Ultrastructure of free neuromasts of Bathygobius fuscus (gobiidae) and canal neuromasts of Apogon cyanosoma (apogonidae)

Light and electron microscopic observations were made on the lateral line organs of the free neuromasts of the goby Bathygobius fuscus and the canal neuromasts of the cardinal fish Apogon cyanosoma. As in other lateral line systems, each neuromast consists of hair cells, supporting cells and mantle supporting cells, the whole being covered by a cupula. In B. fuscus the free neuromasts are mounted on papillae and have hair cells with stereocilia up to 2.5 μm long and a single kinocilium at least 25 μm long. Each neuromast is covered by a vane-like cupula that can be divided into two regions. The central region over the sensory area contains columns of myelin-like figures. These figures are absent from the outer region covering the mantle. The canal neuromasts of A. cyanosoma are diamond-shaped with up to 1,500 hair cells. The cupula is unusual in having a channel that lies over the sensory region. The hair cells have up to 45 stereocilia, the tallest reaching 2.5 μm, and a kinocilium at least 5 μm long. Tip links are shown for the first time between rows of stereocilia of the hair cells of lateral line neuromasts. The presence of tip links has now been demonstrated for all acousticolateral hair cell systems.     

Fine structure of the ordinary lateral line organ

Summary The lateral line organ of the spotted shark is characterized by its semi-cylindrical shape. Each organ (neuromast) is so closely apposed to the next that the individual neuromasts are almost continuous. The neuromast is composed of receptor cells, supporting cells and mantle cells. The receptor cells bear one kinocilium and up to 40 stereocilia. Bi-directional arrangement of the receptor cells as occurs in teleosts was demonstrated. Afferent and efferent nerve endings were found at the base of the receptor cells. The supporting cells extend from the basal lamina to the free surface. Long microvilli and a cilium-like ciliary rod project from the top of each supporting cell. The cell contains relatively few elements of the Golgi apparatus and little rough endoplasmic reticulum, but mitochondria and filaments are abundant. The mantle cell limits the lateral margin of the neuromast. It is distinguished from the supporting cell because of its long crescent-shaped nucleus and scarce, short microvilli. Myelinated nerve fibres are found in the subepithelial connective tissue but not in the epithelium.The fine structure of the shark lateral line organ suggests that this organ is in an intermediated step of evolution between that of lamprey and teleost.     

The cell coat of the sensory and supporting cells of the rainbow trout saccular macula as demonstrated by reaction with ruthenium red and tannic acid.

The surface of most cells is covered by glycoconjugates. The composition and thickness of the surface coat varies among different cell types. The purpose of the present study was to demonstrate the presence of and to characterize the cell coat surrounding the cells in the saccular macula of the rainbow trout. Tissues were fixed in Karnovsky's fixative containing either ruthenium red (0.5, 1, or 2%) or tannic acid (1, 2, or 4%). The apical surface of the sensory and supporting cells reacted with both agents. Varying the concentration of the compounds within a certain range did not significantly affect the degree of tissue staining. Whereas ruthenium red staining was distributed evenly along the luminal surface of the epithelium and along the length of the stereocilia, tannic acid formed electron-dense clumps on the luminal surface of sensory and non-sensory cells and in the basal region of the macular epithelium. The stereocilia of the sensory cells also exhibited tannic acid-positive, electrondense precipitate, particularly near the distal ends of these processes, while uniform staining of the plasma membrane was seen along their lengths. The results of this study suggest that the trout saccular macula is provided with extracellular microenvironments which may be necessary for functional integrity.     

The ultrastructure of the paratympanic organ (Vitali) in Gallus domesticus: preliminary studies

The sensory epithelium of the paratypanic organ (Vitali) was studied by means of the electron microscope. Two kinds of cells are present. One type extends from the basement membrane to the surface of the epithelium; their nuclei are arranged close to the connective tissue and are surrounded by a pale cytoplasm. The distal part of these cells, which are denser and richer in organelles, possess microvilli. The cells of the second type are located above the basement membrane and are found between the upper parts of the cells of the first type. Their cytoplasm is rich in small round vesicles, free ribosomes and cisternae of rough endoplasmic reticulum are present especially in the infranuclear zone. The apical part contains a Golgi apparatus lysosomes and multive sicular bodies. At the apex each cell possesses a cuticular plate numerous stereocilia and one kinocilium. The stereocilia become increasingly longer from one side of the cell surface to the other and the kinocilium is situated on the side where the stereocilia are longest. Nervous fibers are present in the epithelium and are in close contact with the cells of the second type. The cells we have described are remarkably similar to the supporting and hair cells of the vestibular sensory epithelium.     

