Masako Takeda

Supporting cells as phagocytes in the olfactory epithelium after bulbectomy

Macrophages are known to be phagocytes in the olfactory epithelium of adult rats. The participation of other cell types in phagocytosis in association with the cell death process was examined in the olfactory epithelium after unilateral bulbectomy of neonatal mice. The terminal deoxynucleotidyl transferase (TdT)‐mediated biotinylated dUTP nick end‐labeling (TUNEL) method revealed that the process of olfactory cell death consists of acute and chronic periods. The number of apoptotic cell profiles on the operated side peaked at 1 day, and the percentage of labeled cell profiles was 13.6%. The number of dying cells rapidly decreased at 3 days and decreased further at 5 days. Only 3% of the cells were labeled at 5 days. The percentage of dying cells increased again at the end of first postoperative week and remained two‐ to four‐fold higher than control values for 2 months (4.7–5.3%). Electron micrographs of sections from early postbulbectomy stages (1–7 days) showed that as many as 30% of supporting cell profiles contained apoptotic bodies, cellular debris, and phagosomes in the cytoplasm. The number of supporting cell profiles containing phagosomes declined to a plateau 2 weeks following bulbectomy and remained at 8–12% of the supporting cell population for 2 months. The results indicate that supporting cells in the olfactory epithelium play a significant role in phagocytosis in both acute and chronic periods of cell death after bulbectomy in newborn mice. However, supporting cells are not the exclusive phagocytic cell type in the bulbectomized epithelium; a small number of macrophages was also observed. Moreover, the phagocytosis by supporting cells was observed in unperturbed epithelium in the early stages during postnatal development.

Basal cells in the mouse olfactory epithelium after axotomy: immunohistochemical and electron-microscopic studies

SummaryThe olfactory epithelium of mice after axotomy was investigated to clarify the stem cells of olfactory cells by double immunostaining using antikeratin (MA903) and anti-bromodeoxyuridine (BrdU) antibodies and by conventional electron microscopy. When a single dose of BrdU was given to mice 9 days after axotomy, immunostaining for BrdU was found in the globose basal cells which were negative for MA903, but not in the basal cells proper which were positive for MA903. The BrdU-immunoreactive cells increased 3-to 6-fold over the number of these cells in the controls, indicating active cell proliferation. At other postoperative days (4 and 14 days), fewer BrdU-immunoreactive cells were found. Furthermore, three pulses of BrdU resulted in numerous BrdU-immunolabelings in the globose basal cells and a few in the basal cells proper. There was no detectable difference in the number of labeled basal cells proper in operated and unoperated mice. In the electron micrographs 9 days after axotomy, the basal cells proper, flat-shaped in unoperated mice, appeared cylindrical or pyramidal in shape and the globose basal cells often lay between the basal cells proper. In unoperated controls, the globose basal cells were located above the flat-shaped basal cells proper. The results suggest that the stem cells of the olfactory cells are globose basal cells and not basal cells proper, and that the shape of basal cells proper changes in relation to the active proliferation of stem cells.

Effect of monoamines on the taste buds in the mouse

SummaryMouse taste buds were investigated following administration of monoamines and their precursors by fluorescence and electron microscopy. The appearance of fluorescent cells within the taste bud and the ultrastructural changes of vesicles in the gustatory cells were due to the treatment of 5-hydroxytryptophan. Small dense-cored vesicles (30–60 nm in diameter) appeared throughout the cytoplasm and accumulated especially at the presynaptic membranes of afferent synapses. Large dense-cored vesicles (80–100 nm) increased twice in number, and electron densities of their cores became more dense as compared with untreated mice. Fluorescent cells appeared in the taste bud of l-DOPA treated mice, whereas no ultrastructural changes were observed. These results suggest that the gustatory cells of the taste bud are capable of taking up and storing monoamines, which might act as neurotransmitters from the gustatory cells to the nerves.

Apoptosis in mouse taste buds after denervation.

