Defects in the Alternative Splicing-Dependent Regulation of REST Cause Deafness.

The DNA-binding protein REST forms complexes with histone deacetylases (HDACs) to repress neuronal genes in non-neuronal cells. In differentiating neurons, REST is downregulated predominantly by transcriptional silencing. Here we report that post-transcriptional inactivation of REST by alternative splicing is required for hearing in humans and mice. We show that, in the mechanosensory hair cells of the mouse ear, regulated alternative splicing of a frameshift-causing exon into the Rest mRNA is essential for the derepression of many neuronal genes. Heterozygous deletion of this alternative exon of mouse Rest causes hair cell degeneration and deafness, and the HDAC inhibitor SAHA (Vorinostat) rescues the hearing of these mice. In humans, inhibition of the frameshifting splicing event by a novel REST variant is associated with dominantly inherited deafness. Our data reveal the necessity for alternative splicing-dependent regulation of REST in hair cells, and they identify a potential treatment for a group of hereditary deafness cases.

Variants of human CLDN9 cause mild to profound hearing loss.

Hereditary deafness is clinically and genetically heterogeneous. We investigated deafness segregating as a recessive trait in two families. Audiological examinations revealed an asymmetric mild to profound hearing loss with childhood or adolescent onset. Exome sequencing of probands identified a homozygous c.475G>A;p.(Glu159Lys) variant of CLDN9 (NM_020982.4) in one family and a homozygous c.370_372dupATC;p.(Ile124dup) CLDN9 variant in an affected individual of a second family. Claudin 9 (CLDN9) is an integral membrane protein and constituent of epithelial bicellular tight junctions (TJs) that form semipermeable, paracellular barriers between inner ear perilymphatic and endolymphatic compartments. Computational structural modeling predicts that substitution of a lysine for glutamic acid p.(Glu159Lys) alters one of two cis-interactions between CLDN9 protomers. The p.(Ile124dup) variant is predicted to locally misfold CLDN9 and mCherry tagged p.(Ile124dup) CLDN9 is not targeted to the HeLa cell membrane. In situ hybridization shows that mouse Cldn9 expression increases from embryonic to postnatal development and persists in adult inner ears coinciding with prominent CLDN9 immunoreactivity in TJs of epithelia outlining the scala media. Together with the Cldn9 deaf mouse and a homozygous frameshift of CLDN9 previously associated with deafness, the two bi-allelic variants of CLDN9 described here point to CLDN9 as a bona fide human deafness gene.

The phenotypic landscape of a Tbc1d24 mutant mouse includes convulsive seizures resembling human early infantile epileptic encephalopathy.

Epilepsy, deafness, onychodystrophy, osteodystrophy and intellectual disability are associated with a spectrum of mutations of human TBC1D24. The mechanisms underlying TBC1D24-associated disorders and the functions of TBC1D24 are not well understood. Using CRISPR-Cas9 genome editing, we engineered a mouse with a premature translation stop codon equivalent to human S324Tfs*3, a recessive mutation of TBC1D24 associated with early infantile epileptic encephalopathy (EIEE). Homozygous S324Tfs*3 mice have normal auditory and vestibular functions but show an abrupt onset of spontaneous seizures at postnatal day 15 recapitulating human EIEE. The S324Tfs*3 variant is located in an alternatively spliced micro-exon encoding six perfectly conserved amino acids incorporated postnatally into TBC1D24 protein due to a micro-exon utilization switch. During embryonic and early postnatal development, S324Tfs*3 homozygotes produce predominantly the shorter wild-type TBC1D24 protein isoform that omits the micro-exon. S324Tfs*3 homozygotes show an abrupt onset of seizures at P15 that correlates with a developmental switch to utilization of the micro-exon. A mouse deficient for alternative splice factor SRRM3 impairs incorporation of the Tbc1d24 micro-exon. Wild-type Tbc1d24 mRNA is abundantly expressed in the hippocampus using RNAscope in situ hybridization. Immunogold electron microscopy using a TBC1D24-specific antibody revealed that TBC1D24 is associated with clathrin-coated vesicles and synapses of hippocampal neurons, suggesting a crucial role of TBC1D24 in vesicle trafficking important for neuronal signal transmission. This is the first characterization of a mouse model of human TBC1D24-associated EIEE that can now be used to screen for antiepileptogenic drugs ameliorating TBCID24 seizure disorders.

