Calbindin D28k-like immunoreactivity during the formation of the enamel-free area in the rat molar teeth

Previous studies have demonstrated the presence of calbindin D28k in the ameloblasts derived from the inner enamel epithelium. The occlusal surfaces of the rodent molars partly lack the enamel covering, which is referred to as enamel-free area (EFA). In the present study, we compared the immunohistochemical localization of calbindin D28k-like immunoreactivity (CB-LI) in the cells at the EFA (EFA cells) and ameloblasts of the rat molar teeth at the light microscopic level. CB-LI was strong in the ameloblasts of the presecretory through the protective stages, while it was faint at the late secretory to transitional stages. However, some mature ameloblasts lacked the immunoreactivity. On the other hand, the majority of EFA cells showed distinct polarization and elongation that were absent in few cells at the early stage of EFA formation. At all stages, the EFA cells adjacent to the ameloblasts showed CB-LI, however, some cells adjacent to the mature ameloblasts lacked the reaction. Intensive CB-LI was demonstrated in EFA cells at the reduced enamel epithelium. These immunohistochemical findings suggest EFA cells have cytochemical properties similar to those of ameloblasts.     

Calbindin D28k‐immunoreactive afferent nerve endings in the laryngeal mucosa

The distribution of the calbindin D28k in the laryngeal sensory structures was studied by immunohistochemistry, immunoelectronmicroscopy, and double immunofluorescence with calretinin‐immunoreactivity. Moreover, origin of the nerve endings were observed using retrograde tracer, fast blue. Immunoreactivity for calbindin D28k was found in the various types of nerve endings in the larynx, namely, laminar nerve endings, nerve endings associated with the taste buds, intraepithelial nerve endings, and endocrine cells. The laminar endings with calbindin D28k‐immunoreactivity were observed in the subepithelial connective tissue. In some endings, terminals were expanded. The laminar endings were also observed in the perichondrium of the epiglottic cartilage. In the epiglottic and arytenoid epithelia, thick nerve fibers with calbindin D28k‐immunoreactivity ascending to taste buds and intragemmal nerve fibers were also observed. Within the epithelial layer, intraepithelial free nerve endings with calbindin D28k‐immunoreactivity were observed. Furthermore, diffuse endocrine cells were observed within the laryngeal epithelium. By immunoelectron microscopy, immunoreaction products in the endings mentioned above were localized in the cytoplasm of the axon terminals and nerve fibers which contained with numerous mitochondria. Out of the 100 laminar endings, 18 endings were immunopositive for both calbindin D28k and calretinin, 33 were positive for calbindin D28k but negative for calretinin, and 49 were positive for only calretinin in the double immunofluorescence microscopy. The nerve fibers associated with the taste buds and the free nerve endings, which immunostained for calbindin D28k, were not stained with antibody against calretinin. After injection of the fast blue in the laryngeal mucosa, fast blue‐labeled cells were mainly observed in the nodose ganglia. Of the total number of labeled cell in the nodose and dorsal root ganglia at the level C1 to Th2, 65.1% occurred in nodose ganglia (572/879, n = 6). In the nodose ganglia, 79.7% of labeled cells (456/572) were immunoreacted for calbindin D28k. The distribution of calbindin D28k‐immunoreactivity may be differnt from that of calretinin. It is suggested that calbindin D28k have regulatory role on intracellular calcium concentration in the laryngeal sensory corpuscles. Anat Rec 259:237–247, 2000. © 2000 Wiley‐Liss, Inc.     

