Retinoic acid synthesis and autoregulation mediate zonal patterning of vestibular organs and inner ear morphogenesis.

Retinoic acid (RA), a vitamin A (retinol) derivative, has pleiotropic functions during embryonic development. The synthesis of RA requires two enzymatic reactions: oxidation of retinol into retinaldehyde by alcohol dehydrogenases (ADHs) or retinol dehydrogenases (RDHs); and oxidation of retinaldehyde into RA by aldehyde dehydrogenases family 1, subfamily A (ALDH1as), such as ALDH1a1, ALDH1a2 and ALDH1a3. Levels of RA in tissues are regulated by spatiotemporal expression patterns of genes encoding RA-synthesizing and -degrading enzymes, such as cytochrome P450 26 (Cyp26 genes). Here, we show that RDH10 is important for both sensory and non-sensory formation of the vestibule of the inner ear. Mice deficient in Rdh10 exhibit failure of utricle-saccule separation, otoconial formation and zonal patterning of vestibular sensory organs. These phenotypes are similar to those of Aldh1a3 knockouts, and the sensory phenotype is complementary to that of Cyp26b1 knockouts. Together, these results demonstrate that RDH10 and ALDH1a3 are the key RA-synthesis enzymes involved in vestibular development. Furthermore, we discovered that RA induces Cyp26b1 expression in the developing vestibular sensory organs, which generates the differential RA signaling required for zonal patterning.

Genetic interactions support an inhibitory relationship between bone morphogenetic protein 2 and netrin 1 during semicircular canal formation.

The semicircular canals of the mammalian inner ear are derived from epithelial pouches in which epithelial cells in the central region of each pouch undergo resorption, leaving behind the region at the rim to form a tube-shaped canal. Lack of proliferation at the rim and/or over-clearing of epithelial cells in the center of the pouch can obliterate canal formation. Otic-specific knockout of bone morphogenetic protein 2 (Bmp2) results in absence of all three semicircular canals; however, the common crus and ampullae housing the sensory tissue (crista) are intact. The lack of Bmp2 causes Ntn1 (which encodes netrin 1), which is required for canal resorption, to be ectopically expressed at the canal rim. Ectopic Ntn1 results in reduction of Dlx5 and Lmo4, which are required for rim formation. These phenotypes can be partially rescued by removing one allele of Ntn1 in the Bmp2 mutants, indicating that Bmp2 normally negatively regulates Ntn1 for canal formation. Additionally, non-resorption of the canal pouch in Ntn1-/- mutants is partially rescued by removing one allele of Bmp2 Thus, reciprocal inhibition between Bmp2 and netrin 1 is involved in canal formation of the vestibule.

Fgf10 is required for specification of non-sensory regions of the cochlear epithelium.

The vertebrate inner ear is a morphologically complex sensory organ comprised of two compartments, the dorsal vestibular apparatus and the ventral cochlear duct, required for motion and sound detection, respectively. Fgf10, in addition to Fgf3, is necessary for the earliest stage of otic placode induction, but continued expression of Fgf10 in the developing otic epithelium, including the prosensory domain and later in Kolliker׳s organ, suggests additional roles for this gene during morphogenesis of the labyrinth. While loss of Fgf10 was implicated previously in semicircular canal agenesis, we show that Fgf10(-/+) embryos also exhibit a reduction or absence of the posterior semicircular canal, revealing a dosage-sensitive requirement for FGF10 in vestibular development. In addition, we show that Fgf10(-/-) embryos have previously unappreciated defects of cochlear morphogenesis, including a somewhat shortened duct, and, surprisingly, a substantially narrower duct. The mutant cochlear epithelium lacks Reissner׳s membrane and a large portion of the outer sulcus-two non-contiguous, non-sensory domains. Marker gene analyses revealed effects on Reissner׳s membrane as early as E12.5-E13.5 and on the outer sulcus by E15.5, stages when Fgf10 is expressed in close proximity to Fgfr2b, but these effects were not accompanied by changes in epithelial cell proliferation or death. These data indicate a dual role for Fgf10 in cochlear development: to regulate outgrowth of the duct and subsequently as a bidirectional signal that sequentially specifies Reissner׳s membrane and outer sulcus non-sensory domains. These findings may help to explain the hearing loss sometimes observed in LADD syndrome subjects with FGF10 mutations.

