Makoto J. Tabata

Parathyroid hormone 1 (1–34) acts on the scales and involves calcium metabolism in goldfish

The effect of fugu parathyroid hormone 1 (fugu PTH1) on osteoblasts and osteoclasts in teleosts was examined with an assay system using teleost scale and the following markers: alkaline phosphatase (ALP) for osteoblasts and tartrate-resistant acid phosphatase (TRAP) for osteoclasts. Synthetic fugu PTH1 (1-34) (100pg/ml-10ng/ml) significantly increased ALP activity at 6h of incubation. High-dose (10ng/ml) fugu PTH1 significantly increased ALP activity even after 18h of incubation. In the case of TRAP activity, fugu PTH1 did not change at 6h of incubation, but fugu PTH1 (100pg/ml-10ng/ml) significantly increased TRAP activity at 18h. Similar results were obtained for human PTH (1-34), but there was an even greater response with fugu PTH1 than with human PTH. In vitro, we demonstrated that both the receptor activator of the NF-κB ligand in osteoblasts and the receptor activator NF-κB mRNA expression in osteoclasts increased significantly by fugu PTH1 treatment. In an in vivo experiment, fugu PTH1 induced hypercalcemia resulted from the increase of both osteoblastic and osteoclastic activities in the scale as well as the decrease of scale calcium contents after fugu PTH1 injection. In addition, an in vitro experiment with intramuscular autotransplanted scale indicated that the ratio of multinucleated osteoclasts/mononucleated osteoclasts in PTH-treated scales was significantly higher than that in the control scales. Thus, we concluded that PTH acts on osteoblasts and osteoclasts in the scales and regulates calcium metabolism in goldfish.

Cell size-specific appearance of neuropeptide Y in the trigeminal ganglion following peripheral axotomy of different branches of the mandibular nerve of the rat

The effect of peripheral axotomy of the mental nerve (MN) and the cutaneous branch of the mylohyoid nerve (MhN) on the appearance of neuropeptide Y-like immunoreactivity (NPY-IR) in cells in the trigeminal ganglion of the rat was examined with combined retrograde-tracing and immunohistochemistry. Retrograde-tracing with True Blue (TB) revealed that the cell-size spectrum of the trigeminal cells sending peripheral processes to the MN (TB MN cells) ranged from 75.9 to 1560.5 microns2 (or from 9.8 to 44.6 microns in diameter); approximately 53% of TB MN cells were 300-600 microns2. TB MhN cells ranged from 47.7 to 1261.5 microns2 (or from 7.8 to 40.1 microns in diameter); 56% of TB MhN cells were < 300 microns2. In the normal trigeminal ganglion, there were no NPY-IR cells. 14 days after MN transection, approximately 35% of TB MN cells displayed NPY-IR. The distribution of the cross-sectional areas of NPY-IR cells after MN transection was very similar to that of TB MN cells. Transection of MhN also induced the appearance of NPY-IR in the trigeminal ganglion but to a lesser extent (approximately 17% of TB MhN cells). The distribution of the cross-sectional areas of NPY-IR cells after MhN transection was similar to that of NPY-IR cells after MN transection. These results indicate that injury-evoked NPY-IR is specific for the medium- and large-sized ganglion cells.

Expression of cytokeratin 14 in ameloblast-lineage cells of the developing tooth of rat, both in vivo and in vitro

In the search for a cell marker useful for studying tooth development, immunohistochemical studies using antibodies against cytokeratin 14 (K14), c-Met/hepatocyte growth factor receptor and amelogenin were carried out in the developing tooth of the newborn rat and in primary cultured cells of the ameloblast lineage, including inner enamel epithelium cells, preameloblasts and ameloblasts, prepared from the mandibular incisors of postnatal 7-day-old rats. The appearance of K14 was cell- and differentiation-stage specific, i.e. there was a weak expression signal within inner enamel epithelial cells that were in the proliferating stage, and there were strong signals within preameloblasts and ameloblasts that were in the post-proliferating and amelogenesis stages, respectively. In the culture system, c-Met appeared in all cells, whereas K14 and amelogenin appeared mainly in clustered cells that were considered to be in the post-proliferating stage. K14 was detected earlier than amelogenin, and it was also confirmed by immunofluorostaining that c-Met, K14 and amelogenin were coexpressed in ameloblasts. These findings indicate that K14 is a good new marker for ameloblast-lineage cells during rat tooth development both in vivo and in vitro.