Immunolocalization of calbindin D28k and vitamin D receptor during root formation of murine molar teeth

Cells in the epithelial rest of Malassez (ERM cells) express calbindin D28k (CB); however, the hormonal regulation of CB in ERM cells remains to be elucidated. We investigated the immunohistochemical localization of CB and 1,25-dihydroxyvitamin D3 receptor (VDR) during root formation of mouse molar teeth in order to clarify whether the expression of CB in ERM cells is dependent on vitamin D. At the early stage of root formation (postnatal (PN) days 10-14), both CB- and VDR-immunoreactive cells were observed intermittently along the root surface. In the apical portion, almost all CB-immunoreactive cells showed VDR immunoreactivity; however, VDR-immunoreactive cells in the most apical portion were immunonegative for CB. In the middle and cervical portions, the distributions of the two proteins were completely different. At the late stage of root formation (PN28d) and in adult animals, CB immunoreactivity was distributed in cells found along the acellular cementum at the bifurcation region, as well as between the dentin and cellular cementum in the apical portion (although these lacked immunoreactivity for VDR). The present results indicate that CB expression in newly disrupted cells from Hertwig's epithelial root sheath occurs in a vitamin-D dependent manner, whereas the expression of CB in mature ERM cells may be independent of vitamin D.     

Alteration in the expression level of calbindin D28k in the periodontal ligament of the rat molar during experimental tooth movement.

The present immunohistochemical study was designed to investigate changes in the distribution and expression level of calbindin D28k in the periodontal ligament during experimental tooth movement in the rat molar to clarify the physiological role of this protein in the ligament. In normal animals, calbindin D28k-like immunoreactivity appeared sparsely in spindle-shaped cells in the alveolar half of the periodontal ligament. Electron microscopic observations showed that these immunoreactive cells were characterized by well-developed rough-surfaced endoplasmic reticulum and phagosomes--which often contained collagen fibers--suggesting that these cells could be categorized as periodontal fibroblasts. Twelve hours following the onset of the experimental tooth movement, cells positive for calbindin D28k increased in number in the periodontal ligament, especially in the alveolar half of the pressured side. Immunoelectron microscopy showed that the calbindin D28k-immunopositive cells had morphological features similar to those of fibroblasts in the normal ligament, and that these cells occasionally made contact with immunonegative macrophage-like cells. Immunopositive cells gradually decreased in number, and the distribution of the cells and intensity of the immunoreactivity returned to normal levels by 14 days following the induction of the experimental tooth movement. The present results suggest that calbindin D28k plays an important role in the homeostasis and cyto-protection of fibroblasts in the periodontal ligament at the initial phase of experimental tooth movement.     

Analysis of parvalbumin and calbindin D28k-immunoreactive neurons in dorsal root ganglia of rat in relation to their cytochrome oxidase and carbonic anhydrase content

Histochemical and immunohistochemical techniques were used to determine relationships between the parvalbumin or calbindin D28k content and the cytochrome oxidase or carbonic anhydrase activity of neurons in lumbar dorsal root ganglia in rat. Subpopulations of dorsal root ganglion neurons that displayed parvalbumin- or calbindin D28k-immunoreactivity were classified as containing either light, moderate or dense histochemical reaction product for cytochrome oxidase and either a positive or negative reaction for carbonic anhydrase. It was found that approximately 90% of all parvalbumin and calbindin D28k-immunoreactive cells exhibited dense staining for cytochrome oxidase and that 87% of parvalbumin- and 76% of calbindin D28k-immunoreactive cells were positive for carbonic anhydrase. Conversely, 85% of all cells with a dense cytochrome oxidase reaction contained parvalbumin and calbindin D28k. Although not quantified, it appeared that many, but not all, carbonic anhydrase-positive cells contained parvalbumin or calbindin D28k. These results indicate the existence of a subpopulation of primary sensory neurons that contains parvalbumin and calbindin D28k and that expresses high levels of cytochrome oxidase and carbonic anhydrase activity. It is suggested that primary afferent neurons with this cytochemical profile transmit a sensory modality that requires them to discharge rapidly and/or frequently. The existence of a subpopulation of carbonic anhydrase-positive cells that lack immunoreactivity for parvalbumin or calbindin D28k suggests that the role of carbonic anhydrase in some sensory neurons is unrelated to functions requiring these calcium binding proteins.     