Ultrastructure of the lateral-line sense organs of the ratfish,Chimaera monstrosa

Summary The ultrastructure of the lateral-line neuromasts in the ratfish, Chimaera monstrosa is described. The neuromasts rest at the bottom of open grooves and consist of sensory, supporting, basal and mantle cells. Each sensory cell is equipped with sensory hairs consisting of a single kinocilium and several stereocilia. There are several types of sensory hair arrangement, and cells with a particular arrangement form patches within the neuromast. There are two types of afferent synapse. The most common afferent synapse has a presynaptic body and is typically associated with an extensive system of anastomosing tubules on the presynaptic side. When the tubules are absent, vesicles surround the presynaptic body. These synapses are often associated into synaptic fields, containing up to 35 synaptic sites. The second type of afferent synapse does not have a presynaptic body and is not associated with the tubular system. The afferent synapses of the second type do not form synaptic fields and are uncommon. The efferent synapses are either associated with a postsynaptic sac or more commonly with a strongly osmiophilic postsynaptic membrane. The accessory cells are similar to those in the acoustico-lateralis organs of other aquatic vertebrates. A possibility of movement of the presynaptic bodies and of involvement of the tubular system in the turnover of the transmitter is discussed. A comparison of the hair tuft types in the neuromasts of Ch. monstrosa with those in the labyrinth of the goldfish and of the frog is attempted.     

Structure of outer hair cell stereocilia links in the chinchilla

The structure of side, tip, and “attachment” links of chinchilla outer hair cell (OHC) stereocilia was studied by transmission and scanning electron microscopy using tannic acid and Cuprolinic blue histochemical procedures. Tannic acid, which interacts with many different types of proteins and glycoproteins irrespective of their electrical charge, showed strong reactivity for the central area of the side links and weak reactivity for the marginal area of these links adjacent to the stereocilia membrane. Tannic acid treatment revealed the tip links as thin strands, about 5 nm thick. Attachment links were poorly visualized after tannic acid treatment and appeared as sparse filamentous strands at tips of the tallest OHC stereocilia. Cuprolinic blue, at a high critical electrolyte concentration, reacted with strongly negative, primarily sulfated, carbohydrate residues of glycoconjugate macromolecules. In contrast to the tannic acid treatment, the central portions of the OHC stereocilia side links were unstained after Cuprolinic blue treatment; however, membrane-associated ends of these links were darkly stained. The tip links showed a similar appearance as after tannic acid treatment; however, Cuprolinic blue revealed an electron-dense substructure at both ends of its insertion into the stereocilia. Cuprolinic blue reactive structures were also observed as attachment links only at the tips of the OHC stereocilia of the tallest row in each bundle. These structures formed a crown-like array around the tip of each stereocilium. Their primary function appears to be attachment of type B fibrils of the tectorial membrane to the tallest OHC stereocilia. Cuprolinic blue reactive structures of the side, tip, and attachment links appear to contain acidic, sulfated residues of proteoglycans or glycoproteins. These structures may function as connective elements between the stereocilia links and the hair cell cytoskeleton.     

Development of lateral line organs in leptocephali of the freshwater eel Anguilla japonica (Teleostei,Anguilliformes)

A study of the ontogeny of the lateral line system in leptocephali of the Japanese eel Anguilla japonica reveals the existence of three morphologically different types of lateral line organs. Type I is a novel sensory organ with hair cells bearing a single kinocilium, lacking stereocilia, distributed mainly on the head of larvae, and morphologically different from typical superficial neuromasts of the lateral line system. Its developmental sequence suggests that it may be a presumptive canal neuromast. Type II is an ordinary superficial neuromast, common in other teleost larvae, which includes presumptive canal neuromasts that first appear on the trunk and accessory superficial neuromasts that later appear on the head and trunk. Type III is a very unusual neuromast located just behind the orbit, close to the otic vesicle, with radially oriented hair cells, suggesting that these serve as multiple axes of sensitivity for mechanical stimuli. The behavior of larval eels suggests that the radially oriented neuromasts may act as the sole mechanosensory organ until the ordinary superficial neuromasts develop. The finding that larval eels possess a well-developed mechanosensory system suggests the possibility that they are also capable of perceiving weak environmental mechanical stimuli, like other teleost larvae.     

