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.     

Cytodifferentiation in the accessory glands of Tenebrio molitor. XI. Transitional cell types during establishment of pattern

The bean-shaped accessory glands of male Tenebrio consist of a single-layered epithelium which is surrounded by a muscular coat. The epithelial layer, which produces precursors of the wall of the spermatophore, contains eight secretory cell types. Each secretory cell type is in one or more homogenous patches, and discharges granules which form one layer of the eight-layered secretory plug. Maturation begins in cell types 4, 7, and 6 on the last pupal day. A newly identified cell (type 8) in the posterolateral epithelium matures last. Cells of individual types mature in synchrony, and their secretory granules “ripen” in a sequence that is characteristic for each type. As the secretory cells of each patch mature, unusual short-lived cells appear at interfaces between patches. In some cases the secretory granules in these boundary cells have ultrastructural features which are mixtures of the definitive characteristics of granules in adjacent cell types. The transitional cell types disappear at 3–4 days after eclosion. Intermediate cell types are absent in the mature gland and boundaries between the patches are distinct. The transitional cells may form granules of intermediate structural characteristics as a dual response to cellular interaction with adjacent and previously differentiated secretory cells.     

Relationship of saccular ultrastructure to otolith growth in the teleost Oreochromis niloticus

The sacculus of Oreochromis niloticus is anatomically separated from the utriculus and semicircular canals. The saccular wall is composed of the sensory epithelium, transitional epithelia, and squamous epithelium. Cellular granules are abundant in the sensory and transitional epithelia but scarce in the squamous epithelium. Over the dorsal side of the dorsal transitional epithelium there exists an oval patch of cells with distinctive microvilli. New finding is a shallow groove which extends from the anterior end of the sensory epithelium approximately halfway down along the ventral perimacular transitional epithelium. Small vesicles, which appear “empty” under transmission electron microscopy (TEM), are aggregated in the posterior region of the groove. These small vesicles are also present in both the sensory and transitional epithelia. A second kind of vesicle is comparatively large and appears filled with stainable contents. These vesicles are restricted to the sensory region. Both kinds of vesicles appear to be involved in apical secretion and possibly provide the otolithic membrane with fibers. The otolithic membrane is composed of a gelatinuous layer and subcupular meshwork. The meshwork appears to contribute to the formation of the otolith. The small empty vesicles appear to originate in sensory and transitional epithelial cells and may form the subcupular meshwork. The larger filled vesicles are derived predominantly from sensory cells in the sensory epithelium and appear to contribute to the gelatinuous layer of otoliths. © 1992 Wiley-Liss, Inc.     

Anterior and intermediate pituitary tissues express claudin 4 in follicle stellate cells and claudins 2 and 5 in endothelial cells

Follicle-stellate cells are pituitary non-granular cells that are arranged between secretory cells or organized in follicles with small lumens. Cells from the follicles exhibit the typical phenotype of a transporting epithelium, including apical microvilli with a cilium and tight junctions. Freeze-fracture electron microscopy images show that the tight junctions consist of 5–7 anastomosing strands and that cultured follicle-stellate cells develop a trans-epithelial electrical resistance characteristic of “tight” epithelia. Here, we investigate the molecular composition of the tight junction from follicle stellate cells. We found that the rat anterior pituitary lobe expresses mRNAs for claudins 2, 4 and 5; the proteins of all these claudins are observed in the anterior lobe, whereas the intermediate lobe expresses claudins 2 and 5 and the posterior lobe contains only claudin 5. Follicle-stellate cells, identified by their protein marker S100β, expresses claudin 4 in the apical membrane, in co-localization with dipeptidyl-peptidase and near acetylated β-tubulin. Claudin 4 partially co-localizes with E-cadherin, indicating that a fraction of the protein is located in the basolateral domain. Follicle-stellate-enriched cell cultures develop patches of polygonal cells expressing claudin 4 and E-cadherin, encircled by extensive monolayers of fusiform cells. Claudin 2 stains specifically blood vessels, identified by claudin 5 and VE-cadherin labels. Thus, follicles in the anterior pituitary consist of “tight” epithelia that can carry out intense vectorial transport, together with a high cation movement in blood vessels, possibly related to the ion requirements of excitable secretory cells for hormone secretion.     

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.     

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.     

Evidence concerning the morphogenesis of saccular receptors in the bullfrog (Rana catesbeiana)

A dichotomy of hair-cell types has been found in the bullfrog sacculus, and considerable evidence supports the view that one type (“peripheral”) is transformed during macular growth to the other type (“central”). Between the periphery and the center of the macula, one finds a gradation of form from “peripheral” to “central” type. Occasionally in adults and more often in stage-26 tadpoles one finds the presumably younger peripheral type of hair cell occurring well beyond the limits of the macula proper. The apparent morphogenic sequence for saccular hair cells is (1) development of a kinocillum on an endolymphatic epithelial cell, (2) gradual transformation of microvilli into stereocilia, (3) growth of the stereocilia and development of kinociliary bulb, (4) achievement of final size and form.     

