Developmental gene expression profiling along the tonotopic axis of the mouse cochlea

The mammalian cochlear duct is tonotopically organized such that the basal cochlea is tuned to high frequency sounds and the apical cochlea to low frequency sounds. In an effort to understand how this tonotopic organization is established, we searched for genes that are differentially expressed along the tonotopic axis during neonatal development. Cochlear tissues dissected from P0 and P8 mice were divided into three equal pieces, representing the base, middle and apex, and gene expression profiles were determined using the microarray technique. The gene expression profiles were grouped according to changes in expression levels along the tonotopic axis as well as changes during neonatal development. The classified groups were further analyzed by functional annotation clustering analysis to determine whether genes associated with specific biological function or processes are particularly enriched in each group. These analyses identified several candidate genes that may be involved in cochlear development and acquisition of tonotopy. We examined the expression domains for a few candidate genes in the developing mouse cochlea. Tnc (tenacin C) and Nov (nephroblastoma overexpressed gene) are expressed in the basilar membrane, with increased expression toward the apex, which may contribute to graded changes in the structure of the basilar membrane along the tonotopic axis. In addition, Fst (Follistatin), an antagonist of TGF-β/BMP signaling, is expressed in the lesser epithelial ridge and at gradually higher levels towards the apex. The graded expression pattern of Fst is established at the time of cochlear specification and maintained throughout embryonic and postnatal development, suggesting its possible role in the organization of tonotopy. Our data will provide a good resource for investigating the developmental mechanisms of the mammalian cochlea including the acquisition of tonotopy.     

EphA4 misexpression alters tonotopic projections in the auditory brainstem

Auditory pathways contain orderly representations of frequency selectivity, which begin at the cochlea and are transmitted to the brainstem via topographically ordered axonal pathways. The mechanisms that establish these tonotopic maps are not known. Eph receptor tyrosine kinases and their ligands, the ephrins, have a demonstrated role in establishing topographic projections elsewhere in the brain, including the visual pathway. Here, we have examined the function of these proteins in the formation of auditory frequency maps. In birds, the first central auditory nucleus, n. magnocellularis (NM), projects tonotopically to n. laminaris (NL) on both sides of the brain. We previously showed that the Eph receptor EphA4 is expressed in a tonotopic gradient in the chick NL, with higher frequency regions showing greater expression than lower frequency regions. Here we misexpressed EphA4 in the developing auditory brainstem from embryonic day 2 (E2) through E10, when NM axons make synaptic contact with NL. We then evaluated topography along the frequency axis using both anterograde and retrograde labeling in both the ipsilateral and contralateral NM-NL pathways. We found that after misexpression, NM regions project to a significantly broader proportion of NL than in control embryos, and that both the ipsilateral map and the contralateral map show this increased divergence. These results support a role for EphA4 in establishing tonotopic projections in the auditory system, and further suggest a general role for Eph family proteins in establishing topographic maps in the nervous system.     

Frequency sensitivity of auditory neurons in the Caiman cochlear nucleus

Summary The characteristic frequencies of single auditory neurons in Caiman crocodilus (South American Alligator) range from 70 to 2,900 Hz. These neurons in the cochlear nuclei show a striking tonotopic organization which parallels that in birds. The sensitivity curve of all neurons and the number of neurons in each frequency range show features similar to those of birds and mammals.Supported by NSF. grant GB 5697. I thank Dr. Mark Konishi for overseeing this work.     

Conductance and block of hair-cell mechanotransducer channels in transmembrane channel–like protein mutants