Epithelial cytodifferentiation and extracellular matrix formation in enamel-free areas of the occlusal cusp during development of mouse molars: Light and electron microscopic studies

Mandibular first molars in mice ranging in age from 18 days prenatal to 5 days postnatal were used for light and electron microscopic examinations of the enamel-free area (EFA) during development of the occlusal cusp (mesiobuccal cusp). Notable morphological changes in the inner enamel epithelium and the cells of the stratum intermedium were observed. At prenatal age of 18 days, the inner enamel epithelium of the EFA (EFA epithelium) was composed of a layer of columnar cells and covered by the cells of the stratum intermedium. Two days after birth, the EFA epithelium was made up largely of preameloblasts, with mitochondria located in the proximal side of the cells toward the stratum intermedium. The cells of the stratum intermedium were irregularly shaped, with wide intercellular spaces between them. At a postnatal age of 3 days, most of the EFA epithelial cells resembled maturation-stage ameloblasts, being short and columnar in shape and having nuclei located in their proximal side. Distal cell membranes were folded, and mitochondria were scattered throughout the cytoplasm. In 4-day-old mice, the EFA epithelium was found to be formed of short columnar or cuboidal cells with distinct intercellular spaces. The cells of the stratum intermedium could no longer be detected, and cells of the EFA epithelium could not be distinguished from those of the stellate reticulum. Odontoblasts of the EFA were arranged and polarized parallel to the basal lamina, and odontoblastic processes extended toward the cusp tip. The orientation of thin and thick collagen fibers within predentin and dentin was also parallel to the basal lamina. Even after dentin mineraliza tion, disrupted basal lamina and long, aperiodic, fine fibrils were found between the epithelium and the dentin. Following the disappearance of the basal lamina and fine fibrils, stippled material and crystals appeared on the dentin surface. The mineralized matrix, which x-ray microanalytical energy peaks identified as containing calcium and phosphorus, was continuous with enamel in the distal slope of the cusp at the cusp tip. Thus, the inner enamel epithelium of the EFA differentiated into secretory cells capable of enamel-like matrix formation. In conclusion, development of EFA in mouse molars seems to be a system suitable for use in studying both the involvement of extracellular matrix, including basal lamina constituents, in ameioblast differentiation, and the role of the cells of the stratum intermedium, together with the ameloblasts, in comprising a single functional unit responsible for enamel formation.     

Expression of Heat-Shock Protein 25 Immunoreactivity in the Dental Pulp and Enamel Organ During Odontogenesis in the Rat Molar

The present immunocytochemical study reports on the expression of heat-shock protein (Hsp) 25 during odontogenesis in rat molars from postnatal 1 to 100 days. Hsp 25 immunoreactivity (IR) appeared in the immature dental mesenchymal cells and the differentiating and differentiated odontoblasts. At 30 days, the coronal odontoblasts retained intense Hsp 25-IR, whereas the odontoblasts in the root and floor pulp were initially weak or negative but increased in IR in the later stages, indicating that the expression of Hsp 25 reflects the differentiation status of odontoblasts. During amelogenesis, the secretory ameloblasts were Hsp 25 immunopositive and the enamel free area (EFA) cells showed intense Hsp 25-IR when they developed a ruffled border. Ruffle-ended ameloblasts (RA) also consistently showed intense Hsp 25-IR, but smooth-ended ameloblasts (SA) showed weak IR. These data suggest that Hsp 25 is related to the formation and maintenance of the ruffled border of RA and EFA cells.     

Expression of heat-shock protein 25 immunoreactivity in the dental pulp and enamel organ during odontogenesis in the rat molar

The present immunocytochemical study reports on the expression of heat-shock protein (Hsp) 25 during odontogenesis in rat molars from postnatal 1 to 100 days. Hsp 25 immunoreactivity (IR) appeared in the immature dental mesenchymal cells and the differentiating and differentiated odontoblasts. At 30 days, the coronal odontoblasts retained intense Hsp25-IR, whereas the odontoblasts in the root and floor pulp were initially weak or negative but increased in IR in the later stages, indicating that the expression of Hsp 25 reflects the differentiation status of odontoblasts. During amelogenesis, the secretory ameloblasts were Hsp 25 immunopositive and the enamel free area (EFA) cells showed intense Hsp 25-IR when they developed a ruffled border. Ruffle-ended ameloblasts (RA) also consistently showed intense Hsp 25-IR, but smooth ended ameloblasts (SA) showed weak IR. These data suggest that Hsp 25 is related to the formation and maintenance of the ruffled border of RA and EFA cells.     

