Differentiation of the inner ear sensory epithelia of Proteus anguinus (Urodela,amphibia)

The stages of differentiation of the inner ear sensory epithelia of the neotenous cave urodele, Proteus anguinus, was studied with light and electron microscopy. Comparative ultrastructural analysis among specimens of different sizes confirms that new sensory cells may be generated throughout life, particularly along the periphery of the saccular macula. The inner ear of Proteus contains at least four types of sensory cells that differ in their apical ciliary part. The lungs and air-filled buccal cavity may function as transducers of sound pressure in underwater conditions. Sound waves might be transmitted from the buccal cavity to the connected oval window. The very complex orientation of the sensory hair cells of the saccular macula and the large overlying saccular otoconial mass suggest that this macula facilitates orientation of Proteus in its underground aqueous habitat.     

The Formation of Otoliths in the Frog Rana esculenta. Scanning Electron Microscopic and X-Ray Diffraction Studies

Two crystal forms of calcium carbonate were observed: calcite (utricle) and aragonite (saccule, lagena, endolymphatic sac). The first step in otolith formation is the appearance of organic structures in the macula. The subsequent step is characterized by fast growing primitive crystals with a prismatic habitus that successively transform into adult or mature crystals. With the metamorphosis, the aragonite crystals of the endolymphatic organ show clear signs of erosion that can be related to a process of CaCO₃ mobilization from such deposits.     

Sequence similarity between stereocilin and otoancorin points to a unified mechanism for mechanotransduction in the mammalian inner ear

Background  

Interaction between hair cells and acellular gels of the mammalian inner ear, the tectorial and otoconial membranes, is crucial for mechanoreception. Recently, otoancorin was suggested to be a mediator of gel attachment to nonsensory cells, but the molecular components of the interface between gels and sensory cells remain to be identified.     

Development of the supporting cells and structures derived from them in the inner ear of the grass frog,Rana temporaria (Amphibia,Anura)

Summary The inner ear of Rana t. temporaria comprises sensory structures with various special functions, i.e., the detection of spatial orientation (utricle, saccule, lagena), of rotation (ampullae), and of acoustic signals (amphibian and basilar papillae). In each of these structures, there is a sensory epithelium made up of hair (sensory) cells and supporting cells. As the supporting cells differentiate, they produce the organic matrix of the otoconia in the gravity-sensing organs, the ground substance of the cupulae in the ampullae, and the ground substance of the tectorial membranes in the auditory papillae. The supporting cells associated with these various derivative structures have correspondingly different cytoplasmic properties. The preotoconia are formed by extrusion; the otoconia develop from these filamentous precursors by growth and calcium deposition. The organic material that forms the cupulae and tectorial membranes is released from the supporting cells by exocytosis. The organization of this material into the ground substance is initiated mainly around the distal ends of the hair-cell kinocilia, eventually giving rise to the marked morphological differences that distinguish the cupulae from the tectorial membranes.Abbreviations bb basal body - c cilia - ca crista ampullaris - ch chromosome - cu cupula - d dictyosome - hc hair cell - kc kinocilia - ld lipid droplet - m mitochondrion - ma main axis - mb multilamellated body - mc macula communis - mi mitosis - mv microvillus - n nucleus - on organic net - pa amphibian papilla - pb basilar papilla - pg pigment granule - po preotoconia - rer rough endoplasmic reticulum - s saccule - sc supporting cell - sci stereocilia - sd spot desmosome - t tegmentum - tf tonofilaments - tj tight junction - tm tectorial membrane - yp yolk platelet     

Gene targeting reveals the role of Oc90 as the essential organizer of the otoconial organic matrix

A critical part of the functional development of our peripheral balance system is the embryonic formation of otoconia, composite crystals that overlie and provide optimal stimulus input to the sensory epithelium of the gravity receptor in the inner ear. To date neither the functions of otoconial proteins nor the processes of crystal formation are clearly defined. Using gene targeting and protein analysis strategies, we demonstrate that the predominant mammalian otoconin, otoconin-90/95 (Oc90), is essential for formation of the organic matrix of otoconia by specifically recruiting other matrix components, which includes otolin, a novel mammalian otoconin that we identified to be in wildtype murine otoconia. We show that this matrix controls otoconia growth and morphology by embedding the crystallites during seeding and growth. During otoconia development, the organic matrix forms prior to CaCO3 deposition and provides optimal calcification efficiency. Histological and ultrastructural examinations show normal inner ear epithelial morphology but reduced acellular matrices, including otoconial, cupular and tectorial membranes, in Oc90 null mice, likely due to an absence of Oc90 and a profound reduction of otolin. Our data demonstrate the critical roles of otoconins in otoconia seeding, growth and anchoring and suggest mechanistic similarities and differences between otoconia and bone calcification.     