Transmembrane channel–like (TMC) proteins TMC1 and TMC2 are crucial to the function of the mechanotransducer (MT) channel of inner ear hair cells, but their precise function has been controversial. To provide more insight, we characterized single MT channels in cochlear hair cells from wild-type mice and mice with mutations in Tmc1, Tmc2, or both. Channels were recorded in whole-cell mode after tip link destruction with BAPTA or after attenuating the MT current with GsMTx-4, a peptide toxin we found to block the channels with high affinity. In both cases, the MT channels in outer hair cells (OHCs) of wild-type mice displayed a tonotopic gradient in conductance, with channels from the cochlear base having a conductance (110 pS) nearly twice that of those at the apex (62 pS). This gradient was absent, with channels at both cochlear locations having similar small conductances, with two different Tmc1 mutations. The conductance of MT channels in inner hair cells was invariant with cochlear location but, as in OHCs, was reduced in either Tmc1 mutant. The gradient of OHC conductance also disappeared in Tmc1/Tmc2 double mutants, in which a mechanically sensitive current could be activated by anomalous negative displacements of the hair bundle. This “reversed stimulus–polarity” current was seen with two different Tmc1/Tmc2 double mutants, and with Tmc1/Tmc2/Tmc3 triple mutants, and had a pharmacological sensitivity comparable to that of native MT currents for most antagonists, except dihydrostreptomycin, for which the affinity was less, and for curare, which exhibited incomplete block. The existence in the Tmc1/Tmc2 double mutants of MT channels with most properties resembling those of wild-type channels indicates that proteins other than TMCs must be part of the channel pore. We suggest that an external vestibule of the MT channel may partly account for the channel’s large unitary conductance, high Ca²⁺ permeability, and pharmacological profile, and that this vestibule is disrupted in Tmc mutants.     

Tonotopic gradients of Eph family proteins in the chick nucleus laminaris during synaptogenesis

Topographically precise projections are established early in neural development. One such topographically organized network is the auditory brainstem. In the chick, the auditory nerve transmits auditory information from the cochlea to nucleus magnocellularis (NM). NM in turn innervates nucleus laminaris (NL) bilaterally. These projections preserve the tonotopy established at the level of the cochlea. We have begun to examine the expression of Eph family proteins during the formation of these connections. Optical density measurements were used to describe gradients of Eph proteins along the tonotopic axis of NL in the neuropil, the somata, and the NM axons innervating NL at embryonic day 10, when synaptic connections from NM to NL are established. At E10-11, NL dorsal neuropil expresses EphA4 at a higher concentration in regions encoding high frequency sounds, decreasing in concentration monotonically toward the low frequency (caudolateral) end. In the somata, both EphA4 and ephrin-B2 are concentrated at the high frequency end of the nucleus. These tonotopic gradients disappear between E13 and E15, and expression of these molecules is completely downregulated by hatching. The E10-11 patterns run counter to an apparent gradient in dendrite density, as indicated by microtubule associated protein 2 (MAP2) immunolabeling. Finally, ephrin-B2 is also expressed in a gradient in tissue ventral to the NL neuropil. Our findings thus suggest a possible conserved mechanism for establishing topographic projections in diverse sensory systems. These results of this study provide a basis for the functional examination of the role of Eph proteins in the formation of tonotopic maps in the brainstem.     

CDK5 interacts with Slo and affects its surface expression and kinetics through direct phosphorylation

Large-conductance calcium-activated potassium (BK) channels are ubiquitous and play an important role in a number of diseases. In hair cells of the ear, they play a critical role in electrical tuning, a mechanism of frequency discrimination. These channels show variable kinetics and expression along the tonotopic axis. Although the molecular underpinnings to its function in hair cells are poorly understood, it is established that BK channels consist of a pore-forming α-subunit (Slo) and a number of accessory subunits. Here we identify CDK5, a member of the cyclin-dependent kinase family, as an interacting partner of Slo. We show CDK5 to be present in hair cells and expressed in high concentrations in the cuticular plate and in the circumferential zone. In human embryonic kidney cells, we show that CDK5 inhibits surface expression of Slo by direct phosphorylation of Slo. Similarly, we note that CDK5 affects Slo voltage activation and deactivation kinetics, by a direct phosphorylation of T847. Taken together with its increasing expression along the tonotopic axis, these data suggest that CDK5 likely plays a critical role in electrical tuning and surface expression of Slo in hair cells.     

