Immortalized Mouse Inner Ear Cell Lines Demonstrate a Role for Chemokines in Promoting the Growth of Developing Statoacoustic Ganglion Neurons

The target-derived factors necessary for promoting initial outgrowth from the statoacoustic ganglion (SAG) to the inner ear have not been fully characterized. In the present study, conditioned medium from embryonic Immortomouse inner ear cell lines that maintain many characteristics of developing inner ear sensory epithelia were screened for neurite-promoting activity. Conditioned medium found to be positive for promoting SAG neurite outgrowth and neuronal survival was then tested for the presence of chemokines, molecules that have not previously been investigated for promoting SAG outgrowth. One candidate molecule, monocyte chemotactic protein 1 (MCP-1), was detected in the conditioned medium and subsequently localized to mouse hair cells by immunocytochemistry. In vitro studies demonstrated that function-blocking MCP-1 antibodies decreased the amount of SAG neurite outgrowth induced by the conditioned medium and that subsequent addition of MCP-1 protein was able to promote outgrowth when added to the antibody-treated conditioned medium. The use of the Immortomouse cell lines proved valuable in identifying this candidate cofactor that promotes outgrowth of early-stage SAG nerve fibers and is expressed in embryonic hair cells.     

ErbB Expression: The Mouse Inner Ear and Maturation of the Mitogenic Response to Heregulin

In humans, hair cell loss often leads to hearing and balance impairments. Hair cell replacement is vigorous and spontaneous in avians and nonmammalian vertebrates. In mammals, in contrast, it occurs at a very low rate, or not at all, presumably because of a very low level of supporting cell proliferation following injury. Heregulin (HRG), a member of the epidermal growth factor (EGF) family of growth factors, is reported to be a potent mitogen for neonatal rat vestibular sensory epithelium, but its effects in adults are unknown. We report here that HRG- stimulates cell proliferation in organotypic cultures of neonatal, but not adult, mouse utricular sensory epithelia. Our findings support the idea that the proliferative capabilities of the adult mammalian vestibular sensory epithelia differ significantly from that seen in neonatal animals. Immunohistochemistry reveals that HRG-binding receptors (erbBs 2–4) and erbB1 are widely expressed in vestibular and auditory sensory epithelia in neonatal and adult mouse inner ear. The distribution of erbBs in the neonatal and adult mouse ear is consistent with the EGF receptor/ligand family regulating diverse cellular processes in the inner ear, including cell proliferation and differentiation. Present address (Mette Kirkegaard): Department of Zoophysiology, Bld. 131, Universitetsparken, University of Aarhus, Denmark     

CD44 is a Marker for the Outer Pillar Cells in the Early Postnatal Mouse Inner Ear

Cluster of differentiation antigens (CD proteins) are classically used as immune cell markers. However, their expression within the inner ear is still largely undefined. In this study, we explored the possibility that specific CD proteins might be useful for defining inner ear cell populations. mRNA expression profiling of microdissected auditory and vestibular sensory epithelia revealed 107 CD genes as expressed in the early postnatal mouse inner ear. The expression of 68 CD genes was validated with real-time RT-PCR using RNA extracted from microdissected sensory epithelia of cochleae, utricles, saccules, and cristae of newborn mice. Specifically, CD44 was identified as preferentially expressed in the auditory sensory epithelium. Immunohistochemistry revealed that within the early postnatal organ of Corti, the expression of CD44 is restricted to outer pillar cells. In order to confirm and expand this finding, we characterized the expression of CD44 in two different strains of mice with loss- and gain-of-function mutations in Fgfr3 which encodes a receptor for FGF8 that is essential for pillar cell development. We found that the expression of CD44 is abolished from the immature pillar cells in homozygous Fgfr3 knockout mice. In contrast, both the outer pillar cells and the aberrant Deiters’ cells in the Fgfr3 ^{P244R/ +} mice express CD44. The deafness phenotype segregating in DFNB51 families maps to a linkage interval that includes CD44. To study the potential role of CD44 in hearing, we characterized the auditory system of CD44 knockout mice and sequenced the entire open reading frame of CD44 of affected members of DFNB51 families. Our results suggest that CD44 does not underlie the deafness phenotype of the DFNB51 families. Finally, our study reveals multiple potential new cell type-specific markers in the mouse inner ear and identifies a new marker for outer pillar cells.     