Responses to cell loss become restricted as the supporting cells in mammalian vestibular organs grow thick junctional actin bands that develop high stability.

Sensory hair cell (HC) loss is a major cause of permanent hearing and balance impairments for humans and other mammals. Yet, fish, amphibians, reptiles, and birds readily replace HCs and recover from such sensory deficits. It is unknown what prevents replacement in mammals, but cell replacement capacity declines contemporaneously with massive postnatal thickening of F-actin bands at the junctions between vestibular supporting cells (SCs). In non-mammals, SCs can give rise to regenerated HCs, and the bands remain thin even in adults. Here we investigated the stability of the F-actin bands between SCs in ears from chickens and mice and Madin-Darby canine kidney cells. Pharmacological experiments and fluorescence recovery after photobleaching (FRAP) of SC junctions in utricles from mice that express a γ-actin-GFP fusion protein showed that the thickening F-actin bands develop increased resistance to depolymerization and exceptional stability that parallels a sharp decline in the cell replacement capacity of the maturing mammalian ear. The FRAP recovery rate and the mobile fraction of γ-actin-GFP both decreased as the bands thickened with age and became highly stabilized. In utricles from neonatal mice, time-lapse recordings in the vicinity of dying HCs showed that numerous SCs change shape and organize multicellular actin purse strings that reseal the epithelium. In contrast, adult SCs appeared resistant to deformation, with resealing responses limited to just a few neighboring SCs that did not form purse strings. The exceptional stability of the uniquely thick F-actin bands at the junctions of mature SCs may play an important role in restricting dynamic repair responses in mammalian vestibular epithelia.

Vestibular development in marsupials and monotremes.

The young of marsupials and monotremes are all born in an immature state, followed by prolonged nurturing by maternal lactation in either a pouch or nest. Nevertheless, the level of locomotor ability required for newborn marsupials and monotremes to reach the safety of the pouch or nest varies considerably: some are transferred to the pouch or nest in an egg (monotremes); others are transferred passively by gravity (e.g. dasyurid marsupials); some have only a horizontal wriggle to make (e.g. peramelid and didelphid marsupials); and others must climb vertically for a long distance to reach the maternal pouch (e.g. diprotodontid marsupials). In the present study, archived sections of the inner ear and hindbrain held in the Bolk, Hill and Hubrecht collections at the Museum für Naturkunde, Berlin, were used to test the relationship between structural maturity of the vestibular apparatus and the locomotor challenges that face the young of these different mammalian groups. A system for staging different levels of structural maturity of the vestibular apparatus was applied to the embryos, pouch young and hatchlings, and correlated with somatic size as indicated by greatest body length. Dasyurids are born at the most immature state, with the vestibular apparatus at little more than the otocyst stage. Peramelids are born with the vestibular apparatus at a more mature state (fully developed semicircular ducts and a ductus reuniens forming between the cochlear duct and saccule, but no semicircular canals). Diprotodontids and monotremes are born with the vestibular apparatus at the most mature state for the non-eutherians (semicircular canals formed, maculae present, but vestibular nuclei in the brainstem not yet differentiated). Monotremes and marsupials reach the later stages of vestibular apparatus development at mean body lengths that lie within the range of those found for laboratory rodents (mouse and rat) reaching the same vestibular stage.

Cell type-specific transcriptome analysis reveals a major role for Zeb1 and miR-200b in mouse inner ear morphogenesis.

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

Analysis of miR-376 RNA cluster members in the mouse inner ear.

