Kuniaki Toyoshima

Establishment of Dental Epithelial Cell Line (HAT-7) and the Cell Differentiation Dependent on Notch Signaling Pathway

Rat incisors grow continuously throughout life. Producing a variety of dental epithelial cells is performed by stem cells located in the cervical loop of the incisor apex. To study the mechanisms for cell differentiation, we established a dental epithelial cell line (HAT-7) originating from a cervical loop epithelium of a rat incisor. Immunochemical studies showed that HAT-7 produced the cells expressing amelogenin, ameloblastin, or alkaline phosphatase (ALP). To illustrate a role of Notch signaling in the determinant of the cell fate, we examined expression patterns of Notch1 and Jagged1 in HAT-7 density dependently. At lower cell density, Notch1- or Jagged1-expressing cells were not seen. However, when they were fully confluent, cells began to express Notch1 or Jagged1 strongly. Some ALP-positive cells were almost consistent with Notch1-expressing cells but not Jagged1-expressing cells. These results suggested that the determinant of direction of differentiation was associated with Notch signaling pathway.

Identification of Merkel Cells by an Antibody to Villin

Merkel cells represent a population of epithelial cells in the skin and oral mucosa. Although Merkel cells are reliably distinguishable from other epithelial cells at the ultrastructural level, these cells are usually not discernible by standard light microscopy and need special techniques for their identification. Villin is an actin-crosslinking protein that is associated with the actin filament cores of brush border microvilli. In this study we show that an antibody against villin is an excellent marker of Merkel cells and their microvilli even at the light microscopic level. The surrounding keratinocytes and subepithelial connective tissue cells do not show any significant affinity for the antibody against villin. Confocal laser micrographs reconstructed from serial images 0.5 μm thick of Merkel cells that were immunostained with villin clearly reveal the three-dimensional morphology of Merkel cells and their microvilli. The presence of villin in Merkel cell microvilli lends support to the idea that these cells might have a mechanoreceptor function.

Merkel cells are responsible for the initiation of taste organ morphogenesis in the frog.

Taste organs in the frog have a distinctive cell type located exclusively in the basal portion. In the same fashion as type III cells in mammalian taste buds, these basal cells show immunoreactivity for serotonin antibody. Further, these cells are morphologically similar to epidermal Merkel cells. To determine the significance of these serotonergic basal cells, we examined the early development of taste organs during metamorphosis of the frog by focusing on the origin and possible roles of serotonergic basal cells. For convenience of description, five stages of development (metamorphic stage to climax stages A–D) are defined. In the metamorphic stage, a few noninnervated Merkel cells appear at the upper layer of the lingual epithelium. No neuronal elements are seen in the epithelium at this stage. At climax stages A–B, immature fungiform papillae become discernible in the dorsal surface of the tongue, where the Merkel cells are located. Merkel cells then move downward and extend their cytoplasmic processes toward the basal lamina. These cells are identified by their intense immunoreactivity for serotonin. During the later stages, many nerve fibers in the subepithelial connective tissue approach the epithelium containing Merkel cells. At climax stages C–D, Merkel cells extend cytoplasmic processes along the basal lamina toward the center of the newly forming fungiform papillae. The morphology of these Merkel cells exactly coincides with that of serotonergic basal cells in adult taste organs. Profuse exocytotic release of dense‐cored granules of Merkel cells toward the nerve fibers through the basal lamina is frequently seen in these stages. The present study indicates that serotonergic basal cells are derived from intraepithelial Merkel cells, which act as target sites for growing nerves and may be responsible for the initiation of taste organ morphogenesis. J. Comp. Neurol. 406:129–140, 1999.

Comparative ultrastructure of intercalated ducts in major salivary glands: a review

Intercalated ducts (IDs) connect the secretory endpieces of mammalian major salivary glands to the prominent intralobular striated ducts or, in the case of some groups of rodents, to the granular convoluted tubules (GCT). Because they have a nondescript appearance in conventional histological preparations, they generally are given short shrift in standard histology texts. Early electron microscopic studies of salivary glands dealt mainly with common laboratory animals and the impression was gained that IDs have a rather mundane structure. Our comparative ultrastructural studies of salivary glands include the parotid and submandibular glands (and in many cases, the sublingual gland) of more than 250 mammalian species. Most of these have not been described previously and are included here as ‘‘unpublished.’’ About 90% of these were collected and field-fixed for transmission electron micros

