Gene targeting reveals the role of Oc90 as the essential organizer of the otoconial organic matrix

A critical part of the functional development of our peripheral balance system is the embryonic formation of otoconia, composite crystals that overlie and provide optimal stimulus input to the sensory epithelium of the gravity receptor in the inner ear. To date neither the functions of otoconial proteins nor the processes of crystal formation are clearly defined. Using gene targeting and protein analysis strategies, we demonstrate that the predominant mammalian otoconin, otoconin-90/95 (Oc90), is essential for formation of the organic matrix of otoconia by specifically recruiting other matrix components, which includes otolin, a novel mammalian otoconin that we identified to be in wildtype murine otoconia. We show that this matrix controls otoconia growth and morphology by embedding the crystallites during seeding and growth. During otoconia development, the organic matrix forms prior to CaCO3 deposition and provides optimal calcification efficiency. Histological and ultrastructural examinations show normal inner ear epithelial morphology but reduced acellular matrices, including otoconial, cupular and tectorial membranes, in Oc90 null mice, likely due to an absence of Oc90 and a profound reduction of otolin. Our data demonstrate the critical roles of otoconins in otoconia seeding, growth and anchoring and suggest mechanistic similarities and differences between otoconia and bone calcification.     

A mouse model for benign paroxysmal positional vertigo with genetic predisposition for displaced otoconia

Abnormal formation of otoconia, the biominerals of the inner ear, results in balance disorders. The inertial mass of otoconia activates the underlying mechanosensory hair cells in response to change in head position primarily during linear and rotational acceleration. Otoconia associate exclusively with the two gravity receptors, the utricle and saccule. The cristae sensory epithelium is associated with an extracellular gelatinous matrix known as cupula, equivalent to otoconia. During head rotation, the inertia of endolymphatic fluids within the semicircular canals deflects the cupula of the corresponding crista and activates the underlying mechanosensory hair cells. It is believed that detached free‐floating otoconia particles travel ectopically to the semicircular canal and cristae and are the culprit for benign paroxysmal positional vertigo (BPPV). The Slc26a4 mouse mutant harbors a missense mutation in pendrin. This mutation leads to impaired transport activity of pendrin and to defects in otoconia composition and distribution. All Slc26a4 ^loop/loop homozygous mutant mice are profoundly deaf but show inconsistent vestibular deficiency. A panel of behavioral tests was utilized in order to generate a scoring method for vestibular function. A pathological finding of displaced otoconia was identified consistently in the inner ears of mutant mice with severe vestibular dysfunction. In this work, we present a mouse model with a genetic predisposition for ectopic otoconia with a clinical correlation to BPPV. This unique mouse model can serve as a platform for further investigation of BPPV pathophysiology, and for developing novel treatment approaches in a live animal model.     

Each otoconia polymorph has a protein unique to that polymorph

1. Otoconia, contained within the vestibular portion of the inner ear, are mineralized by one of three polymorphs of calcium carbonate. Each otoconial polymorph contains a unique, major protein. 2. The major protein of calcitic otoconia of members of different vertebrate classes, Amphibia (African clawed frog) and Mammalia (rat), have similar molecular weights. 3. The major protein of calcitic rat otoconia and of vateritic otoconia of the gar may be calcium binding proteins. No protein from the other polymorph, aragonite, appear to have this characteristic.     

In Vitro Calcite Crystal Morphology Is Modulated by Otoconial Proteins Otolin-1 and Otoconin-90

Otoconia are formed embryonically and are instrumental in detecting linear acceleration and gravity. Degeneration and fragmentation of otoconia in elderly patients leads to imbalance resulting in higher frequency of falls that are positively correlated with the incidence of bone fractures and death. In this work we investigate the roles otoconial proteins Otolin-1 and Otoconin 90 (OC90) perform in the formation of otoconia. We demonstrate by rotary shadowing and atomic force microscopy (AFM) experiments that Otolin-1 forms homomeric protein complexes and self-assembled networks supporting the hypothesis that Otolin-1 serves as a scaffold protein of otoconia. Our calcium carbonate crystal growth data demonstrate that Otolin-1 and OC90 modulate in vitro calcite crystal morphology but neither protein is sufficient to produce the shape of otoconia. Coadministration of these proteins produces synergistic effects on crystal morphology that contribute to morphology resembling otoconia.     

