Hair cells without supporting cells: further studies in the ear of the zebrafish mind bomb mutant

Each sensory hair cell in the ear is normally surrounded by supporting cells, which separate it from the next hair cell. In the mind bomb mutant, as a result of a failure of lateral inhibition, cells that would normally become supporting cells differentiate as hair cells instead, creating sensory patches that consist of hair cells only. This provides a unique opportunity to pinpoint the functions for which supporting cells are required in normal hair cell development. We find that hair cells in the mutant develop an essentially normal cytoskeleton, with a correctly structured hair bundle and well-defined planar polarity, and form apical junctional complexes with one another in standard epithelial fashion. They fail, however, to form a basal lamina or to adhere properly to the adjacent non-sensory epithelial cells, which overgrow them. The hair cells are eventually expelled from the ear epithelium into the underlying mesenchyme, losing their hair bundles in the process. It is not clear whether they undergo apoptosis: many cells staining strongly with the TUNEL procedure are seen but do not appear apoptotic by other criteria. Supporting cells, therefore, are needed to hold hair cells in the otic epithelium and, perhaps, to keep them alive, but are not needed for the construction of normal hair bundles or to give the hair bundles a predictable polarity. Moreover, supporting cells are not absolutely required as a source of materials for otoliths, which, though small and deformed, still develop in their absence.     

The homeobox gene Emx2 underlies middle ear and inner ear defects in the deaf mouse mutant pardon

The semi-dominantly inherited mouse mutation pardon (Pdo) was isolated due to the lack of a Preyer reflex (ear flick) in response to sound from a large-scale N -ethyl- N -nitrosourea (ENU) mutagenesis programme. Dissection of the middle ear revealed malformations in all three ossicles, rendering the ossicular chain incomplete. Hair cell counts in the apical turn of the organ of Corti revealed a significant 22.7% increase in the number of outer hair cells. Raised compound action potential thresholds in Pdo/+ mutants suggested a combined sensorineural/conductive hearing loss. We show that a missense mutation in the homeobox gene Emx2 is responsible for these defects, identifying a new function for this gene in the development of specific structures in the ear.     

Microtubules in inner ear hair cells of the snake

Electron microscopy of snake inner ear sensory epithelia fixed in a microtubule stabilizing fixation revealed a narrow connection between the sensory hair rootlets and microtubules. Also, in the synaptic region microtubules were very frequently present.     

Hair cell replacement in the avian inner ear following two exposures to intense sound

Summary The present study is concerned with the degree to which the avian cochlea retains the capacity to regenerate hair cells following repeated exposures to intense sound. Two groups of chicks were exposed once to an intense pure tone for 48 h at either 2 or 16 days of age. In a third group, they were exposed to the same stimulus at both ages, Structural alterations of the auditory epithelium were assessed both qualitatively and quantitatively at 0, 12 or 26 days following the single exposure at 2 days of age, and at 0 or 12 days following the single or second exposure at 16 days of age. The numbers of hair cells lost in the twice-exposed birds and those exposed once at 2 days of age were approximately 24 and 36%, respectively, and were not significantly different. Interestingly, the single exposure at 16 days of age caused greater hair cell loss (61%). Twelve days after overstimulation, the hair cell population in all experimental groups returned to near normal levels due to the emergence of new hair cells. This observation of hair cell replacement extends the early findings that birds are able to repair their acoustically damaged ears after either a single or repeated overexposure.     

Affinity of Sendai virus for the inner ear of mice.

The direct immunofluorescent antibody method revealed that Sendai virus, which was thought to be most prevalent in respiratory diseases in mice, infected the inner ear of mice when the virus was injected intracerebrally. Specific immunofluorescence that indicated the presence of antigens of Sendai virus was observed in both perilymphatic and endolymphatic structures. The routes of virus spread are discussed. This experimental study, using newborn mice and Sendai virus, may be useful for the investigation of the pathogenesis of viral labyrinthitis not only because of the high frequency of inner ear infection, but also because of long-time survivors which facilitate prolonged observation.     

Second branchial arch lineages of the middle ear of wild-type and Hoxa2 mutant mice.