Possible role of serotonin in Merkel-like basal cells of the taste buds of the frog, Rana nigromaculata

Merkel‐like basal cells in the taste buds of the frog were examined by fluorescence histochemistry, immunohistochemistry and electron microscopy. There were about 16–20 basal cells arranged in a radial fashion at the base of each taste bud. These cells were strongly immunopositive for serotonin antiserum. They were characterised by the presence of numerous dense‐cored granules in the cytoplasm ranging from 80 to 120 nm in diameter, and of microvilli protruding from the cell surface. For 4 mo after sensory denervation by cutting the gustatory nerves, all cell types of the taste bud were well preserved and maintained their fine structure. Even at 4 mo after denervation, the basal cells exhibited a strong immunoreaction with serotonin antiserum. To investigate the function of serotonin in the basal cells in taste bud function, serotonin deficiency was induced by administration of p‐chlorophenylalanine (PCPA), an inhibitor of tryptophan hydroxylase, and of p‐chloroamphetamine (PCA), a depletor of serotonin. After administration of these agents to normal and denervated frogs for 2 wk, a marked decrease, or complete absence, of immunoreactivity for serotonin was observed in the basal cells. Ultrastructurally, degenerative changes were observed in both types of frog; numerous lysosome‐like myelin bodies were found in all cell types of the taste buds. The number of dense‐cored granules in the basal cells also was greatly decreased by treatment with these drugs. Serotonin in Merkel‐like basal cells appears to have a trophic role in maintenance of the morphological integrity of frog taste bud cells.

Dense-Cored Vesicles and Unusual Lamellar Bodies in Type III Gustatory Cells in Taste Buds of Rabbit Foliate Papillae

Some type III cells in taste buds of rabbit foliate papillae have greatly increased numbers of dense-cored vesicles. Such cells also contain unusual lamellar bodies that resemble those in alveolar type II cells; they consist of alternating dense and light layers with a periodicity of about 4.4 nm. The precise relationship between the vesicles and the lamellar bodies is unknown.

Ultrastructure of the salivary glands in the midtongue of the common vampire bat, Desmodus rotundus

All examined mammals have at least two sets of lingual salivary glands: von Ebners glands and Webers glands. A third set, the glands of Blandin and Nuhn, is present in the tongues of some but not all mammals. Vampire bats, Desmodus rotundus, are unusual in that they possess another set of lingual glands, these being in the midtongue region.

Occurrence of melanosome-containing Merkel cells in mammalian oral mucosa

The occurrence and fine structure of Merkel cells with melanosomes in the labial epithelium of the dog and cat were studied by electron microscopy. These melanosomes were polarized in the Merkel cell cytoplasm opposite to the major aggregation of dense-cored granules and the closely abutting nerve terminal. None of melanosomes was found in the Golgi complex region where a few immature dense-cored granules were observed. This may suggest that the melanosomes are not produced in the Merkel cell, but are taken from the melanocytes like in keratinocytes. The possible phagocytotic ability of Merkel cells is discussed.

Isolation, partial purification, and ultrastructure of taste bud cells from rabbit foliate papillae

A method is described for obtaining large numbers of isolated taste bud cells from lingual epithelium of rabbit foliate papillae. The isolation method is based on isopyenic sedimentation in a Percoll gradient. The purification of taste bud cells was evaluated by electron microscopy and by immunohistochemistry using CK 20 antibody. The cytology of the isolated taste bud cells remained very similar to in situ cells. The type III cells, which are regarded as gustatory cells, retained their characteristic dense-cored granules in the cytoplasm. This method will permit study of various parameters of taste bud cell biology.

The Ultrastructural and Cytochemical Studies on the Taste Organs of the Frog

The ultrastructural and cytochemical studies were made on the taste organs of bull frogs (Rana catesbiana). The taste organ contained three different types of cells: mucous, taste, and Merkel cells. Mucous cells contained many secretory granules in the supranuciear regions, which were positive to alcian blue and PA-TCH. It seemed that the functions of the mucous cells were wetting and cleansing the outer surface of the organ and circulating of the tastants in co-operation with the ciliated cells surrounding the organ. Twe types of the taste cells could be distinguished by the apical fine structures. One terminated in microvilli ancl the other in rod-shaped process. The characteristics of these taste cells were the presenee of large-cored vesicles, ranging from 90-140nm in diameter, in their basal cytoplasms and synaptic contacts with nerve fibers.