Physical mapping of the mouse tilted locus identifies an association between human deafness loci DFNA6/14 and vestibular system development

The tilted (tlt) mouse carries a recessive mutation causing vestibular dysfunction. The defect in tlt homozygous mice is limited to the utricle and saccule of the inner ear, which completely lack otoconia. Genetic mapping of tlt placed it in a region orthologous with human 4p16.3-p15 that contains two loci, DFNA6 and DFNA14, responsible for autosomal dominant, nonsyndromic hereditary hearing impairment. To identify a possible relationship between tlt in mice and DFNA6 and DFNA14 in humans, we have refined the mouse genetic map, assembled a BAC contig spanning the tlt locus, and developed a comprehensive comparative map between mouse and human. We have determined the position of tlt relative to 17 mouse chromosome 5 genes with orthologous loci in the human 4p16.3-p15 region. This analysis identified an inversion between the mouse and human genomes that places tlt and DFNA6/14 in close proximity.     

Aminoglycoside-Induced Hair Cell Death of Inner Ear Organs Causes Functional Deficits in Adult Zebrafish (Danio rerio)

Aminoglycoside antibiotics, like gentamicin, kill inner ear sensory hair cells in a variety of species including chickens, mice, and humans. The zebrafish (Danio rerio) has been used to study hair cell cytotoxicity in the lateral line organs of larval and adult animals. Little is known about whether aminoglycosides kill the hair cells within the inner ear of adult zebrafish. We report here the ototoxic effects of gentamicin on hair cells in the saccule, the putative hearing organ, and utricle of zebrafish. First, adult zebrafish received a single 30 mg/kg intraperitoneal injection of fluorescently-tagged gentamicin (GTTR) to determine the distribution of gentamicin within inner ear sensory epithelia. After 4 hours, GTTR was observed in hair cells throughout the saccular and utriclar sensory epithelia. To assess the ototoxic effects of gentamicin, adult zebrafish received a single 250 mg/kg intraperitoneal injection of gentamicin and, 24 hours later, auditory evoked potential recordings (AEPs) revealed significant shifts in auditory thresholds compared to untreated controls. Zebrafish were then euthanized, the inner ear fixed, and labeled for apoptotic cells (TUNEL reaction), and the stereociliary bundles of hair cells labeled with fluorescently-tagged phalloidin. Whole mounts of the saccule and utricle were imaged and cells counted. There were significantly more TUNEL-labeled cells found in both organs 4 hours after gentamicin injection compared to vehicle-injected controls. As expected, significantly fewer hair cell bundles were found along the rostral-caudal axis of the saccule and in the extrastriolar and striolar regions of the utricle in gentamicin-treated animals compared to untreated controls. Therefore, as in other species, gentamicin causes significant inner ear sensory hair cell death and auditory dysfunction in zebrafish.     

The otoconia of the guinea pig utricle: internal structure, surface exposure, and interactions with the filament matrix

A unique feature of the vertebrate gravity receptor organs, the saccule and utricle, is the mass of biomineral structures, the otoconia, overlying a gelatinous matrix also called "otoconial membrane" on the surface of the sensory epithelium. In mammals, otoconia are deposits of calcium carbonate in the form of composite calcite crystals. We used quick-freezing, deep etching to examine the otoconial mass of the guinea pig utricle. The deep-etching step exposed large expanses of intact and fractured otoconia, showing the fine structure and relationship between their internal crystal structure, their surface components, and the filament matrix in which they are embedded. Each otoconium has a compact central core meshwork of filaments and a composite outer shell of ordered crystallites and macromolecular aggregates. A distinct network of 20-nm beaded filaments covers the surface of the otoconia. The otoconia are interconnected and secured to the gelatinous matrix by surface adhesion and by confinement within a loose interotoconial filament matrix. The gelatinous matrix is a dense network made of yet another type of filament, 22 nm in diameter, which are cross-linked by shorter filaments, characteristically 11 nm in diameter. Our freeze-etching data provide a structural framework for considering the molecular nature of the components of the otoconial complex, their mechanical properties, and the degree of biological versus chemical control of otoconia biosynthesis.     

