of Hohenheim, Garbenstrasse 30, 70593 Stuttgart, Federal Republic of Germany.     

Hypergravity decreases carbonic anhydrase-reactivity in inner ear maculae of fish

Previous investigations revealed that fish inner ear otolith growth depends on the amplitude and the direction of gravity. Both otolith total size, otolith bilateral size-asymmetry and the total and bilateral calcium-incorporation are also affected by gravity. Hypergravity, e.g., slows down otolith growth and diminishes bilateral otolith asymmetry as compared to 1 g control specimens raised in parallel. Since the enzyme carbonic anhydrase (CA) plays a prominent role in otolithic calcification, the reactivity of inner ear CA during otolith growth under hypergravity was investigated. CA-reactivity was demonstrated histochemically and densitometrically on sections of inner ear maculae of larval cichlid fish (Oreochromis mossambicus), that were kept for 6 hrs in a 3 g hypergravity centrifuge. The total unilateral macular CA-reactivity and the bilateral difference in CA between the left and the right maculae were significantly lower in 3 g animals than in 1g controls. The result is in complete agreement with previous studies indicating that a regulatory mechanism, which adjusts otolith size and asymmetry towards the gravity vector, acts via activation/deactivation of macular CA.     

Effects of linear acceleration on fish behavior and eye movements.

Behavioral responses and eye movements of fish during linear acceleration were reviewed. It is known that displacement of otoliths in the inner ear leads to body movements and/or eye movements. On the ground, the utriculus of the vestibular system is stimulated by otolith displacement caused by gravitational and inertial forces during horizontal acceleration of whole body. When the acceleration is imposed on the fish's longitudinal axis, the fish showed nose-down and nose-up posture for tailward and noseward displacement of otolith respectively. These responses were understood that the fish aligned his longitudinal body axis in a plane perpendicular to the direction of resultant force vector acting on the otoliths. When the acceleration was sideward, the fish rolled around his longitudinal body axis so that his back was tilted against the direction in which the inertial force acted on the otoliths. Linear acceleration applied to fish's longitudinal body axis evoked torsional eye movement. Direction of torsion coincided with the direction of acceleration, which compensate the change of resultant force vector produced by linear acceleration and gravity. Torsional movement of left and right eye coordinated with each other. In normal fish, both sinusoidal and rectangular acceleration of 0.1G could evoke clear eye torsion. Though the amplitude of response increased with increasing magnitude of acceleration up to 0.5 G, the torsion angle did not fully compensate the angle calculated from gravity and linear acceleration. Removal of the otolith on one side reduced the response amplitude of both eyes. The torsion angle evoked by rectangular acceleration was smaller than that evoked by sinusoidal acceleration in both normal and unilaterally labyrinthectomized fish. These results suggest that eye torsion of fish include both static and dynamic components.     

Analysis of the soluble matrix of vaterite otoliths of juvenile herring (Clupea harengus): do crystalline otoliths have less protein?

Otoliths are calcium carbonate concretions laid down in the inner ear of fish and used in fish age estimation. Otoliths precipitate in the form of aragonite but aberrant precipitation may result in vaterite formation instead of aragonite. Vaterite otoliths are more translucent than aragonite. The quantity of HCl-soluble proteins (SP) was measured in the vaterite otoliths and their aragonite pairs of one year old reared herring Clupea harengus to assess the changes induced by the precipitation of vaterite in the amount of soluble proteins in the otolith. Results showed that vaterite otoliths had as much soluble proteins as their aragonite pairs (p>0.05). Due to the lower density of the vaterite, vaterite otoliths were lighter than their aragonite pairs (p<0.05) which explained that protein concentrations were significantly higher (p<0.05) than in aragonite otoliths. These results indicate that the precipitation of vaterite in otoliths did not affect the inclusion of soluble proteins. Furthermore, they suggest that soluble proteins do not affect the translucent or opaque appearance of otoliths. Differences in translucency may instead be caused by the amounts of insoluble proteins or by differences in the physical properties of proteins. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the otolith proteins revealed two bands at 50 and 62 kDa in both aragonite and vaterite otoliths suggesting that the precipitation of vaterite in the otolith is not controlled by either of these two proteins present in the otolith.     