Induction of Fos protein in the rat trigeminal nucleus complex during an experimental tooth movement

The induction and temporal changes of Fos protein in the rat spinal trigeminal nucleus complex during experimental tooth movement were studied immunohistochemically. Separating elastics were unilaterally inserted between the upper molars. The animals were perfused at 0, 1, 2, 4, 8, 12 and 24 h thereafter, and the brains containing the mesencephalic trigeminal nucleus and the spinal trigeminal nucleus complex were then removed. Cells showing Fos immunoreactivity were observed in the superficial layers of the subnucleus caudalis on the ipsilateral side except at 0 and 24 h. The numbers of the immunoreactive cells peaked 2 and 4 h after the insertion. On the contralateral side, only a few immunoreactive cells were observed in the subnucleus caudalis of the 1-, 2- and 4-h groups. The subnucleus interporalis and the subnucleus oralis of the spinal trigeminal nucleus complex, the principal trigeminal nucleus and the mesencephalic trigeminal nucleus of the experimental animals and all the trigeminal nuclei of the control animals contained no immunopositive cells. Thus experimental tooth movement induced a Fos protein in the superficial layers of the subnucleus caudalis of the spinal trigeminal nucleus complex. The subnucleus caudalis may modulate pain induced by tooth movement.

Fibronectin accelerates the growth and differentiation of ameloblast lineage cells in vitro.

During tooth development, the growth and differentiation of ameloblast lineage (AL) cells are regulated by epithelial–mesenchymal interactions. To examine the dynamic effects of components of the basement membrane, which is the extracellular matrix (ECM) lying between the epithelium and mesenchyme, we prepared AL cells from the epithelial layer sheet of mandibular incisors of postnatal day 7 rats and cultured them on plates coated with type IV collagen, laminin-1, or fibronectin. The growth of AL cells was supported by type IV collagen and fibronectin but not by laminin-1 in comparison with that on type I collagen as a reference. Clustering and differentiation of AL cells were observed on all matrices examined. AL cells showed normal growth and differentiation at low cell density on fibronectin but not on type I collagen. Furthermore, the population of cytokeratin 14-positive cells on fibronectin was lower than that on other ECM components, suggesting that fibronectin may be a modulator to accelerate the differentiation of AL cells. After the cells had been cultured for 9 days on fibronectin, crystal-like structures were observed. These structures overlaid the cell clusters and were positive for von Kossa staining. These findings indicate that each matrix component has a regulative role in the proliferation and differentiation of AL cells and that fibronectin causes the greatest acceleration of AL cell differentiation.

Parathyroid hormone-related peptide is involved in protection against invasion of tooth germs by bone via promoting the differentiation of osteoclasts during tooth development

In order to elucidate the role of parathyroid hormone-related peptide (PTHrP) in tooth development, we treated tooth germ explants of mouse molars with antisense phosphorothioate-oligodeoxynucleotide (ODN) against PTHrP. Antisense ODN-treatment of the explants resulted in the invasion of the tooth germs by bone. The number of tartrate-resistant acid phosphatase (TRAP)-positive cells around the tooth germs in antisense ODN-treated explants was much lower than that of the control explants. Electron microscopic examination suggested that the antisense ODN-treatment inhibited differentiation of osteoclasts. Treatment of the explants with bisphosphonate or vitamin K2, inhibitors of the differentiation of osteoclasts, induced the invasion by bone into the tooth germs as observed in the antisense ODN-treated explants. The results obtained suggest that PTHrP is involved in the mechanism protecting tooth germs from bone invasion by promoting the differentiation of osteoclasts around them.

Monohydroxylated polycyclic aromatic hydrocarbons inhibit both osteoclastic and osteoblastic activities in teleost scales

AIMS We previously demonstrated that monohydroxylated polycyclic aromatic hydrocarbons (OHPAHs) bound to a human estrogen receptor (ER) by a yeast two-hybrid assay, but polycyclic aromatic hydrocarbons did not have a binding activity. Therefore, the direct effect of 3-hydroxybenz[a]anthracene (3-OHBaA) and 4-hydroxybenz[a]anthracene (4-OHBaA) on osteoclasts and osteoblasts in teleosts was examined. As a negative control, 1-hydroxypyrene (1-OHPy), which has no binding activity to human ER, was used. MAIN METHODS The effect of OHPAHs on osteoclasts and osteoblasts was examined by an assay system using teleost scale as each marker: tartrate-resistant acid phosphatase for osteoclasts and alkaline phosphatase for osteoblasts. Changes in cathepsin K (an osteoclastic marker) and insulin-like growth factor-I (IGF-I) (an osteoblastic marker) mRNA expressions in 4-OHBaA-treated goldfish scales were examined by using a reverse transcription-polymerase chain reaction. KEY FINDINGS In both goldfish (a freshwater teleost) and wrasse (a marine teleost), the osteoclastic activity in the scales was significantly suppressed by 3-OHBaA and 4-OHBaA, although 1-OHPy did not affect the osteoclastic activity. In reference to osteoblasts, the osteoblastic activity decreased with both 3-OHBaA and 4-OHBaA and did not change with the 1-OHPy treatment. However, 17beta-estradiol (E(2)) significantly increased both the osteoclastic and osteoblastic activities in the scales of both goldfish and wrasse. The mRNA expressions of both cathepsin K and IGF-I decreased in the 4-OHBaA-treated scales but increased in the E(2)-treated scales. SIGNIFICANCE The current data are the first to demonstrate that 3-OHBaA and 4-OHBaA inhibited both osteoclasts and osteoblasts and disrupted the bone metabolism in teleosts.