Modeling Hair Cell Tuning by Expression Gradients of Potassium Channel β Subunits

The receptor potential of sensory hair cells arises from the gating of mechanosensitive cation channels, but its amplitude and time course also depend on the number and kinetics of voltage-gated ion channels in each cell. Prominent among these are “BK” potassium channels encoded by the slo gene that support electrical tuning in some hair cells. Hair cells tuned to low frequencies have slowly gating BK channels, whereas those of higher-frequency hair cells gate more rapidly. Alternative splicing of the slo gene mRNA that encodes the pore-forming α subunit can alter BK channel kinetics, and gating is dramatically slowed by coexpression with modulatory β subunits. The effect of the β subunit is consistent with low-frequency tuning, and β mRNA is expressed at highest levels in the low frequency apex of the bird’s auditory epithelium. How might an expression gradient of β subunits contribute to hair cell tuning? The present work uses a computational model of hair cell-tuning based on the functional properties of BK channels expressed from hair cell α and βslo cDNA. The model reveals that a limited tonotopic gradient could be achieved simply by altering the fraction of BK channels in each hair cell that are combined with β subunits. However, complete coverage of the tuning spectrum requires kinetic variants in addition to those modeled here.     

Lack of Bdnf and TrkB signalling in the postnatal cochlea leads to a spatial reshaping of innervation along the tonotopic axis and hearing loss

Members of the neurotrophin gene family and their high-affinity Trk receptors control innervation of the cochlea during embryonic development. Lack of neurotrophin signalling in the cochlea has been well documented for early postnatal animals, resulting in a loss of cochlear sensory neurones and a region-specific reduction of target innervation along the tonotopic axis. However, how reduced neurotrophin signalling affects the innervation of the mature cochlea is currently unknown. Here, we have analysed the consequences of a lack of the TrkB receptor and its ligand, the neurotrophin brain-derived neurotrophic factor (Bdnf), in the late postnatal or adult cochlea using mouse mutants. During early postnatal development, mutant animals show a lack of afferent innervation of outer hair cells in the apical part of the cochlea, whereas nerve fibres in the basal part are maintained. Strikingly, this phenotype is reversed during subsequent maturation of the cochlea, which results in a normal pattern of outer hair cell innervation in the apex and loss of nerve fibres at the base in adult mutants. Measurements of auditory brain stem responses of these mice revealed a significant hearing loss. The observed innervation patterns correlate with opposing gradients of Bdnf and Nt3 expression in cochlear neurones along the tonotopic axis. Thus, the reshaping of innervation may be controlled by autocrine signalling between neurotrophins and their receptors in cochlear neurones. Our results indicate a substantial potential for re-innervation processes in the mature cochlea, which may also be of relevance for treatment of hearing loss in humans.     

Expression of α and β parvalbumin is differentially regulated in the rat organ of corti during development

The expression of two calcium‐binding proteins of the parvalbumin (PV) family, the α isoform (αPV) and the β isoform known as oncomodulin (OM), was investigated in the rat cochlea during postnatal development and related to cholinergic efferent innervation. Using RT‐PCR analysis, we found that OM expression begins between postnatal day 2 (P2) and P4, and peaks as early as P10, while αPV mRNA begins expression before birth and remains highly expressed into the adult period. Both in situ hybridization and immunoreactivity confirm that OM is uniquely expressed by the outer hair cells (OHCs) in the rat cochlea and occurs after efferent innervation along the cochlear spiral between P2 and P4. In contrast to OM expression, αPV immunoreactivity is expressed in both inner hair cells (IHCs) and OHCs at birth. Following olivocochlear efferent innervation, OHCs demonstrate weak OM immunoreactivity beginning at P5 and diminished αPV immunoreactivity after P10. In organ cultures isolated prior to the efferent innervation of OHCs, OM immunoreactivity failed to develop in OHCs, but αPV immunoreactivity remained present in both IHCs and OHCs. In contrast, organ cultures isolated after efferent innervation of OHCs show OHCs with low levels of OM immunoreactivity and high levels of αPV immunoreactivity. This study suggests that OM and αPV are differentially regulated in OHCs during cochlear development. Our findings further raise the possibility that the expression of PV proteins in OHCs may be influenced by efferent innervation. © 2003 Wiley Periodicals, Inc. J Neurobiol 58: 479–492, 2004     