Ultrastructure of the cardiac and pyloric glands of the gastric mucosa of the South African hedgehog,Atelerix frontalis

The cardiac and pyloric glands in the gastric mucosa of the South African hedgehog, Atelerix frontalis, are described. The cardiac area of the stomach contains proper cardiac glands and lacks undifferentiated fundic glands. The cardiac glands are simple tubular, coiled, and lined with columnar cells ultrastructurally similar to those of the gastric surface epithelium. Secretory granules with varying electron densities fill the apical cytoplasm of these cells. In contrast to other mammals, these glands lack mucous neck cells. The neck of the pyloric glands contains only a single cell type, whereas the basal regions of these glands contain “light” and “dark” cells. The secretory granules in the “dark” cells and the pyloric neck cells have a moderate electron density and often contain an electron dense core. An electron-lucent cytoplasm with numerous polysomes is characteristic of the “light” cells. Some “light” cells contain electron-dense granules in the apical cytoplasm. The presence of only neutral mucins in the cardiac gland cells denotes the absence of mucous neck cells. The acidic mucins within the pyloric neck cells seem to indicate that these cells are mucous neck cells, whereas the neutral mucins within the basally located pyloric gland cells show at least a partial functional difference from the pyloric neck cells. © 1993 Wiley-Liss, Inc.     

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.     

Olfactory metamorphosis in the coastal tailed frog Ascaphus truei (Amphibia,Anura, Leiopelmatidae)

The structure of the olfactory organ in larvae and adults of the basal anuran Ascaphus truei was examined using light micrography, electron micrography, and resin casts of the nasal cavity. The larval olfactory organ consists of nonsensory anterior and posterior nasal tubes connected to a large, main olfactory cavity containing olfactory epithelium; the vomeronasal organ is a ventrolateral diverticulum of this cavity. A small patch of olfactory epithelium (the “epithelial band”) also is present in the preoral buccal cavity, anterolateral to the choana. The main olfactory epithelium and epithelial band have both microvillar and ciliated receptor cells, and both microvillar and ciliated supporting cells. The epithelial band also contains secretory ciliated supporting cells. The vomeronasal epithelium contains only microvillar receptor cells. After metamorphosis, the adult olfactory organ is divided into the three typical anuran olfactory chambers: the principal, middle, and inferior cavities. The anterior part of the principal cavity contains a “larval type” epithelium that has both microvillar and ciliated receptor cells and both microvillar and ciliated supporting cells, whereas the posterior part is lined with an “adult‐type” epithelium that has only ciliated receptor cells and microvillar supporting cells. The middle cavity is nonsensory. The vomeronasal epithelium of the inferior cavity resembles that of larvae but is distinguished by a novel type of microvillar cell. The presence of two distinct types of olfactory epithelium in the principal cavity of adult A. truei is unique among previously described anuran olfactory organs. A comparative review suggests that the anterior olfactory epithelium is homologous with the “recessus olfactorius” of other anurans and with the accessory nasal cavity of pipids and functions to detect water‐borne odorants. J. Morphol. 2011. © 2011 Wiley Periodicals, Inc.     

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.     

Morphology of the macula neglecta in sharks of the genus Carcharhinus

Ears from several species of carcharhinid sharks were studied by gross dissection, light microscopy, transmission electron microscopy, and scanning electron microscopy. Structures along a possible sound transmission path to the ear are described, but main consideration is given to the structure of the macula neglecta. The macula neglecta is composed of two patches of sensory epithelium which line part of the posterior canal duct. In an adult shark the larger of these contains 224,000 sensory hair cells oriented so as to detect forces directed posteroventrolaterally in the duct. The smaller patch contains 43,000 hair cells oriented so as to detect oppositely directed forces. These receptor cells project through numerous small terminals to a total for both patches of 4,700 myelinated nerve fibers. Cytostructural variations throughout the hair cell population are also reported. Estimated acoustic properties of the tissues in this complex and the processing potential of the neural elements are interpreted as suggestive of auditory function. A mechanism based on the geometry of the receptor arrays is proposed to explain behaviorally observed instantaneous sound localization from the farfield. Evolution of the macula neglecta is reviewed, and evidence for homology of the macula neglecta and amphibian papilla is presented.     

