Tectorial structures on the inner ear sensory epithelia of Proteus anguinus (Amphibia,caudata)

The tectorial structures of the inner ear of the proteid salamander Proteus anguinus were studied with transmission and scanning electron microscopy in order to analyze the ultrastructure of the otoconial membranes and otoconial masses of the maculae and the tectorial membrane of the papilla amphibiorum. Both otoconial and tectorial membranes consist of two parts: (1) a compact part and (2) a fibrillar part that joins the membrane with the sensory epithelium. Masses of otoconia occupy the lumina above these membranes. There are two types of calcium carbonate crystals in the otoconial masses within the inner ear of Proteus anguinus. The relatively small otoconial mass of the utricular macula occupies an area no greater than the diameter of the sensory epithelium, and it is composed of calcite crystals. On the other hand, the enormous otoconial masses of the saccular macula and the lagenar macula are composed of aragonite crystals. In the sacculus and lagena, globular structures 2–9 m?m in diameter were discovered on the lower surfaces of the otoconial masses above the sensory epithelia. These globules show a progression from smooth-surfaced, small globules to large globules with spongelike, rough surfaces. It is hypothesized that these globules are precursors of the aragonite crystals and that calcite crystals develop similarly in the utriculus. The presence of globular precursors in adult animals suggests that the formation of new crystals in the otoconial membranes of the sacculus and lagena of Proteus is a continuous, ongoing process.     

Hair Cell Polarization in the Flatfish Inner Ear

The hair cell polarization of the various sensory epithelia in the inner ear was examined in two species of flatfish, the Plaice (Pleuronectes platessa) and the Dab (Limanda limanda). The hair cells in the macula utriculi are polarized in the pattern usually seen for this macula in vertebrates. In the macula sacculi and macula lagenae the hair cell polarization is different from that hitherto described from bony fishes and other vertebrates. The polarization seen in these maculae in the flatfish explains their ability to sense movements in all directions, which is necessary if these sensory areas are the most important inner ear organs in the regulation of postural orientation.     

Development of the inner ear of the brown trout (Salmo trutta fario): I. Gross morphology and sensory cell proliferation

The gross development of the trout inner ear between embryonic and juvenile stages was studied by light microscopy. The otocyst has already formed in 3–4 mm embryos. The semicircular canals begin to separate from the utriculo-saccular cavity in 6 mm embryos, the anterior canal first, then the posterior and the horizontal canal later. The formation of the saccular cavity begins in 7 mm embryos, whereas that of the lagena occurs in 18 mm fry. The first macular primordia appear before the separation of cavities. The anterior and horizontal crests arise from the primordium of the utricular macula, and the posterior crest, macula lagena, and macula neglecta arise from that of the saccular macula. The macula lagena and macula neglecta appear later. The sensory areas of the labyrinth and the number of receptor cells grow continuously between the embryonic and juvenile stages. © 1993 Wiley-Liss, Inc.     

Ultrastructural study of the saccular epithelium of the inner ear of two teleosts, Oncorhynchus mykiss and Psetta maxima

The secretory cells and ionocytes of the saccular epithelium of the inner ear of trout (Oncorhynchus mykiss) and turbot (Psetta maxima) have been studied by electron microscopy. In these species, the saccular epithelium may be subdivided into four zones: the “macula”, the “meshwork area”, the “patches area”, and the “intermediate area”. In addition to the sensory “hair cells” and their supporting cells, the macula contains, at its periphery, “granular cells” that have the ultrastructural characteristics of secretory cells. The “meshwork area” around the macula contains large ionocytes endowed with pseudopods, many mitochondria, and three intracytoplasmic membrane systems (endoplasmic reticulum, tubular, and vesicular systems). The patches area, located at some distance from the macula, consists of groups of small mitochondria-rich ionocytes characterized by infoldings of their lateral plasma membrane. In the intermediate area, the size and organelle-content of cells decrease from the meshwork area to the patches area. There is no significant difference in cell composition or structure of the saccular epithelium between the trout and the turbot. The secreting cells might be involved in secretion of endolymph and formation of the otolith, whereas the ionocytes probably regulate the ionic composition of the endolymph.     