Expression analysis and tissue distribution of two 14-3-3 proteins in silkworm (Bombyx mori)

14-3-3 proteins, which have been identified in a wide variety of eukaryotes, are highly conserved acidic proteins. In this study, we identified two genes in silkworm that encode 14-3-3 proteins (Bm14-3-3ζ and Bm14-3-3ε). Category of two 14-3-3 proteins was identified according to phylogenetic analysis. Bm14-3-3ζ shared 90% identity with that in Drosophila, while Bm14-3-3ε shared 86% identity with that in Drosophila. According to Western blot and real time PCR analysis, the Bm14-3-3ζ expression levels are higher than Bm14-3-3ε in seven tissues and in four silkworm developmental stages examined. Bm14-3-3ζ was expressed during every stage of silkworm and in every tissue of the fifth instar larvae that was examined, but Bm14-3-3ε expression was not detected in eggs or heads of the fifth instar larvae. Both 14-3-3 proteins were highly expressed in silk glands. These results suggest that Bm14-3-3ζ expression is universal and continuous, while Bm14-3-3ε expression is tissue and stage-specific. Based on tissue expression patterns and the known functions of 14-3-3 proteins, it may be that both 14-3-3 proteins are involved in the regulation of gene expression in silkworm silk glands.     

Molecular characterization of anion exchangers in the cochlea

Anion exchange proteins (AE) in the inner ear have been the focus of attention for some time. They have been suggested to play a role as anion exchangers for the regulation of endolymphatic pH or as anion exchangers and anchor proteins for the maintenance of the shape and turgor of outer hair cells, and they also have been discussed as a candidate protein for motile hair cell responses that follow high-frequency stimulation. The existence of anion exchangers in hair cells and the specific isoforms which are expressed in hair cells and the organ of Corti is controversial. Using a polyclonal antibody to AE1 (AB1992, Chemicon), we immunoprecipitated a 100 kDa AE polypeptide in isolated outer hair cells which, due to its glycosylation, is comprised of AE2 than AE1 isoforms. We confirmed AE2 expression in outer hair cells with the help of subtype-specific monoclonal and polyclonal antibodies to AE, AE subtype-specific primers and AE subtype-specific cDNA and found glycosylated truncated as well as full-length AE2 isoforms. No AE1 or AE3 subtypes were noted in outer hair cells. In contrast, AE2 and AE3 but not AE1 subtypes were seen in supporting cells of the organ of Corti. Their expression preceded the development of cochlear function, coincident with the establishment of the endocochlear potential and the differentiation of supporting cells. While most developmental processes in the inner ear usually begin in the basal cochlear turn, the AE2 expression in outer hair cells (but not that of AE2 and AE3 in supporting cells) progressed from the apical to the basal cochlear turn, reminiscent of the maturation of frequency-dependency. Irrespective of their presumed individual role as either anion exchanger, anchor protein or motility protein, the differential expression and developmental profile of these proteins suggest a most important role of anion exchange proteins in the development of normal hearing. These findings may also provide novel insights into AE function in general.     

<Emphasis Type="Italic">Lmx1a</Emphasis> is required for segregation of sensory epithelia and normal ear histogenesis and morphogenesis