Neurod1与内耳感觉神经及毛细胞发育的相关研究

本文主要介绍了Neurod1在内耳感觉神经元及毛细胞发育中的相关研究。Neurod1是b HLH转录因子家族中的重要一员,它能促进感觉神经元前体细胞分化为成熟感觉神经元细胞。在内耳中,Neurod1主要表达于感觉神经元细胞,部分感觉上皮也有表达。Neurod1基因条件性敲除小鼠可出现听力及平衡障碍,其内耳感觉神经元细胞大量缺失,同时内耳中的传入及传出神经也存在缺陷。Neurod1基因敲除小鼠螺旋神经节中还可出现异位毛细胞的生长。在内耳发育过程中,Neurod1基因与多种基因相互调控,协同促进内耳发育成熟。如Neurog1、Atoh1、Sox2、Nhlh1等。     

Permeation of Fluorophore-Conjugated Phalloidin into Live Hair Cells of the Inner Ear Is Modulated by P2Y Receptors

Phalloidin, a toxin isolated from the death cap mushroom, Amanita phalloides, binds to filamentous actin with high affinity, and this has made fluorophore-conjugated phalloidin a useful tool in cellular imaging. Hepatocytes take up phalloidin via the liver-specific organic anion transporting polypeptide 1b2, but phalloidin does not permeate most living cells. Rapid entry of styryl dyes into live hair cells has been used to evaluate function, but the usefulness of those fluorescence dyes is limited by broad and fixed absorption spectra. Since phalloidin can be conjugated to fluorophores with various spectra, we investigated whether it would permeate living hair cells. When we incubated mouse utricles in 66 nM phalloidin-CF488A and followed that by washes in phalloidin-free medium, we observed that it entered a subset of hair cells and labeled entire hair bundles fluorescently after 20 min. Incubations of 90 min labeled nearly all the hair bundles. When phalloidin-treated utricles were cultured for 24 h after washout, the label disappeared from the hair cells and progressively but heterogeneously labeled filamentous actin in the supporting cells. We investigated how phalloidin may enter hair cells and found that P2 receptor antagonists, pyridoxalphosphate-6-azophenyl-2′, 4′-disulfonic acid and suramin, blocked phalloidin entry, while the P2Y receptor ligands, uridine-5’-diphosphate and uridine-5’-triphosphaste, stimulated uptake. Consistent with that, the P2Y6 receptor antagonist, MRS 2578, decreased phalloidin uptake. The results show that phalloidin permeates live hair cells through a pathway that requires metabotropic P2Y receptor signaling and suggest that phalloidin can be transferred from hair cells to supporting cells in culture.     

Differences Between the Negatively Activating Potassium Conductances of Mammalian Cochlear and Vestibular Hair Cells

Cochlear and type I vestibular hair cells of mammals express negatively activating potassium (K⁺) conductances, called g _K,n and g _K,L respectively, which are important in setting the hair cells resting potentials and input conductances. It has been suggested that the channels underlying both conductances include KCNQ4 subunits from the KCNQ family of K⁺ channels. In whole-cell recordings from rat hair cells, we found substantial differences between g _K,n and g _K,L in voltage dependence, kinetics, ionic permeability, and stability during whole-cell recording. Relative to g _K,L, g _K,n had a significantly broader and more negative voltage range of activation and activated with less delay and faster principal time constants over the negative part of the activation range. Deactivation of g _K,n had an unusual sigmoidal time course, while g _K,L deactivated with a double-exponential decay. g _K,L, but not g _K,n, had appreciable permeability to Cs⁺. Unlike g _K,L, g _K,ns properties did not change (wash out) during the replacement of cytoplasmic solution with pipette solution during ruptured-patch recordings. These differences in the functional expression of g _K,n and g _K,L channels suggest that there are substantial differences in their molecular structure as well.     