Calbindin‐D28K Immunoreactivity in the Human Cerebellar Cortex

Calbindin‐D28k (CB) is a calcium‐binding protein largely distributed in the cerebellum of various species of vertebrates. As regards the human cerebellar cortex, precise data on the distribution of CB have not yet been reported. Aim of the present work was to analyze the distribution of CB in postmortem samples of human cerebellar cortex using light microscopy immunohistochemical techniques. Immunoreactivity to CB was detected within neuronal bodies and processes distributed in all cortex layers. In the molecular layer, the immunoreactivity was observed in subpopulations of stellate and basket neurons. In the Purkinje neuron layer, the immunoreactivity was observed in practically all the Purkinje neurons. In the granular layer, the immunoreactivity was observed in subpopulations of granules, of Golgi neurons, and also of other types of large neurons (candelabrum, Lugaro neurons, etc.). Immunoreactivity to CB was also observed in axon terminals distributed throughout the cortex according to layer‐specific patterns of distribution. The qualitative and quantitative patterns of distribution of CB showed no difference among the different lobes of the cerebellar cortex. This study reports that CB is expressed by different neuron types, both inhibitory (GABAergic) and excitatory (glutamatergic), involved in both intrinsic and extrinsic circuits of the human cerebellar cortex. The study provides further insights on the functional role of CB and on the neuronal types of the cerebellar cortex in which it is expressed. Anat Rec, 297:1306–1315, 2014. © 2014 Wiley Periodicals, Inc.     

Localization of RELM‐β/FIZZ2 Is Associated with Cementum Formation

Resistin‐like molecule‐β/found in inflammatory zone 2 (RELM‐β/FIZZ2) is a cysteine‐rich secretory protein that is localized in the epithelium of the gastrointestinal tract and lung alveoli. Previous reports have suggested that this protein regulates glucose metabolism and inflammation. In the present study, to analyze the involvement of RELM‐β/FIZZ2 in tooth development, we immunohistochemically examined the localization of RELM‐β/FIZZ2 in tooth germs of embryonic days (E) 15‐20 and postnatal days (P) 7‐42 rats. RELM‐β/FIZZ2 was hardly detected in the tooth germ at the bud (E15) stage. However, at the cap (E17) and bell (E20) stages, this protein was detectable in the inner enamel epithelium; whereas cells in the other parts of the enamel organ including the outer enamel epithelium and stellate reticulum did not show the immunoreactivity. During the root formation stage (P14‐28), cells in Hertwig's epithelial root sheath (HERS) localized RELM‐β/FIZZ2. Intense immunoreactivity was also seen in the matrix of the root dentin facing the HERS and the dental follicle. This reactivity was not present on the more upwardly located dentin surface. In contrast, cementum matrix positive for osteopontin and bone sialoprotein was observed on the dentin instead of immunoreactivity for RELM‐β/FIZZ2. Osterix‐positive cells, indicating cementoblast progenitors, were also detected in the dental follicle near the matrix positive for RELM‐β/FIZZ2. These results suggest that RELM‐β/FIZZ2 secreted by the inner enamel epithelium was mainly localized in the matrix at the surface of the apical root dentin and might be involved in cementogenesis. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:1865–1874, 2017. © 2017 Wiley Periodicals, Inc.     