Scanning electron microscopic and X-ray diffraction studies of otoconia in the lizard Podarcis s. sicula

Summary The otoliths of embryos and young animals of the lizard Podarcis s. sicula were studied by X-ray diffraction and scanning electron microscopy. Two types of crystal that give different X-ray diffraction patterns were found in the membranous labyrinth of Podarcis. The crystals consist of calcite or aragonite and are easily distinguished by scanning electron microscopy because of their different morphology. The two calcium carbonate crystal forms are not mixed at random but are present in the embryo from the very beginning in specific sites. The endolymphatic sac contains aragonite crystals while the saccule contains calcite crystals adjacent to the wall, in addition to a preponderance of aragonite crystals. The utricle and lagena contain only calcite crystals. The presence of two crystal forms of calcium carbonate in the membranous labyrinth are discussed in terms of differing genetic and functional significance.     

Extracellular matrices associated with the apical surfaces of sensory epithelia in the inner ear: molecular and structural diversity

The ultrastructure and molecular composition of the extracellular matrices that are associated with the apical surfaces of the mechanosensory epithelia in the mouse inner ear are compared. A progressive increase in molecular and structural organization is observed, with the cupula being the simplest, the otoconial membrane exhibiting an intermediate degree of complexity, and the tectorial membrane being the most elaborate of the three matrices. These differences may reflect changes that occurred in the acellular membranes of the inner ear as a mammalian hearing organ arose during evolution from a simple equilibrium receptor. A comparison of the molecular composition of the acellular membranes in the chick inner ear suggests the auditory epithelium and the striolar region of the maculae are homologous, indicating the basilar papilla may have evolved from the striolar region of an otolithic organ. A comparison of the tectorial membranes in the chick cochlear duct and the mouse cochlea reveals differences in the structure of the noncollagenous matrix in the two species that may result from differences in the stochiometry of alpha- and beta-tectorin and/or differences in the post-translational modification of alpha-tectorin. This comparison also indicates that the appearance of collagen in the mammalian tectorial membrane may have been a major step in the evolution of an electromechanically tuned vertebrate hearing organ that operates over an extended frequency range.     

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.     

Scanning electron microscopic observations on the inner ear of the skate,Raja ocellata

The inner ear of the skate, Raja ocellata, was examined by scanning electron microscopy. The otolithic membranes have a gelatinous component and an endogenous class of otoconia. Cupulae are reticulate in form. The morphology and polarization of sensory cell hair bundles are described for the various regions of the labyrinth, and are compared with published observations on other species. In the otolithic maculae, the more centrally located receptor cells generally have longer sterecolia than the peripheral cells. The hair bundles of the lacinia are similar to those of the central portion of the sacculus and differed from those of the rest of the utricular macula. Hair bundles in the peripheral regions of all maculae and cristae are similar. The polarization pattern of the utriculus is similar to that of teleosts, while that of the lagena is less clearly dichotomized. The receptor cells of most of the sacculus are oriented in a bivertical direction, with cells in the anterior portion, and a few in the posterior region, being aligned longitudinally. The significance of morphology and polarization with respect to the functions of the otolithic organs is discussed. The relationship of cell processes of the ampullary receptors to the cupula is briefly considered.     

Molecular cloning of chick beta-tectorin, an extracellular matrix molecule of the inner ear