Mutations in phosphoribosyl pyrophosphate synthetase 1 (PRPS1) are associated with a spectrum of non-syndromic to syndromic hearing loss. PRPS1 transcript levels have been shown to be regulated by the microRNA-376 genes. The long primary RNA transcript of the miR-376 RNA cluster members undergo extensive and simultaneous A → I editing at one or both of two specific sites (+4 and +44) in particular human and mouse tissues. The PRPS1 gene, which contains target sites for the edited version of miR-376a-5p within its 3'UTR, has been shown to be repressed in a tissue-specific manner. To investigate whether the transcription of Prps1 is regulated by miR-376 cluster members in the mouse inner ear, we first quantified the expression of the mature miR-376 RNAs by quantitative real-time-PCR. The spatio-temporal patterns of miR-376 expression were assessed by in situ hybridization. Finally, we examined whether A →I editing of pri-miR-376 RNAs occurs in mouse inner ear by direct sequencing. Our data showed that the miR-376a-3p, b-3p, c-3p are present in mouse embryonic inner ears and intensive expression of miR-376a-3p/b-3p was detected in the sensory epithelia and ganglia of both auditory and vestibular portions of the inner ear. In adult inner ear, the expression of miR-376a-3p/b-3p is restricted within ganglion neurons of auditory and vestibular systems as well as the cells in the stria vascularis. Only unedited pri-miR-376 RNAs were detected in the cochlea suggesting that the activity of PRPS1 in the inner ear may not be regulated through the editing of miR-376 cluster.

MicroRNAs are essential for development and function of inner ear hair cells in vertebrates.

MicroRNAs (miRNAs) inhibit the translation of target mRNAs and affect, directly or indirectly, the expression of a large portion of the protein-coding genes. This study focuses on miRNAs that are expressed in the mouse cochlea and vestibule, the 2 inner ear compartments. A conditional knock-out mouse for Dicer1 demonstrated that miRNAs are crucial for postnatal survival of functional hair cells of the inner ear. We identified miRNAs that have a role in the vertebrate developing inner ear by combining miRNA transcriptome analysis, spatial and temporal expression patterns, and bioinformatics. Microarrays revealed similar miRNA profiles in newborn-mouse whole cochleae and vestibules, but different temporal and spatial expression patterns of six miRNAs (miR-15a, miR-18a, miR-30b, miR-99a, miR-182, and miR-199a) may reflect their roles. Two of these miRNAs, miR-15a-1 and miR-18a, were also shown to be crucial for zebrafish inner ear development and morphogenesis. To suggest putative target mRNAs whose translation may be inhibited by selected miRNAs, we combined bioinformatics-based predictions and mRNA expression data. Finally, we present indirect evidence that Slc12a2, Cldn12, and Bdnf mRNAs may be targets for miR-15a. Our data support the hypothesis that inner ear tissue differentiation and maintenance are regulated and controlled by conserved sets of cell-specific miRNAs in both mouse and zebrafish.

Pivotal role of hmx2 and hmx3 in zebrafish inner ear and lateral line development.

hmx2 (nkx5.2) and hmx3 (nkx5.1) are highly conserved homeobox transcription factors required for mouse inner ear development. We have identified four hmx genes that are expressed in developing mechanosensory organs in zebrafish. Knockdown of both hmx2 and hmx3 disrupts formation of the mechanosensory neuromasts and also leads to impaired vestibular function in which utricular maculae fail to develop and the utricular otolith gradually fuses with the saccular otolith. We demonstrate that pax5, known to be required for development of the utricular maculae, is expressed downstream of hmx2 and hmx3. In addition, we show that FGF signaling regulates expression of hmx2 and hmx3 in the otic vesicle, and conversely, hmx2 and hmx3 maintain the expression of fgf ligands, thus revealing a novel tissue-specific feedback mechanism. Our data suggest that hmx2 and hmx3 act as cell autonomous factors required redundantly for cell fate specification and differentiation during inner ear and lateral line development.

