Yoshiro Takano

Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation.

The composite structure of the mammalian skull, which forms predominantly via intramembranous ossification, requires precise pre- and post-natal growth regulation of individual calvarial elements. Disturbances of this process frequently cause severe clinical manifestations in humans. Enhanced DNA binding by a mutant MSX2 homeodomain results in a gain of function and produces craniosynostosis in humans. Here we show that Msx2-deficient mice have defects of skull ossification and persistent calvarial foramen. This phenotype results from defective proliferation of osteoprogenitors at the osteogenic front during calvarial morphogenesis, and closely resembles that associated with human MSX2 haploinsufficiency in parietal foramina (PFM). Msx2−/− mice also have defects in endochondral bone formation. In the axial and appendicular skeleton, post-natal deficits in Pth/Pthrp receptor (Pthr) signalling and in expression of marker genes for bone differentiation indicate that Msx2 is required for both chondrogenesis and osteogenesis. Consistent with phenotypes associated with PFM, Msx2-mutant mice also display defective tooth, hair follicle and mammary gland development, and seizures, the latter accompanied by abnormal development of the cerebellum. Most Msx2-mutant phenotypes, including calvarial defects, are enhanced by genetic combination with Msx1 loss of function, indicating that Msx gene dosage can modify expression of the PFM phenotype. Our results provide a developmental basis for PFM and demonstrate that Msx2 is essential at multiple sites during organogenesis.

The distribution and origin of substance P-like immunoreactivity in the rat molar pulp and periodontal tissues.

Rat mandibles were fixed in Zamboni fixative and demineralized in a mixture of EDTA and fixative. Substance P-like immunoreactivity was demonstrated by indirect immunofluorescence in molar pulp, periodontal ligament and gingiva. Substance P (SP) containing nerve fibres with varicosities were observed in the pulp horn and root pulp in general located around blood vessels. Some SP-containing fibres penetrated into the predentine and dentine. In the periodontal ligament, SP fibres were localized along the blood vessels in the middle and apical regions. Many SP-containing fibres were associated with the blood vessels in the lamina propria of gingiva. After inferior alveolar nerve section, SP-positive nerve fibres in the pulp and periodontal ligament disappeared completely. In gingiva the number of SP fibres decreased but not all fibres disappeared. Removal of the superior cervical ganglion did not affect the distribution of SP-containing nerve fibres.

The teleost intervertebral region acts as a growth center of the centrum: In vivo visualization of osteoblasts and their progenitors in transgenic fish

The vertebral column is a defined feature of vertebrates. In birds and mammals, the sclerotome yields cartilaginous material for the vertebral column. In teleosts, however, it remains uncertain whether the sclerotome participates in vertebral column formation. To investigate osteoblast development in the teleost, we established transgenic systems that allow in vivo observation of osteoblasts and their progenitors marked by fluorescence of DsRed and enhanced green fluorescent protein (EGFP), respectively. In twist‐EGFP transgenic medaka, EGFP‐positive cells first appeared in the ventromedial portion of respective somites corresponding to the sclerotome, migrated dorsally around the notochord, and concentrated in the intervertebral regions. Ultrastructural analysis of the intervertebral regions revealed that some of these cells were directly located on the osteoidal surface of the perichordal centrum, and enriched with rough endoplasmic reticulum in their cytoplasm. By using the double transgenic medaka of twist‐EGFP and osteocalcin‐DsRed, we clarified that the EGFP‐positive cells in the intervertebral region differentiated into mature osteoblasts expressing the DsRed. In vivo bone labeling in fact confirmed active matrix formation and mineralization of the perichordal centrum exclusively in the intervertebral region of zebrafish larvae as well as medaka larvae. These findings strongly suggest that the teleost intervertebral region acts as a growth center of the perichordal centrum, where the sclerotome‐derived cells differentiate into osteoblasts. Developmental Dynamics 236:3031–3046, 2007.

