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.     

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.     

A compendium of mouse knockouts with inner ear defects

Genetically engineered strains of mice, modified by gene targeting (knockouts), are increasingly being employed as alternative effective research tools in elucidating the genetic basis of human deafness. An impressive array of auditory and vestibular mouse knockouts is already available as a valuable resource for studying the ontogenesis, morphogenesis and function of the mammalian inner ear. This article provides a current catalog of mouse knockouts with inner ear morphogenetic malformations and hearing or balance deficits resulting from ablation of genes that are regionally expressed in the inner ear and/or within surrounding tissues, such as the hindbrain, neural crest and mesenchyme.     

The Candidate Splicing Factor Sfswap Regulates Growth and Patterning of Inner Ear Sensory Organs

The Notch signaling pathway is thought to regulate multiple stages of inner ear development. Mutations in the Notch signaling pathway cause disruptions in the number and arrangement of hair cells and supporting cells in sensory regions of the ear. In this study we identify an insertional mutation in the mouse Sfswap gene, a putative splicing factor, that results in mice with vestibular and cochlear defects that are consistent with disrupted Notch signaling. Homozygous Sfswap mutants display hyperactivity and circling behavior consistent with vestibular defects, and significantly impaired hearing. The cochlea of newborn Sfswap mutant mice shows a significant reduction in outer hair cells and supporting cells and ectopic inner hair cells. This phenotype most closely resembles that seen in hypomorphic alleles of the Notch ligand Jagged1 (Jag1). We show that Jag1; Sfswap compound mutants have inner ear defects that are more severe than expected from simple additive effects of the single mutants, indicating a genetic interaction between Sfswap and Jag1. In addition, expression of genes involved in Notch signaling in the inner ear are reduced in Sfswap mutants. There is increased interest in how splicing affects inner ear development and function. Our work is one of the first studies to suggest that a putative splicing factor has specific effects on Notch signaling pathway members and inner ear development.     

Establishment of a knock-in mouse model with the SLC26A4 c.919-2A>G mutation and characterization of its pathology

Recessive mutations in the SLC26A4 gene are a common cause of hereditary hearing impairment worldwide. Previous studies have demonstrated that different SLC26A4 mutations may have different pathogenetic mechanisms. In the present study, we established a knock-in mouse model (i.e., Slc26a4(tm1Dontuh/tm1Dontuh) mice) homozygous for the c.919-2A>G mutation, which is a common mutation in East Asians. Mice were then subjected to audiologic assessment, a battery of vestibular evaluations, and inner ear morphological studies. All Slc26a4(tm1Dontuh/tm1Dontuh) mice revealed profound hearing loss, whereas 46% mice demonstrated pronounced head tilting and circling behaviors. There was a significant difference in the vestibular performance between wild-type and Slc26a4(tm1Dontuh/tm1Dontuh) mice, especially those exhibiting circling behavior. Inner ear morphological examination of Slc26a4(tm1Dontuh/tm1Dontuh) mice revealed an enlarged endolymphatic duct, vestibular aqueduct and sac, atrophy of stria vascularis, deformity of otoconia in the vestibular organs, consistent degeneration of cochlear hair cells, and variable degeneration of vestibular hair cells. Audiologic and inner ear morphological features of Slc26a4(tm1Dontuh/tm1Dontuh) mice were reminiscent of those observed in humans. These features were also similar to those previously reported in both knock-out Slc26a4(-/-) mice and Slc26a4(loop/loop) mice with the Slc26a4 p.S408F mutation, albeit the severity of vestibular hair cell degeneration appeared different among the three mouse strains.     

Genetic background modifies inner ear and eye phenotypes of jag1 heterozygous mice

Mice heterozygous for missense mutations of the Notch ligand Jagged1 (Jag1) exhibit head-shaking behavior indicative of an inner ear vestibular defect. In contrast, mice heterozygous for a targeted deletion of the Jag1 gene (Jag1del1) do not demonstrate obvious head-shaking behavior. To determine whether the differences in inner ear phenotypes were due to the types of Jag1 mutations or to differences in genetic background, we crossed Jag1del1 heterozygous mice onto the same genetic background as the missense mutants. This analysis revealed that variation of the Jag1 mutant inner ear phenotype is caused by genetic background differences and is not due to the type of Jag1 mutation. Genome scans of N2 backcross mice identified a significant modifier locus on chromosome 7, as well as a suggestive locus on chromosome 14. We also analyzed modifiers of an eye defect in Jag1del1 heterozygous mice from this same cross.     