Exposure to hypergravity during specific developmental periods differentially affects metabolism and vestibular reactions in adult C57BL /6j mice.

The development of the posturo-motor control of movement is conditioned by Earth's gravity. Missing or altered gravity during the critical periods of development delays development and induces durable changes in the vestibular, cerebellar, or muscular structures, but these are not consistently mirrored at a functional level. The differences in the time schedule of vestibular and motor development could contribute to this inconstancy. To investigate the influence of gravity on the development of vestibular and locomotor functions, we analysed the performance of adult mice subjected to hypergravity during the time covering either the vestibular or locomotor development. The mice were centrifuged at 2 g from embryonic day (E) 0 to postnatal day (P) 10 (PRE), from P10 to P30 (POST), from E0 to P30 (FULL), and from E7 to P21. Their muscular force, anxiety level, vestibular reactions, and aerobic capacity during treadmill training were then evaluated at the age of 2 and 6 months. The performance of young adults varied in relation to the period of exposure to hypergravity. The mice that acquired locomotion in hypergravity (POST and FULL) showed a lower forelimb force and delayed vestibular reactions. The mice centrifuged from conception to P10 (PRE) showed a higher aerobic capacity during treadmill training. The differences in muscular force and vestibular reactions regressed with age, but the metabolic changes persisted. These results confirmed that early exposure to hypergravity induces qualitative changes depending on the period of exposure. They validated, at a functional level, the existence of several critical periods for adaptation to gravity.

Bmp4 is essential for the formation of the vestibular apparatus that detects angular head movements.

Angular head movements in vertebrates are detected by the three semicircular canals of the inner ear and their associated sensory tissues, the cristae. Bone morphogenetic protein 4 (Bmp4), a member of the Transforming growth factor family (TGF-beta), is conservatively expressed in the developing cristae in several species, including zebrafish, frog, chicken, and mouse. Using mouse models in which Bmp4 is conditionally deleted within the inner ear, as well as chicken models in which Bmp signaling is knocked down specifically in the cristae, we show that Bmp4 is essential for the formation of all three cristae and their associated canals. Our results indicate that Bmp4 does not mediate the formation of sensory hair and supporting cells within the cristae by directly regulating genes required for prosensory development in the inner ear such as Serrate1 (Jagged1 in mouse), Fgf10, and Sox2. Instead, Bmp4 most likely mediates crista formation by regulating Lmo4 and Msx1 in the sensory region and Gata3, p75Ngfr, and Lmo4 in the non-sensory region of the crista, the septum cruciatum. In the canals, Bmp2 and Dlx5 are regulated by Bmp4, either directly or indirectly. Mechanisms involved in the formation of sensory organs of the vertebrate inner ear are thought to be analogous to those regulating sensory bristle formation in Drosophila. Our results suggest that, in comparison to sensory bristles, crista formation within the inner ear requires an additional step of sensory and non-sensory fate specification.

Lmx1a maintains proper neurogenic, sensory, and non-sensory domains in the mammalian inner ear.

Lmx1a is a LIM homeodomain-containing transcription factor, which is required for the formation of multiple organs. Lmx1a is broadly expressed in early stages of the developing inner ear, but its expression is soon restricted to the non-sensory regions of the developing ear. In an Lmx1a functional null mutant, dreher (dr(J)/dr(J)), the inner ears lack a non-sensory structure, the endolymphatic duct, and the membranous labyrinth is poorly developed. These phenotypes are consistent with Lmx1a's role as a selector gene. More importantly, while all three primary fates of the inner ear - neural, sensory, and non-sensory - are specified in dr(J)/dr(J), normal boundaries among these tissues are often violated. For example, the neurogenic domain of the ear epithelium, from which cells delaminate to form the cochleovestibular ganglion, is expanded. Within the neurogenic domain, the demarcation between the vestibular and auditory neurogenic domains is most likely disrupted as well, based on the increased numbers of vestibular neuroblasts and ectopic expression of Fgf3, which normally is associated specifically with the vestibular neurogenic region. Furthermore, aberrant and ectopic sensory organs are observed; most striking among these is vestibular-like hair cells located in the cochlear duct.