Ion transporters in secretory and cyclically modulating ameloblasts: a new hypothesis for cellular control of preeruptive enamel maturation

Mature enamel consists of densely packed and highly organized large hydroxyapatite crystals. The molecular machinery responsible for the formation of fully matured enamel is poorly described but appears to involve oscillative pH changes at the enamel surface. We conducted an immunohistochemical investigation of selected transporters and related proteins in the multilayered rat incisor enamel organ. Connexin 43 (Cx-43) is found in papillary cells and ameloblasts, whereas Na(+)-K(+)-ATPase is heavily expressed during maturation in the papillary cell layer only. Given the distribution of Cx-43 channels and Na(+)-K(+)-ATPase, we suggest that ameloblasts and the papillary cell layer act as a functional syncytium. During enamel maturation ameloblasts undergo repetitive cycles of modulation between ruffle-ended (RA) and smooth-ended (SA) ameloblast morphologies. Carbonic anhydrase II and vacuolar H(+)-ATPase are expressed simultaneously at the beginning of the maturation stage in RA cells. The proton pumps are present in the ruffled border of RA and appear to be internalized during the SA stage. Both papillary cells and ameloblasts express plasma membrane acid/base transporters (AE2, NBC, and NHE1). AE2 and NHE1 change position relative to the enamel surface as localization of the tight junctions changes during ameloblast modulation cycles. We suggest that the concerted action of the papillary cell layer and the modulating ameloblasts regulates the enamel microenvironment, resulting in oscillating pH fluctuations. The pH fluctuations at the enamel surface may be required to keep intercrystalline spaces open in the surface layers of the enamel, enabling degraded enamel matrix proteins to be removed while hydroxyapatite crystals grow as a result of influx of calcium and phosphate ions.

Gene Expression of Growth Differentiation Factors in the Developing Periodontium of Rat Molars

Growth and differentiation factors (GDF) 5, 6, and 7 are known to play roles in tendon and ligament formation, and are therefore probably involved in the formation of periodontal ligament. In this study, we sought to determine temporal and spatial expression of GDF-5, -6, and -7 mRNA in developing periodontal tissue of rat molars using in situ hybridization. GDF gene expression in the periodontal ligament was first detected in cells associated with the initial process of periodontal ligament fiber bundle formation. Gene signals were also detected in cells located along the alveolar bone and cementum surfaces, the insertion sites of periodontal ligaments, during the course of root formation. GDF expression in these cells were down-regulated after completion of root formation. Our results appeared to suggest the involvement of GDF-5, -6, and -7 in the formation of the dental attachment apparatus.

Correlation of45Ca incorporation with maturation ameloblast morphology in the rat incisor

SummaryRats were injected with45Ca and horseradish peroxidase to determine the patterns of45Ca incorporation into incisor enamel and the morphological types of the overlying maturation ameloblasts.45Ca autoradiography showed no differences in the patterns of incorporation into enamel between routinely embedded and freeze-dried specimens. Enamel overlaid by ruffle-ended ameloblasts was much more heavily labeled while that overlaid by smooth-ended ameloblasts showed only moderate labeling. The observations lend further support to the hypothesis that the ruffle-ended cells are very active in mineralizing enamel and that the smooth-ended cells are in a passive, restorative phase.

Ca-ATPase and ALPase activities at the initial calcification sites of dentin and enamel in the rat incisor

SummaryEnzymatic activities of calcium-magnesium dependent adenosine triphosphatase (Ca-ATPase) and nonspecific alkaline phosphatase (ALPase) were localized at the initial calcification sites of dentin and enamel of rat incisor teeth using electron-microscopic cytochemistry.Ca-ATPase was localized in the Golgi cisternae, cytoplasmic vesicles and along the outer surface of the presecretory and secretory ameloblasts, whereas it was totally absent from the odontoblasts in the pulp. Inversely, ALPase reaction was localized along the outer surface of the odontoblasts, but almost completely absent from the ameloblasts.Diffuse extracellular reactions of both enzymes were distributed throughout the unmineralized fibrous matrix of mantle dentin in which a large number of matrix vesicles were scattered. Both Ca-ATPase and ALPase reactions, which appeared in the matrix vesicles in the process of formation of mantle dentin, became most conspicuous at the site of initial dentin calcification. At this stage, an intense Ca-ATPase reaction also appeared along some of the collagen fibrils adjacent to the reactive matrix vesicles. No ALPase reaction was localized along these Ca-ATPase reactive collagen fibrils.Our observations suggest strongly that Ca-ATPase in the matrix vesicles originates from the inner enamel epithelium and/or preameloblasts whereas ALPase originates from the odontoblasts in the pulp. The importance of the coexistence of both enzymes for the control of initial calcification of dental hard tissues is suggested.

