Tooth morphogenesis results from reciprocal interactions between oral epithelium and ectomesenchyme culminating in the formation of mineralized tissues, enamel, and dentin. During this process, epithelial cells differentiate into enamel-secreting ameloblasts. Ameloblastin, an enamel matrix protein, is expressed by differentiating ameloblasts. Here, we report the creation of ameloblastin-null mice, which developed severe enamel hypoplasia. In mutant tooth, the dental epithelium differentiated into enamel-secreting ameloblasts, but the cells were detached from the matrix and subsequently lost cell polarity, resumed proliferation, and formed multicell layers. Expression of Msx2, p27, and p75 were deregulated in mutant ameloblasts, the phenotypes of which were reversed to undifferentiated epithelium. We found that recombinant ameloblastin adhered specifically to ameloblasts and inhibited cell proliferation. The mutant mice developed an odontogenic tumor of dental epithelium origin. Thus, ameloblastin is a cell adhesion molecule essential for amelogenesis, and it plays a role in maintaining the differentiation state of secretory stage ameloblasts by binding to ameloblasts and inhibiting proliferation.

Cartilage plays an important role in mechanical load resistance and in skeletal structure support. It also serves as the skeletal template for endochondral ossification by which most bones in the body, such as long bones, are formed. In endochondral ossification, cartilage development is initiated by mesenchymal cell condensation, followed by a series of proliferation and differentiation processes. Cells undergoing condensation differentiate into chondrocytes, which then proliferate, produce type II collagen and form the proliferative zone of the cartilage molds. As development proceeds, chondrocytes in the center of the cartilage molds (prehypertrophic zone) cease proliferating and differentiate into type X collagen-producing hypertrophic chondrocytes to form the hypertrophic zone. Terminally differentiated hypertrophic chondrocytes mineralize the surrounding matrix. Eventually these cells die by apoptosis and are replaced by osteoblasts that form trabecular bone.The regulation of chondrocyte proliferation and differentiation must be tightly coordinated to allow formation of properly sized cartilage and bone (1). Parathyroid hormone-related peptide (PTHrP) 2 and parathyroid hormone (PTH) sustain chondrocyte proliferation and delay differentiation of the growth plate (2). PTHrP is expressed by perichondrial cells and chondrocytes in the upper region of growing cartilage. Mutant mice that are deficient in PTHrP (3), PTH (4), or its receptor (5) have short proliferative zones and accelerated chondrocyte differentiation, which results in abnormal endochondral bone formation. In contrast, mice that overexpress PTHrP have enlarged proliferative zones and delayed chondrocyte terminal differentiation (6). Humans with an activating mutation in the PTH/ PTHrP receptor develop Jansen metaphyseal chondrodysplasia, characterized by disorganization of the growth plates and delayed chondrocyte terminal differentiation (7). These results suggest that PTH/PTHrP signaling regulates skeletal development by promoting cell proliferation and inhibiting hypertrophic differentiation of chondrocytes.The binding of PTH/PTHrP to its receptor activates both G s and G q family heterotrimeric G proteins (8, 9). The activation of G s is necessary for cAMP production and protein kinase A (PKA) activation, which leads to phosphorylation of the cAMPresponse element-binding (CREB) family of transcription factors. CREB then induces genes such as the cyclin D1 and cyclin A genes. The activated cyclin/cyclin-dependent kinases in turn phosphorylate the retinoblastoma protein and its relative factors, which then dissociates the E2F transcription factor and subsequently activates the target genes necessary for DNA replication and cell cycle progression. Thus, CREB is a direct target of PKA and a downstream target of PTH/PTHrP/cAMP signaling and is required for chondrocyte proliferation (10, 11). How proliferation signaling is down-regulated in the prehypertrophic zone to stop proliferation and allow the switch to the postmitotic state is not well unde...

