Changshan Wu

Cellular and molecular mechanisms of synovial joint and articular cartilage formation.

Abstract:  Synovial joints and articular cartilage play crucial roles in the skeletal function, but relatively little is actually known about their embryonic development. Here we first focused on the interzone, a thin mesenchymal cell layer forming at future joint sites that is widely thought to be critical for joint and articular cartilage development. To determine interzone cell origin and fate, we microinjected the vital fluorescent dye DiI at several peri‐joint sites in chick limbs and monitored the behavior and fate of labeled cells over time. Peri‐joint mesenchymal cells located immediately adjacent to incipient joints migrated, became part of the interzone, and were eventually found in epiphyseal articular layer and joint capsule. Interzone cells isolated and reared in vitro expressed typical phenotypic markers, including GDF‐5, Wnt‐14, and CD‐44, and differentiated into chondrocytes over time. To determine the molecular mechanisms of articular chondrocyte formation, we carried out additional studies on the ets transcription factor family member ERG and its alternatively spliced variant C‐1‐1 that we previously found to be expressed in developing avian articular chondrocytes. We cloned the human counterpart of avian C‐1‐1 (ERGp55Δ81) and conditionally expressed it in transgenic mice under cartilage‐specific Col2 gene promotor‐enhancer control. The entire transgenic mouse limb chondrocyte population exhibited an immature articular‐like phenotype and a virtual lack of growth plate formation and chondrocyte maturation compared to wild‐type littermate. Together, our studies reveal that peri‐joint mesenchymal cells take part in interzone and articular layer formation, interzone cells can differentiate into chondrocytes, and acquisition of a permanent articular chondrocyte phenotype is aided and perhaps dictated by ets transcription factor ERG.

Temporomandibular joint formation and condyle growth require Indian hedgehog signaling

The temporomandibular joint (TMJ) is essential for jaw function, but the mechanisms regulating its development remain poorly understood. Because Indian hedgehog (Ihh) regulates trunk and limb skeletogenesis, we studied its possible roles in TMJ development. In wild‐type mouse embryos, Ihh expression was already strong in condylar cartilage by embryonic day (E) 15.5, and expression of Ihh receptors and effector genes (Gli1, Gli2, Gli3, and PTHrP) indicated that Ihh range of action normally reached apical condylar tissue layers, including polymorphic chondroprogenitor layer and articular disc primordia. In Ihh−/− embryos, TMJ development was severely compromised. Condylar cartilage growth, polymorphic cell proliferation, and PTHrP expression were all inhibited, and growth plate organization and chondrocyte gene expression patterns were abnormal. These severe defects were partially corrected in double Ihh−/−/Gli3−/− mutants, signifying that Ihh action is normally modulated and delimited by Gli3 and Gli3R in particular. Both single and double mutants, however, failed to form an articular disc primordium, normally appreciable as an independent condensation between condylar apex and neighboring developing temporal bone in wild‐type. This failure persisted at later stages, leading to complete absence of a normal functional disc and lubricin‐expressing joint cavities. In summary, Ihh is very important for TMJ development, where it appears to regulate growth and elongation events, condylar cartilage phenotype, and chondroprogenitor cell function. Absence of articular disc and joint cavities in single and double mutants points to irreplaceable Ihh roles in formation of those critical TMJ components. Developmental Dynamics 236:426–434, 2007.

Indian hedgehog and syndecans-3 coregulate chondrocyte proliferation and function during chick limb skeletogenesis

Hedgehog proteins exert critical roles in embryogenesis and require heparan sulfate proteoglycans (HS‐PGs) for action. Indian hedgehog (Ihh) is produced by prehypertrophic chondrocytes in developing long bones and regulates chondrocyte proliferation and other events, but it is not known whether it requires HS‐PGs for function. Because the HS‐PG syndecan‐3 is preferentially expressed by proliferating chondrocytes, we tested whether it mediates Ihh action. Primary chick chondrocyte cultures were treated with recombinant Ihh (rIhh‐N) in absence or presence of heparinase I or syndecan‐3 neutralizing antibodies. While rIhh‐N stimulated proliferation in control cultures, it failed to do so in heparinase‐ or antibody‐treated cultures. In reciprocal gain‐of‐function studies, chondrocytes were made to overexpress syndecan‐3 by an RCAS viral vector. Cells became more responsive to rIhh‐N, but even this response was counteracted by heparinase or antibody treatment. To complement the in vitro data, RCAS viral particles were microinjected in day 4–5 chick wing buds and effects of syndecan‐3 misexpression were monitored over time. Syndecan‐3 misexpression led to widespread chondrocyte proliferation and, interestingly, broader expression and distribution of Ihh. In addition, the syndecan‐3 misexpressing skeletal elements were short, remained cartilaginous, lacked osteogenesis, and exhibited a markedly reduced expression of collagen X and osteopontin, products characteristic of hypertrophic chondrocytes and bone cells. The data are the first to indicate that Ihh action in chondrocyte proliferation involves syndecan‐3 and to identify a specific member of the syndecan family as mediator of hedgehog function. Developmental Dynamics 229:607–617, 2004.