Dark hair cells in the inner ear of the rainbow trout. A study of the influence of different fixation methods

The occurrence of dark staining cells in different tissues has been suggested to be artefactual and caused during the fixation process. In inner ear sensory epithelia, dark hair cells (DHC) have been suggested to be apoptotic cells. We have examined whether dark cells represent dying cells or whether they are the results of fixation artefacts. The effects of buffer osmolarity and different fixation methods on the incidence of dark hair cells in the inner ear macula sacculi of the rainbow trout (Oncorhynchus mykiss) were investigated by light and electron microscopy. Glutaraldehyde in phosphate buffer with osmolarities of 0, 135, 225, 425, and 560 mosmol were used for fixation by immersion. For comparison, fixation by vascular perfusion as well as the effects of mechanical injury and delayed fixation were studied. DHC were found in all examined saccular maculae except for the delayed fixation protocol where almost all the sensory cells were lost. The number of DHC accounted for 2.5–12.9‰ of the sensory cells. Neither the buffer osmolarity nor the fixation method had significant effects on the frequencies of DHC. Mitotic cell division events were seen exclusively in the apical cell strata of the sensory epithelium. The DHC are suggested to be associated with apoptosis rather than fixation artefacts.     

The Notch ligand JAG1 is required for sensory progenitor development in the mammalian inner ear

In mammals, six separate sensory regions in the inner ear are essential for hearing and balance function. Each sensory region is made up of hair cells, which are the sensory cells, and their associated supporting cells, both arising from a common progenitor. Little is known about the molecular mechanisms that govern the development of these sensory organs. Notch signaling plays a pivotal role in the differentiation of hair cells and supporting cells by mediating lateral inhibition via the ligands Delta-like 1 and Jagged (JAG) 2. However, another Notch ligand, JAG1, is expressed early in the sensory patches prior to cell differentiation, indicating that there may be an earlier role for Notch signaling in sensory development in the ear. Here, using conditional gene targeting, we show that the Jag1 gene is required for the normal development of all six sensory organs within the inner ear. Cristae are completely lacking in Jag1-conditional knockout (cko) mutant inner ears, whereas the cochlea and utricle show partial sensory development. The saccular macula is present but malformed. Using SOX2 and p27^kip1 as molecular markers of the prosensory domain, we show that JAG1 is initially expressed in all the prosensory regions of the ear, but becomes down-regulated in the nascent organ of Corti by embryonic day 14.5, when the cells exit the cell cycle and differentiate. We also show that both SOX2 and p27^kip1 are down-regulated in Jag1-cko inner ears. Taken together, these data demonstrate that JAG1 is expressed early in the prosensory domains of both the cochlear and vestibular regions, and is required to maintain the normal expression levels of both SOX2 and p27^kip1. These data demonstrate that JAG1-mediated Notch signaling is essential during early development for establishing the prosensory regions of the inner ear.     

Sensory surface of the saccule and lagena in the ears of ostariophysan fishes

The inner ears of a few fishes in the teleost superorder Ostariophysi are structurally unlike those of most other teleosts. Scanning electron microscopy was used to determine if other ostariophysans share these unusual features. Examined were the families Cyprinidae, Characidae, and Gymnotidae (all of the series Otophysi), and Chanidae (of the sister series Anotophysi), representing the four major ostariophysan lineages, the auditory organs of which have not yet been well described. Among the Otophysi, the saccular and lagenar otolith organs are similar to those reported for other ostariophysans. The lagena is generally the larger of the two organs. The saccular sensory epithelium (macula) contains long ciliary bundles on the sensory hair cells in the caudal region, and short bundles in the rostral region. The saccule and the lagena each have hair cells organized into two groups having opposing directional orientations. In contrast, Chanos, the anotophysan, has a saccular otolith larger than the lagenar otolith, and ciliary bundles that are more uniform in size over most of its saccular macula. Most strikingly, its saccular macula has hair cells organized into groups oriented in four directions instead of two, in a pattern very similar to that in many nonostariophysan teleosts. We suggest that the bi-directional pattern seen consistently in the Otophysi is a derived development related to particular auditory capabilities of these species.     

