Tenomodulin (TeM) is a type II transmembrane glycoprotein containing a C-terminal anti-angiogenic domain and is predominantly expressed in tendons and ligaments. Here we report that TeM expression is closely associated with the appearance of tenocytes during chick development and is positively regulated by Scleraxis (Scx). At stage 23, when Scx expression in the syndetome has extended to the tail region, TeM was detectable in the anterior eight somites. At stage 25, TeM and Scx were both detectable in the regions adjacent to the myotome. Double positive domains for these genes were flanked by a dorsal TeM single positive and a ventral Scx single positive domain. At stage 28, the expression profile of TeM in the axial tendons displayed more distinct morphological features at different levels of the vertebrae. At stage 32 and later, Scx and TeM showed similar expression profiles in developing tendons. Retroviral expression of Scx resulted in the significant upregulation of TeM in cultured tenocytes, but not in chondrocytes. In addition, the misexpression of RCAS-cScx by electroporation into the hindlimb could not induce the generation of additional tendons, but did result in the upregulation of TeM expression in the tendons at stage 33 and later. These findings suggest that TeM is a late marker of tendon formation and that Scx positively regulates TeM expression in a tendon cell lineage-dependent manner.

SRY-box containing gene 9 (Sox9) and scleraxis (Scx) regulate cartilage and tendon formation, respectively. Here we report that murine Scx+/Sox9+ progenitors differentiate into chondrocytes and tenocytes/ligamentocytes to form the junction between cartilage and tendon/ligament. Sox9 lineage tracing in the Scx+ domain revealed that Scx+ progenitors can be subdivided into two distinct populations with regard to their Sox9 expression history: Scx+/Sox9+ and Scx+/Sox9− progenitors. Tenocytes are derived from Scx+/Sox9+ and Scx+/Sox9− progenitors. The closer the tendon is to the cartilaginous primordium, the more tenocytes arise from Scx+/Sox9+ progenitors. Ligamentocytes as well as the annulus fibrosus cells of the intervertebral discs are descendants of Scx+/Sox9+ progenitors. Conditional inactivation of Sox9 in Scx+/Sox9+ cells causes defective formation in the attachment sites of tendons/ligaments into the cartilage, and in the annulus fibrosus of the intervertebral discs. Thus, the Scx+/Sox9+ progenitor pool is a unique multipotent cell population that gives rise to tenocytes, ligamentocytes and chondrocytes for the establishment of the chondro-tendinous/ligamentous junction.

Abstract. The regulatory role of parathyroid hormone (PTH)/PTH-related peptide (PTHrP) signaling has been implicated in embryonic skeletal development. Here, we studied chondrogenic differentiation of the mouse embryonal carcinoma-derived clonal cell line ATDC5 as a model of chondrogenesis in the early stages of endochondral bone development. ATDC5 cells retain the properties of chondroprogenitor cells, and rapidly proliferate in the presence of 5% FBS. Insulin (10 ixg/ml) induced chondrogenic differentiation of the cells in a postconfluent phase through a cellular condensation process, resulting in the formation of cartilage nodules, as evidenced by expression of type II collagen and aggrecan genes. We found that differentiated cultures of ATDC5 cells abundantly expressed the high affinity receptor for PTH (M r ~'-~80 kD; K d = 3.9 nM; 3.2 x 105 sites/cell). The receptors on differentiated cells were functionally active, as evidenced by a PTH-dependent activation of adenylate cyclase. Specific binding of PTH to cells markedly increased with the formation of cartilage nodules, while undifferentiated cells failed to show specific binding of PTH. Northern blot analysis indicated that expression of the PTH/PTHrP receptor gene became detectable at the early stage of chondrogenesis of ATDC5 cells, preceding induction of aggrecan gene expression. Expression of the PTH/PTHrP receptor gene was undetectable in undifferentiated cells. The level of PTH/PTHrP receptor mRNA was markedly elevated parallel to that of type II collagen mRNA. These lines of evidence suggest that the expression of functional PTH/PTHrP receptor is associated with the onset of chondrogenesis. In addition, activation of the receptor by exogenous PTH or PTHrP significantly interfered with cellular condensation and the subsequent formation of cartilage nodules, suggesting a novel site of PTHrP action. IT has been suggested that when limb mesenchymal cells move away from the influence of the apical ectodermal ridge, their cAMP content increases, triggering chondrogenic differentiation (38). The response of limb mesenchymal cells to dibutyryl cAMP is a responsive property of the cells, which changes temporally and spatially during limb differentiation (57). Elevation of cAMP content of the subridge mesenchymal cells precludes the necessity of cells passing through a condensation phase before overt cartilage formation (38). Thus, the increase in intracellular cAMP is hypothesized to be a key cellular response to cellular condensation that is a prerequisite for limb chondrogenesis (46,49). We previously reported that parathyroid hormone (PTH) 1 stimulates sulfate incorporation into cartilagecharacteristic proteoglycans followed by elevation of intracellular cAMP in primary growth plate chondrocytes (23,59,60). This stimulation of sulfate incorporation was mainly caused by elongation of glycosaminoglycan chains rather than increased synthesis of proteoglycan core protein (23). Chondrocytes respond to PTH through the adenylate cyclase-coupled surface recepto...