Sensory structures at the surface of fish skin. II. Lateralis System

Scanning electron microscopy shows the form of the cupulae of free neuromasts in two species of teleost fish, and gives information about the organization of the free neuromasts in teleosts and lampreys. In lampreys some neuromasts were found to lack the surrounding moat and the flanking hillocks characteristic of the lateral line organs previously described in these fish. In all cases, the sensory cells had the kinocilium aligned with respect to the stereocilia on the longer axis of the neuromast surface, thus enabling the direction of effective stimulation of the free neuromasts to be deduced from their morphological arrangement.     

Interconnections between the stereovilli of the fish inner ear

Summary We investigated at the electron-microscopic level the hair bundles of the macula organs in the inner ear of two species of teleost fish (Rutilus rutilus, Scardinius erythrophthalmus). All hairs (stereovilli) are interconnected by extracellular material that, similar to the cell coat, is well contrasted by application of tannic acid/osmium.The kinocilium is connected to the longest stereovilli via filaments. Filaments also connect the stereovilli at their bases. More distally all stereovilli are linked by thin filaments or very short solid connectors.The interconnections of the stereovilli in fish show greater variations, and are different in structural detail and distribution, compared to the interconnections described for amphibians and mammals. The interconnections are discussed in the context of their structural and functional significance and variations in terms of organ- and species specificity. Certain forms of interconnections are tentatively interpreted to be characteristic for developing hair bundles.     

Fine structure of the lateral-line organ of the common eel,Anguilla japonica

Summary The sensory epithelium of the lateral line organ of the common eel consists of two types of cells, (sensory and supporting). The sensory cell bears a kinocilium together with about 40 to 60 stereocilia on its surface. The kinocilium is situated either at rostral or at caudal margin of this cilial group. Such polarity of the cilial group of one cell is inverse to that of an adjacent cell.Two types of crystal-like inclusions exist in the sensory cells, consisting of granules 100 Å in diameter. Granules in one type are arranged regularly whereas those in the other rather irregularly.Two types of nerve endings exist at the base of sensory cells: one is predominant in number and contains few vesicles, accompanied by a dense spherical body surrounded by small vesicles in the sensory cell and the other is rare in number and contains many vesicles, accompanied by a small flat sac just beneath the plasma membrane of the sensory cell.The supporting cells contain numerous mitochondria, a well developed Golgi apparatus and rough-surfaced endoplasmic reticulum, and surround a sensory cell completely. Physiologic significance of some of these components is discussed.     

Electron Microscope Observations on in Vitro Cultures of the Isolated Fowl Embryo Otocyst

In vitro cultures of isolated fowl embryo otocysts were studied with the electron microscope. Hair cells of the developing organ of Corti and crista ampullaris have been examined with particular reference to the structure of the cilia and of the cell membrane. Two types of hair cells could be distinguished on the basis whether or not they possessed a "kinocilium" and "stereocilia," or "stereocilia" only. The cytoplasmic membranes were simple and there were no multiple vesicular layers in any of the hair cells. The supporting elements consisted of supporting cells flanking the hair cells, fibroblasts, and the cartilaginous otic capsule. Both the cochlear and vestibular sensory area showed rich innervation by mainly non-myelinated fibers with partial myelinization in others. There were well developed ganglion cells present. Bare axons penetrated the basement membrane and spread, amongst the supporting cells sheltering them, to the base of the hair cells where they formed bud-shaped nerve endings but, at the stage of development examined, no calyces. These in vitro cultures of the isolated fowl embryo otocyst provided convenient and suitable material for the electron microscope study of the sensory epithelium of the ear and revealed further that the isolated fowl embryo otocyst possesses great powers of self-differentiation also at the ultrastructural level.     

Structure of outer hair cell stereocilia side and attachment links in the chinchilla cochlea.