Expression and changes of calbindin D-28k immunoreactivity in the ventral horn after transient spinal cord ischemia in rabbits

We examined ischemia-related changes of calbindin D-28k (CB) immunoreactivity in L(7) of the spinal ventral horn after transient spinal cord ischemia in rabbits. In the sham-operated group, CB immunoreactivity was not present in the spinal ventral horn, but CB immunoreactivity was detectable in the dorsal horn. CB immunoreactivity was detectable in the ventral horn at 30 min after ischemia: the CB immunoreactivity was found in glial cells identified as astrocytes. At 1 h after ischemia, CB immunoreactivity was highest and present at a few somata located in the lamina VII as well as many glial cells. CB immunoreactivity was lower in the lamina VII at 3 h after ischemia compared to 1 h post-ischemic group. By 2 days after ischemia, CB immunoreactivity was decreased in this region. In addition, the result of Western blot result showed the pattern of CB expression similar to that of immunohistochemistry. In conclusion, the ischemia-related changes of CB immunoreactivity in neurons and glial cells in the ischemic spinal ventral horn in rabbits may be related to modulation of intracellular calcium following transient ischemia.     

Immunohistochemical demonstration of amelogenin penetration toward the dental pulp in the early stages of ameloblast development in rat molar tooth germs

In order to examine the synthesis and secretion of enamel protein by ameloblasts in their early stages of development, immunohistochemical localization was carried out at light and electron microscopic levels using a monoclonal antibody produced in a preliminary experiment. Materials used were tooth germs of mandibular first molars of rats at 0-5 days after birth. Immunoblot analysis after two-dimensional electrophoresis revealed that antigen molecules recognized by the monoclonal antibody were amelogenins of 26-28 kDa (pI, 6.6-7.0). An immunohistochemical examination using this monoclonal antibody demonstrated that the presecretory ameloblasts in their early stages of differentiation both synthesized amelogenin and secreted through a classical merocrine secretory pathway. In some presecretory ameloblasts as well as ameloblasts we observed the distended cisternae of rough endoplasmic reticulum (rER) which demonstrated heterogenous immunolabelling. The immunolabellings were also detected in the predentin as well as the intercellular spaces of odontoblasts and dental pulp cells which indicated penetration of amelogenin from the presecretory ameloblast layer to the dental pulp. The presence of coated pits at the plasma membrane of odontoblasts in close proximity to enamel protein along with the immunolabelling of lysosomes of the odontoblasts suggests the phagocytosis of the enamel protein into the odontoblasts. These observations suggest the possibility that the penetration of enamel protein toward the dental pulp and odontoblasts plays a role in the interaction between ameloblasts and odontoblasts.     

Postnatal development of parvalbumin and calbindin D28K immunoreactivities in the cerebral cortex of the rat

Parvalbumin and calbindin D28k immunoreactivities were examined in the neocortex of the rat during postnatal development. Parvalbumin-immunoreactive nonpyramidal neurons first appear in layer V and later in layers VI and IV, and then in II and III. Immunoreactive terminals forming baskets surrounding unlabelled somata appear about 2 days later. The first parvalbumin-immunoreactive neurons appear in the retrosplenial and cingulate cortices, and the rostral region of the primary somatosensory cortex at postnatal days 8 or 9 (P8–P9). These regions are followed by the primary visual, primary auditory and motor cortices at P11. Parvalbumin immunoreactivity appears last in the secondary areas of the sensory regions and association cortices. Adult patterns are reached at the end of the 3rd week. Calbindin D28K-immunoreactive nonpyramidal neurons are found at birth in all cortical layers excepting the molecular layer. The intensity of the immunoreaction increases during the first 8 or 11 days of postnatal life, first in the inner and later in the upper cortical layers, following, therefore, an inside-out gradient. Heavily-labelled calbindin D28K-immunoreactive nonpyramidal cells dramatically decrease in number from P11 to P15 due mainly to a decrease of the multipolar subtypes. This suggests that two populations of calbindin D28k-immunoreactive nonpyramidal neurons are produced in the neocortex during postnatal development: one population of neurons transitorily expresses calbindin D28k immunoreactivity; the other population is composed of neurons that are permanently calbindin D28k immunoreactive. In addition to heavily labelled nonpyramidal cells, a band of weakly labelled pyramid-like neurons progressively appears in layers II and III throughout the cerebral cortex, beginning in layer IV in the somatosensory cortex by the end of the 2st week. Adult patterns are reached at the end of the 3rd week. These results indicate that parvalbumin and calbindin D28k immunoreactivities in the cerebral neocortx follow different characteristic patterns during postnatal development. The appearance of parvalbumin immunoreactivity correlates with the appearance of the related functional activity in the different cortical regions, and, probably, with the appearance of inhibitory activity in the neocortex. On the other hand, the early appearance of calbindin D28k immunoreactivity in the neocortex may be related to the early appearance of calbindin immunoreactivity in many other brain regions, and suggests another, as yet unknown, role for this calcium-binding protein during development of the cerebral cortex.     