Ephrin-B2 governs morphogenesis of endolymphatic sac and duct epithelia in the mouse inner ear

Control over ionic composition and volume of the inner ear luminal fluid endolymph is essential for normal hearing and balance. Mice deficient in either the EphB2 receptor tyrosine kinase or the cognate transmembrane ligand ephrin-B2 (Efnb2) exhibit background strain-specific vestibular-behavioral dysfunction and signs of abnormal endolymph homeostasis. Using various loss-of-function mouse models, we found that Efnb2 is required for growth and morphogenesis of the embryonic endolymphatic epithelium, a precursor of the endolymphatic sac (ES) and duct (ED), which mediate endolymph homeostasis. Conditional inactivation of Efnb2 in early-stage embryonic ear tissues disrupted cell proliferation, cell survival, and epithelial folding at the origin of the endolymphatic epithelium. This correlated with apparent absence of an ED, mis-localization of ES ion transport cells relative to inner ear sensory organs, dysplasia of the endolymph fluid space, and abnormally formed otoconia (extracellular calcite-protein composites) at later stages of embryonic development. A comparison of Efnb2 and Notch signaling-deficient mutant phenotypes indicated that these two signaling systems have distinct and non-overlapping roles in ES/ED development. Homozygous deletion of the Efnb2 C-terminus caused abnormalities similar to those found in the conditional Efnb2 null homozygote. Analyses of fetal Efnb2 C-terminus deletion heterozygotes found mis-localized ES ion transport cells only in the genetic background exhibiting vestibular dysfunction. We propose that developmental dysplasias described here are a gene dose-sensitive cause of the vestibular dysfunction observed in EphB–Efnb2 signaling-deficient mice.     

Embryonic ionocytes in the European sea bass (Dicentrarchus labrax): Structure and functionality

Early ionocytes have been studied in the European sea bass (Dicentrarchus labrax) embryos. Structural and functional aspects were analyzed and compared with those observed in the same conditions (38 ppt) in post hatching stages. Immunolocalization of Na⁺/K⁺‐ATPase (NKA) in embryos revealed the presence of ionocytes on the yolk sac membrane from a stage 12 pair of somites (S), and an original cluster around the first gill slits from stage 14S. Histological investigations suggested that from these cells, close to the future gill chambers, originate the ionocytes observed on gill arches and gill filaments after hatching. Triple immunocytochemical staining, including NKA, various Na⁺/K⁺/2Cl^? cotransporters (NKCCs) and the chloride channel “cystic fibrosis transmembrane regulator” (CFTR), point to the occurrence of immature and mature ionocytes in early and late embryonic stages at different sites. These observations were completed with transmission electronic microscopy. The degree of functionality of ionocytes is discussed according to these results. Yolk sac membrane ionocytes and enteric ionocytes seem to have an early role in embryonic osmoregulation, whereas gill slits tegumentary ionocytes are presumed to be fully efficient after hatching.     

Morphological aspects of Culex quinquefasciatus salivary glands

The salivary glands of Culex quinquefasciatus female mosquitoes are paired organs composed of two lateral lobes with proximal and distal secretory portions, and a medial lobe. All portions comprise a simple epithelium that surrounds a salivary duct. In the apical portion of the medial lobe, non-secretory cells strongly resemble cells involved in ion and water transport. The general architecture of the secretory portions is similar between lobes. The appearance of the secretory material and the morphological aspect of the apical cell membrane are the most distinctive features among the three secretory portions. Cells in the lateral proximal lobe display thin membrane projections extending into a translucent and finely filamentous secretory product. At the lateral distal portion, the apical cell membrane forms an intricate meshwork that encloses a dark secretory product. Medial lobe secretory cells also contain secretory cavities surrounded by intracytoplasmic vesicles, all containing a very dark and uniform product. Scattered cells holding numerous vacuoles, some of them containing a small and electron-dense granule eccentrically located and resembling those of the diffuse endocrine system, are frequently observed in the periphery of all secretory portions. Immunofluorescence assays revealed that the distal portion of the lateral lobes contains apyrase, an enzyme putatively responsible for platelet aggregation inhibition, diffusely distributed in the cell cytoplasm.     

On the Polarization and Innervation of the Pars Inferior Sensory Epithelia of the Herring Labyrinth

The macula sacculi and the macula lagenae of the herring, Clupea harengus L., were examined by light microscopy, the macula lagenae is large compared to what is normal among non-ostariophysan fishes, the morphological polarization of the hair cells in the inferior maculae shows a pattern which is similar to that usually seen in teleost fishes. The fibres in the nerves supplying the macula sacculi and the macula lagenae were counted and their diameters measured. The ramulus saccularis is divided in two separate ramuli innervating populations of hair cells with different morphological polarization. The saccular rostral nerve trunk contains 1800–2300 fibres, with 1300–1800 fibres in the caudal nerve trunk. The lagenar nerve is composed of 2100–4000 fibres. The fibre diameters are 1–14 μm in all ramuli. Silver staining of the nerve axoplasm reveals a unique differentiation of the maculae, which can be divided into a central area surrounded by a peripheral part. The hair cells in the central area are innervated by thick nerve fibres (5–14 μm diameter) as well as a few thin nerve fibres (about 1 μm diameter), while the receptor cells in the peripheral area are exclusively innervated by thin fibres having diameters of 2 μm or less.     