Preliminary Characterization of Voltage-Activated Whole-Cell Currents in Developing Human Vestibular Hair Cells and Calyx Afferent Terminals

We present preliminary functional data from human vestibular hair cells and primary afferent calyx terminals during fetal development. Whole-cell recordings were obtained from hair cells or calyx terminals in semi-intact cristae prepared from human fetuses aged between 11 and 18 weeks gestation (WG). During early fetal development (11–14 WG), hair cells expressed whole-cell conductances that were qualitatively similar but quantitatively smaller than those observed previously in mature rodent type II hair cells. As development progressed (15–18 WG), peak outward conductances increased in putative type II hair cells but did not reach amplitudes observed in adult human hair cells. Type I hair cells express a specific low-voltage activating conductance, G _K,L. A similar current was first observed at 15 WG but remained relatively small, even at 18 WG. The presence of a “collapsing” tail current indicates a maturing type I hair cell phenotype and suggests the presence of a surrounding calyx afferent terminal. We were also able to record from calyx afferent terminals in 15–18 WG cristae. In voltage clamp, these terminals exhibited fast inactivating inward as well as slower outward conductances, and in current clamp, discharged a single action potential during depolarizing steps. Together, these data suggest the major functional characteristics of type I and type II hair cells and calyx terminals are present by 18 WG. Our study also describes a new preparation for the functional investigation of key events that occur during maturation of human vestibular organs.     

Maintained Expression of the Planar Cell Polarity Molecule Vangl2 and Reformation of Hair Cell Orientation in the Regenerating Inner Ear

The avian inner ear possesses a remarkable ability to regenerate sensory hair cells after ototoxic injury. Regenerated hair cells possess phenotypes and innervation that are similar to those found in the undamaged ear, but little is known about the signaling pathways that guide hair cell differentiation during the regenerative process. The aim of the present study was to examine the factors that specify the orientation of hair cell stereocilia bundles during regeneration. Using organ cultures of the chick utricle, we show that hair cells are properly oriented after having regenerated entirely in vitro and that orientation is not affected by surgical removal of the striolar reversal zone. These results suggest that the orientation of regenerating stereocilia is not guided by the release of a diffusible morphogen from the striolar reversal zone but is specified locally within the regenerating sensory organ. In order to determine the nature of the reorientation cues, we examined the expression patterns of the core planar cell polarity molecule Vangl2 in the normal and regenerating utricle. We found that Vangl2 is asymmetrically expressed on cells within the sensory epithelium and that this expression pattern is maintained after ototoxic injury and throughout regeneration. Notably, treatment with a small molecule inhibitor of c-Jun-N-terminal kinase disrupted the orientation of regenerated hair cells. Both of these results are consistent with the hypothesis that noncanonical Wnt signaling guides hair cell orientation during regeneration.     

Development of the Gerbil Inner Ear Observed in the Hemicochlea

A frequency-dependent change in hearing sensitivity occurs during maturation in the basal gerbil cochlea. This change takes place during the first week after the onset of hearing. It has been argued that the mass of a given cochlear segment decreases during development and thus increases the best frequency. Changes in mass during cochlear maturation have been estimated previously by measuring the changes in cochlear dimensions. Fixed, dehydrated, embedded, or sputter-coated tissues were used in such work. However, dehydration of the tissue, a part of most histological techniques, results in severe distortion of some aspects of cochlear morphology. The present experiments, using a novel preparation, the hemicochlea, show that hydrated structures, such as the tectorial membrane and the basilar membrane hyaline matrix, are up to 100% larger than estimated previous studies. Therefore, the hemicochlea was used to study the development of cochlear morphology in the gerbil between the day of birth and postnatal day 19. We used no protocols that would have resulted in severe distortion of cochlear elements. Consequently, a detailed study of cochlear morphology yields several measures that differ from previously published data. Our experiments confirm growth patterns of the cochlea that include a period of remarkably rapid change between postnatal day 6 and 8. The accelerated growth starts in the middle of the cochlea and progresses toward the base and the apex. In particular, the increase in height of Deiters' cells dominated the change, "pushing" the tectorial membrane toward scala vestibuli. This resulted in a shape change of the tectorial membrane and the organ of Corti. The tectorial membrane was properly extended above the outer hair cells by postnatal day 12. This time coincides with the onset of hearing. The basilar membrane hyaline matrix increased in thickness, whereas the multilayered tympanic cover layer cells decreased to a single band of cells by postnatal day 19. Before and after the period of rapid growth, the observed gross morphological changes are rather small. It is unlikely that dimensional changes of cochlear structures between postnatal days 12 and 19 contribute significantly in the remapping of the frequency-place code in the base of the cochlea. Instead, structural changes affecting the stiffness of the cochlear partition might be responsible for the shift in best frequency.     