The structure and symmetry of chinchilla outer hair cell (OHC) stereocilia side and attachment links were investigated by transmission electron microscopy using tannic acid and Cuprolinic blue histochemical procedures. The side links run laterally between and across the rows of the stereocilia and connect the stereocilia together within the bundle. Attachment links form a crown-like array around the tips of only the tallest OHC stereocilia and attach these stereocilia to the Type B fibrils of the tectorial membrane. Computer averaging of the side links from tannic acid-treated tissues showed a central dense region of the link between adjacent stereocilia and a smaller dense portion at the plasma membrane end of the link. Computer averaging of Cuprolinic blue-treated tissues showed low electron density of the central region of the link, and the plasma membrane ends of the link were electron dense. After tannic acid treatment, the attachment links showed a diffused radial distribution around the tips of the tallest OHC stereocilia. After Cuprolinic blue treatment, the attachment links appeared as electron-dense, membrane-bound granular structures arranged with radial symmetry. The central regions of the side links are reactive to tannic acid. These regions appear to contain neutral and basic residues of proteins and participate in side-by-side association of the side links in regular aggregates. Cuprolinic blue-reactive regions of the side and attachment links appear to contain acidic sulfated residues of glycoproteins or proteoglycans, which may be involved in the attachment of these links to the stereocilium membrane.     

Electron microscope observations on in vitro cultures of the isolated fowl embryo otocyst

In vitro cultures of isolated fowl embryo otocysts were studied with the electron microscope. Hair cells of the developing organ of Corti and crista ampullaris have been examined with particular reference to the structure of the cilia and of the cell membrane. Two types of hair cells could be distinguished on the basis whether or not they possessed a "kinocilium" and "stereocilia," or "stereocilia" only. The cytoplasmic membranes were simple and there were no multiple vesicular layers in any of the hair cells. The supporting elements consisted of supporting cells flanking the hair cells, fibroblasts, and the cartilaginous otic capsule. Both the cochlear and vestibular sensory area showed rich innervation by mainly non-myelinated fibers with partial myelinization in others. There were well developed ganglion cells present. Bare axons penetrated the basement membrane and spread, amongst the supporting cells sheltering them, to the base of the hair cells where they formed bud-shaped nerve endings but, at the stage of development examined, no calyces. These in vitro cultures of the isolated fowl embryo otocyst provided convenient and suitable material for the electron microscope study of the sensory epithelium of the ear and revealed further that the isolated fowl embryo otocyst possesses great powers of self-differentiation also at the ultrastructural level.     

Hair cell regeneration in the chick inner ear following acoustic trauma: ultrastructural and immunohistochemical studies

The regeneration of hair cells in the chick inner ear following acoustic trauma was examined using transmission electron microscopy. In addition, the localization of proliferation cell nuclear antigen (PCNA) and basic fibroblast growth factor (b-FGF) was demonstrated immunohistochemically. The auditory sensory epithelium of the normal chick consists of short and tall hair cells and supporting cells. Immediately after noise exposure to a 1500-Hz pure tone at a sound pressure level of 120 decibels for 48 h, all the short hair cells disappeared in the middle region of the auditory epithelium. Twelve hours to 1 day after exposure, mitotic cells, binucleate cells and PCNA-positive supporting cells were observed, and b-FGF immunoreactivity was shown in the supporting cells and glial cells near the habenula perforata. Spindle-shaped hair cells with immature stereocilia and a kinocilium appeared 3 days after exposure; these cells had synaptic connections with the newly developed nerve endings. The spindle-shaped hair cell is considered to be a transitional cell in the lineage of the supporting cell to the mature short hair cell. These results indicate that, after acoustic trauma, the supporting cells divide and differentiate into new short hair cells via spindle-shaped hair cells. Furthermore, it is suggested that b-FGF is related to the proliferation of the supporting cells and the extension of the nerve fibers.     

Actin filaments, stereocilia and hair cells of the bird cochlea. VI. How the number and arrangement of stereocilia are determined.