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Pannexin 3 functions as an essential protein for Ca2+ and ATP transport and cell–cell communication during osteoblast differentiation

In tooth morphogenesis, the dental epithelium and mesenchyme interact reciprocally for growth and differentiation to form the proper number and shapes of teeth. We previously identified epiprofin (Epfn), a gene preferentially expressed in dental epithelia, differentiated ameloblasts, and certain ectodermal organs. To identify the role of Epfn in tooth development, we created Epfn-deficient mice (Epfn ؊/؊ ). Epfn ؊/؊ mice developed an excess number of teeth, enamel deficiency, defects in cusp and root formation, and abnormal dentin structure. Mutant tooth germs formed multiple dental epithelial buds into the mesenchyme. In Epfn ؊/؊ molars, rapid proliferation and differentiation of the inner dental epithelium were inhibited, and the dental epithelium retained the progenitor phenotype. Formation of the enamel knot, a signaling center for cusps, whose cells differentiate from the dental epithelium, was also inhibited. However, multiple premature nonproliferating enamel knot-like structures were formed ectopically. These dental epithelial abnormalities were accompanied by dysregulation of Lef-1, which is required for the normal transition from the bud to cap stage. Transfection of an Epfn vector promoted dental epithelial cell differentiation into ameloblasts and activated promoter activity of the enamel matrix ameloblastin gene. Our results suggest that in Epfn-deficient teeth, ectopic nonproliferating regions likely bud off from the self-renewable dental epithelium, form multiple branches, and eventually develop into supernumerary teeth. Thus, Epfn has multiple functions for cell fate determination of the dental epithelium by regulating both proliferation and differentiation, preventing continuous tooth budding and generation.

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We identified a cDNA clone for epiprofin, which is preferentially expressed in teeth, by differential hybridization using DNA microarrays from an embryonic day 19.5 mouse molar cDNA library. Sequence analysis revealed that this cDNA encodes a member of the Krü ppellike factor family containing three characteristic C 2 H 2 -type zinc finger motifs. The full-length cDNA was obtained by the 5 Cap capture method. Except for its 5 -terminal sequence, the epiprofin mRNA sequence is almost identical to the predicted sequence of Krü ppellike factor 14/Sp6 (specificity protein 6), which was previously identified in expressed sequence tag data bases and GenBank TM by an Sp1 zinc finger DNA-binding domain search (Scohy, S., Gabant, P., Van Reeth, T., Hertveldt, V., Dreze, P. L., Van Vooren, P., Riviere, M., Szpirer, J., and Szpirer, C. (2000) Genomics 70, 93-101). This sequence difference is due to differences in the assignment of the location of exon 1. In situ hybridization revealed that epiprofin mRNA is expressed by proliferating dental epithelium, differentiated odontoblast, and also hair follicle matrix epithelium. In addition, whole mount in situ hybridization showed transient expression of epiprofin mRNA in cells of the apical ectodermal ridge in developing limbs and the posterior neuropore. Transfection of an epiprofin expression vector revealed that this molecule is localized in the nucleus and promotes cell proliferation. Thus, epiprofin is a highly cell-and tissue-specific nuclear protein expressed primarily by proliferating epithelial cells of teeth, hair follicles, and limbs that may function in the development of these tissues by regulating cell growth.

Articular cartilage and synovial joints are critical for skeletal function, but the mechanisms regulating their development are largely unknown. In previous studies we found that the ets transcription factor ERG and its alternatively-spliced variant C-1-1 have roles in joint formation in chick. Here, we extended our studies to mouse. We found that ERG is also expressed in developing mouse limb joints. To test regulation of ERG expression, beads coated with the joint master regulator protein GDF-5 were implanted close to incipient joints in mouse limb explants; this led to rapid and strong ectopic ERG expression. We cloned and characterized several mammalian ERG variants and expressed a human C-1-1 counterpart (hERG3Delta81) throughout the cartilaginous skeleton of transgenic mice, using Col2a1 gene promoter/enhancer sequences. The skeletal phenotype was severe and neonatal lethal, and the transgenic mice were smaller than wild type littermates and their skeletons were largely cartilaginous. Limb long bone anlagen were entirely composed of chondrocytes actively expressing collagen IX and aggrecan as well as articular markers such as tenascin-C. Typical growth plates were absent and there was very low expression of maturation and hypertrophy markers, including Indian hedgehog, collagen X and MMP-13. The results suggest that ERG is part of molecular mechanisms leading chondrocytes into a permanent developmental path and become joint forming cells, and may do so by acting downstream of GDF-5.