Abstract.Apoptotic cells in the taste buds of mouse circumvallate papillae after the sectioning of bilateral glossopharyngeal nerves were examined by the method of DNA nick-end labeling (TUNEL), together with standard electron microscopy. The taste buds decreased in number and size 3–11 days after denervation and disappeared at 11 days. The TUNEL method revealed only a few positively stained nuclei in normal taste buds but, in those of mice 1–5 days after denervation, the number of positive nuclei had increased to 3–5 times that of taste buds from normal mice. Electron-microscopic observation after denervation demonstrated taste bud cells containing condensed and fragmentary nuclei in a cytoplasm with increased density. The results show that taste bud cells under normal conditions die by apoptosis at the end of their life span, and that gustatory nerve sectioning causes apoptosis of taste bud cells with taste buds decreasing in number and ultimately disappearing.

Basal cells in the mouse olfactory epithelium during development : immunohistochemical and electron-microscopic studies

The basal region in the developing olfactory epithelium of mice was investigated by double immunostaining using anti-keratin (MA903) and anti-bromodeoxyuridine antibodies. Basal cells proper, which are MA903 positive, and globose basal cells, which are MA903 negative, were differentiated in the basal region on embryonic day 18 to postnatal day 1. At this stage, BrdU-labeling was found more in the glubose basal cells than in the basal cells proper. The labeled globose basal cells increased in number and reached the maximum on postnatal day 3, and decreased in number on postnatal day 14. The labeled basal cells proper was concentrated near the border with the respiratory epithelium on postnatal day 1, but they were found in small numbers in other regions of olfactory epithelium during later stages of development. Active division of globose basal cells accompanied the 1.5-fold increase in the thickness of the olfactory epithelium, which is caused by the increase in the number of olfactory cells, during development. It is suggested that during late embryonic and postnatal days olfactory cells originate from globose basal cells, not from basal cells proper. The division of basal cells proper may contribute to an 8-fold increase in the surface area of the olfactory epithelium during development.

Expression of E- and P-cadherin during tooth morphogenesis and cytodifferentiation of ameloblasts

Abstractu2002Cell-cell adhesion is fundamental in morphogenesis and is known to be mediated by several groups of cell adhesion molecules. Cadherins are a group of such molecules involved in the Ca2+-dependent cell-cell adhesion mechanism and are found in most kinds of tissue. In this study using indirect immunofluorescence microscopy, we analyzed the distribution of two kinds of cadherins, E- and P-cadherin, in developing tooth germs. In the molar tooth germs at the early bud stage, marginal cells of the epithelial tooth bud expressed both E- and P-cadherin, whereas central cells expressed only E-cadherin. At the cap stage, in addition to the cells of the inner and outer enamel epithelium, which outline the enamal organ, cells of the enamel knot, which is thought to control tooth morphogenesis, strongly expressed P-cadherin. The expression of P-cadherin was prominent in the inner enamel epithelium during the early to mid bell stage, and was also evident in the non-dividing cell masses at future cusp tips, which are the so-called secondary enamel knots. In the tooth germ at the late bell stage when the cells of the inner enamel epithelium began to polarize to differentiate into ameloblasts, the polarizing ameloblasts lost P-cadherin and strongly expressed E-cadherin. However, E-cadherin was also lost from polarized ameloblasts at later stages. The stratum intermedium and the stellate reticulum were E-cadherin positive from the bell stage onward even at the stages when the ameloblasts became E-cadherin negative again. These results suggest that the differential expression of E- and P-cadherin during morphogenetic stages plays a role in the regulation of tooth morphogenesis, whereas alteration of E-cadherin expression during later stages of tooth development is related to differentiation and function of the ameloblasts and other cells supporting amelogenesis.

Keratins in the developing olfactory epithelia

At embryonic day 14, the supporting cells of the olfactory epithelium already contained tonofilaments which terminated in the desmosomes, and were stained by antikeratin antibodies of RGE53 and MA902, indicating the presence of 45 and 52.5 kDa keratins. The basal cells were identified at postnatal day 1 by the appearance of a few filaments, and stained by PKK2 antikeratin antibody which reacts with 40, 46, 48, and 54 kDa keratins, and by CKB1 antikeratin antibody which reacts with 50 kDa keratin. At postnatal day 14, the basal cells possessed densely aggregated bundles of filaments and reacted with KL1 and MA902 antikeratin antibodies, indicating the appearance of 56 and 52.5 kDa keratins. The basal cells showed a columnar or pyramidal shape changing into a flat shape during postnatal development. The olfactory cells remained unstained by antikeratin antibodies throughout their development.