Three-Dimensional Measurements of Pharyngeal Airway in Patients with Unilateral Cleft Lip and Palate.

The aim of this study was to investigate 3-dimensional (3D) airway volume in patients with unilateral cleft lip and palate (UCLP) using computed tomography (CT). The study population comprised 15 UCLP patients (UCLP group) scheduled to receive alveolar bone grafts and 15 with impacted teeth (control group). The clinical requirements for a CT scan were met in both groups. Measurements were recorded from 3D reconstructions of Digital Imaging and Communications in Medicine data obtained from the CT images. Airway volume, cross-sectional area, and linear and angular measurements were recorded. Airway volume and cross-sectional area showed no significant difference between the two groups. The narrowest section of the airway in the UCLP group was tighter than that in the control group, however (p=0.017). The results of this study suggest that this difference in the measurements of the narrowest section of the airway is involved in the particular maxillofacial morphology found in UCLP patients.

Synchronized activation of striatal direct and indirect pathways underlies the behavior in unilateral dopamine-depleted mice.

For more than three decades it has been known, that striatal neurons become hyperactive after the loss of dopamine input, but the involvement of dopamine (DA) D1- or D2-receptor-expressing neurons has only been demonstrated indirectly. By recording neuronal activity using fluorescent calcium indicators in D1 or D2 eGFP-expressing mice, we showed that following dopamine depletion, both types of striatal output neurons are involved in the large increase in neuronal activity generating a characteristic cell assembly of particular neurons that dominate the pattern. When we expressed channelrhodopsin in all the output neurons, light activation in freely moving animals, caused turning like that following dopamine loss. However, if the light stimulation was patterned in pulses the animals circled in the other direction. To explore the neuronal participation during this stimulation we infected normal mice with channelrhodopsin and calcium indicator in striatal output neurons. In slices made from these animals, continuous light stimulation for 15 s induced many cells to be active together and a particular dominant group of neurons, whereas light in patterned pulses activated fewer cells in more variable groups. These results suggest that the simultaneous activity of a large dominant group of striatal output neurons is intimately associated with parkinsonian symptoms.

A mutation in the mouse ttc26 gene leads to impaired hedgehog signaling.

The phenotype of the spontaneous mutant mouse hop-sterile (hop) is characterized by a hopping gait, polydactyly, hydrocephalus, and male sterility. Previous analyses of the hop mouse revealed a deficiency of inner dynein arms in motile cilia and a lack of sperm flagella, potentially accounting for the hydrocephalus and male sterility. The etiology of the other phenotypes and the location of the hop mutation remained unexplored. Here we show that the hop mutation is located in the Ttc26 gene and impairs Hedgehog (Hh) signaling. Expression analysis showed that this mutation led to dramatically reduced levels of the Ttc26 protein, and protein-protein interaction assays demonstrated that wild-type Ttc26 binds directly to the Ift46 subunit of Intraflagellar Transport (IFT) complex B. Although IFT is required for ciliogenesis, the Ttc26 defect did not result in a decrease in the number or length of primary cilia. Nevertheless, Hh signaling was reduced in the hop mouse, as revealed by impaired activation of Gli transcription factors in embryonic fibroblasts and abnormal patterning of the neural tube. Unlike the previously characterized mutations that affect IFT complex B, hop did not interfere with Hh-induced accumulation of Gli at the tip of the primary cilium, but rather with the subsequent dissociation of Gli from its negative regulator, Sufu. Our analysis of the hop mouse line provides novel insights into Hh signaling, demonstrating that Ttc26 is necessary for efficient coupling between the accumulation of Gli at the ciliary tip and its dissociation from Sufu.

[Anesthetic Management of an Infant who Underwent Awake-intubation for Her Pharyngeal Injury Caused by a Toothbrush].

A 2-year-and-4-month-old female infant, 12 kg in weight and 90 cm in height fell off from a table, which was about 1 m height with a toothbrush in her mouth without her parents noticing. Urgent CT scan showed that it penetrated the left side of her oropharyngeal wall to the bifurcation of her right carotid artery. According to the initial assessment, carotid artery seemed intact and there seemed to be no sign of CNS involvement. She underwent general anesthesia for further investigation and operation. We could detect vocal code with ease by inserting Glidescope between her tongue and the toothbrush. After the intubation, we administered fentanyl 25 µg rocuronium 15 mg and sevoflulane 3-5% to her, and then she underwent arteriography. The neurosurgeon found no sign of major arterial injury nor traumatic aneurysm nor CNS involvement. She went to the ICU intubated after the removal of the toothbrush. She was extubated 5 days after operation. One of the benefits of the Glidescope is that we can share the visual image, and we chose it this time. When we expect a difficult airway during management for oropharyngeal trauma, we have to consider the way to manage the airway.

