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.

Rabbit chondrocyte cultures on plastic dishes are capable of depositing a cartilaginous matrix, although the matrix does not calcify unless high levels of phosphate are added to the medium. In the present study, we cultivated a pelleted mass of, rabbit growth-plate chondrocytes in the presence of Eagle's minimum essential medium supplemented with 10% fetal bovine serum and 50 ,sg of ascorbic acid per ml in a plastic centrifuge tube. These cells proliferated for several generations and then reorganized into a cartilage-like tissue that calcified without additional phosphate. The deposition of minerals was observed only after synthesis of a short-chain collagen and alkaline phosphatase. Serum factors were required for the increases in alkaline phosphatase and calcium contents. 5-Bromo-2'-deoxyuridine abolished the increases in uronic acid, alkaline phosphatase, and calcium contents. Transforming growth factor J3, at very low concentrations, suppressed the expression of the mineralization-related phenotype by chondrocytes. These results suggest that cartilugematrix calcification can be controlled by growth factor(s) and that chondrocytes induce the mineralization of extracellular matrix when terminal differentiation is permitted in the absence of an artificial substrate.The calcification of the extracellular matrix in growth-plate cartilage is a key event that leads to longitudinal growth ofthe skeleton. Growth-plate cartilage is organized into three cellular zones containing proliferating, maturing, and hypertrophic cells. Maturing chondrocytes synthesize and deposit large amounts of cartilage-specific proteoglycans and type II collagen. The calcification process is confined to the hypertrophic zone where mineralization is initiated in matrix vesicles (1, 2). Hypertrophic chondrocytes produce a shortchain (type X) collagen (3,4) and alkaline phosphatase (4-6), some of which is associated with matrix vesicles (4, 6).Hypertrophic cells eventually die, and the zone is invaded by capillary sprouts. Vascular invasion is believed to play an important role in the onset of mineralization (see refs. 7 and 8 for review), but it remains unclear whether hypertrophic chondrocytes have the capacity to induce extracellular matrix mineralization in the absence of vascular invasion.Chondrocyte cultures have been used extensively during the past 30 years to study the control of their phenotypic expression. However; there have been few studies demonstrating cartilage-matrix calcification in a cell culture system. It has been reported that embryonic chicken chondrocytes (9-11) and rat growth-plate chondrocytes (12) in mass cultures on plastic dishes produce an abundant extracellular matrix, although the matrix does not calcify unless high levels of phosphate (3-fold over the level in standard medium) are included (11,12). The mineral deposition with high phosphate should be regarded as a physicochemical phenomenon (12). Because of the lack of a good in vitro system, no information about the regulation of cartilage calcificati...

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.

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.

Bone modeling is the central system controlling the formation of bone including bone growth and shape in early development, in which bone is continuously resorbed by osteoclasts and formed by osteoblasts. However, this system has not been well documented, because it is difficult to trace osteoclasts and osteoblasts in vivo during development. Here we showed the important role of osteoclasts in organogenesis by establishing osteoclast-specific transgenic medaka lines and by using a zebrafish osteoclast-deficient line. Using in vivo imaging of osteoclasts in the transgenic medaka carrying an enhanced GFP (EGFP) or DsRed reporter gene driven by the medaka TRAP (Tartrate-Resistant Acid Phosphatase) or Cathepsin K promoter, respectively, we examined the maturation and migration of osteoclasts. Our results showed that mononuclear or multinucleated osteoclasts in the vertebral body were specifically localized at the inside of the neural and hemal arches, but not at the vertebral centrum. Furthermore, transmission electron microscopic (TEM) analyses revealed that osteoclasts were flat-shaped multinucleated cells, suggesting that osteoclasts initially differentiate from TRAP-positive mononuclear cells residing around bone. The zebrafish panther mutant lacks a functional c-fms (receptor for macrophage colony-stimulating factor) gene crucial for osteoclast proliferation and differentiation and thus has a low number of osteoclasts. Analysis of this mutant revealed deformities in both its neural and hemal arches, which resulted in abnormal development of the neural tube and blood vessels located inside these arches. Our results provide the first demonstration that bone resorption during bone modeling is essential for proper development of neural and vascular systems associated with fish vertebrae.

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.

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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.