Ultrastructure of the endolymphatic sac in the larva of the Japanese red-bellied newt Cynops pyrrhogaster

The ultrastructure of the endolymphatic sac (ES) of the late stage larva of the Japanese red-bellied newt, Cynops pyrrhogaster (stage 57), was examined by light and transmission electron microscopy. The two endolymphatic sacs are located at the dorsal-medial side of the otic vesicle on the dorsal-lateral side of the midbrain in the cranial cavity. The wall of the sac is composed of a layer of cubical epithelial cells with loose, interposed intercellular spaces. The sac contains a large luminal cavity, in which endolymph and numerous otoconia are present. The epithelial cells of different portions of the sac have a similar structure. These cells contain an abundance of cytoplasmic organelles, including ribosomes, Golgi complexes, and numerous vesicles. Two types of vesicles are found in the epithelial cells: the “floccular” vesicle and the “granular” vesicle. The floccular vesicles are located in the supra- and lateral-nuclear cytoplasm and contain flocccular material. The granular vesicles have a fine granular substance and are usually situated apposed to the apical cell membrane. The granular vesicles are suggested to be secreted into the lumen, while the floccular vesicles are thought to be absorbed from the lumen and conveyed to the intercellular spaces by the epithelial cells. The apical surfaces of the epithelial cells bear numerous microvilli. Apparently floating cells, which bear long microvilli on the free surfaces, are observed in the lumen of the ES. Based on the fine structure, the function of the endolymphatic sac of the newt Cynops pyrrhogaster is discussed.     

Ultrastructure of the lung of the rattlesnake,Crotalus viridis oreganus

Scanning and transmission electron microscopic observations were made on the rattlesnake lung, which has the form of a cigar-shaped bag enclosing a large axial air chamber. The lungs were fixed by tracheal instillation of fixative to preserve the structural features of inflated lungs. An open tracheal groove along the ventral aspect of the lung is the only structural “airway” present. The wall of the lung has two histologically distinct regions: anteriorly, a respiratory portion, where up to three generations of septa subdivide the wall into cup-shaped gas-exchange chambers, termed faveoli; and posteriorly, a simple, thin-walled saccular portion. The epithelium lining the internal surface of the lung is composed of several cell types: (1) ciliated cells; (2) type I pneumonocytes; (3) type II pneumonocytes, secretory cells characterized by the presence of lamellar bodies; and (4) serous epithelial cells, secretory cells characterized by the presence of homogeneous, densely staining secretory granules. However, the distinctiveness of the secretory cell types in the snake lung is blurred because intermediate-appearing cells have both the lamellar body and homogenous type of secretory granule. The nonepithelial components of the pulmonary wall and septa consist of blood vessels and lymphatics, smooth muscle cells and fibroblasts, embedded in a matrix of extracellular connective tissue fibers. Tubular myelin figures were observed in the faveolar lining layer.     

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.     

Adenylate cyclase and carbonic anhydrase in the semicircular canal epithelium of the frog Rana esculenta

Summary Because the secretion of endolymph has been localized in the ampullar part of the frog semicircular canal, we attempted to determine by cytochemical methods the ultrastructural localization of two enzymes that are assumed to play a role in endolymph secretion: carbonic anhydrase and adenylate cyclase. Functionally, the epithelium of the frog semicircular canal can be schematically divided into three areas: sensory (crista ampullaris), secretory (dark cells), and non-sensory and nonsecretory (transitional and undifferentiated cells) areas. Carbonic anhydrase activity was widely distributed in dark cells. Dark cell labeling disappeared in the presence of acetazolamide. The other cells of the canal did not show any carbonic anhydrase labeling except for the supporting cells of the sensory cells. Adenylate cyclase activity was found on the basolateral and apical membranes of dark cells, and on the apical membrane of sensory cells; weak labeling was also observed in the other epithelial cells. In the apical membrane of the dark cells, adenylate cyclase labeling was dependent on the presence of vasotocin, the frog antidiuretic hormone. The dark cells of the frog semicircular canal thus possess the enzyme equipment needed for the secretion of endolymph and its possible hormonal regulation.