Our current understanding of the evolution of the mammalian middle ear was first suggested by embryological studies from the 19th century. Here, site-specific recombinase-mediated lineage tracing was used to define the second branchial arch contribution to the middle ear of wild-type and Hoxa-2 mutant embryos. The processus brevis of the malleus was found to arise from second arch tissues, making it the likely homologue of the retroarticular process of nonmammalian tetrapods. The second arch also formed a portion of the otic capsule. In light of avian lineage studies, second arch cells were probably incorporated into the otic capsule before avian and mammalian lineages diverged. In Hoxa2 mutant embryos, middle ear skeletal duplications occurred at sites where first and second arch elements are normally apposed. The dorsoventral positions at which second arch skeletal elements formed and the early migration of second arch neural crest cells were not altered by the absence of Hoxa2 function.     

Hair cell orientation and a possible intraepithelial growth of the macula lagenae in the inner ear of the European eel

The hair cell orientation of the macula lagenae in eels at three different stages of life was examined with the light microscope. Part of the posterior periphery of all three stages was occupied by a few rows of sensory cells having their kinocilium pointing in a direction opposite to the direction of the adjacent sensory cells. The width of this peripheral belt remained the same at all three stages of life, in spite of a considerable growth of the sensory epithelium, and the belt was always confined to the margin. This indicates an intraepithelial growth of at least part of the neuroepithelium of the lagenar macula of the European eel.     

Atorvastatin slows down the deterioration of inner ear function with age in mice

Statins have revolutionized the treatment of hypercholesterolemia due to their ability to inhibit cholesterol biosynthesis. Their immunomodulatory and anti-inflammatory effects and positive effects on the treatment of atherosclerosis and its complications are well known. Here, we describe the effects of statins on the treatment of presbycusis in C57BL/6J mice. In this strain with accelerated aging, we demonstrate that animals treated with atorvastatin (10mg/kg per day in chow diet) for 2 months showed larger amplitudes of distortion product otoacoustic emissions (DPOAE) than did the non-treated control group. This finding indicates a better survival of outer hair cell function in the inner ear of C57BL/6J mice. The observed decreased expression of intercellular and vascular adhesion molecules in the aortic wall of atorvastatin-treated animals suggests that reducing endothelial inflammatory effects may contribute to the positive effect of atorvastatin on the amplitudes of DPOAE by influencing the blood supply to the inner ear. No such beneficial effect of statins was found in apoE(-/-) mice treated with atorvastatin under the same conditions. Our results suggest that statins could also slow down the age-related deterioration of hearing in man.     

The role of Zic genes in inner ear development in the mouse: Exploring mutant mouse phenotypes

Background: Murine Zic genes (Zic1–5) are expressed in the dorsal hindbrain and in periotic mesenchyme (POM) adjacent to the developing inner ear. Zic genes are involved in developmental signaling pathways in many organ systems, including the ear, although their exact roles haven't been fully elucidated. This report examines the role of Zic1, Zic2, and Zic4 during inner ear development in mouse mutants in which these Zic genes are affected. Results: Zic1/Zic4 double mutants don't exhibit any apparent defects in inner ear morphology. By contrast, inner ears from Zic2^kd/kd and Zic2^Ku/Ku mutants have severe but variable morphological defects in endolymphatic duct/sac and semicircular canal formation and in cochlear extension in the inner ear. Analysis of otocyst patterning in the Zic2^Ku/Ku mutants by in situ hybridization showed changes in the expression patterns of Gbx2 and Pax2. Conclusions: The experiments provide the first genetic evidence that the Zic genes are required for morphogenesis of the inner ear. Zic2 loss‐of‐function doesn't prevent initial otocyst patterning but leads to molecular abnormalities concomitant with morphogenesis of the endolymphatic duct. Functional hearing deficits often accompany inner ear dysmorphologies, making Zic2 a novel candidate gene for ongoing efforts to identify the genetic basis of human hearing loss. Developmental Dynamics 243:1487–1498, 2014. © 2014 Wiley Periodicals, Inc.     