Beginning in 8-day embryos, stereocilia sprout from the apical surface of hair cells apparently at random. As the embryo continues to develop, the number of stereocilia increases. By 10 1/2 days the number is approximately the same as that encountered extending from mature hair cells at the same relative positions in the adult cochlea. Surprisingly, over the next 2-3 days the number of stereocilia continues to increase so that hair cells in a 12-day embryo have 1 1/2 to 2 times as many stereocilia as in adult hair cells. In short, there is an overshoot in stereociliary number. During the same period in which stereocilia are formed (9-12 days) the apical surface of each hair cell is filled with closely packed stereocilia; thus the surface area is proportional to the number of stereocilia present per hair cell, as if these features were coupled. The staircase begins to form in a 10-day embryo, with what will be the tallest row beginning to elongate first and gradually row after row begins to elongate by incorporation of stereocilia at the foot of the staircase. Extracellular connections or tip linkages appear as the stereocilia become incorporated into the staircase. After a diminutive staircase has formed, eg. in a 12-day embryo, the remaining stereocilia located at the foot of the staircase begin to be reabsorbed, a process that occurs during the next few days. We conclude that the hair cell determines the number of stereocilia to form by filling up the available apical surface area with stereocilia and then, by cropping back those that are not stabilized by extracellular linkages, arrives at the appropriate number. Furthermore, the stereociliary pattern, which changes from having a round cross-sectional profile to a rectangular one, is generated by these same linkages which lock the stereocilia into a precise pattern. As this pattern is established, we envision that the stereocilia flow over the apical surface until frozen in place by the formation of the cuticular plate in the apical cell cytoplasm.     

Development of the hair bundle and mechanotransduction

This review focuses on the cellular and molecular mechanisms underlying the development of the sensory hair bundle, an apical specialisation of the hair cell that is essential for mechanotransduction. The structure, function and development of the hair bundle is described, with an emphasis on the properties and possible roles played by the different link types that interconnect the individual elements of the hair bundle - the multiple stereocilia and the single kinocilium. Studies of mouse and zebrafish mutants have revealed that several classes of molecule are required for the genesis and maintenance of hair-bundle structure. These include cell surface molecules that are associated with the different hair-bundle links, along with myosin motors, scaffolding proteins and an actin cross-linker. Finally we consider how differences in the form and shape of hair bundles within and between different sensory organs are generated.     

Actin filaments, stereocilia, and hair cells of the bird cochlea. I. Length, number, width, and distribution of stereocilia of each hair cell are related to the position of the hair cell on the cochlea

Located on the sensory epithelium of the sickle-shaped cochlea of a 7- to 10-d-old chick are approximately 5,000 hair cells. When the apical surface of these cell is examined by scanning microscopy, we find that the length, number, width, and distribution of the stereocilia on each hair cell are predetermined. Thus, a hair cell located at the distal end of the cochlea has 50 stereocilia, the longest of which are 5.5 microns in length and 0.12 microns in width, while those at the proximal end number 300 and are maximally 1.5 microns in length and 0.2 micron in width. In fact, if we travel along the cochlea from its distal to proximal end, we see that the stereocilia on successive hair cells gradually increase in number and width, yet decrease in length. Also, if we look transversely across the cochlea where adjacent hair cells have the same length and number of stereocilia (they are the same distance from the distal end of the cochlea), we find that the stereocilia of successive hair cells become thinner and that the apical surface area of the hair cell proper, not including the stereocilia, decreases from a maximum of 80 microns2 to 15 microns2. Thus, if we are told the length of the longest stereocilium on a hair cell and the width of that stereocilium, we can pinpoint the position of that hair cell on the cochlea in two axes. Likewise, if we are told the number of stereocilia and the apical surface of a hair cell, we can pinpoint the location of that cell in two axes. The distribution of the stereocilia on the apical surface of the cell is also precisely determined. More specifically, the stereocilia are hexagonally packed and this hexagonal lattice is precisely positioned relative to the kinocilium. Because of the precision with which individual hair cells regulate the length, width, number, and distribution of their cell extensions, we have a magnificent object with which to ask questions about how actin filaments that are present within the cell are regulated. Equally interesting is that the gradient in stereociliary length, number, width, and distribution may play an important role in frequency discrimination in the cochlea. This conclusion is amplified by the information presented in the accompanying paper (Tilney, L.G., E.H. Egelman, D.J. DeRosier, and J.C. Saunders, 1983, J. Cell Biol., 96:822- 834) on the packing of actin filaments in this stereocilia.     