Factors associated with antisocial behavior in patients with developmental disorder.

Objective: This study investigated the factors associated with antisocial behavior (AB) in children with developmental disorder and effective treatments. Methods: Participants were 110 schoolchildren with developmental disorder and with or without accompanying AB who visited our hospital between October 2009 and October 2012. Among the children with AB, those who exhibited one or more symptoms of conduct disorder (CD) were assigned to the CD subgroup. We examined the background characteristics, past history, type of antisocial behavior, and symptom improvement after treatment in the children with AB and compared the relevant factors with children with developmental disorder without AB. Results: Of the 110 participants, 72 (65.5%) did not exhibit AB and 38 (34.5%) did, 7 (5.5%) of whom fulfilled the criteria for CD. Compared to the children without AB, the children with AB showed a significantly higher occurrence of attention deficit/hyperactivity disorder (AD/HD), maltreatment, institutionalization due to maltreatment, parental mental/psychological problems, and family instability. After medical treatment combined with social-skills training and parental education, 22 of the 38 children with AB showed improved behavior. In the CD subgroup, 4 children were diagnosed with AD/HD and 3 with pervasive developmental disorder, and none of the 7 improved with treatment. Conclusion: AB was associated with AD/HD, maltreatment, institutionalization, parental mental/psychological problems, and family instability. The most effective therapy was parental education. Children with AB need early intervention given that those who already exhibited symptoms of CD showed little improvement with treatment.

Prenatal Counseling on Prenatal Diagnosis of Cleft Lip and/or Cleft Palate at Tokyo Dental College Ichikawa General Hospital.

Remarkable technological advances have been made in the field of medicine in recent years, one result of which is that a prenatal diagnosis of cleft lip and/or cleft palate (CL/P) is now possible. In this situation, it is extremely important to provide the parents with mental care from the moment they are informed. Here, we describe cases of CL/P treated at our hospital and how such a diagnosis and prenatal counseling are handled. A survey was carried out on 4 cases seen at our department between April 2013 and March 2014. Patients are referred to our department from local or our own obstetrics clinics on a prenatal diagnosis of CL/P based on findings from ultrasonography. If the case is a referral from outside, the patient will first be seen at our own obstetrics department. Our department may then be subsequently requested to provide the parents with prenatal counseling. Effort is made to reassure the parents that postnatal support will be provided, right from the start. Next, the multidisciplinary nature of the treatment process is explained. However, only the essential outline is given at first so as to avoid inducing unnecessary anxiety. A response is also given to any questions the parents may have. Our experience of giving such care leads us to believe that improvements are required in the way that explanations and assistance are provided. The number of cases in which prenatal counseling is required is expected to increase in future.

A mutation in the Srrm4 gene causes alternative splicing defects and deafness in the Bronx waltzer mouse.

Sensory hair cells are essential for hearing and balance. Their development from epithelial precursors has been extensively characterized with respect to transcriptional regulation, but not in terms of posttranscriptional influences. Here we report on the identification and functional characterization of an alternative-splicing regulator whose inactivation is responsible for defective hair-cell development, deafness, and impaired balance in the spontaneous mutant Bronx waltzer (bv) mouse. We used positional cloning and transgenic rescue to locate the bv mutation to the splicing factor-encoding gene Ser/Arg repetitive matrix 4 (Srrm4). Transcriptome-wide analysis of pre-mRNA splicing in the sensory patches of embryonic inner ears revealed that specific alternative exons were skipped at abnormally high rates in the bv mice. Minigene experiments in a heterologous expression system confirmed that these skipped exons require Srrm4 for inclusion into the mature mRNA. Sequence analysis and mutagenesis experiments showed that the affected transcripts share a novel motif that is necessary for the Srrm4-dependent alternative splicing. Functional annotations and protein-protein interaction data indicated that the encoded proteins cluster in the secretion and neurotransmission pathways. In addition, the splicing of a few transcriptional regulators was found to be Srrm4 dependent, and several of the genes known to be targeted by these regulators were expressed at reduced levels in the bv mice. Although Srrm4 expression was detected in neural tissues as well as hair cells, analyses of the bv mouse cerebellum and neocortex failed to detect splicing defects. Our data suggest that Srrm4 function is critical in the hearing and balance organs, but not in all neural tissues. Srrm4 is the first alternative-splicing regulator to be associated with hearing, and the analysis of bv mice provides exon-level insights into hair-cell development.

