Zona Pellucida Domain-Containing Protein β-Tectorin is Crucial for Zebrafish Proper Inner Ear Development

BACKGROUND: The zona pellucida (ZP) domain is part of many extracellular proteins with diverse functions from structural components to receptors. The mammalian β-tectorin is a protein of 336 amino acid residues containing a single ZP domain and a putative signal peptide at the N-terminus of the protein. It is 1 component of a gel-like structure called the tectorial membrane which is involved in transforming sound waves into neuronal signals and is important for normal auditory function. β-Tectorin is specifically expressed in the mammalian and avian inner ear. METHODOLOGY/PRINCIPAL FINDINGS: We identified and cloned the gene encoding zebrafish β-tectorin. Through whole-mount in situ hybridization, we demonstrated that β-tectorin messenger RNA was expressed in the otic placode and specialized sensory patch of the inner ear during zebrafish embryonic stages. Morpholino knockdown of zebrafish β-tectorin affected the position and number of otoliths in the ears of morphants. Finally, swimming behaviors of β-tectorin morphants were abnormal since the development of the inner ear was compromised. CONCLUSIONS/SIGNIFICANCE: Our results reveal that zebrafish β-tectorin is specifically expressed in the zebrafish inner ear, and is important for regulating the development of the zebrafish inner ear. Lack of zebrafish β-tectorin caused severe defects in inner ear formation of otoliths and function

Simulation of the Response of the Inner Hair Cell Stereocilia Bundle to an Acoustical Stimulus

Mammalian hearing relies on a cochlear hydrodynamic sensor embodied in the inner hair cell stereocilia bundle. It is presumed that acoustical stimuli induce a fluid shear-driven motion between the tectorial membrane and the reticular lamina to deflect the bundle. It is hypothesized that ion channels are opened by molecular gates that sense tension in tip-links, which connect adjacent stepped rows of stereocilia. Yet almost nothing is known about how the fluid and bundle interact. Here we show using our microfluidics model how each row of stereocilia and their associated tip links and gates move in response to an acoustical input that induces an orbital motion of the reticular lamina. The model confirms the crucial role of the positioning of the tectorial membrane in hearing, and explains how this membrane amplifies and synchronizes the timing of peak tension in the tip links. Both stereocilia rotation and length change are needed for synchronization of peak tip link tension. Stereocilia length change occurs in response to accelerations perpendicular to the oscillatory fluid shear flow. Simulations indicate that nanovortices form between rows to facilitate diffusion of ions into channels, showing how nature has devised a way to solve the diffusive mixing problem that persists in engineered microfluidic devices

Mammalian Otolin: A Multimeric Glycoprotein Specific to the Inner Ear that Interacts with Otoconial Matrix Protein Otoconin-90 and Cerebellin-1

The mammalian otoconial membrane is a dense extracellular matrix containing bio-mineralized otoconia. This structure provides the mechanical stimulus necessary for hair cells of the vestibular maculae to respond to linear accelerations and gravity. In teleosts, Otolin is required for the proper anchoring of otolith crystals to the sensory maculae. Otoconia detachment and subsequent entrapment in the semicircular canals can result in benign paroxysmal positional vertigo (BPPV), a common form of vertigo for which the molecular basis is unknown. Several cDNAs encoding protein components of the mammalian otoconia and otoconial membrane have recently been identified, and mutations in these genes result in abnormal otoconia formation and balance deficits.Here we describe the cloning and characterization of mammalian Otolin, a protein constituent of otoconia and the otoconial membrane. Otolin is a secreted glycoprotein of ∼70 kDa, with a C-terminal globular domain that is homologous to the immune complement C1q, and contains extensive posttranslational modifications including hydroxylated prolines and glycosylated lysines. Like all C1q/TNF family members, Otolin multimerizes into higher order oligomeric complexes. The expression of otolin mRNA is restricted to the inner ear, and immunohistochemical analysis identified Otolin protein in support cells of the vestibular maculae and semi-circular canal cristae. Additionally, Otolin forms protein complexes with Cerebellin-1 and Otoconin-90, two protein constituents of the otoconia, when expressed in vitro. Otolin was also found in subsets of support cells and non-sensory cells of the cochlea, suggesting that Otolin is also a component of the tectorial membrane.Given the importance of Otolin in lower organisms, the molecular cloning and biochemical characterization of the mammalian Otolin protein may lead to a better understanding of otoconial development and vestibular dysfunction

Cell Type–Specific Transcriptome Analysis Reveals a Major Role for Zeb1 and miR-200b in Mouse Inner Ear Morphogenesis

Cellular heterogeneity hinders the extraction of functionally significant results and inference of regulatory networks from wide-scale expression profiles of complex mammalian organs. The mammalian inner ear consists of the auditory and vestibular systems that are each composed of hair cells, supporting cells, neurons, mesenchymal cells, other epithelial cells, and blood vessels. We developed a novel protocol to sort auditory and vestibular tissues of newborn mouse inner ears into their major cellular components. Transcriptome profiling of the sorted cells identified cell type–specific expression clusters. Computational analysis detected transcription factors and microRNAs that play key roles in determining cell identity in the inner ear. Specifically, our analysis revealed the role of the Zeb1/miR-200b pathway in establishing epithelial and mesenchymal identity in the inner ear. Furthermore, we detected a misregulation of the ZEB1 pathway in the inner ear of Twirler mice, which manifest, among other phenotypes, malformations of the auditory and vestibular labyrinth. The association of misregulation of the ZEB1/miR-200b pathway with auditory and vestibular defects in the Twirler mutant mice uncovers a novel mechanism underlying deafness and balance disorders. Our approach can be employed to decipher additional complex regulatory networks underlying other hearing and balance mouse mutants