Structure and Innervation of the Inner Ear Sensory Organs in an Otophysine Fish,the Upside–down Catfish (Synodontis nigriventrisDavid)

All the sensory epithelia of the inner ear in the upside–down catfish (Synodontis nigriventrisDavid) were examined by light microscopy. The morphology of the membranous labyrinth and the orientation of the hair cells is similar to what has been found in other otophysine fishes. The sensory cells are of variable size both inter– and intraepithelially; particularly the macula sacculi is equipped with heterogeneous receptors. Regional differences in the hair cell density are presented for all the otolith organs plus the papilla neglecta. Nerve stainings reveal regional differentiation. The central areas are innervated by stout and stubbly nerve endings intermingled with a few thin nerve fibres while the peripheral parts are reached exclusively by thin axons. In the anterior region of the macula sacculi are found unique cup–shaped axon terminations which surround the basal parts of a single or a few sensory cells. The number and diameter range of the myelinated nerve fibres as well as the hair cell/axon ratio are presented. Electron microscopy demonstrates the presence of unmyelinated axons in all inner ear nerve ramuli.     

The ultrastructure and innervation of the ear of the gar, Lepisosteus osseus

The endorgans of the inner ear of the gar were examined using transmission and scanning electron microscopy as well as nerve staining. The ultrastructure of the sensory hair cells and supporting cells of the gar ear are similar to cells in other bony fishes, whereas there are significant differences between the gar and other bony fishes in the orientations patterns of the sensory hair cells on the saccular and lagenar sensory epithelia. The saccular sensory epithelium has two regions, a main region and a secondary region ventral to the main region. The ciliary bundles on the main region are divided into two groups, one oriented dorsally and the other ventrally. Furthermore, as a result of curvature of the saccular sensory epithelium, the dorsal and ventral ciliary bundles on the rostral portion of the epithelium are rotated ninety degrees and are thus oriented on the animal's rostro-caudal axis. Hair cells on the secondary region are generally oriented ventrally. The lagenar epithelium has three groups of sensory hair cells. The groups on the rostral and caudal ends of the macula are oriented dorsally, whereas the middle group is oriented ventrally. Hair cell orientations on the utricular epithelium and macula neglecta are similar to those in other bony fishes. Nerve fiber diameters can be divided into three size classes, 1-8 microns, 9-13 microns, and 14 microns or more, with the smallest size class containing the majority of fibers. The distribution of the various classes of fiber diameters is not the same in nerve branches to each of the end organs. Similarly, the ratio of hair cells to axons differs in each end organ. The highest hair cell to axon ratio is in the utricle (23:1) and the smallest is in the macula neglecta (7:1). The number of sensory hair cells far exceed the number of eighth nerve axons in all sensory epithelia.     

The developing lamprey ear closely resembles the zebrafish otic vesicle: otx1 expression can account for all major patterning differences

The inner ear of adult agnathan vertebrates is relatively symmetric about the anteroposterior axis, with only two semicircular canals and a single sensory macula. This contrasts with the highly asymmetric gnathostome arrangement of three canals and several separate maculae. Symmetric ears can be obtained experimentally in gnathostomes in several ways, including by manipulation of zebrafish Hedgehog signalling, and it has been suggested that these phenotypes might represent an atavistic condition. We have found, however, that the symmetry of the adult lamprey inner ear is not reflected in its early development; the lamprey otic vesicle is highly asymmetric about the anteroposterior axis, both morphologically and molecularly, and bears a striking resemblance to the zebrafish otic vesicle. The single sensory macula originates as two foci of hair cells, and later shows regions of homology to the zebrafish utricular and saccular maculae. It is likely, therefore, that the last common ancestor of lampreys and gnathostomes already had well-defined otic anteroposterior asymmetries. Both lamprey and zebrafish otic vesicles express a target of Hedgehog signalling, patched, indicating that both are responsive to Hedgehog signalling. One significant distinction between agnathans and gnathostomes, however, is the acquisition of otic Otx1 expression in the gnathostome lineage. We show that Otx1 knockdown in zebrafish, as in Otx1(-/-) mice, gives rise to lamprey-like inner ears. The role of Otx1 in the gnathostome ear is therefore highly conserved; otic Otx1 expression is likely to account not only for the gain of a third semicircular canal and crista in gnathostomes, but also for the separation of the zones of the single macula into distinct regions.     