Tooth-type specific expression of dHAND/Hand2: possible involvement in murine lower incisor morphogenesis.

Abstract. dHAND/Hand2 is a basic helix-loop-helix transcription factor required for the development of the heart, pharyngeal arches, and vasculature and is expressed during embryogenesis. However, there are no reports on the involvement of the dHAND gene in tooth development. In the present study, the expression of dHAND was examined in developing tooth germs of mice. The dHAND gene was expressed in the mesenchyme of the presumptive incisor region of the lower jaw at an early stage and in the mesenchyme of the lower incisor tooth germ at a later stage. However, the dHAND gene was not expressed in the upper incisor region or the upper and lower molar regions during jaw development. Treatment of tooth germ explants of lower incisors with antisense oligodeoxinucleotide (ODN) against dHAND prevented the differentiation of tooth germ cells, including ameloblasts and odontoblasts, the formation of dentin and enamel, and the proliferation of tooth germ cells and increased the apoptosis of tooth germ cells, suggesting that dHAND is essential for these cells during development. On the other hand, the treatment of tooth germ explants of upper incisor and upper or lower molars did not induce severe effects on their development. Treatment of the explants with basic fibroblast growth factor in association with antisense ODN partially rescued them from the effects of antisense ODN. The present results suggest that the dHAND gene plays important roles in type-specific development of lower incisors, and that basic fibroblast growth factor is involved downstream of the dHAND pathway in tooth germ cells.

Immunohistochemical localization of calbindin D28k during root formation of rat molar teeth.

Abstract The present study was undertaken to examine the localization of calbindin D28k (CB)-like immunoreactivity (-LI) during the root formation of the rat molar. In the adult rat, CB-LI was detected in some of the cells of the epithelial rest of Malassez at the bifurcational region and in certain cells between the root dentin and cementum at the apical region. These cells had indented nuclei and many tonofilaments, and cementocytes lacked CB-LI. Moreover, CB-LI was observed in the periodontal fibroblasts in the alveolar half of the apical region. During root formation, the cells in the Hertwig’s epithelial root sheath (HERS) lacked CB-LI, but most fragmented cells along the root surface began to express CB-LI when HERS was disrupted. Preodontoblasts and odontoblasts at the apical portion of the root also showed CB-LI. After the formation of cellular cementum, the CB-immunoreactive (-IR) cells were entrapped between the root dentin and cementum in the apical portion of the root. The number of CB-IR cells at the root surface decreased gradually, while that between the root dentin and cementum increased. The fibroblasts in the periodontal ligament began to express CB-LI after commencement of the occlusion, and the number and the staining intensity of CB-IR fibroblasts increased gradually with the passage of time. The present results suggest that CB may play an important role in the survival of the epithelial cells, in the cellular responses of periodontal fibroblasts against mechanical forces caused by the occlusion, and in the initial mineralization by the odontoblasts through the regulation of intracellular Ca2+ concentration.

Tooth replacement and putative odontogenic stem cell niches in pharyngeal dentition of medaka (Oryzias latipes)

The small-sized teleost fish medaka, Oryzias latipes, has as many as 1000 pharyngeal teeth undergoing continuous replacement. In this study, we sought to identify the tooth-forming units and determine its replacement cycles, and further localize odontogenic stem cell niches in the pharyngeal dentition of medaka to gain insights into the mechanisms whereby continuous tooth replacement is maintained. Three-dimensional reconstruction of pharyngeal epithelium and sequential fluorochrome labeling of pharyngeal bones and teeth indicated that the individual functional teeth and their successional teeth were organized in families, each comprising up to five generations of teeth and successional tooth germs, and that the replacement cycle of functional teeth was approximately 4 weeks. BrdU label/chase experiments confirmed the existence of clusters of label-retaining epithelial cells at the posterior end of each tooth family where the expression of pluripotency marker Sox2 was confirmed by in situ hybridization. Label-retaining cells were also identified in the mesoderm immediately adjacent to the posterior end of each tooth family. These data suggest the importance of existence of slow-cycling dental epithelial cells and Sox2 expressions at the posterior end of each tooth family to maintain continuous tooth formation and replacement in the pharyngeal dentition of medaka.