Interleukin-17 is involved in orthodontically induced inflammatory root resorption in dental pulp cells.

INTRODUCTION: The objectives of this study were (1) to investigate the expressions of interleukin (IL)-17, RANKL (the receptor activator of NF-kappaB ligand), and osteoprotegerin (OPG) in root resorption areas during experimental tooth movement in rats, and (2) to determine the effect of IL-17 on the expressions of RANKL and OPG mRNA from human dental pulp cells. METHODS: Twelve male 6-week-old Wistar rats were subjected to an orthodontic force of 50 g to induce a mesially tipping movement of the maxillary first molars for 7 days. The expression levels of tartrate resistant acid phosphatase (TRAP), interleukin (IL)-17, IL-17 receptor (IL-17R), receptor activator of nuclear factor-kappa B ligand (RANKL), and OPG proteins were determined in dental pulp by immunohistochemical analysis. Furthermore, the effects of IL-17 on the expressions of RANKL and OPG mRNA were investigated using human dental pulp cells in vitro. RESULTS: In the experimental tooth movements in vivo, resorption lacunae with multinucleated cells were observed in the 50-g group. The immunoreactivities for IL-17, IL-17R, and RANKL were detected in dental pulp tissues subjected to the orthodontic force on day 7. Moreover, IL-17 increased the mRNA expression of RANKL from human dental pulp cells in vitro. CONCLUSIONS: The results of this study suggest that IL-17 and RANKL may be involved in the process of orthodontically induced inflammatory root resorption in dental pulp cells.

Quantification of negative aesthetic ratings in primary unrepaired unilateral cleft lip infants by modified paired comparison method.

OBJECTIVE: To quantify negative aesthetic ratings in primary unrepaired cleft lip infants by further refining our previously reported paired comparison method. DESIGN: The Thurstone paired comparison method was used to quantify negative aesthetic ratings of plaster facial models selected and ordered according to a table of paired random numbers. PATIENTS, PARTICIPANTS: A total of 30 facial models of unrepaired incomplete unilateral cleft lip infants were used in this study. Raters comprised 20 oral surgeons and anesthesiologists. RESULTS: Quantification of aesthetic ratings for the 30 face models of unrepaired incomplete unilateral cleft lip infants was obtained. The ratings ranged from 0 to 5.08. CONCLUSION: Quantification of the aesthetic ratings for the 30 face models will be used as objective variables in a multivariate analysis in the third stage of this ongoing study.

Correlation between main pancreatic duct diameter measurements: Special pancreatic ultrasonography versus magnetic resonance cholangiopancreatography.

Main pancreatic duct (MPD) dilatation is reported to be a risk factor for pancreatic cancer (PC). Although magnetic resonance cholangiopancreatography (MRCP) and ultrasonographic modalities are valuable for monitoring the pancreas, there is limited information on the efficacy of different imaging modalities in measuring MPD diameter. To improve pancreatic imaging, we developed a specialized ultrasound approach focusing on the pancreas (special pancreatic US). We aimed to examine the correlation between MPD diameter measurements using special pancreatic US versus MRCP. We retrospectively reviewed the clinical data of patients with MPD dilation (≥2.5 mm) via special pancreatic US used for screening at our institution between January 2020 and October 2022 and included patients who underwent magnetic resonance imaging 2 months before and after pancreatic US. The MPD diameter on MRCP was measured at the pancreatic locus, where the maximum MPD diameter was obtained on special pancreatic US. This study included 96 patients, with a median interval of 8.5 days between the date of special pancreatic US and the date of undergoing MRCP. MPD dilatation and/or pancreatic cysts were diagnosed in 86 patients, PC in 5 patients, and other diseases in 5 patients. The median MPD diameter, measured using special pancreatic US, was 3.4 mm (interquartile range: 2.9-4.9 mm), whereas it was 3.5 mm using MRCP (interquartile range: 2.8-4.5 mm). There were strong positive correlations between MPD diameter measured on special pancreatic US and that measured on MRCP (R = 0.925, P < .001). This study revealed strong positive correlations between the MPD diameter measurements using special pancreatic US and MRCP. MPD diameter measurements from each imaging method can be helpful during follow-up in individuals at a high risk of PC.