Disturbed tooth development in parathyroid hormone-related protein (PTHrP)-gene knockout mice

Parathyroid hormone-related protein (PTHrP) is involved in epithelial-mesenchymal cell interactions during development of various tissues and organs. Tooth germ development is a classical model for this interaction. In tooth germs, PTHrP is expressed in the enamel organ (epithelial component), whereas its major receptor, the type I PTH/PTHrP receptor is expressed in cells of the alveolar bone and dental follicle (mesenchymal components). To clarify the role of PTHrP during fetal tooth germ development, PTHrP gene-knockout mice were used for histochemical and ultrastructural analysis. In wild-type mice, osteoclastic cells were aligned predominantly in the inner aspects of the alveolar bone surrounding the developing tooth germs throughout the late embryonic (after embryonic, 17.5 days) and neonatal animals examined. In contrast, osteoblasts were predominant in corresponding areas of fetal homozygous PTHrP-gene knockout mice with only occasional osteoclasts. In such areas, cell-free surfaces showing cement line-like tartrate-resistant acid phosphatase (TRAP) reactions were frequently observed. In neonatal homozygous mice, bone spicules were often shown to penetrate and/or compress the enamel organ and caused partial destruction of the tooth germs. Osteoclasts were few in number in the inner aspects of the alveolar bone, and had poorly developed ruffled border. No morphological abnormality was noted in cells of the tooth germs proper. On bone surfaces away from developing tooth germs, functional osteoclasts with structural features similar to those in wild-type mice were observed in homozygous mice. These observations suggest that PTHrP is required to maintain an appropriate spatiotemporal arrangement of bone cells and osteoclast function, which are necessary for the normal development of tooth germ and alveolar bone encasing the tooth germ. The observation also demonstrates that PTHrP deficiency affects the structure and function of osteoclasts exclusively those located in the vicinity of the growing tooth germ.

RESPONSES OF IMMUNOCOMPETENT CELLS TO CAVITY PREPARATION IN RAT MOLARS : AN IMMUNOHISTOCHEMICAL STUDY USING OX6-MONOCLONAL ANTIBODY

Responses of immunocompetent cells, especially class II major histocompatibility complex (MHC) antigen-expressing cells, were investigated after cavity preparation in the erupted upper first molar teeth of rats, by immunohistochemistry using OX6-monoclonal antibody. In control teeth, OX6-immunopositive cells were predominantly located beneath the odontoblast layer in the dental pulp. Cavity preparation caused an acute edematous reaction between the injured odontoblasts and predentin, and most of OX6-immunopositive cells in the affected site shifted away from the pulp-dentin border. After 12-24 hours, many OX6-immunopositive cells accumulated along the pulp-dentin border and extended their cytoplasmic processes into the exposed dentinal tubules. After 72 hours, newly differentiated odontoblasts replaced the degenerated odontoblasts, and few OX6-immunopositive cells remained along the pulp-dentin border. Our data suggest that some of the class II MHC antigen-expressing cells in the dental pulp participate in the initial defense reaction and presumably serve as a biological sensor for the external stimuli arriving through the exposed dentinal tubules.

Enamel Mineralization and the Role of Ameloblasts in Calcium Transport

Amelogenesis is a dynamic and unique process of cell-matrix interactions in that matrix synthesis, degradation and resorption all proceed simultaneously, coupled with mineral depositions in a compartment between ameloblasts and dentin or dental papilla. Accumulation of data suggest the role of ameloblasts in tooth morphogenesis and matrix formation, but no fully acceptable explanation has been given concerning the role of ameloblasts in calcium transport. In this article, old and new points of issue raised regarding the role of ameloblasts in calcium acquisition are reviewed and possible mechanisms whereby the ameloblasts prevent the rise of cytosolic calcium while actively or less actively transporting calcium are elaborated upon based on recent findings.