At embryonic day 8.5, the LIM-homeodomain factor Lmx1a is expressed throughout the otic placode but becomes developmentally restricted to non-sensory epithelia of the ear (endolymphatic duct, ductus reuniens, cochlea lateral wall). We confirm here that the ears of newborn dreher (Lmx1a ^dr) mutants are dysmorphic. Hair cell markers such as Atoh1 and Myo7 reveal, for the first time, that newborn Lmx1a mutants have only three sensory epithelia: two enlarged canal cristae and one fused epithelium comprising an amalgamation of the cochlea, saccule, and utricle (a “cochlear-gravistatic” endorgan). The enlarged anterior canal crista develops by fusion of horizontal and anterior crista, whereas the posterior crista fuses with an enlarged papilla neglecta that may extend into the cochlear lateral wall. In the fused endorgan, the cochlear region is distinguished from the vestibular region by markers such as Gata3, the presence of a tectorial membrane, and cochlea-specific innervation. The cochlea-like apex displays minor disorganization of the hair and supporting cells. This contrasts with the basal half of the cochlear region, which shows a vestibular epithelium-like organization of hair cells and supporting cells. The dismorphic features of the cochlea are also reflected in altered gene expression patterns. Fgf8 expression expands from inner hair cells in the apex to most hair cells in the base. Two supporting cell marker proteins, Sox2 and Prox1, also differ in their cellular distribution between the base and the apex. Sox2 expression expands in mutant canal cristae prior to their enlargement and fusion and displays a more diffuse and widespread expression in the base of the cochlear region, whereas Prox1 is not detected in the base. These changes in Sox2 and Prox1 expression suggest that Lmx1a expression restricts and sharpens Sox2 expression, thereby defining non-sensory and sensory epithelium. The adult Lmx1a mutant organ of Corti shows a loss of cochlear hair cells, suggesting that the long-term maintenance of hair cells is also disrupted in these mutants. This work was supported by grants from the NCRR/COBRE (P20 RR 018788; D.H.N.) and NIH (RO1 DC 005590; B.F.). Parts of this investigation were conducted in a facility constructed with support of a Research Facilities Improvement Program Grant from the National Center for Research Resources, National Institutes of Health. We acknowledge the use of the confocal microscope facility of the NCCB, supported by EPSCoR EPS-0346476 (CFD 47.076), and of the University of Nebraska microarray facility, supported by NCRR/COBRE.     

Transient expression of the t-isoform of plastins/fimbrin in the stereocilia of developing auditory hair cells

The transduction of auditory signals by cochlear hair cells depends upon the integrity of hair cell stereociliary bundles. Stereocilia contain a central core of actin filaments, cross-linked by actin bundling proteins. In the cochlea, the two proteins described to date as responsible for the spatial arrangement of actin filaments in sterocilia are fimbrin and the recently discovered espin. Fimbrin (the chick homolog of human I-plastin) belongs to the plastins/fimbrin family that includes two additional isoforms of plastins, T- and L-plastin. In the present study, we used isoform specific antibodies to investigate the presence of the T- and L-isoforms of plastin/fimbrin in the adult and developing rat cochlea. We found that T-plastin, but not L-plastin, is expressed in the rat cochlea. During postnatal development of the rat organ of Corti, T-plastin can be detected in the core of stereocilia from early stages of hair cell differentiation, and its expression gradually increases in stereocilia as hair cells mature. However, as opposed to other actin-binding proteins expressed in stereocilia, T-plastin is absent from the stereocilia of mature hair cells. Such temporally restricted expression strengthens the idea of functional differences between plastins isoforms, and suggests that T-plastin could have a specific role in stereocilia formation.     

Basic maps in the auditory midbrain

Topologic maps at consecutive levels of sensory pathways indicate behaviorally relevant features of stimuli at increasing degrees of complexity. In the auditory system, except for tonotopic maps, the nature of represented features is unknown. In a model analogous to visual map formation we show that in the auditory midbrain, layers of neurons with preference to the same frequency (isofrequency planes) may hold maps of two basic, mutually orthogonal parameters--instantaneous amplitude and phase--of basilar membrane displacement at the cochlear location responding to that frequency. The proposed neural tuning to frequency, amplitude, and phase implies that sound is transformed into specific temporal trajectories of neural activation, with consequences for experimental design and interpretation of neural response behavior.     