Calbindin D28k-immunoreactive afferent nerve endings in the laryngeal mucosa

The distribution of the calbindin D28k in the laryngeal sensory structures was studied by immunohistochemistry, immunoelectronmicroscopy, and double immunofluorescence with calretinin-immunoreactivity. Moreover, origin of the nerve endings were observed using retrograde tracer, fast blue. Immunoreactivity for calbindin D28k was found in the various types of nerve endings in the larynx, namely, laminar nerve endings, nerve endings associated with the taste buds, intraepithelial nerve endings, and endocrine cells. The laminar endings with calbindin D28k-immunoreactivity were observed in the subepithelial connective tissue. In some endings, terminals were expanded. The laminar endings were also observed in the perichondrium of the epiglottic cartilage. In the epiglottic and arytenoid epithelia, thick nerve fibers with calbindin D28k-immunoreactivity ascending to taste buds and intragemmal nerve fibers were also observed. Within the epithelial layer, intraepithelial free nerve endings with calbindin D28k-immunoreactivity were observed. Furthermore, diffuse endocrine cells were observed within the laryngeal epithelium. By immunoelectron microscopy, immunoreaction products in the endings mentioned above were localized in the cytoplasm of the axon terminals and nerve fibers which contained with numerous mitochondria. Out of the 100 laminar endings, 18 endings were immunopositive for both calbindin D28k and calretinin, 33 were positive for calbindin D28k but negative for calretinin, and 49 were positive for only calretinin in the double immunofluorescence microscopy. The nerve fibers associated with the taste buds and the free nerve endings, which immunostained for calbindin D28k, were not stained with antibody against calretinin. After injection of the fast blue in the laryngeal mucosa, fast blue-labeled cells were mainly observed in the nodose ganglia. Of the total number of labeled cell in the nodose and dorsal root ganglia at the level C1 to Th2, 65.1% occurred in nodose ganglia (572/879, n = 6). In the nodose ganglia, 79.7% of labeled cells (456/572) were immunoreacted for calbindin D28k. The distribution of calbindin D28k-immunoreactivity may be differnt from that of calretinin. It is suggested that calbindin D28k have regulatory role on intracellular calcium concentration in the laryngeal sensory corpuscles.     

Differential expression patterns of the tight junction-associated proteins occludin and claudins in secretory and mature ameloblasts in mouse incisor

Tight junctions (TJs) function primarily as a barrier against paracellular transport between epithelial cells and are composed mainly of occludin (OLD) and claudins (CLDs). The CLD family consists of 24 members that show tissue- or cell-specific expression. Ameloblasts, which originate from the oral epithelium, form enamel, and enamel proteins and minerals are transported across the ameloblastic layer during amelogenesis. We immunohistochemically examined the distribution patterns of TJs in ameloblasts by observing the expression patterns of OLD and CLDs (CLD-1 to CLD-10). Secretory ameloblasts contained OLD and CLD-1, -8, and -9 at the distal end of the cell. In mature ameloblasts, OLD and CLD-1, -6, -7, -8, -9, and -10 were present mainly at both the distal and proximal ends of the cell, regardless of whether the ameloblasts were ruffle-ended or smooth-ended. Mature ameloblasts in which only the proximal ends were stained for OLD and CLDs were also found. These results indicate that the expression patterns of CLDs and the distribution patterns of TJs change drastically between the secretory and mature ameloblast stages, suggesting that these patterns reflect the different functions of these cells, specifically in the transport of proteins and ions for enamel formation.     