During the process of endochondral bone formation, proliferating chondrocytes give rise to hypertrophic cells, which then deposit a mineralized matrix to form calcified cartilage prior to replacement by bone. Previously, we reported that a clonal cell line, ATDC5, undergoes efficient chondrogenic differentiation through a cellular condensation stage. Here we report that the differentiated ATDC5 cells became hypertrophic at the center of cartilage nodules, when the cells ceased to grow. Formation of hypertrophic chondrocytes took place in association with type X collagen gene expression and a dramatic elevation of alkaline phosphate (ALPase) activity. After 5 weeks of culture, mineralization of the culture could be discerned as Alizarin red-positive spots, which spread throughout the nodules even in the absence of ␤-glycerophosphate. Electron microscopy and electron probe microanalysis revealed that calcification was first initiated at matrix vesicles in the territorial matrix and that it advanced progressively along the collagen fibers in a manner similar to that which occurs in vivo. The infrared spectrum of the mineralized nodules indicated two absorption doublets around 1030 cm ؊1 and 600 cm ؊1, which are characteristic of apatitic mineral. Calcifying cultures of ATDC5 cells retained responsiveness to parathyroid hormone (PTH): PTH markedly inhibited elevation of ALPase activity and calcification in the culture in a dose-dependent manner. Thus, we demonstrated that ATDC5 cells keep track of the multistep differentiation process encompassing the stages from mesenchymal condensation to calcification in vitro. ATDC5 cells provide an excellent model to study the molecular mechanism underlying regulation of cartilage differentiation during endochondral bone

Adolescent idiopathic scoliosis (AIS) is the most common pediatric skeletal disease. We previously reported a locus on chromosome 10q24.31 associated with AIS susceptibility in Japanese using a genome-wide association study (GWAS) consisting of 1,033 cases and 1,473 controls. To identify additional AIS-associated loci, we expanded the study by adding X-chromosome SNPs in the GWAS and increasing the size of the replication cohorts. Through a stepwise association study including 1,819 cases and 25,939 controls, we identified a new susceptibility locus on chromosome 6q24.1 in Japanese (P = 2.25 × 10(-10); odds ratio (OR) = 1.28). The most significantly associated SNP, rs6570507, was in GPR126 (encoding G protein-coupled receptor 126). Its association was replicated in Han Chinese and European-ancestry populations (combined P = 1.27 × 10(-14); OR = 1.27). GPR126 was highly expressed in cartilage, and the knockdown of gpr126 in zebrafish caused delayed ossification of the developing spine. Our results should provide insights into the etiology and pathogenesis of AIS.