Characterization of inner ear sensory hair cells after rapid-freezing and freeze-substitution

Summary In order to determine the ultrastructural organization of normal cells and to understand better the anatomical substrates for outer hair cell motility, cryofixation was performed on the sensory epithelium of the inner ear of guinea pigs prior to substitution of frozen water with organic solvents containing chemical fixatives. In this way cells would not be altered by the direct application of the chemicals commonly used for preservation, which are also known to cause fixation-induced shape changes in outer hair cells. Following rapid freezing and freeze-substitution, preservation of cells within the isolated sensory epithelium containing the organ of Corti was similar to that seen in conventionally fixed cells. However, in rapidly frozen and freeze-substituted outer hair cells the cytoplasm and the cellular membranes differed from those seen in conventionally fixed preparations. The cytoplasmic matrix was densely packed with filaments and stained homogeneously, suggesting better preservation of the cytoskeleton and less extraction of the soluble ground substance. Cell membranes were smooth, indicating that fixation-induced shape changes and shrinkage had been avoided. The subsurface cisternal system of intracellular membranes lining the lateral wall of the outer hair cells was composed of continuous, tightly packed, parallel rows of membranous lamellae. Thus rapid-freezing and freeze-substitution are important techniques by which structural alterations correlated with outer hair cell motility can be separated from fixation-induced cell shape changes.     

Developmental acquisition of sensory transduction in hair cells of the mouse inner ear

Sensory transduction in hair cells requires assembly of membrane-bound transduction channels, extracellular tip-links and intracellular adaptation motors with sufficient precision to confer nanometer displacement sensitivity. Here we present evidence based on FM1-43 fluorescence, scanning electron microscopy and RT-PCR that these three essential elements are acquired concurrently between embryonic day 16 and 17, several days after the appearance of hair bundles, and that their acquisition coincides with the onset of mechanotransduction.     

Embryogenesis of the mammalian inner ear

Summary The development of the tectorial membrane in the basal coil of the cochlea has started already in the 15th gestational day inner ear and has reached a considerable thickness and maturation at birth. The development of the tectorial membrane occurs synchronously in in vivo labyrinths and the in vitro material cultured to an age corresponding to birth. At least during this part of the development the formation of the tectorial membrane is independent of the specific composition of endolymph. In the in vivo material a secretory maximum was reached on the 18th gestational day, whereafter the secretory activity was low, especially after birth. In the in vitro specimens, however, a rather constant secretion of material occurred also post partum, which indicates a lack of control mechanisms during in vitro conditions. A complete maturation of the tectorial membrane did not occur in vitro. When passing the point of time corresponding to birth, in the in vitro inner ear explants the gross structure of the tectorial membrane is only slightly changed. In vivo a mature configuration of the tectorial membrane is observed on the 14th DAB (day after birth).Supported by grants from Karolinska Institutet, the Ragnar & Torsten Söderberg Foundation and the Swedish Medical Research Council (12X-720)     

Oxidative imbalance in the aging inner ear

The mammalian inner ear loses its sensory cells with advancing age, accompanied by a functional decrease in balance and hearing. This study investigates oxidant stress in the cochlea of aging male CBA/J mice. Glutathione-conjugated proteins, markers of H2O2-mediated oxidation, began to increase at 12 months of age; 4-hydroxynonenal and 3-nitrotyrosine, products of hydroxyl radical and peroxynitrite action, respectively, were elevated by 18 months. Immunoreactivity to these markers was stronger in the supporting cells (Deiters and pillar cells) than the sensory cells and appeared later (23 months) in spiral ganglion cells and in the stria vascularis and spiral ligament. Conversely, antioxidant proteins (AIF) and enzymes (SOD2) decreased by 18 months in the organ of Corti (including the sensory cells) and spiral ganglion cells but not in the stria vascularis. These results suggest the presence of different reactive oxygen species and differential time courses of oxidative changes in individual tissues of the aging cochlea. An imbalance of redox status may be a component of age-related hearing loss.     

Cell replacement therapy in the inner ear

The mammalian inner ear is vulnerable to genetic disorders and aging, as well as to injuries caused by overstimulation, ototoxic drugs, and viral infections. Due to the poor regeneration of the sensory epithelium and the spiral ganglion neurons in the adult mammalian inner ear, cell replacement therapy strategies have been proposed to compensate for degeneration and loss of sensory and neuronal cells. Transplantation of stem cells and embryonic neurons into the inner ear has revealed that exogenous cells can survive, migrate, differentiate, and extend neuritic projections in the auditory system of adult mammals. These results suggest that cell replacement therapy could provide an effective future treatment alternative for hearing loss and other inner ear disorders.