Pax2 and Pax8 cooperate in mouse inner ear morphogenesis and innervation.

BACKGROUND: Pax2;5;8 transcription factors play diverse roles in vertebrate and invertebrate organogenesis, including the development of the inner ear. Past research has suggested various cochlear defects and some vestibular defects in Pax2 null mice but the details of the cochlear defects and the interaction with other Pax family members in ear development remain unclear. RESULTS: We show that Pax2;8 double null mice do not develop an ear past the otocyst stage and show little to no sensory as well as limited and transient neuronal development, thus indicating that these two family members are essential for overall ear morphogenesis and sustained neurosensory development. In support of functional redundancy between Pax proteins, Pax2 can be substituted by a Pax5 minigene, a gene normally not expressed in the embryonic mouse ear. There is no detectable morphological defect in Pax8 null mice suggesting that Pax2 expression can compensate for Pax8. Conversely, Pax8 cannot compensate for Pax2 leading to a cochlear phenotype not fully appreciated previously: Cochlear development is delayed until E15.5 when the cochlea extrudes as a large sack into the brain case. Immunocytochemistry and tracing from the brain show that a cochlear spiral ganglia form as a small addition to the inferior vestibular ganglion. However, the empty cochlear sack, devoid of any sensory epithelium development as indicated by the absence of Sox2 or MyoVII expression, nevertheless develop a dense innervation network of small neurons situated in the wall of the cochlear sack. CONCLUSIONS: Combined these data suggest that Pax2 is needed for organ of Corti formation and is directly or indirectly involved in the coordination of spiral ganglion formation which is partially disrupted in the Pax2 null ears. All three Pax genes can signal redundantly in the ear with their function being determined primarily by the spatio-temporal expression driven by the three distinct promoters of these genes.

Glial but not neuronal development in the cochleo-vestibular ganglion requires Sox10.

The cochleo-vestibular ganglion contains neural crest-derived glial cells and sensory neurons that are derived from the neurogenic otic placode. Little is known about the molecular mechanisms that regulate the tightly orchestrated development of this structure. Here, we report that Sox10, a high-mobility group DNA-binding domain transcription factor that is required for the proper development of neural crest cell derivatives, is specifically expressed in post-migratory neural crest cells in the cochleo-vestibular ganglion. Using Sox10-deficient mice, we demonstrate that this transcription factor is essential for the survival, but not the generation, of the post-migratory neural crest cells within the inner ear. In the absence of these neural crest-derived cells, we have investigated the survival of the otocyst-derived auditory neurons. Surprisingly, auditory neuron differentiation, sensory target innervation and survival are conserved despite the absence of glial cells. Moreover, brain-derived neurotrophic factor expression is increased in the hair cells of Sox10-deficient mice, a compensatory mechanism that may prevent spiral ganglion neuronal cell death. Taken together, these data suggest that in the absence of neural crest-derived glial cells, an increase trophic support from hair cells promotes the survival of spiral ganglion neurons in Sox10 mutant mice.

Development of spontaneous activity and response properties of primary lagenar neurons in the chick.