Conformational analysis of chiral supramolecular aggregates: modeling the subtle difference between hydrogen and deuterium.

A detailed analysis of the conformational states of self-assembled, stereoselectively deuterated benzene-1,3,5-tricarboxamides ((S,S,S)-D-BTAs) reveals four different conformers for the supramolecular polymers. The relative amount of the conformers depends on the solvent structure and the temperature. With the help of a model, the thermodynamic parameters that characterize the different conformational states were quantified as well as the amount of the species that occur at different stages of the polymerization process. The results show that small changes in the stability between different types of conformers formed by (S,S,S)-D-BTAs­in the order of a few J mol(­1)­arise from the combination of interactions between the solvent/supramolecular aggregate, temperature, and solvent structure. While the introduction of a deuterium label allows to sensitively probe the solvophobic effects in the supramolecular aggregation, a rationalization of the observed effects on a molecular level is not yet straightforward but is proposed to result from subtle effects in the vibrational enthalpy and entropy terms of the isotope effect.

Mesoscale modulation of supramolecular ureidopyrimidinone-based poly(ethylene glycol) transient networks in water.

In natural systems, highly synergistic non-covalent interactions among biomolecular components exert mesoscopic control over hierarchical assemblies. We herein present a multicomponent self-assembly strategy to tune hierarchical supramolecular polymer architectures in water using highly affine and directional ureidopyrimidinone-poly(ethylene glycol)s (UPy-PEG). Using scattering methods and oscillatory rheology, we observe the structural and mechanical regulation of entangled monofunctional UPy-PEG fibrils by cross-linking bifunctional UPy-PEG fibrils. This supramolecular mixing approach opens the door to a range of subtly distinct materials for chemical and biological applications.

Postoperative evaluation of grafted bone in alveolar cleft using three-dimensional computed tomography data.

Objective : Postoperative evaluation of bone formation in the alveolar cleft by computed tomography imaging has been reported. We quantitatively evaluated bone grafts in the alveolar cleft preoperatively and postoperatively using three-dimensional data and superimposition of images. Subjects : A total of 12 patients with complete unilateral cleft lip and palate (six left-sided and six right-sided) were studied. Methods : Helical computed tomography scans were taken immediately before surgery and at 6 months after surgery and the DICOM files obtained were processed using Mimics and 3-matic software for three-dimensional data analysis. The preoperative and postoperative computed tomography data were superimposed, and the position and length of the unerupted canines and width of the alveolar cleft measured. Results : Strong and significant correlations were observed between bone formation in the alveolar cleft bone graft region and preoperative canine position (r  =  -.766, p < .01) and canine length (r  =  .681, p < .05). Stepwise multiple regression analysis demonstrated that only the preoperative canine position was independently and significantly correlated with bone formation in the alveolar cleft. Conclusion : These results indicate that the optimal timing for surgery is when the canine cusp is close to the alveolar plane.

Mirror-symmetry-breaking in poly[(9,9-di-n-octylfluorenyl- 2,7-diyl)-alt-biphenyl] (PF8P2) is susceptible to terpene chirality, achiral solvents, and mechanical stirring.

Solvent chirality transfer of (S)-/(R)-limonenes allows the instant generation of optically active PF8P2 aggregates with distinct circular dichroism (CD)/circularly polarized luminescence (CPL) amplitudes with a high quantum yield of 16-20%. The present paper also reports subtle mirror-symmetry-breaking effects in CD-/CPL-amplitude and sign, CD/UV-vis spectral wavelengths, and photodynamics of the aggregates, though the reasons for the anomaly are unsolved. However, these photophysical properties depend on (i) the chemical natures of chiral and achiral molecules when used in solvent quantity, (ii) clockwise and counterclockwise stirring operations, and (iii) the order of addition of limonene and methanol to the chloroform solution.

Main pancreatic duct dilatation and pancreatic cysts in relatives and spouses of patients with pancreatic cancer.