The tectorial membrane is an extracellular matrix lying over the apical surface of the auditory epithelium. Immunofluorescence studies have suggested that some proteins of the avian tectorial membrane, the tectorins, may be unique to the inner ear (Killick, R., C. Malenczak, and G. P. Richardson. 1992. Hearing Res. 64:21-38). The cDNA and deduced amino acid sequences for chick beta-tectorin are presented. The cDNA encodes a protein of 36,902.6 D with a putative signal sequence, four potential N-glycosylation sites, 13 cysteines, and a hydrophobic COOH terminus. Western blots of two-dimensional gels using antibodies to a synthetic peptide confirm the identity of the cDNA. Southern and Northern analysis suggests that beta-tectorin is a single-copy gene only expressed in the inner ear. The predicted COOH terminus is similar to that of glycosylphosphatidylinositol-linked proteins, and antisera raised to this region react with in vitro translation products of the cDNA clone but not with mature beta-tectorin. These data suggest beta- tectorin is synthesized as a glycosylphosphatidyl-inositol-linked precursor, targeted to the apical surface of the sensory epithelium by the lipid moiety, and then further processed. Sequence analysis indicates the predicted protein possesses a zona pellucida domain, a sequence that is common to a limited number of other matrix-forming proteins and may be involved in the formation of filaments. In the cochlear duct, beta-tectorin is expressed in the basilar papilla, in the clear cells and the cuboidal cells, as well as in the striolar region of the lagena macula. The expression of beta-tectorin is associated with hair cells that have an apical cell surface specialization known as the 275-kD hair cell antigen restricted to the basal region of the hair bundle, suggesting that matrices containing beta-tectorin are required to drive this hair cell type.     

Cupulin Is a Zona Pellucida-Like Domain Protein and Major Component of the Cupula from the Inner Ear

The extracellular membranes of the inner ear are essential constituents to maintain sensory functions, the cupula for sensing torsional movements of the head, the otoconial membrane for sensing linear movements and accelerations like gravity, and the tectorial membrane in the cochlea for hearing. So far a number of structural proteins have been described, but for the gelatinous cupula precise data are missing. Here, we describe for the first time a major proteinogenic component of the cupula structure with an apparent molecular mass of 45 kDa from salmon. Analyses of respective peptides revealed highly conserved amino-acid sequences with identity to zona pellucida-like domain proteins. Immunohistochemistry studies localized the protein in the ampulla of the inner ear from salmon and according to its anatomical appearance we identified this glycoprotein as Cupulin. Future research on structure and function of zona pellucida-like domain proteins will enhance our knowledge of inner ear diseases, like sudden loss of vestibular function and other disturbances.     

Otopetrin 1 is required for otolith formation in the zebrafish Danio rerio

Orientation with respect to gravity is essential for the survival of complex organisms. The gravity receptor is one of the phylogenetically oldest sensory systems, and special adaptations that enhance sensitivity to gravity are highly conserved. The fish inner ear contains three large extracellular biomineral particles, otoliths, which have evolved to transduce the force of gravity into neuronal signals. Mammalian ears contain thousands of small particles called otoconia that serve a similar function. Loss or displacement of these structures can be lethal for fish and is responsible for benign paroxysmal positional vertigo (BPPV) in humans. The distinct morphologies of otoconial particles and otoliths suggest divergent developmental mechanisms. Mutations in a novel gene Otopetrin 1 (Otop1), encoding multi-transmembrane domain protein, result in nonsyndromic otoconial agenesis and a severe balance disorder in mice. Here we show that the zebrafish, Danio rerio, contains a highly conserved gene, otop1, that is essential for otolith formation. Morpholino-mediated knockdown of zebrafish Otop1 leads to otolith agenesis without affecting the sensory epithelium or other structures within the inner ear. Despite lack of otoliths in early development, otolith formation partially recovers in some fish after 2 days. However, the otoliths are malformed, misplaced, lack an organic matrix, and often consist of inorganic calcite crystals. These studies demonstrate that Otop1 has an essential and conserved role in the timing of formation and the size and shape of the developing otolith.     

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.     

The lagena (the third otolith endorgan in vertebrates)

In this review, the structure and functions of the lagena (the third otolith organ) in an evolutionary lineage of the vertebrates are described and discussed. The lagenar macula appears first in the posterior part of the sacculus of elasmobranchs; in these animals, the lagena is considered to be involved in the balance support (orientation with respect to the gravitation force). The lagena as a separate endorgan has been described in teleost fishes; in some species, the lagena is connected with the sacculus, while in other species the interrelations of these structures can be dissimilar. The lagena supplements the functions of the sacculus; in fishes (animals with no special organ of hearing), it is involved in discrimination of sound oscillations, identification of the gravitation vector, and orientation in the course of movements within the vertical plane. In amphibians, the lagena is localized in the posterior part of the sacculus, near the auditory structures; it performs mostly vestibular and (to a much lesser extent) auditory functions. In amniotes, the lagena was first separated from the sacculus; it is localized in the cochlear canal, distally with respect to the hearing organ. Information on the functions of the lagena in amniotes is rather limited and contradictory. Central projections of this organ have been examined practically only in birds. Lagenar afferents project to the vestibular nuclei and cerebellum, while some fibers come to the auditory nuclei of the medulla. The lagena in birds can be related to their navigation abilities (birds are supposed to be capable of orienting within the magnetic field of the Earth due to the magnetic properties of the lagenar otoconia; this structure can also provide detection of movements along the vertical axis. The close proximity between the otolithic and auditory endorgans in the cochlear canal of amniotes can be indicative of the functional significance of these interrelations. This aspect, however, remains at present undiscovered. In mammals (except Monotremata), there is no lagena as an independent endorgan. Neirofiziologiya/Neurophysiology, Vol. 40, No. 2, pp. 160–178, March–April, 2008.     