Immunohistochemical localization of two otolith matrix proteins in the otolith and inner ear of the rainbow trout,<Emphasis Type="Italic"> Oncorhynchus mykiss</Emphasis>: comparative aspects between the adult inner ear and embryonic otocysts

The fish otolith consists mainly of calcium carbonate and organic matrices, the latter of which may play important roles in the process of otolith formation. We previously identified two otolith matrix proteins, named otolith matrix protein-1 (OMP-1) and otolin-1, from the rainbow trout, Oncorhynchus mykiss, and the chum salmon, O. keta. In this study, recombinant proteins corresponding to OMP-1 and otolin-1 were synthesized using yeast and bacterial expression systems, respectively, to produce specific antibodies against each protein. Immunohistochemical analysis using these antisera revealed that in the otoliths of adult fish, OMP-1 and otolin-1 were colocalized along the daily rings possibly formed by alternate deposition of calcium carbonate and organic matrices. In the adult inner ear, OMP-1 was produced at most of the saccular epithelium, while otolin-1 was produced at a limited part of cylindrical cells located at the marginal zone of the sensory epithelium. In the embryonic inner ear, these proteins had already existed in the otolith primordia when calcification had commenced. In addition, otolin-1 was localized in the fibrous materials connecting otolith primordia and sensory epithelium at this stage. These results indicate that these proteins are required as essential components for otolith formation and calcification.     

Ultrastructure of the sensory formations of the internal ear in the common frog

By means of scanning electron microscopy method, sensory formations of the membranous labyrinth has been studied in the frog (Rana temporaria). The form of sensory fields and morphological peculiarities of the hair cells are described. For the saccular macula, amphibian and basilar papillae, the number of the hair cells is calculated, orientation of the hair cell poles is demonstrated.     

Gross and ultrastructural development of the saccule of the toadfish Opsanus tau

The development of the saccule of the inner ear in the toadfish was studied using light and scanning electron microscopy. Development was studied from the early embryo (2-3 days postfertilization), when the otocyst first forms, to the early-aged juvenile when the development of the inner ear approximates that of the adult (4 weeks postfertilization). The ultrastructural features examined included the morphological sequence of ciliary bundle growth, the development of orientation patterns of the ciliary bundles, and the relation of the ultrastructural development to overall gross development. Gross development may be divided into four distinct morphological stages. Stage I encompasses the time from initial formation of the otocyst until the start of stage II, which is the stage when the pars inferior begins migrating ventrally. In stage III the pars inferior continues to elongate ventrally. Stage IV starts when the pars inferior elongates in a rostral and caudal direction. The ear attains its adult shape in stage IV. The differentiation of the sensory cells begins during stage I. During the early part of stage I, a small cilium is found on the apical surface of each cell throughout the otocyst. In the middle and late periods of stage I, a few microvillous buds add to the surface of the cells that already have a kinocilium. These early ciliary bundles are clustered on the rostral-ventral and caudal walls of the otocyst. There is no clear patterning to the orientation of these ciliary bundles. In stage II the ventral stretching of the labyrinth wall causes a spreading of the clustered bundles along the ventral and medial walls of the pars inferior. The orientation of the ciliary bundles has no distinct pattern. In stage III the orientations of the ciliary bundles appear adultlike, although there are so few ciliary bundles that it is difficult to make a definite determination. During stage IV, hair cells with an adultlike horizontal and vertical orientation pattern are found on the rostral and caudal sections of the saccular macula, respectively. The transition region lying between these areas has ciliary bundles with various orientations.     

Residual microRNA expression dictates the extent of inner ear development in conditional Dicer knockout mice

Inner ear development requires coordinated transformation of a uniform sheet of cells into a labyrinth with multiple cell types. While numerous regulatory proteins have been shown to play critical roles in this process, the regulatory functions of microRNAs (miRNAs) have not been explored. To demonstrate the importance of miRNAs in inner ear development, we generated conditional Dicer knockout mice by the expression of Cre recombinase in the otic placode at E8.5. Otocyst-derived ganglia exhibit rapid neuron-specific miR-124 depletion by E11.5, degeneration by E12.5, and profound defects in subsequent sensory epithelial innervations by E17.5. However, the small and malformed inner ear at E17.5 exhibits residual and graded hair cell-specific miR-183 expression in the three remaining sensory epithelia (posterior crista, utricle, and cochlea) that closely corresponds to the degree of hair cell and sensory epithelium differentiation, and Fgf10 expression required for morphohistogenesis. The highest miR-183 expression is observed in near-normal hair cells of the posterior crista, whereas the reduced utricular macula demonstrates weak miR-183 expression and develops presumptive hair cells with numerous disorganized microvilli instead of ordered stereocilia. The correlation of differential and delayed depletion of mature miRNAs with the derailment of inner ear development demonstrates that miRNAs are crucial for inner ear neurosensory development and neurosensory-dependent morphogenesis.     