Differential Distribution of Stem Cells in the Auditory and Vestibular Organs of the Inner Ear

The adult mammalian cochlea lacks regenerative capacity, which is the main reason for the permanence of hearing loss. Vestibular organs, in contrast, replace a small number of lost hair cells. The reason for this difference is unknown. In this work we show isolation of sphere-forming stem cells from the early postnatal organ of Corti, vestibular sensory epithelia, the spiral ganglion, and the stria vascularis. Organ of Corti and vestibular sensory epithelial stem cells give rise to cells that express multiple hair cell markers and express functional ion channels reminiscent of nascent hair cells. Spiral ganglion stem cells display features of neural stem cells and can give rise to neurons and glial cell types. We found that the ability for sphere formation in the mouse cochlea decreases about 100-fold during the second and third postnatal weeks; this decrease is substantially faster than the reduction of stem cells in vestibular organs, which maintain their stem cell population also at older ages. Coincidentally, the relative expression of developmental and progenitor cell markers in the cochlea decreases during the first 3 postnatal weeks, which is in sharp contrast to the vestibular system, where expression of progenitor cell markers remains constant or even increases during this period. Our findings indicate that the lack of regenerative capacity in the adult mammalian cochlea is either a result of an early postnatal loss of stem cells or diminishment of stem cell features of maturing cochlear cells.     

Effects of Chronic Furosemide Treatment and Age on Cell Division in the Adult Gerbil Inner Ear

Atrophy of the stria vascularis and spiral ligament and an associated decrease in the endocochlear potential (EP) are significant factors in age-related hearing loss (presbyacusis). To model this EP decrease, furosemide was delivered into the round-window niche of young adult gerbils by osmotic pump for seven days, chronically reducing the EP by 30–40 mV. Compound action potential (CAP) thresholds were correspondingly reduced by 30–40 dB SPL at high frequencies. Two weeks after withdrawal of furosemide, the treated ears showed an EP recovery of up to 20–30 mV along with a similar recovery of CAP thresholds. The influence of cell division on furosemide-induced and age-related decline of the EP was examined using a mitotic tracer, bromodeoxyuridine (BrdU). Cell proliferation was examined in three groups: young control, furosemide-treated, and aged cochleas. Sections immunostained for BrdU were bleached with H₂O₂ to eliminate ambiguities with melanin pigment in the inner ear. Cell types positively labeled for BrdU in all three groups included Schwann cells in Rosenthal's canal; glial cells in the osseous spiral lamina; fibrocytes in the limbus, sacculus, and spiral ligament (SL); epithelial cells in Reissner's and round-window membranes; intermediate cells in the stria vascularis; and vascular endothelial cells. Quantitative analysis showed that the mean number of BrdU-positive (BrdU+) intermediate cells in the stria did not differ significantly among the three groups. In contrast, there was a significant increase of BrdU + fibrocytes in the SL of furosemide-treated animals as compared to the young control group. Moreover, there was a significant decrease in labeled fibrocytes in the aged versus the young ears, particularly among the type II and type IV subtypes. The results suggest that the increased fibrocyte turnover in the SL after furosemide treatment may be related to the recovery of EP and CAP thresholds, supporting the hypothesis that fibrocyte proliferation may be essential for maintaining the EP and cochlear function in normal and damaged cochleas. Moreover, the decreased turnover of SL fibrocytes with age may be a contributing factor underlying the lateral wall pathology and consequent EP loss that often accompanies presbyacusis.     