A Myo7a mutation cosegregates with stereocilia defects and low-frequency hearing impairment

A phenotype-driven approach was adopted in the mouse to identify molecules involved in ear development and function. Mutant mice were obtained using N-ethyl-N-nitrosourea (ENU) mutagenesis and were screened for dominant mutations that affect hearing and/or balance. Heterozygote headbanger (Hdb/+) mutants display classic behavior indicative of vestibular dysfunction including hyperactivity and head bobbing, and they show a Preyer reflex in response to sound but have raised cochlear thresholds especially at low frequencies. Scanning electron microscopy of the surface of the organ of Corti revealed abnormal stereocilia bundle development from an early age that was more severe in the apex than the base. Utricular stereocilia were long, thin, and wispy. Homozygotes showed a similar but more severe phenotype. The headbanger mutation has been mapped to a 1.5-cM region on mouse Chromosome 7 in the region of the unconventional myosin gene Myo7a, and mutation screening revealed an A>T transversion that is predicted to cause an isoleucine-to-phenylalanine amino acid substitution (I178F) in a conserved region in the motor-encoding domain of the gene. Protein analysis revealed reduced levels of myosin VIIa expression in inner ears of headbanger mice. Headbanger represents a novel inner ear phenotype and provides a potential model for low-frequency-type human hearing loss.These authors contributed equally to this study.     

The Spinner-IBMM mouse is a new spontaneous mutant in the Tmie gene

A recessively inherited, spontaneous mutation named Spinner-IBMM (SI) was identified in a transgenic mouse colony in our institute. SI mutant mice displayed hyperactivity, including a severe circling behavior, ataxia and inability to swim. Gene mapping revealed that the causative gene was located on a 35 Mb DNA fragment on chromosome 9. Candidate genes sequencing in this DNA fragment identified a new mutant allele in the Tmie gene. The identified mutant is characterized by a nucleotide deletion in exon 5, leading to a frameshift and a premature STOP codon. It has been reported that inactivating mutations in the mouse Tmie gene result in an identical phenotype, probably resulting from defects in the inner ear. However, the exact function of the Tmie protein in the ear and other organs is still unknown. The analysis of this new mouse mutant could contribute to a better understanding of Tmie functions in vivo in the ear and other organs.     

Tailchaser (Tlc): A new mouse mutation affecting hair bundle differentiation and hair cell survival

We have undertaken a phenotypic approach in the mouse to identifying molecules involved in inner ear function by N-ethyl-N-nitrosourea mutagenesis followed by screening for new dominant mutations affecting hearing or balance. The pathology and genetic mapping of the first of these new mutants, tailchaser (Tlc), is described here. Tlc/+ mutants display classic behavioural symptoms of a vestibular dysfunction, including head-shaking and circling. Behavioural testing of ageing mice revealed a gradual deterioration of both hearing and balance function, indicating that the pathology caused by the Tlc mutation is progressive, similar to many dominant nonsyndromic deafnesses in humans. Based on scanning electron microscopy (SEM) studies, Tlc clearly plays a developmental role in the hair cells of the cochlea since the stereocilia bundles fail to form the characteristic V-shape pattern around the time of birth. By young adult stages, Tlc/+ outer hair bundles are grossly disorganised although inner hair bundles appear relatively normal by SEM. Increased compound action potential thresholds revealed that the Tlc/+ cochlear hair cells were not functioning normally in young adults. Similar to inner hair cells, the hair bundles of the vestibular hair cells also do not appear grossly disordered. However, all types of hair cells in the Tlc/+ inner ear eventually degenerate, apparently regardless of the degree of organisation of their hair bundles. We have mapped the Tlc mutation to a 12 cM region of chromosome 2, between D2Mit164 and D2Mit423. Based on the mode of inheritance and map location, Tlc appears to be a novel mouse mutation affecting both hair cell survival and stereocilia bundle development.     

TRPA1 contributes to cold, mechanical, and chemical nociception but is not essential for hair-cell transduction

TRPA1, a member of the transient receptor potential (TRP) family of ion channels, is expressed by dorsal root ganglion neurons and by cells of the inner ear, where it has proposed roles in sensing sound, painful cold, and irritating chemicals. To test the in vivo roles of TRPA1, we generated a mouse in which the essential exons required for proper function of the Trpa1 gene were deleted. Knockout mice display behavioral deficits in response to mustard oil, to cold ( approximately 0 degrees C), and to punctate mechanical stimuli. These mice have a normal startle reflex to loud noise, a normal sense of balance, a normal auditory brainstem response, and normal transduction currents in vestibular hair cells. TRPA1 is apparently not essential for hair-cell transduction but contributes to the transduction of mechanical, cold, and chemical stimuli in nociceptor sensory neurons.     