Otx1 and Otx2 activities are required for the normal development of the mouse inner ear

The Otx1 and Otx2 genes are two murine orthologues of the Orthodenticle (Otd) gene in Drosophila. In the developing mouse embryo, both Otx genes are expressed in the rostral head region and in certain sense organs such as the inner ear. Previous studies have shown that mice lacking Otx1 display abnormal patterning of the brain, whereas embryos lacking Otx2 develop without heads. In this study, we examined, at different developmental stages, the inner ears of mice lacking both Otx1 and Otx2 genes. In wild-type inner ears, Otx1, but not Otx2, was expressed in the lateral canal and ampulla, as well as part of the utricle. Ventral to the mid-level of the presumptive utricle, Otx1 and Otx2 were co-expressed, in regions such as the saccule and cochlea. Paint-filled membranous labyrinths of Otx1-/- mutants showed an absence of the lateral semicircular canal, lateral ampulla, utriculosaccular duct and cochleosaccular duct, and a poorly defined hook (the proximal part) of the cochlea. Defects in the shape of the saccule and cochlea were variable in Otx1-/- mice and were much more severe in an Otx1-/-;Otx2(+/)- background. Histological and in situ hybridization experiments of both Otx1-/- and Otx1-/-;Otx2(+/)- mutants revealed that the lateral crista was absent. In addition, the maculae of the utricle and saccule were partially fused. In mutant mice in which both copies of the Otx1 gene were replaced with a human Otx2 cDNA (hOtx2(1)/ hOtx2(1)), most of the defects associated with Otx1-/- mutants were rescued. However, within the inner ear, hOtx2 expression failed to rescue the lateral canal and ampulla phenotypes, and only variable rescues were observed in regions where both Otx1 and Otx2 are normally expressed. These results suggest that both Otx genes play important and differing roles in the morphogenesis of the mouse inner ear and the development of its sensory organs.     

Inactivation of NADPH oxidase organizer 1 results in severe imbalance

Otoconia are biominerals of the vestibular system that are indispensable for the perception of gravity. Despite their importance, the process of otoconia genesis is largely unknown. Reactive oxygen species (ROS) have been recognized for their toxic effects in antimicrobial host defense as well as in aging and carcinogenesis. Enzymes evolved for ROS production belong to the recently discovered NADPH oxidase (Nox) enzyme family . Here we show that the inactivation of a regulatory subunit, NADPH oxidase organizer 1 (Noxo1), resulted in the severe balance deficit seen in the spontaneous mutant "head slant" (hslt) mice whose phenotype was rescued by Noxo1 transgenes. Wild-type Noxo1 was expressed in the vestibular and cochlear epithelia and was required for ROS production by an oxidase complex. In contrast, the hslt mutation of Noxo1 was biochemically inactive and led to an arrest of otoconia genesis, characterized by a complete lack of calcium carbonate mineralization and an accumulation of otoconial protein, otoconin-90/95 (OC-90/95). These results suggest that ROS generated by a Noxo1-dependent vestibular oxidase are critical for otoconia formation and may be required for interactions among otoconial components. Noxo1 mutants implicate a constructive developmental role for ROS, in contrast to their previously described toxic effects.     