Here, we report for the first time developmental changes in spontaneous activity and in response properties of single nerve fibers from the macular chick lagena. Such aspects are important in order to get insight into the functional role of the lagena which remains undetermined. For this purpose, we used intracellular and extracellular single-unit recording techniques in an isolated inner ear preparation from the chicken at ages E15 and P1. At E15, afferent fibers displayed a low irregular spontaneous discharge rate (41 +/- 14 spikes/s, CV = 1.17 +/- 0.1), which was replaced by regular high frequency spontaneous activity at P1 (CV = 0.48 +/- 0.8, 89 +/- 27 spikes/s). During the developmental period including E15, the percentage of silent neurons was 60% while that of P1 was 40%. The synaptic activity was higher at E15 than at P1. The action potential waveform generated at E15 had small amplitude and derivative depolarization, and consequently, a large duration in correlation with respect to action potential waveform at P1 (respectively: 53 +/- 2 vs. 65 +/- 3 mV, 60 +/- 11 vs. 109 +/- 20 mV/ms, 3.6 +/- 0.4 vs. 1.1 +/- 0.12 ms). In addition, we recognized two response dynamics to the injection of current steps: phasic, or rapidly adapting neurons and tonic, or slowly adapting neurons. Our results indicate similar developmental processes for the lagena as described for the vestibular system in other species, in agreement with the known morphological characteristics of this otholitic end organ. The presence of more than one subtype of afferent neuron also correlates with previous reports on vestibular afferents with analogous electrophysiological properties, strongly suggesting the vestibular nature of the lagena.

A symphony of inner ear developmental control genes.

The inner ear is one of the most complex and detailed organs in the vertebrate body and provides us with the priceless ability to hear and perceive linear and angular acceleration (hence maintain balance). The development and morphogenesis of the inner ear from an ectodermal thickening into distinct auditory and vestibular components depends upon precise temporally and spatially coordinated gene expression patterns and well orchestrated signaling cascades within the otic vesicle and upon cellular movements and interactions with surrounding tissues. Gene loss of function analysis in mice has identified homeobox genes along with other transcription and secreted factors as crucial regulators of inner ear morphogenesis and development. While otic induction seems dependent upon fibroblast growth factors, morphogenesis of the otic vesicle into the distinct vestibular and auditory components appears to be clearly dependent upon the activities of a number of homeobox transcription factors. The Pax2 paired-homeobox gene is crucial for the specification of the ventral otic vesicle derived auditory structures and the Dlx5 and Dlx6 homeobox genes play a major role in specification of the dorsally derived vestibular structures. Some Micro RNAs have also been recently identified which play a crucial role in the inner ear formation.

Migration and fate of vestibular melanocytes during the development of the human inner ear.

Melanocytes are present in various parts of the inner ear, including the stria vascularis in the cochlea and the dark cell areas in the vestibular organs, where they contribute to endolymph homeostasis. Developmental studies describing the distribution of vestibular melanocytes are scarce, especially in humans. In this study, we investigated the distribution and maturation of the vestibular melanocytes in relation to the developing dark cell epithelium in inner ear specimens from week 5 to week 14 of development and in surgical specimens of the adult ampulla. Vestibular melanocytes were located around the utricle and the ampullae of the semicircular canals before week 7 and were first seen underneath the transitional zones and dark cell areas between week 8 and week 10. At week 10, melanocytes made intimate contact with epithelial cells, interrupting the local basement membrane with their dendritic processes. At week 11, most melanocytes were positioned under the dark cell epithelia. No melanocytes were seen around or in the saccule during all investigated developmental stages. The dark cell areas gradually matured and showed an adult immunohistochemical profile of the characteristic ion transporter protein Na+ /K+ -ATPase α1 by week 14. Furthermore, we investigated the expression of the migration-related proteins ECAD, PCAD, KIT, and KITLG in melanocytes and dark cell epithelium. This is the first study to describe the spatiotemporal distribution of vestibular melanocytes during the human development and thereby contributes to understanding normal vestibular function and pathophysiological mechanisms underlying vestibular disorders.

Retinoic acid degradation shapes zonal development of vestibular organs and sensitivity to transient linear accelerations.