Expression of neural cell adhesion molecule (NCAM) during the first molar development in the mouse

NCAM, the neural cell adhesion molecule, was immunolocalized in the mandibular first molar tooth germ of the mouse. NCAM was first detected in the tooth germ of the late bud stage, where only the cells in the outer part of the condensed mesenchyme (primitive dental follicle) exhibited faint immunoreactivity. The entire dental follicle was intensely immunostained for NCAM from cap stage to the stage when root formation started. During root formation, NCAM disappeared from the follicular tissue surrounding the cervical root as well as from the part covering the crown top. This loss of NCAM proceeded in the direction of the root apex, but even after the tooth had achieved functional occlusion, NCAM was still expressed by the mesenchymal cells adjacent to the root apex. On the other hand, NCAM was negative in the dental papilla until birth. After birth, NCAM-immunoreactivity appeared in the basal portion of the dental papilla, but this NCAM-positive area gradually diminished in width during the root elongation. Instead, another NCAM-positive zone appeared in the core of the pulp during root formation. Even in the tooth that had already erupted, the pulp core contained cells that were strongly positive for NCAM immunostaining. In addition to its expression in the above two mesenchymal cell lineages, NCAM was transiently expressed by epithelial components of the tooth germ, some of the cells of the dental lamina and the enamel organ. The results suggest that NCAM participates in several processes of tooth development.

Keratin filaments of epithelial and taste-bud cells in the circumvallate papillae of adult and developing mice.

SummaryKeratin filaments of epithelial- and taste-bud cells in the circumvallate papillae of adult and developing mice were studied by immunocytochemistry using monoclonal antikeratin antibodies (PKK2 and PKK3) and by conventional electron microscopy. Elongated cells (type-I,-II, and-III cells) of the taste buds were stained by PKK3 antibody, which reacts with 45-kdalton keratin, whereas basal cells of the taste buds and surrounding epithelial cells showed negative staining with PKK3. Such PKK3-reactive cells occurred at 0 day after birth, when taste-buds first appeared in the dorsal surface epithelium of the papillae. Thus 45-kdalton keratin seems to be an excellent immunocytochemical marker for identifying taste-bud cells. Epithelial cells in all layers of the trench wall and basal layer cells of the dorsal surface contained densely aggregated bundles of keratin filaments that reacted with PKK2 antibody, but not with PKK3. In contrast, taste-bud cells and spinous and granular layer cells of the dorsal surface possessed loose aggregated bundles of filaments that reacted with PKK3, but not with PKK2. These results suggest that the aggregation and distribution pattern of keratin filaments may reflect differences in the keratin subtypes that comprise these filaments.

Gene expression of β–catenin is up-regulated in inner dental epithelium and enamel knots during molar tooth morphogenesis in the mouse

Beta–catenin is a multi–functional molecule that is involved in both cell–cell adhesion and signaling. We analyzed changes in β–catenin gene expression during mouse molar tooth development by in situ hybridization. Prominent up–regulation of the expression of this gene was evident exclusively in the enamel knot at the early cap stage. During the cap and bell stages, the enamel knot, inner dental epithelium, and differentiating stratum intermedium expressed the β–catenin gene more strongly than other parts of the enamel organ. During these stages, the strength of the gene expression changed heterogeneously within the inner dental epithelium and stratum intermedium. However, the heterogeneity was not evident at the late bell stage, when the cells in the inner dental epithelium had differentiated into ameloblasts at the cusp tip. No spatiotemporal change in β–catenin gene expression was apparent in the dental papilla except for the cells that differentiated into odontoblasts, which became negative for the expression of the gene after their differentiation. Thus, the up-regulated expression of the β–catenin gene was strongly associated with epithelial morphogenesis. These findings raise the possibility that the up–regulation of the gene expression and the stabilization of the protein by Wnt signaling play a role in the regulation of the activities of β–catenin in tooth morphogenesis.