Differential Expression of the Tight Junction Proteins,Claudin‐1, Claudin‐4, Occludin,ZO‐1, and PAR3, in the Ameloblasts of Rat Upper Incisors

Tight junctions (TJs) create a paracellular permeability barrier to restrict the passage of ions, small solutes, and water. Ameloblasts are enamel‐forming cells that sequentially differentiate into preameloblasts, secretory, transition, and ruffle‐ended and smooth‐ended maturation ameloblasts (RAs and SAs). TJs are located at the proximal and distal ends of ameloblasts. TJs at the distal ends of secretory ameloblasts and RAs are well‐developed zonula occludens, but other TJs are moderately developed but incomplete zonula occludens (ZO) or less‐developed macula occludens. We herein examined the immunofluorescence localization of TJ proteins, 10 claudin isoforms, occludin, ZO‐1, and PAR3, a cell polarity‐related protein, in ameloblasts of rat upper incisors. ZO‐1 and claudin‐1 were detected at both ends of all ameloblasts except for the distal ends of SAs. Claudin‐4 and occludin were detected at both ends of transition and maturation ameloblasts except for the distal ends of SAs. PAR3 was detected at the proximal TJs of all ameloblasts and faintly at the distal TJs of early RAs. These results indicate that functional zonula occludens formed at the distal ends of the secretory ameloblasts and RAs consisted of different TJ proteins. Therefore, the distal TJs of secretory ameloblasts and RAs may differentially regulate the paracellular permeability to create a microenvironment suitable for enamel deposition and enamel maturation, respectively. In addition, PAR3 may be principally involved in the formation and maintenance of the proximal, but not distal, TJs. Anat Rec, 291:577–585, 2008. © 2008 Wiley‐Liss, Inc.     

Immunolocalization of enamel proteins during amelogenesis in the cat

Amelogenesis in the cat has been suggested to closely resemble enamel formation in human teeth. In order to further characterize the sequence of events leading to enamel formation in the cat, the expression and distribution of enamel proteins throughout amelogenisis were examined by postembedding immunocytochemistry using an antibody to mouse amelogenins and the high resolution protein A-gold technique. Enamel proteins were first immunodetected in ameloblasts and in the extracellular matrix during the presecretory stage. Secretory stage ameloblasts showed the most intense cellular reactivity. In these cells, protein synthetic organelles, secretory granules, and large lysosome-like structures were all intensely labeled. Extracellulary, numerous gold particles were observed over enamel and over patches of material found at the baso-lateral surfaces of these ameloblasts. During the early maturation stage, the protein synthetic organelles and secretory granules of ameloblasts still showed some immunoreactivity, although the most conspicuous labeling at this later stage was found over enamel and over material present amoung the extensive apical membrane infoldings of ruffle-ended ameloblasts. Qualitative analysis of lysosome-like elements in ameloblasts suggested that their frequency and immunoreactivity in the maturation stage were relatively lower than in the secretory stage, where some groups of cells often showed numerous large labeled structures. The enamel matrix was intensely labeled at all stages; however, cervical-occlusal and surface-depth gradients were readily apparent by conventional staining and by quantitative analysis of immunolabeling in the late secretory and early maturation stages. These data suggest that the cellular and extracellular distribution of enamel proteins in the cat is generally similar to that reported in other species, although some particularities were observed, perhaps reflecting variation in the timing of developmental parameters. © 1992 Wiley-Liss, Inc.     

Immunolocalization of enamel proteins during amelogenesis in the cat.