Expression of neurotrophins and Trk receptors in the developing,adult, and regenerating avian cochlea

We studied the expression of neurotrophins and their Trk receptors in the chicken cochlea. Based on in situ hybridization, brain-derived neurotrophic factor (BDNF) is the major neurotrophin there, in contrast to the mammalian cochlea, where neurotrophin-3 (NT-3) predominates. NT-3 mRNA labeling was weak and found only during a short time period in the early cochles. During embryogenesis, BDNF mRNA was first seen in early differentiating hair cells. Afferent cochlear neurons expressed trkB mRNA from the early stages of gangliogenesis onward. In accordance, in vitro, BDNF promoted survival of dissociated neurons and stimulated neuritogenesis from ganglionic explants. High levels of BDNF mRNA in hair cells and trkB mRNA in cochlear neurons persisted in the mature cochlea. In addition, mRNA for the truncated TrkB receptor was expressed in nonneuronal cells, specifically in supporting cells, located adjacent to the site of BDNF synthesis and nerve endings. Following acoustic trauma, regenerated hair cells acquired BDNF mRNA expression at early stages of differentiation. Truncated trkB mRNA was lost from supporting cells that regenerated into hair cells. High levels of BDNF mRNA persisted in surviving hair cells and trkB mRNA in cochlear neurons after noise exposure. These results suggest that in the avian cochlea, peripheral target-derived BDNF contributes to the onset and maintenance of hearing function by supporting neuronal survival and regulating the (re)innervation process. Truncated TrkB receptors may regulate the BDNF concentration available to neurites, and they might have an important role during reinnervation. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 1019–1033, 1997     

The role of Math1 in inner ear development: Uncoupling the establishment of the sensory primordium from hair cell fate determination

During embryonic development of the inner ear, the sensory primordium that gives rise to the organ of Corti from within the cochlear epithelium is patterned into a stereotyped array of inner and outer sensory hair cells separated from each other by non-sensory supporting cells. Math1, a close homolog of the Drosophila proneural gene atonal, has been found to be both necessary and sufficient for the production of hair cells in the mouse inner ear. Our results indicate that Math1 is not required to establish the postmitotic sensory primordium from which the cells of the organ of Corti arise, but instead is limited to a role in the selection and/or differentiation of sensory hair cells from within the established primordium. This is based on the observation that Math1 is only expressed after the appearance of a zone of non-proliferating cells that delineates the sensory primordium within the cochlear anlage. The expression of Math1 is limited to a subpopulation of cells within the sensory primordium that appear to differentiate exclusively into hair cells as the sensory epithelium matures and elongates through a process that probably involves radial intercalation of cells. Furthermore, mutation of Math1 does not affect the establishment of this postmitotic sensory primordium, even though the subsequent generation of hair cells is blocked in these mutants. Finally, in Math1 mutant embryos, a subpopulation of the cells within the sensory epithelium undergo apoptosis in a temporal gradient similar to the basal-to-apical gradient of hair cell differentiation that occurs in the cochlea of wild-type animals.     

Expression of alpha and beta parvalbumin is differentially regulated in the rat organ of corti during development

The expression of two calcium-binding proteins of the parvalbumin (PV) family, the alpha isoform (alphaPV) and the beta isoform known as oncomodulin (OM), was investigated in the rat cochlea during postnatal development and related to cholinergic efferent innervation. Using RT-PCR analysis, we found that OM expression begins between postnatal day 2 (P2) and P4, and peaks as early as P10, while alphaPV mRNA begins expression before birth and remains highly expressed into the adult period. Both in situ hybridization and immunoreactivity confirm that OM is uniquely expressed by the outer hair cells (OHCs) in the rat cochlea and occurs after efferent innervation along the cochlear spiral between P2 and P4. In contrast to OM expression, alphaPV immunoreactivity is expressed in both inner hair cells (IHCs) and OHCs at birth. Following olivocochlear efferent innervation, OHCs demonstrate weak OM immunoreactivity beginning at P5 and diminished alphaPV immunoreactivity after P10. In organ cultures isolated prior to the efferent innervation of OHCs, OM immunoreactivity failed to develop in OHCs, but alphaPV immunoreactivity remained present in both IHCs and OHCs. In contrast, organ cultures isolated after efferent innervation of OHCs show OHCs with low levels of OM immunoreactivity and high levels of alphaPV immunoreactivity. This study suggests that OM and alphaPV are differentially regulated in OHCs during cochlear development. Our findings further raise the possibility that the expression of PV proteins in OHCs may be influenced by efferent innervation.