Gravity Receptor Aging in the CBA/CaJ Strain: A Comparison to Auditory Aging

The CBA/CaJ mouse strain is commonly used as a control as it has no known genetic mutations affecting the inner ear, maintains hearing sensitivity throughout life, and serves as a background for creating new genetic strains. The purpose of the present study was to characterize the effects of age and gender on gravity receptor function and compare functional changes between auditory and vestibular modalities. Vestibular-evoked potentials (VsEPs), auditory brainstem responses (ABRs), and distortion product otoacoustic emissions were measured in 131 mice. VsEP thresholds deteriorated an average of 0.39 dB re: 1.0 g/ms per month and at the oldest ages (18–23 months old) showed an average loss of 49% of VsEP dynamic range. No significant gender differences were found for VsEPs. ABR thresholds increased by an average of 1.35, 1.38, and 1.15 dB pe SPL per month for ABR stimulus frequencies of 8, 16, and 32 kHz, respectively, demonstrating an average decrease in auditory dynamic range of 25–35% at advanced ages. Both modalities declined with age. Age-related decreases in gravity receptor sensitivity should be considered when using the CBA/CaJ strain for vestibular studies.     

哇巴因对小鼠内耳神经胶质细胞的影响

目的 研究哇巴因对小鼠内耳神经胶质细胞的影响,为干细胞移植治疗感音神经性聋的研究奠定基础.方法 成年雌性SPF级CBA/J小鼠60只,随机分为实验组和对照组,每组30只;实验组动物接受哇巴因(3 mM)经圆窗渗透给药,对照组给予等量生理盐水,于给药后7、14和30天用免疫组织荧光染色法观察位于耳蜗螺旋神经节内的内耳神经胶质细胞(inner ear glial cells,IEGs)的变化.结果 给药后7、14、30天实验组耳蜗各回螺旋神经节内可见内耳神经胶质细胞存活,但数量减少,排列紊乱;与对照组相比,哇巴因给药后7天实验组耳蜗各回神经胶质细胞的数量及密度即显著减少,给药后14天及30天后胶质细胞数量进一步减少;给药后30天各回螺旋神经节内内耳神经胶质细胞数降至最低,与实验组给药后7天、14天及与同时间点对照组比较显著减少,差异有统计学意义(P<0.05).结论 哇巴因经圆窗渗透给药可直接造成CBA/J小鼠内耳螺旋神经节内神经胶质细胞的急性进行性损伤.     

A Sox10rtTA/+ Mouse Line Allows for Inducible Gene Expression in the Auditory and Balance Organs of the Inner Ear

Genetic mouse models provide invaluable tools for discerning gene function in vivo. Tetracycline-inducible systems (Tet-On/Off) provide temporal and cell-type specific control of gene expression, offering an alternative or even complementary approach to existing Cre/LoxP systems. Here we characterized a Sox10^rtTA/+ knock-in mouse line which demonstrates inducible reverse tetracycline trans-activator (rtTA) activity and Tet-On transgene expression in the inner ear following induction with the tetracycline derivative doxycycline (Dox). These Sox10^rtTA/+ mice do not exhibit any readily observable developmental or hearing phenotypes, and actively drive Tet-On transgene expression in Sox10 expressing cells in the inner ear. Sox10^rtTA/+ activity was revealed by multiple Tet-On reporters to be nearly ubiquitous throughout the membranous labyrinth of the developing inner ear, and notably absent from hair cells, tympanic border cells, and ganglion neurons following postnatal Dox inductions. Interestingly, Dox-induced Sox10^rtTA/+ activity declined with induction age, where Tet-On reporters became uninducible in adult cochlear epithelium. Co-administration of the loop diuretic furosemide was able to rescue Dox-induced reporter expression, though this method also caused significant cochlear hair cell loss. Surprisingly, Sox10^rtTA/+ driven reporter expression in the cochlea persists for at least 54 days after cessation of neonatal induction, presumably due to the persistence of Dox within inner ear tissues. These findings highlight the utility of the Sox10^rtTA/+ mouse line as a powerful tool for functional genetic studies of the auditory and balance organs in vivo, but also reveal some important considerations that must be adequately controlled for in future studies that rely upon Tet-On/Off systems.     