Although main pancreatic duct dilatation and pancreatic cysts are risk factors for developing pancreatic cancer, limited data exist regarding these findings in relatives and spouses of pancreatic cancer patients. The frequency of these findings was examined using long-term follow-up data and transabdominal ultrasonography focusing on the pancreas. We prospectively enrolled 184 relatives and spouses of pancreatic cancer patients and performed special pancreatic ultrasonography to detect main pancreatic duct dilatation and pancreatic cysts. First-degree relatives (148 participants) of patients with pancreatic cancer were significantly younger than the spouses (36 participants; 41 vs. 65 years old). The frequency of ultrasonographic findings was significantly different between the relative (8.8%) and spouse (33.3%) groups. Main pancreatic duct dilatation and pancreatic cysts were observed in seven (4.7%) and seven (4.7%) participants in the relative group, and in nine (25.0%) and five (13.9%) participants in the spouse group, respectively. On multivariate analysis, age was an independent risk factor for the ultrasonographic findings. The frequency of ultrasonographic findings was significantly higher in spouses than in first-degree relatives of patients with pancreatic cancer and was strongly influenced by the age gap between the groups. Main pancreatic duct dilatation was frequently observed, especially in the spouse group.

CmpR is important for circadian phasing and cell growth.

In the cyanobacterium Synechococcus elongatus PCC 7942, the circadian clock entrains to a daily light/dark cycle. The transcription factor Pex is abundant under dark conditions and represses kaiA transcription to fine-tune the KaiC-based core circadian oscillator. The transcription of pex also increases during exposure to darkness; however, its mechanism is unknown. We performed a molecular genetic study by constructing a pex expression bioluminescent reporter and screening for brightly luminescent mutants by random insertion of a drug resistance gene cassette in the reporter genome. One mutant contained an insertion of an antibiotic resistance cassette in the cmpR locus, a transcriptional regulator of inorganic carbon concentration. Insertions of the cassette in the remaining two mutant genomes were in the genes encoding flavodoxin and a putative partner of an ABC transporter with unknown function (ycf22). We further analyzed the cmpR mutant to examine whether CmpR directly or indirectly targeted pex expression. In the cmpR mutant, the pex mRNA level was 1.8-fold that of the wild type, and its circadian peak phase in bioluminescence rhythm occurred 5 h later. Moreover, a high-light stress phenotype was present in the colony. The abnormalities were complemented by ectopic induction of the native gene. However, the cmpR/pex double mutation partly suppressed the phase abnormality (2.5 h). In vitro DNA binding analysis of CmpR showed positive binding to the psbAII promoter, but not to any pex DNA. We postulate that the phenotypes of cmpR-deficient cells were attributable mainly to a feeble metabolic and/or redox status.

A claudin-9-based ion permeability barrier is essential for hearing.

Hereditary hearing loss is one of the most common birth defects, yet the majority of genes required for audition is thought to remain unidentified. Ethylnitrosourea (ENU)-mutagenesis has been a valuable approach for generating new animal models of deafness and discovering previously unrecognized gene functions. Here we report on the characterization of a new ENU-induced mouse mutant (nmf329) that exhibits recessively inherited deafness. We found a widespread loss of sensory hair cells in the hearing organs of nmf329 mice after the second week of life. Positional cloning revealed that the nmf329 strain carries a missense mutation in the claudin-9 gene, which encodes a tight junction protein with unknown biological function. In an epithelial cell line, heterologous expression of wild-type claudin-9 reduced the paracellular permeability to Na+ and K+, and the nmf329 mutation eliminated this ion barrier function without affecting the plasma membrane localization of claudin-9. In the nmf329 mouse line, the perilymphatic K+ concentration was found to be elevated, suggesting that the cochlear tight junctions were dysfunctional. Furthermore, the hair-cell loss in the claudin-9-defective cochlea was rescued in vitro when the explanted hearing organs were cultured in a low-K+ milieu and in vivo when the endocochlear K+-driving force was diminished by deletion of the pou3f4 gene. Overall, our data indicate that claudin-9 is required for the preservation of sensory cells in the hearing organ because claudin-9-defective tight junctions fail to shield the basolateral side of hair cells from the K+-rich endolymph. In the tight-junction complexes of hair cells, claudin-9 is localized specifically to a subdomain that is underneath more apical tight-junction strands formed by other claudins. Thus, the analysis of claudin-9 mutant mice suggests that even the deeper (subapical) tight-junction strands have biologically important ion barrier function.