Amelogenesis in the cat has been suggested to closely resemble enamel formation in human teeth. In order to further characterize the sequence of events leading to enamel formation in the cat, the expression and distribution of enamel proteins throughout amelogenesis were examined by postembedding immunocytochemistry using an antibody to mouse amelogenins and the high resolution protein A-gold technique. Enamel proteins were first immunodetected in ameloblasts and in the extracellular matrix during the presecretory stage. Secretory stage ameloblasts showed the most intense cellular reactivity. In these cells, protein synthetic organelles, secretory granules, and large lysosome-like structures were all intensely labeled. Extracellularly, numerous gold particles were observed over enamel and over patches of material found at the baso-lateral surfaces of these ameloblasts. During the early maturation stage, the protein synthetic organelles and secretory granules of ameloblasts still showed some immunoreactivity, although the most conspicuous labeling at this later stage was found over enamel and over material present among the extensive apical membrane infoldings of ruffle-ended ameloblasts. Qualitative analysis of lysosome-like elements in ameloblasts suggested that their frequency and immunoreactivity in the maturation stage were relatively lower than in the secretory stage, where some groups of cells often showed numerous large labeled structures. The enamel matrix was intensely labeled at all stages; however, cervical-occlusal and surface-depth gradients were readily apparent by conventional staining and by quantitative analysis of immunolabeling in the late secretory and early maturation stages. These data suggest that the cellular and extracellular distribution of enamel proteins in the cat is generally similar to that reported in other species, although some particularities were observed, perhaps reflecting variation in the timing of developmental parameters.     

Prenatal expression of growth hormone receptor/binding protein and insulin-like growth factor-I (IGF-I) in the enamel organ

Immunohistochemistry was used to study the ontogeny of GH receptor/binding protein (GHR/BP) and IGF-I from the 13-day-old embryo (E13) to the E19 rat fetus in the developing incisor and molar. Analysis of serial sections revealed diffuse staining of GHR/BP and IGF-I at the bud and early cap stages within both the mesenchyme of the dental papilla and the ectodermal-erived enamel organ. Just before transition to the cap stage, immunoreactivity of GHR/BP and IGF-I increased in the epithelial bud and extended to the condensed dental mesenchyme. At the cap stage, the dental epithelium showed an intense expression of GHR/BP and IGF-I, whereas the dental mesenchymal cells showed very weak staining. The inner enamel epithelium and the outer enamel epithelium were positive for both GHR/BP and IGF-I in the bell stage. Differentiating ameloblasts, odontoblasts and the secretory ameloblasts and odontoblasts continued to express GHR/BP and IGF-I in incisors. These findings support the premise that growth hormone and IGF-I may play a role in embryonic tooth development by regulating the epithelial-mesenchymal interactions that influence events in growth and cytodifferentiation.     

Cooperation of nectin‐1 and nectin‐3 is required for normal ameloblast function and crown shape development in mouse teeth

Nectins are immunoglobulin‐like cell adhesion proteins and their interactions recruit various cell–cell junctions. Mutations in human NECTIN‐1 cause an ectodermal dysplasia syndrome, but Nectin‐1 null mice have only slight defects in teeth, suggesting compensation by other nectin(s). We observed overlapping expression of nectin‐3 with nectin‐1 and enamel abnormality in the nectin‐3 mutant. We, therefore, generated nectin‐1;nectin‐3 compound mutants. However, all teeth developed and no significant dental abnormalities were observed before birth. At postnatal day 10, the upper molars of compound mutants exhibited conical crown shape and retarded enamel maturation. Nectin‐1 was expressed in ameloblasts whereas nectin‐3 was expressed in neighboring stratum intermedium cells at this stage. The immunohistochemical localization and electron microscopical observations indicated that the desmosomal junctions between stratum intermedium and ameloblasts were significantly reduced. These results suggest that heterophilic interaction between nectin‐1 and nectin‐3 recruits desmosomal junctions, and that these are required for proper enamel formation. Developmental Dynamics 239:2558–2569, 2010. © 2010 Wiley‐Liss, Inc.