Here we report on the structure, expression, and function of a novel cartilage-specific gene coding for a 17-kDa small, highly charged, and secreted protein that we termed Ucma (unique cartilage matrix-associated protein). The protein is processed by a furin-like protease into an N-terminal peptide of 37 amino acids and a C-terminal fragment (Ucma-C) of 74 amino acids. Ucma is highly conserved between mouse, rat, human, dog, clawed frog, and zebrafish, but has no homology to other known proteins. Remarkable are 1-2 tyrosine sulfate residues/molecule and dense clusters of acidic and basic residues in the C-terminal part. In the developing mouse skeleton Ucma mRNA is expressed in resting chondrocytes in the distal and peripheral zones of epiphyseal and vertebral cartilage. Ucma is secreted into the extracellular matrix as an uncleaved precursor and shows the same restricted distribution pattern in cartilage as Ucma mRNA. In contrast, antibodies prepared against the processed C-terminal fragment located Ucma-C in the entire cartilage matrix, indicating that it either diffuses or is retained until chondrocytes reach hypertrophy. During differentiation of an MC615 chondrocyte subclone in vitro, Ucma expression parallels largely the expression of collagen II and decreases with maturation toward hypertrophic cells. Recombinant Ucma-C does not affect expression of chondrocyte-specific genes or proliferation of chondrocytes, but interferes with osteogenic differentiation of primary osteoblasts, mesenchymal stem cells, and MC3T3-E1 pre-osteoblasts. These findings suggest that Ucma may be involved in the negative control of osteogenic differentiation of osteochondrogenic precursor cells in peripheral zones of fetal cartilage and at the cartilage-bone interface.Elucidation of molecular mechanisms underlying chondrocyte differentiation is not only important for our understanding of skeletal development, but also of particular interest for our knowledge on the behavior of chondrocytes following articular cartilage damage during cartilage repair and treatment of degenerative cartilage diseases. Initial steps of chondrogenesis, i.e. the formation of a cartilage blastema from limb bud mesenchymal cells, include cell condensation and onset of chondrocyte differentiation marked by the expression of cartilage-specific matrix proteins such as aggrecan, collagen II, IX, and XI and others (1, 2). These events are regulated by the orchestrated action of several growth factors including BMPs, Wnt factors, FGFs, and the transcription factors Sox5,6, and 9 (3,4). Further steps of chondrocyte growth, maturation, and replacement by bone in the growth plate of long bones, ribs, and vertebrae during endochondral ossification can be defined by the stepwise onset or decline of differentially expressed genes: collagen II and Sox9 for resting and proliferating, FGFR3 for proliferating and prehypertrophic, Ihh and PTHrP receptor for prehypertrophic, collagen X for hypertrophic, and Runx2, osteocalcin, and MMP13 for late hypertrophic chondrocytes (...
During endochondral ossiWcation hypertrophic chondrocytes in the growth plate of fetal long bones, ribs and vertebrae play a key role in preparing growth plate cartilage for replacement by bone. In order to establish a reporter gene mouse to facilitate functional analysis of genes expressed in hypertrophic chondrocytes in this process, Col10a1-BAC reporter gene mouse lines were established expressing LacZ speciWcally in hypertrophic cartilage under the control of the complete Col10a1 gene. For this purpose, a bacterial artiWcial chromosome (BAC RP23-192A7) containing the entire murine Col10a1 gene together with 200 kb Xanking sequences was modiWed by inserting a LacZ-Neo cassette into the second exon of Col10a1 by homologous recombination in E. coli. Transgenic mice containing between one and seven transgene copies were generated by injection of the puriWed BACCol10a1-lLacZ DNA. X-gal staining of newborns and embryos revealed strong and robust LacZ activity exclusively in hypertrophic cartilage of the fetal and neonatal skeleton of the transgenic oVspring. This indicates that expression of the reporter gene in its proper genomic context in the BAC Col10a1 environment is independent of the integration site and reXects authentic Col10a1 expression in vivo. The Col10a1 speciWc BAC recombination vector described here will enable the speciWc analysis of eVector gene functions in hypertrophic cartilage during skeletal development, endochondral ossiWcation, and fracture callus healing.