The ears of butterflyfishes (Chaetodontidae): ‘hearing generalists' on noisy coral reefs?

Analysis of the morphology of all three otolithic organs (sacculus, lagena and utriculus), including macula shape, hair cell morphology, density, orientation pattern, otolith morphology and the spatial relationships of the swimbladder and ear, reveals that butterflyfishes in the genera Chaetodon (which has anterior swimbladder horns) and Forcipiger (which lacks anterior swimbladder horns) both demonstrate the ear morphology typical of teleosts that lack otophysic connections, fishes that have traditionally been considered to be 'hearing generalists'.     

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.     

The ultrastructure of the calcium carbonate balance organs of the inner ear: an ultra-high resolution electron microscopy study

The balance organs of the inner ear of vertebrates, found as single, large growths of aragonite ('otoliths') in fish and small clumped masses ('otoconia') of either aragonite (amphibians) or calcite (mammals), have long been regarded as polycrystalline and single crystals respectively. The use of ultra-high resolution electron microscopy and electron diffraction to study comparatively crushed samples of these biominerals and samples of geological calcium carbonates, as examples of pure inorganic crystals, reveals that the biological structures are composed of microcrystals joined together by organic matrices to form composite crystals. Such structures either grow to a finite, controlled size (otoconia) or have daily growth patterns (otoliths). Mechanisms of growth are proposed to link these seemingly different patterns varying only in the number of nucleation sites and the degree of biological as against chemical control over the growth.     

Fgf19 expression patterns in the developing chick inner ear

The inner ear is a complex sensorial structure with hearing and balance functions. A key aim of developmental biology is to understand the molecular and cellular mechanisms involved in the induction, patterning and innervation of the vertebrate inner ear. These developmental events could be mediated by the expression of regulating genes, such as the members of the family of Fibroblast Growth Factors (Fgfs). This work reports the detailed spatial and temporal patterns of Fgf19 expression in the developing inner ear from otic cup (stage 14) to 8 embryonic days (stage 34). In the earliest stages, Fgf19 and Fgf8 expressions determine two subdomains within the Fgf10-positive proneural-sensory territory. We show that, from the earliest stages, the Fgf19 expression was detected in the acoustic-vestibular ganglion and the macula utriculi. The Fgf19 gene was also strongly, but transiently, expressed in the macula lagena, whereas the macula neglecta never expressed this gene in the period analysed. The Fgf19 expression was also clearly observed in some borders of various sensory elements. These results could be useful from further investigations into the role of FGF19 in otic patterning.     

The membranous labyrinth and its innervation inChaetodon trifasciatus (Pisces,Teleostei, Chaetodontidae)

Synopsis In the butterflyfishChaetodon trifasciatus, the labyrinth is characterized by its elevated form and especially the size of the vertical canals, the almost circular form of the horizontal canal and its posterior opening not directly in the utriculus but in the common pillar of the two vertical canals. There is an almost complete separation between utriculus and sacculus which are only linked by a virtual pore. The lagena, which is medially situated to the posterior part of the sacculus, is separated from it by an incomplete vertical wall. There are two maculae neglectae, the anterior macula being situated in the pore separating utriculus from sacculus and filling this pore, the posterior in a gutter of the floor of the utriculus. A long and narrow endolymphatic canal, originating from the sacculus close to the communication with the utriculus, follows the common pillar of the two vertical canals and widens into an endolymphatic sac at the top of the membranous labyrinth. The innervation of the labyrinth is made by the acoustic ganglion, which is connected to the brain by two roots and elongated into three parts: the anterior part innervates the anterior and horizontal cristae and the utricular and saccular maculae; the middle part innervates the macula sacculae and the macula neglecta 1; the posterior part innervates the macula neglecta II, the macula lagenae and the posterior crista. The important size of the vertical canals and the almost circular form of the horizontal canal may reflect very precise locomotory aptitudes.