Establishment of hair bundle polarity and orientation in the developing vestibular system of the mouse

The morphological development of the vestibular maculae in the mouse was studied in order to identify elements that may determine how hair-bundle polarity is established. Utricles and saccules develop in parallel. Hair-bundles first appear at embryonic day (E) 13.5. They are initially not polarised and have a kinocilium located at the centre of the cell surface surrounded by stereocilia. Polarisation is rapidly established as the kinocilium becomes eccentrically positioned. The orientation of these polarised bundles is initially not random. It varies systematically across the maculae and the general orientation in utricles is the opposite of that in saccules. At E15.5, in both maculae, hair-bundle orientation angles fall into two populations that differ by approximately 180° defining a line of orientation reversal, the position of which varies little during subsequent maturation. Many more immature hair bundles appear at E15.5 suggesting a second wave of hair cell differentiation is initiated. Otoconial membrane is produced simultaneously across the entire width of both maculae, indicating directional growth of the overlying extracellular matrix is unlikely to influence hair-bundle orientation. Growth of both maculae occurs asymmetrically, essentially outwards from the striola, but it is most pronounced after orientation is defined. Microtubules are prominent in hair cells at the earliest stages of their differentiation, but are oriented parallel to the long axis of the cell and, thus, may not have a role in directing hair-bundle polarity. Microfilament assemblies that are aligned parallel to the apical surface and connect to the adherens junctions in supporting cells could provide a “framework” for hair-bundle orientation. The striated rootlets of ciliary centrioles that are aligned parallel to the cell surface with their tips associated with microfilament assemblies at adherens junctions were the only structural asymmetry identified that might influence the development of hair-bundle polarity.     

Identification of a 275-kD protein associated with the apical surfaces of sensory hair cells in the avian inner ear

Immunological techniques have been used to generate both polyclonal and monoclonal antibodies specific for the apical ends of sensory hair cells in the avian inner ear. The hair cell antigen recognized by these antibodies is soluble in nonionic detergent, behaves on sucrose gradients primarily as a 16S particle, and, after immunoprecipitation, migrates as a polypeptide with a relative molecular mass of 275 kD on 5% SDS gels under reducing conditions. The antigen can be detected with scanning immunoelectron microscopy on the apical surface of the cell and on the stereocilia bundle but not on the kinocilium. Double label studies indicate that the entire stereocilia bundle is stained in the lagena macula (a vestibular organ), whereas in the basilar papilla (an auditory organ) only the proximal region of the stereocilia bundle nearest to the apical surface is stained. The monoclonal anti-hair cell antibodies do not stain brain, tongue, lung, liver, heart, crop, gizzard, small intestine, skeletal muscle, feather, skin, or eye tissues but do specifically stain renal corpuscles in the kidney. Experiments using organotypic cultures of the embryonic lagena macula indicate that the antibodies cause a significant increase in the steady-state stiffness of the stereocilia bundle but do not inhibit mechanotransduction. The antibodies should provide a suitable marker and/or tool for the purification of the apical sensory membrane of the hair cell.     

Shaping the mammalian auditory sensory organ by the planar cell polarity pathway

The human ear is capable of processing sound with a remarkable resolution over a wide range of intensity and frequency. This ability depends largely on the extraordinary feats of the hearing organ, the organ of Corti and its sensory hair cells. The organ of Corti consists of precisely patterned rows of sensory hair cells and supporting cells along the length of the snail-shaped cochlear duct. On the apical surface of each hair cell, several rows of actin-containing protrusions, known as stereocilia, form a "V"-shaped staircase. The vertices of all the "V"-shaped stereocilia point away from the center of the cochlea. The uniform orientation of stereocilia in the organ of Corti manifests a distinctive form of polarity known as planar cell polarity (PCP). Functionally, the direction of stereociliary bundle deflection controls the mechanical channels located in the stereocilia for auditory transduction. In addition, hair cells are tonotopically organized along the length of the cochlea. Thus, the uniform orientation of stereociliary bundles along the length of the cochlea is critical for effective mechanotransduction and for frequency selection. Here we summarize the morphological and molecular events that bestow the structural characteristics of the mammalian hearing organ, the growth of the snail-shaped cochlear duct and the establishment of PCP in the organ of Corti. The PCP of the sensory organs in the vestibule of the inner ear will also be described briefly.