Tmprss3, a transmembrane serine protease deficient in human DFNB8/10 deafness, is critical for cochlear hair cell survival at the onset of hearing

Mutations in the type II transmembrane serine protease 3 (TMPRSS3) gene cause non-syndromic autosomal recessive deafness (DFNB8/10), characterized by congenital or childhood onset bilateral profound hearing loss. In order to explore the physiopathology of TMPRSS3 related deafness, we have generated an ethyl-nitrosourea-induced mutant mouse carrying a protein-truncating nonsense mutation in Tmprss3 (Y260X) and characterized the functional and histological consequences of Tmprss3 deficiency. Auditory brainstem response revealed that wild type and heterozygous mice have normal hearing thresholds up to 5 months of age, whereas Tmprss3(Y260X) homozygous mutant mice exhibit severe deafness. Histological examination showed degeneration of the organ of Corti in adult mutant mice. Cochlear hair cell degeneration starts at the onset of hearing, postnatal day 12, in the basal turn and progresses very rapidly toward the apex, reaching completion within 2 days. Given that auditory and vestibular deficits often co-exist, we evaluated the balancing abilities of Tmprss3(Y260X) mice by using rotating rod and vestibular behavioral tests. Tmprss3(Y260X) mice effectively displayed mild vestibular syndrome that correlated histologically with a slow degeneration of saccular hair cells. In situ hybridization in the developing inner ear showed that Tmprss3 mRNA is localized in sensory hair cells in the cochlea and the vestibule. Our results show that Tmprss3 acts as a permissive factor for cochlear hair cells survival and activation at the onset of hearing and is required for saccular hair cell survival. This mouse model will certainly help to decipher the molecular mechanisms underlying DFNB8/10 deafness and cochlear function.     

Hmx homeobox gene function in inner ear and nervous system cell-type specification and development

The Hmx homeobox gene family is comprised of three members in mammals, Hmx1, Hmx2, and Hmx3, which are conserved across the animal kingdom and are part of the larger NKL clustered family of homeobox genes. Expression domains of Hmx genes in distantly related species such as Drosophila and mouse suggest an ancestral function in rostral central nervous system development. During vertebrate evolution, the Hmx genes appear to have been recruited into additional roles in inner ear morphogenesis and specification of vestibular inner ear sensory and supporting cell types. Being derived from a common ancestor, the vertebrate Hmx gene family is thus a strong candidate to investigate functional overlap versus the unique roles played by multiple genes belonging to the same family. The functions of Hmx2 and Hmx3 were investigated via directed gene mutagenesis and the primary regions where Hmx2 and Hmx3 exert their individual functions are consistent with their expression domains, such as the vestibule and uterus. Meanwhile, it is notable that some tissues where both Hmx2 and Hmx3 are extensively expressed were not severely affected in either of the Hmx2 or Hmx3 single mutant mice, suggesting a possible functional overlap existing between these two genes. Compound Hmx2 and Hmx3 double mutant mice showed more severe defects in the inner ear than those displayed by either single knockout. Furthermore, novel abnormalities in the hypothalamic-neuroendocrine system, which were never observed in either of the single mutant mice, confirmed a hypothesis that Hmx2 and Hmx3 also function redundantly to control embryonic development of the central nervous system.     

Requirements for FGF3 and FGF10 during inner ear formation

Members of the fibroblast growth factor (FGF) gene family control formation of the body plan and organogenesis in vertebrates. FGF3 is expressed in the developing hindbrain and has been shown to be involved in inner ear development of different vertebrate species, including zebrafish, Xenopus, chick and mouse. In the mouse, insertion of a neomycin resistance gene into the Fgf3 gene via homologous recombination results in severe developmental defects during differentiation of the otic vesicle. We have addressed the precise roles of FGF3 and other FGF family members during formation of the murine inner ear using both loss- and gain-of-function experiments. We generated a new mutant allele lacking the entire FGF3-coding region but surprisingly found no evidence for severe defects either during inner ear development or in the mature sensory organ, suggesting the functional involvement of other FGF family members during its formation. Ectopic expression of FGF10 in the developing hindbrain of transgenic mice leads to the formation of ectopic vesicles, expressing some otic marker genes and thus indicating a role for FGF10 during otic vesicle formation. Expression analysis of FGF10 during mouse embryogenesis reveals a highly dynamic pattern of expression in the developing hindbrain, partially overlapping with FGF3 expression and coinciding with formation of the inner ear. However, FGF10 mutant mice have been reported to display only mild defects during inner ear differentiation. We thus created double mutant mice for FGF3 and FGF10, which form severely reduced otic vesicles, suggesting redundant roles of these FGFs, acting in combination as neural signals for otic vesicle formation.     