Twisted gastrulation (TSG) is an extracellular modulator of bone morphogenetic protein (BMP) activity and regulates dorsoventral axis formation in early Drosophila and Xenopus development. Studies on tsg-deficient mice also indicated a role of this protein in skeletal growth, but the mechanism of TSG activity in this process has not yet been investigated. Here we show for the first time by in situ hybridization and immunohistochemistry that TSG is strongly expressed in bovine and mouse growth plate cartilage as well as in fetal ribs, vertebral cartilage, and cartilage anlagen of the skull. Furthermore we provide evidence that TSG is directly involved in BMP-regulated chondrocyte differentiation and maturation. In vitro, TSG impaired the dose-dependent BMP-2 stimulation of collagen II and X expression in cultures of MC615 chondrocytes and primary mouse chondrocytes. In the presence of chordin, a BMP antagonist, the inhibitory effect of TSG was further enhanced. TSG also inhibited BMP-2-stimulated phosphorylation of Smad factors in chondrocytes, confirming the role of TSG as a modulator of BMP signaling. For analysis of TSG functions in cartilage development in vivo, the gene was overexpressed in transgenic mice under the control of the cartilage-specific Col2a1 promoter. As a result, Col10a1 expression was significantly reduced in the growth plates of transgenic embryos and newborns in comparison with wild type littermates as shown by in situ hybridization and by real time PCR analysis. The data suggest that TSG is an important modulator of BMP-regulated cartilage development and chondrocyte differentiation.Bone morphogenetic proteins (BMPs) 5 are key regulators in the formation of cartilage models of vertebrate long bones, vertebrae, and ribs and their transition to bone during endochondral ossification (for reviews, see Refs. 1-5). However, the role of BMP factors in cartilage development and chondrocyte differentiation is complex and differs with time and site of chondrogenic differentiation: BMPs control limb outgrowth by negatively modulating apical ectodermal ridge activity (6 -8), whereas BMPs are required for the formation of prechondrogenic mesenchyme and chondrocyte differentiation in the limb (9). The ability of BMP-2, BMP-4, and others to induce chondrogenic differentiation of undifferentiated stems cells in vivo and in vitro has been amply demonstrated (10 -12). In line with this observation is the finding that chondrogenesis in vivo is impaired by blocking BMP activities with noggin (13, 14) or deletion of the BMP receptor (BMPRIB) (15).The role of BMPs during chondrocyte maturation and hypertrophy is also complex and controversially discussed: several studies show that BMP factors such as BMP-6 and BMP-7 stimulate hypertrophic differentiation of chondrocytes and promote collagen X expression (16 -18), thus preparing growth plate cartilage for replacement by endochondral bone. On the other hand, it was reported that BMP-2 and BMP-4 overexpression in developing chick limbs caused delayed hypertroph...
Unresolved questions remain concerning the derivation of the vagina with respect to the relative contributions from the Mü llerian ducts, the urogenital sinus, and the Wolffian ducts. Recent molecular and cellular studies in rodents have opened up a large gap between the level of understanding of vaginal development in mice and understanding of human vaginal development, which is based on histology. To compare the findings in mice with human vaginal development and to address this gap, we analysed molecular characteristics of the urogenital sinus, Wolffian ducts, and Mü llerian ducts in 8-14-week-old human specimens using immunohistochemical methods. The monoclonal antibodies used were directed against cytokeratin (CK) 14, CK19, vimentin, laminin, p63, E-cadherin, caspase-3, Ki67, HOX A13, and BMP-4. The immunohistochemical analysis revealed that, during weeks 8-9, the epithelium of the Mü llerian ducts became positive for p63 as p63-positive cells that originated from the sinus epithelium reached the caudal tip of the fused Mü llerian ducts via the Wolffian ducts. The lumen of the fused Mü llerian ducts was closed by an epithelial plug that contained both vimentin-positive and vimentin-negative cells. Subsequently, the resulting epithelial tube enlarged by proliferation of basal p63-positive cells. The first signs of squamous differentiation were detected during week 14, with the appearance of CK14-positive cells. According to our results, all three components, namely, the urogenital sinus, Wolffian ducts, and Mü llerian ducts, interacted during the formation of the human vagina. The sinus epithelium provided p63-positive cells, the Wollfian ducts acted as a 'transporter', and the Mü llerian ducts contributed the guiding structure for the vaginal anlagen. Epithelial differentiation began at the end of the period studied and extended in a caudo-cranial direction. The present study is one of the first to provide up-to-date molecular correlates for human vaginal development that can be compared with the results of cell biological studies in rodents.
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