The polarisation of hair cells from the ear of the European bass (Dicentrarchus labrax)

The polarisation of ciliary bundles on the macula of the saccule in the European bass (Dicentrarchus labrax L) has been studied using a scanning electron microscope (SEM). These data show that D. labrax possesses ciliary bundles arranged in four dichotomous quadrants with a standard orientation, comparable to hearing generalists from the order Perciformes. The spacing between ciliary bundles was investigated in three size classes of fish, with the results indicating that the addition of receptor cells in the ear of D. labrax continues for at least the first 2 years of development. The lengths of the kinocilia from ciliary bundles in each quadrant of the macula were also studied, and found to be of uniform length. In addition, we look at the internal structure of the afferent using transmission electron microscopy (TEM), revealing the nucleated cell body and peripheral nerve fibres of the saccule consistent with other TEM examinations of saccular ultrastructure. This information is required to gain an insight into the inner ear of D. labrax, as part of a larger study of the morphology and physiology of the hearing systems of both vertebrate and invertebrate marine animals.     

Aminoglycoside-Induced Hair Cell Death of Inner Ear Organs Causes Functional Deficits in Adult Zebrafish (Danio rerio)

Aminoglycoside antibiotics, like gentamicin, kill inner ear sensory hair cells in a variety of species including chickens, mice, and humans. The zebrafish (Danio rerio) has been used to study hair cell cytotoxicity in the lateral line organs of larval and adult animals. Little is known about whether aminoglycosides kill the hair cells within the inner ear of adult zebrafish. We report here the ototoxic effects of gentamicin on hair cells in the saccule, the putative hearing organ, and utricle of zebrafish. First, adult zebrafish received a single 30 mg/kg intraperitoneal injection of fluorescently-tagged gentamicin (GTTR) to determine the distribution of gentamicin within inner ear sensory epithelia. After 4 hours, GTTR was observed in hair cells throughout the saccular and utriclar sensory epithelia. To assess the ototoxic effects of gentamicin, adult zebrafish received a single 250 mg/kg intraperitoneal injection of gentamicin and, 24 hours later, auditory evoked potential recordings (AEPs) revealed significant shifts in auditory thresholds compared to untreated controls. Zebrafish were then euthanized, the inner ear fixed, and labeled for apoptotic cells (TUNEL reaction), and the stereociliary bundles of hair cells labeled with fluorescently-tagged phalloidin. Whole mounts of the saccule and utricle were imaged and cells counted. There were significantly more TUNEL-labeled cells found in both organs 4 hours after gentamicin injection compared to vehicle-injected controls. As expected, significantly fewer hair cell bundles were found along the rostral-caudal axis of the saccule and in the extrastriolar and striolar regions of the utricle in gentamicin-treated animals compared to untreated controls. Therefore, as in other species, gentamicin causes significant inner ear sensory hair cell death and auditory dysfunction in zebrafish.     

A study on the fine structure of the saccular macula of the gold fish

Summary The fine structure of the saccular macula of the gold fish has been studied by means of the electron microscope.The sensory epithelium of the macula consists of sensory cells and supporting cells. The surface of the sensory cell is studded with a group of sensory hairs consisting of one kino-cilium and 50–60 stereocilia. In the dorsal half of the macula, the kino-cilium is located at the dorsal end of the sensory hair group. In the ventral half of the macula, the kino-cilium is located at the ventral end of the sensory hair group. In the intermediary portion of the macula, the sensory cells with opposite polarities are situated side-by-side. The relation between the microphonic potential and the position of the kino-cilium has been discussed.Two types of nerve terminals are found situated on the basal surface of the receptor cells. The one contains no synaptic vesicle and the other contains a cluster of synaptic vesicles and a few cored vesicles. It is considered that the former corresponds to the afferent nerve terminal and the latter to the efferent one.This investigation was supported by NIH Grant NB-06052.The author is very grateful to Prof. Taro Furukawa, Osaka City University for his invaluable advice and discussion.