Circling mouse: possible animal model for deafness

Mutant mice with abnormalities are potentially useful as models for studying human defects. Here we report a group of mice with abnormal behavioral patterns. A new spontaneous mutant mouse exhibited hyperactive behavior at about seven days of age, followed by tight circling behavior. Breeding studies suggest that this mutation is caused by a single gene defect inherited in an autosomal recessive manner. Consequently, this mutation is referred to as a circling (cir) mouse mutation with the gene symbol cir. Auditory test results identified clearly the hearing loss of the cir, compared with wild-type mice. Pathologic studies confirmed developmental defects in cochlea and spiral ganglions that were correlated to the abnormal behavior observed in the cir mice. Thus, cir mice may be useful as a model for studying inner ear abnormalities and deafness in humans.     

Lineage-specific evolution of the vertebrate Otopetrin gene family revealed by comparative genomic analyses

Background  

Mutations in the Otopetrin 1 gene (Otop1) in mice and fish produce an unusual bilateral vestibular pathology that involves the absence of otoconia without hearing impairment. The encoded protein, Otop1, is the only functionally characterized member of the Otopetrin Domain Protein (ODP) family; the extended sequence and structural preservation of ODP proteins in metazoans suggest a conserved functional role. Here, we use the tools of sequence- and cytogenetic-based comparative genomics to study the Otop1 and the Otop2-Otop3 genes and to establish their genomic context in 25 vertebrates. We extend our evolutionary study to include the gene mutated in Usher syndrome (USH) subtype 1G (Ush1g), both because of the head-to-tail clustering of Ush1g with Otop2 and because Otop1 and Ush1g mutations result in inner ear phenotypes.     

Ephrin-B2 governs morphogenesis of endolymphatic sac and duct epithelia in the mouse inner ear

Control over ionic composition and volume of the inner ear luminal fluid endolymph is essential for normal hearing and balance. Mice deficient in either the EphB2 receptor tyrosine kinase or the cognate transmembrane ligand ephrin-B2 (Efnb2) exhibit background strain-specific vestibular-behavioral dysfunction and signs of abnormal endolymph homeostasis. Using various loss-of-function mouse models, we found that Efnb2 is required for growth and morphogenesis of the embryonic endolymphatic epithelium, a precursor of the endolymphatic sac (ES) and duct (ED), which mediate endolymph homeostasis. Conditional inactivation of Efnb2 in early-stage embryonic ear tissues disrupted cell proliferation, cell survival, and epithelial folding at the origin of the endolymphatic epithelium. This correlated with apparent absence of an ED, mis-localization of ES ion transport cells relative to inner ear sensory organs, dysplasia of the endolymph fluid space, and abnormally formed otoconia (extracellular calcite-protein composites) at later stages of embryonic development. A comparison of Efnb2 and Notch signaling-deficient mutant phenotypes indicated that these two signaling systems have distinct and non-overlapping roles in ES/ED development. Homozygous deletion of the Efnb2 C-terminus caused abnormalities similar to those found in the conditional Efnb2 null homozygote. Analyses of fetal Efnb2 C-terminus deletion heterozygotes found mis-localized ES ion transport cells only in the genetic background exhibiting vestibular dysfunction. We propose that developmental dysplasias described here are a gene dose-sensitive cause of the vestibular dysfunction observed in EphB–Efnb2 signaling-deficient mice.     

Deletion of Sema3a or plexinA1/plexinA3 Causes Defects in Sensory Afferent Projections of Statoacoustic Ganglion Neurons

Statoacoustic ganglion (SAG) neurons project sensory afferents to appropriate targets in the inner ear to form functional vestibular and auditory circuits. Neuropilin1 (Npn1), a receptor for class 3 semaphorins, is required to generate appropriate afferent projections in SAG neurons; however, the ligands and coreceptors involved in Npn1 functioning remain unknown. Here we show that both plexinA1 and plexinA3 are expressed by SAG neurons, and plexinA1/plexinA3 double mutant mice show defects in afferent projections of SAG neurons in the inner ear. In control mice, sensory afferents of SAG neurons terminate at the vestibular sensory patches, whereas in plexinA1/plexinA3 double mutants, they extend more dorsally in the inner ear beyond normal vestibular target areas. Moreover, we find that semaphorin3a (Sema3a) is expressed in the dorsal otocyst, and Sema3a mutant mice show defects in afferent projections of SAG neurons similar to those observed in plexinA1/plexinA3 double mutants and in mice lacking a functional Npn1 receptor. Taken together, these genetic findings demonstrate that Sema3a repellent signaling plays a role in the establishment of proper afferent projections in SAG neurons, and this signaling likely occurs through a receptor complex involving Npn1 and either plexinA1 or plexinA3.