Audiological Evaluation of Affected Members from a Dutch DFNA8/12 (TECTA) Family

In DFNA8/12, an autosomal dominantly inherited type of nonsyndromic hearing impairment, the TECTA gene mutation causes a defect in the structure of the tectorial membrane in the inner ear. Because DFNA8/12 affects the tectorial membrane, patients with DFNA8/12 may show specific audiometric characteristics. In this study, five selected members of a Dutch DFNA8/12 family with a TECTA sensorineural hearing impairment were evaluated with pure-tone audiometry, loudness scaling, speech perception in quiet and noise, difference limen for frequency, acoustic reflexes, otoacoustic emissions, and gap detection. Four out of five subjects showed an elevation of pure-tone thresholds, acoustic reflex thresholds, and loudness discomfort levels. Loudness growth curves are parallel to those found in normal-hearing individuals. Suprathreshold measures such as difference limen for frequency modulated pure tones, gap detection, and particularly speech perception in noise are within the normal range. Distortion otoacoustic emissions are present at the higher stimulus level. These results are similar to those previously obtained from a Dutch DFNA13 family with midfrequency sensorineural hearing impairment. It seems that a defect in the tectorial membrane results primarily in an attenuation of sound, whereas suprathreshold measures, such as otoacoustic emissions and speech perception in noise, are preserved rather well. The main effect of the defects is a shift in the operation point of the outer hair cells with near intact functioning at high levels. As most test results reflect those found in middle-ear conductive loss in both families, the sensorineural hearing impairment may be characterized as a cochlear conductive hearing impairment

Characterization of a spontaneous, recessive, missense mutation arising in the Tecta gene

The TECTA gene encodes alpha-tectorin ( TECTA), a major noncollagenous component of the tectorial membrane (TM). In humans, mutations in TECTA lead to either dominant (DFNA8/A12) or recessive (DFNB21) forms of nonsyndromic hearing loss. All missense mutations in TECTA that have been reported thus far are associated with the dominant subtype, whereas those leading to recessive deafness are all inactivating mutations. In this paper, we characterize a spontaneous missense mutation (c.1046C9 > A, p.A349D) arising in the mouse Tecta gene that is, unlike all previously reported missense mutations in TECTA, recessive. The morphological phenotype of the Tecta(A349D/A349D) mouse resembles but is not identical to that previously described for the Tecta(Delta ENT/Delta ENT) mouse. As in the Tecta(Delta ENT/Delta ENT) mouse, the TM is completely detached from the surface of the organ of Corti and spiral limbus, lacks a striated-sheet matrix, and is deficient in both beta-tectorin (Tectb) and otogelin. A significant amount of Tecta is, however, detected in the TM of the Tecta(A349D/A349D) mouse, and numerous, electron-dense matrix granules are seen interspersed among the disorganized collagen fibrils. Mutated Tecta(A349D) is therefore incorporated into the TM but presumably unable to interact with either Tectb or otogelin. The Tecta(A349D/A349D) mouse reveals that missense mutations in Tecta can be recessive and lead to TM detachment and suggests that should similar mutations arise in the human population, they would likely cause deafness

A Novel TECTA Mutation in a Dutch DFNA8/12 Family Confirms Genotype–Phenotype Correlation

A novel TECTA mutation, p.R1890C, was found in a Dutch family with nonsyndromic autosomal dominant sensorineural hearing impairment. In early life, presumably congenital, hearing impairment occurred in the midfrequency range, amounting to about 40 dB at 1 kHz. Speech recognition was good with all phoneme recognition scores exceeding 90%. An intact horizontal vestibuloocular reflex was found in four tested patients. The missense mutation is located in the zona pellucida (ZP) domain of α-tectorin. Mutations affecting the ZP domain of α-tectorin are significantly associated with midfrequency hearing impairment. Substitutions affecting other amino acid residues than cysteines show a significant association with hearing impairment without progression. Indeed, in the present family progression seemed to be absent. In addition, the presently identified mutation affecting the ZP domain resulted in a substantially lesser degree of hearing impairment than was previously reported for DFNA8/12 traits with mutations affecting the ZP domain of α-tectorin

Mid-frequency DFNA8/12 hearing loss caused by a synonymous TECTA mutation that affects an exonic splice enhancer.

Contains fulltext : 69348.pdf (publisher's version ) (Closed access)Autosomal dominant hearing loss is highly heterogeneous. Hearing impairment mainly involves the mid-frequencies (500-2000 Hz) in only a low percentage of the cases. In a Dutch family with autosomal dominant mid-frequency/flat hearing loss, genome-wide SNP analysis combined with fine mapping using microsatellite markers mapped the defect to the DFNA8/12 locus, with a maximum two-point LOD score of 3.52. All exons and intron-exon boundaries of the TECTA gene, of which mutations are causative for DFNA8/12, were sequenced. Only one heterozygous synonymous change in exon 16 (c.5331G>A; p.L1777L) was found to segregate with the hearing loss. This change was predicted to cause the loss of an exonic splice enhancer (ESE). RT-PCR using primers flanking exon 16 revealed, besides the expected PCR product from the wild-type allele, a smaller fragment only in the affected individual, representing part of an aberrant TECTA transcript lacking exon 16. The aberrant splicing is predicted to result in a deletion of 37 amino acids (p.S1758Y/G1759_N1795del) in alpha-tectorin. Subsequently, the same mutation was detected in two out of 36 individuals with a comparable phenotype. Owing to the position of the protein deletion just N-terminal of the zona pellucida (ZP) domain of alpha-tectorin, it is likely that the deletion of 37 amino acids may affect the proteolytic processing, structure and/or function of this domain, which results in a clinical phenotype comparable to that of missense mutations in the ZP domain. In addition, this is the first report of a synonymous mutation that affects an ESE and causes hereditary hearing loss