Each vestibular sensory epithelium in the inner ear is divided morphologically and physiologically into two zones, called the striola and extrastriola in otolith organ maculae, and the central and peripheral zones in semicircular canal cristae. We found that formation of striolar/central zones during embryogenesis requires Cytochrome P450 26b1 (Cyp26b1)-mediated degradation of retinoic acid (RA). In Cyp26b1 conditional knockout mice, formation of striolar/central zones is compromised, such that they resemble extrastriolar/peripheral zones in multiple features. Mutants have deficient vestibular evoked potential (VsEP) responses to jerk stimuli, head tremor and deficits in balance beam tests that are consistent with abnormal vestibular input, but normal vestibulo-ocular reflexes and apparently normal motor performance during swimming. Thus, degradation of RA during embryogenesis is required for formation of highly specialized regions of the vestibular sensory epithelia with specific functions in detecting head motions.

Spatial and temporal segregation of auditory and vestibular neurons in the otic placode.

The otic placode generates the auditory and vestibular sense organs and their afferent neurons; however, how auditory and vestibular fates are specified is unknown. We have generated a fate map of the otic placode and show that precursors for vestibular and auditory cells are regionally segregated in the otic epithelium. The anterior-lateral portion of the otic placode generates vestibular neurons, whereas the posterior-medial region gives rise to auditory neurons. Precursors for vestibular and auditory sense organs show the same distribution. Thus, different regions of the otic placode correspond to particular sense organs and their innervating neurons. Neurons from contiguous domains rarely intermingle suggesting that the regional organisation of the otic placode dictates positional cues to otic neurons. But, in addition, vestibular and cochlear neurogenesis also follows a stereotyped temporal pattern. Precursors from the anterior-lateral otic placode delaminate earlier than those from its medial-posterior portion. The expression of the proneural genes NeuroM and NeuroD reflects the sequence of neuroblast formation and differentiation. Both genes are transiently expressed in vestibular and then in cochlear neuroblasts, while differentiated neurons express Islet1, Tuj1 and TrkC, but not NeuroM or NeuroD. Together, our results indicate that the position of precursors within the otic placode confers identity to sensory organs and to the corresponding otic neurons. In addition, positional information is integrated with temporal cues that coordinate neurogenesis and sensory differentiation.

Visualization of Reissner membrane and the spiral ganglion in human fetal cochlea by micro-computed tomography.

PURPOSE: Although visualization of fine structures in the cochlea such as Reissner membrane (vestibular membrane) is important for elucidation of the mechanism and the establishment of therapy for inner ear diseases, they cannot be visualized by even the most advanced high-resolution medical computed tomography (CT) and magnetic resonance imaging. Visualization of Reissner membrane in dissected animals by micro-magnetic resonance imaging has been reported, but bone could not be visualized. We attempted to visualize human fetal Reissner membrane and the spiral ganglion by micro-focus x-ray CT (micro-CT), which has a spatial resolution several hundred times greater than the conventional medical CT. MATERIALS AND METHODS: Serial tomograms of a dissected pyramis, including the cochlea of human fetuses (stillborn specimens), were obtained by micro-CT, and 3-dimensional reconstruction was performed by a volume-rendering method. RESULTS: Clear tomograms (theoretical spatial resolution, 12.2 x 12.2 microm; slice thickness 77.5 microm) and 3-dimensional reconstructed images (theoretical spatial resolution, 6.8 x 6.8 microm; slice thickness, 40.0 microm) of Reissner membrane and the spiral ganglion with a bony labyrinth (cochlear bone) were successfully obtained for the first time. The thickness of Reissner membrane obtained by the tomogram was 12 microm, which corresponds to the optical macroscopic value from resin-embedded histologic sections. CONCLUSIONS: This study showed that micro-CT enables us to visualize the internal fine structure of the human cochlea. As the success rate of the visualization of Reissner membrane is not high, it is necessary to improve the image quality and contrast resolution of micro-CT to enable stable visualization of fine structures. The development of imaging equipment such as micro-CT for medical use should play an important role in the elucidation of the mechanism and the establishment of therapy for inner ear diseases.