Development of utricular otoliths, but not saccular otoliths, is necessary for vestibular function and survival in zebrafish

We have been studying the consequences of embryonic vestibular dysfunction caused by the monolith (mnl) mutation in zebrafish. mnl is a dominant mutation that specifically inhibits formation of utricular otoliths. However, briefly immobilizing mnl/mnl embryos in agarose with the otic vesicle orientated at certain angles selectively induces or prevents formation of utricular and/or saccular otoliths. With this noninvasive technique, we generated six phenotypic classes of mnl/mnl mutants, designated S-S, U-U, U-S, S-US, U-US, and US-US, depending on which otoliths are present on each side (U, utricular otolith; S, saccular otolith). All mnl/mnl larvae survived through day 10 of development. Thereafter, S-S larvae showed a rapid decline, probably because of starvation, and none survived to adulthood. Survival rates in all other classes of mnl/mnl larvae (those having at least one utricular otolith) were close to normal. The presence or absence of utricular otoliths also correlated with vestibular function during early larval development, as measured by three criteria: First, unlike wild-type larvae, S-S mutant larvae showed almost no detectable counter-rotation of the eyes when tilted tail up or tail down. Second, 95% of S-S mutant larvae never acquired the ability to maintain a balanced dorsal-up posture. Third, although most wild-type larvae responded to gentle prodding by swimming in a straight line, S-S larvae responded by swimming in rapid circles, showing sudden and frequent changes in direction ("zigzagging"), and/or rolling and spiraling. All other phenotypic classes of mnl/mnl larvae behaved normally in these assays. These data demonstrate that bilateral loss of utricular otoliths disrupts the ability to sense gravity, severely impairs balance and motor coordination, and is invariably lethal. The presence of a utricular otolith in at least one inner ear is necessary and sufficient for vestibular function and survival. In contrast, saccular otoliths are dispensable for these functions.     

Cupulin Is a Zona Pellucida-Like Domain Protein and Major Component of the Cupula from the Inner Ear

The extracellular membranes of the inner ear are essential constituents to maintain sensory functions, the cupula for sensing torsional movements of the head, the otoconial membrane for sensing linear movements and accelerations like gravity, and the tectorial membrane in the cochlea for hearing. So far a number of structural proteins have been described, but for the gelatinous cupula precise data are missing. Here, we describe for the first time a major proteinogenic component of the cupula structure with an apparent molecular mass of 45 kDa from salmon. Analyses of respective peptides revealed highly conserved amino-acid sequences with identity to zona pellucida-like domain proteins. Immunohistochemistry studies localized the protein in the ampulla of the inner ear from salmon and according to its anatomical appearance we identified this glycoprotein as Cupulin. Future research on structure and function of zona pellucida-like domain proteins will enhance our knowledge of inner ear diseases, like sudden loss of vestibular function and other disturbances.     

Exposure to thyroid hormone in ovo affects otolith crystallization in rainbow trout Oncorhynchus mykiss

Calcareous otoliths in the inner ears of fishes are necessary for proper hearing and vestibular function. Sagittal otoliths are usually composed of the calcium carbonate polymorph aragonite but may contain the polymorph vaterite, a phenomenon called otolith crystallization. The causes of otolith crystallization are poorly understood. Thyroid hormone (TH) can influence the chemical microenvironment and structure of the inner ear, suggesting that TH may influence otolith crystallization. The present study examined the effect of exogenous TH treatment on sagittal otolith crystallization and growth in larval and juvenile rainbow trout, Oncorhynchus mykiss. In the first experiment, 110?C179?day-old fish raised from TH-treated oocytes had significantly fewer sagittal otoliths containing the crystalline calcium carbonate polymorph vaterite as compared to untreated fish. Vaterite-containing otoliths were significantly longer than those containing the typical polymorph aragonite, although there was no effect of TH treatment on otolith length. In the second experiment, juveniles immersed in an exogenous solution of TH for 6?weeks had slightly longer otoliths (relative to fish length) than age-matched controls, but this effect was not significant. This juvenile population had a very high percentage (88.3?%) of vaterite sagittae overall and this percentage did not change significantly with treatment, suggesting the switch from aragonite to vaterite occurred prior to inclusion of the fish in the study. These results suggest that early manipulation of TH levels may affect calcium carbonate deposition on the otolith but that later TH exposure is unable to restore typical otolith composition.     