Conditional Gene Expression in the Mouse Inner Ear Using Cre-<Emphasis Type="Italic">loxP</Emphasis>

In recent years, there has been significant progress in the use of Cre-loxP technology for conditional gene expression in the inner ear. Here, we introduce the basic concepts of this powerful technology, emphasizing the differences between Cre and CreER. We describe the creation and Cre expression pattern of each Cre and CreER mouse line that has been reported to have expression in auditory and vestibular organs. We compare the Cre expression patterns between Atoh1-CreER^TM and Atoh1-CreER^T2 and report a new line, Fgfr3-iCreER^T2, which displays inducible Cre activity in cochlear supporting cells. We also explain how results can vary when transgenic vs. knock-in Cre/CreER alleles are used to alter gene expression. We discuss practical issues that arise when using the Cre-loxP system, such as the use of proper controls, Cre efficiency, reporter expression efficiency, and Cre leakiness. Finally, we introduce other methods for conditional gene expression, including Flp recombinase and the tetracycline-inducible system, which can be combined with Cre-loxP mouse models to investigate conditional expression of more than one gene.     

Topological and Developmental Gradients of Calbindin Expression in the Chick's Inner Ear

Mobile intracellular calcium buffers play an important role in regulating calcium flux into mechanosensory hair cells and calbindin D-28k is expressed at high levels in the chick's basilar papilla. We have used RT-PCR, in situ hybridization, and immunohistology to demonstrate that calbindin expression varies systematically according to hair cell position and developmental age. RT-PCR using microdissected quarters of the posthatch basilar papilla showed that mRNA levels were lowest in the (low frequency) apex and higher in basal quadrants. In situ hybridization revealed calbindin mRNA in posthatch hair cells and supporting cells, with more intense labeling of hair cells from basal (high frequency) positions. A similar topology was obtained with calbindin antibodies. Neither calbindin riboprobe nor calbindin antibody labeled cochlear neurons. In contrast, a subset of large vestibular neurons and their calyciform endings onto Type I vestibular hair cells were strongly labeled by the calbindin antibody, while vestibular hair cells were negative for calbindin immunoreactivity. Likewise, calbindin in situ hybridization was negative for vestibular hair cells but positive in a subset of larger vestibular neurons. Calbindin mRNA was detected in hair cells of the basal half of the papilla at embryonic day 10 (E10) and calbindin immunoreactivity was detected at E12. Hair cells in the apical half of the papilla had equivalent calbindin expression two days later. Immunoreactivity appeared in abneural supporting cells days later than in hair cells, and not until E20 in neurally located supporting cells. These results demonstrate that calbindin message and protein levels are greater in high-frequency hair cells. This "tonotopic" gradient may result from the stabilization of a basal-to-apical developmental gradient and could be related at least in part to calcium channel expression along this axis.     

Age- and Size-Related Changes in the Inner Ear and Hearing Ability of the Adult Zebrafish (Danio rerio)

Fishes, unlike most other vertebrate groups, continue to add sensory hair cells to their ears for much of their lives. However, it is not clear whether the addition ever stops or how the addition of sensory cells impacts hearing ability. In this article, we tested both questions using the zebrafish, Danio rerio. Our results not only have important implications for understanding the consequences of adding sensory receptors, but these results for normal zebrafish also serve as valuable baseline information for future studies of select mutations on the ear and hearing of this species. Our results show that hair cell production continues in uncrowded zebrafish up to 10 months of age (about one-third of a normal life span), but despite this addition there is no change in hearing sensitivity or bandwidth. Therefore, hearing is not related to the number of sensory cells in the ear in juvenile and adult animals. We also show that despite no net addition of hair cells after about 10 months, hair cells are still being produced, but at a lower rate, presumably to replace cells that are dying. Moreover, crowding of zebrafish has a marked impact on the growth of the fish and on the addition of sensory cells to the ear. We also demonstrate that fish size, not age, is a better indicator of developmental state of zebrafish.