Proliferation of vertebrate inner ear supporting cells

Using two S phase markers, we determined the cell‐cycle behavior of inner ear supporting cells from two species, the chicken and the oscar. The results indicate that chicken utricular supporting cells divide once and do not return to the cell cycle for at least 7 days. In contrast, supporting cell progeny in the oscar saccule return to S phase after 5 days. While both the chicken utricle and oscar saccule show ongoing supporting cell proliferation, these data indicate that there may be a dedicated recycling population of supporting cells in the oscar saccule but not in the chicken utricle that is responsible for hair cell production. An expulsion of proliferative cell progeny in the chicken utricle after 7 days may be a driving force for proliferation, as well as an explanation for why hair cell numbers do not increase in the chicken utricle with age. This was not seen in the oscar saccule, possibly explaining how this end organ increases in size throughout the adult life of the animal. The absence of S phase cell expulsion, however, does not rule out the role of cell death in the oscar saccule. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 527–535, 1999     

Proliferation of vertebrate inner ear supporting cells.

Using two S phase markers, we determined the cell-cycle behavior of inner ear supporting cells from two species, the chicken and the oscar. The results indicate that chicken utricular supporting cells divide once and do not return to the cell cycle for at least 7 days. In contrast, supporting cell progeny in the oscar saccule return to S phase after 5 days. While both the chicken utricle and oscar saccule show ongoing supporting cell proliferation, these data indicate that there may be a dedicated recycling population of supporting cells in the oscar saccule but not in the chicken utricle that is responsible for hair cell production. An expulsion of proliferative cell progeny in the chicken utricle after 7 days may be a driving force for proliferation, as well as an explanation for why hair cell numbers do not increase in the chicken utricle with age. This was not seen in the oscar saccule, possibly explaining how this end organ increases in size throughout the adult life of the animal. The absence of S phase cell expulsion, however, does not rule out the role of cell death in the oscar saccule.     

Otopetrin 1 is required for otolith formation in the zebrafish Danio rerio

Orientation with respect to gravity is essential for the survival of complex organisms. The gravity receptor is one of the phylogenetically oldest sensory systems, and special adaptations that enhance sensitivity to gravity are highly conserved. The fish inner ear contains three large extracellular biomineral particles, otoliths, which have evolved to transduce the force of gravity into neuronal signals. Mammalian ears contain thousands of small particles called otoconia that serve a similar function. Loss or displacement of these structures can be lethal for fish and is responsible for benign paroxysmal positional vertigo (BPPV) in humans. The distinct morphologies of otoconial particles and otoliths suggest divergent developmental mechanisms. Mutations in a novel gene Otopetrin 1 (Otop1), encoding multi-transmembrane domain protein, result in nonsyndromic otoconial agenesis and a severe balance disorder in mice. Here we show that the zebrafish, Danio rerio, contains a highly conserved gene, otop1, that is essential for otolith formation. Morpholino-mediated knockdown of zebrafish Otop1 leads to otolith agenesis without affecting the sensory epithelium or other structures within the inner ear. Despite lack of otoliths in early development, otolith formation partially recovers in some fish after 2 days. However, the otoliths are malformed, misplaced, lack an organic matrix, and often consist of inorganic calcite crystals. These studies demonstrate that Otop1 has an essential and conserved role in the timing of formation and the size and shape of the developing otolith.     

Dlx5 and Dlx6 homeobox genes are required for specification of the mammalian vestibular apparatus

The mammalian inner ear is a complex organ that develops from a surface ectoderm into distinct auditory and vestibular components. Congenital malformation of these two components resulting from single or multiple gene defects is a common clinical occurrence and is observed in patients with split hand/split foot malformation, a malformation which is phenocopied by Dlx5/6 null mice. Analysis of mice lacking Dlx5 and Dlx6 homeobox genes identified their restricted and combined expression in the otic epithelium as a crucial regulator of vestibular cell fates. Otic induction initiates without incident in Dlx5/6(-/-) embryos, but dorsal otic derivatives including the semicircular ducts, utricle, saccule, and endolymphatic duct fail to form. Dlx5 and Dlx6 seem to influence vestibular cell fates by restricting Pax2 and activating Gbx2 and Bmp4 expression domains. Given their proximity to the disease locus and the observed phenotype in Dlx5/6 null mice, Dlx5/6 are likely candidates to mediate the inner ear defects observed in patients with split hand/split foot malformation.