Identification of a novel matrix protein contained in a protein aggregate associated with collagen in fish otoliths

In the biomineralization processes, proteins are thought to control the polymorphism and morphology of the crystals by forming complexes of structural and mineral-associated proteins. To identify such proteins, we have searched for proteins that may form high-molecular-weight (HMW) aggregates in the matrix of fish otoliths that have aragonite and vaterite as their crystal polymorphs. By screening a cDNA library of the trout inner ear using an antiserum raised against whole otolith matrix, a novel protein, named otolith matrix macromolecule-64 (OMM-64), was identified. The protein was found to have a molecular mass of 64 kDa, and to contain two tandem repeats and a Glu-rich region. The structure of the protein and that of its DNA are similar to those of starmaker, a protein involved in the polymorphism control in the zebrafish otoliths [S?llner C, Burghammer M, Busch-Nentwich E, Berger J, Schwarz H, Riekel C & Nicolson T (2003) Science302, 282-286]. (45)Ca overlay analysis revealed that the Glu-rich region has calcium-binding activity. Combined analysis by western blotting and deglycosylation suggested that OMM-64 is present in an HMW aggregate with heparan sulfate chains. Histological observations revealed that OMM-64 is expressed specifically in otolith matrix-producing cells and deposited onto the otolith. Moreover, the HMW aggregate binds to the inner ear-specific short-chain collagen otolin-1, and the resulting complex forms ring-like structures in the otolith matrix. Overall, OMM-64, by forming a calcium-binding aggregate that binds to otolin-1 and forming matrix protein architectures, may be involved in the control of crystal morphology during otolith biomineralization.     

Enigmatic ear stones: what we know about the functional role and evolution of fish otoliths

Otoliths in bony fishes play an important role in the senses of balance and hearing. Otolith mass and shape are, among others, likely to be decisive factors influencing otolith motion and thus ear functioning. Yet our knowledge of how exactly these factors influence otolith motion is incomplete. In addition, experimental studies directly investigating the function of otoliths in the inner ear are scarce and yield partly conflicting results. Herein, we discuss questions and hypotheses on how otolith mass and shape, and the relationship between the sensory epithelium and overlying otolith, influence otolith motion. We discuss (i) the state‐of‐the‐art knowledge regarding otolith function, (ii) gaps in knowledge that remain to be filled, and (iii) future approaches that may improve our understanding of the role of otoliths in ear functioning. We further link these functional questions to the evolution of solid teleost otoliths instead of numerous tiny otoconia as found in most other vertebrates. Until now, the selective forces and/or constraints driving the evolution of solid calcareous otoliths and their diversity in shape in teleosts are largely unknown. Based on a data set on the structure of otoliths and otoconia in more than 160 species covering the main vertebrate groups, we present a hypothetical framework for teleost otolith evolution. We suggest that the advent of solid otoliths may have initially been a selectively neutral ‘by‐product’ of other key innovations during teleost evolution. The teleost‐specific genome duplication event may have paved the way for diversification in otolith shape. Otolith shapes may have evolved along with the considerable diversity of, and improvements in, auditory abilities in teleost fishes. However, phenotypic plasticity may also play an important role in the creation of different otolith types, and different portions of the otolith may show different degrees of phenotypic plasticity. Future studies should thus adopt a phylogenetic perspective and apply comparative and methodologically integrative approaches, including fossil otoliths, when investigating otoconia/otolith evolution and their function in the inner ear.