Expression, gene regulation, and roles of Fisp12/CTGF in developing tooth germs

Odontogenesis involves multiple events, including tissue–tissue interactions, cell proliferation, and cell differentiation, but the underlying mechanisms of regulation are far from clear. Because Fisp12/CTGF is a signaling protein involved in similar events in other systems, we asked whether it is expressed in developing tooth germs and what roles it may have. Indeed, Fisp12/CTGF transcripts were first expressed by dental laminas, invaginating epithelium, and condensing mesenchyme at the bud stage, and then became abundant in enamel knot and preameloblasts. Fisp12/CTGF was present not only in inner dental epithelium but also in stratum intermedium and underlying dental mesenchyme. Fisp12/CTGF expression decreased markedly in secreting ameloblasts. Tissue reconstitution experiments showed that Fisp12/CTGF expression in dental epithelium required interaction with mesenchyme but was maintained by treatment of epithelium with transforming growth factor‐1, a factor regulating Fisp12/CTGF expression in other systems, or with bone morphogenetic protein‐2. Loss‐of‐function studies using CTGF neutralizing antibodies revealed that interference with endogenous factor action in tooth germ explants led to a severe inhibition of proliferation in both epithelium and mesenchyme and a marked delay in cytodifferentiation of ameloblasts and odontoblasts. Treatment of dental epithelial and mesenchymal cells in culture with recombinant CTGF stimulated cell proliferation, whereas treatment with neutralizing antibodies inhibited it. The data demonstrate for the first time that Fisp12/CTGF is expressed during odontogenesis. Expression is confined to specific sites and times, is regulated by epithelial–mesenchymal interactions and critical soluble factors, and appears to be needed for proliferation and differentiation along both ameloblast and odontoblast cell lineages.

Expression and roles of connective tissue growth factor in Meckel's cartilage development

Meckels cartilage is a prominent feature of the developing mandible, but its formation and roles remain unclear. Because connective tissue growth factor (CTGF, CCN2) regulates formation of other cartilages, we asked whether it is expressed and what roles it may have in developing mouse Meckels cartilage. Indeed, CTGF was strongly expressed in anterior, central, and posterior regions of embryonic day (E) 12 condensing Meckels mesenchyme. Expression decreased in E15 newly differentiated chondrocytes but surged again in E18 hypertrophic chondrocytes located in anterior region and most‐rostral half of central region. These cells were part of growth plate‐like structures with zones of maturation resembling those in a developing long bone and expressed such characteristic genes as Indian hedgehog (Ihh), collagen X, MMP‐9, and vascular endothelial growth factor. At each stage examined perichondrial tissues also expressed CTGF. To analyze CTGF roles, mesenchymal cells isolated from E10 first branchial arches were tested for interaction and responses to recombinant CTGF (rCTGF). The cells readily formed aggregates in suspension culture and interacted with substrate‐bound rCTGF, but neither event occurred in the presence of CTGF neutralizing antibodies. In good agreement, rCTGF treatment of micromass cultures stimulated both expression of condensation‐associated macromolecules (fibronectin and tenascin‐C) and chondrocyte differentiation. Expression of these molecules and CTGF itself was markedly up‐regulated by treatment with transforming growth factor‐β1, a chondrogenic factor. In conclusion, CTGF is expressed in highly dynamic manners in developing Meckels cartilage where it may influence multiple events, including chondrogenic cell differentiation and chondrocyte maturation. CTGF may aid chondrogenesis by acting down‐stream of transforming growth factor‐β and stimulating cell–cell interactions and expression of condensation‐associated genes. Developmental Dynamics 231:136–147, 2004.

Development of stratum intermedium and its role as a Sonic hedgehog-signaling structure during odontogenesis.

Stratum intermedium is a transient and subtle epithelial structure closely associated with inner dental epithelium in tooth germs. Little is known about its development and roles. To facilitate analysis, we used bovine tooth germs, predicting that they may contain a more conspicuous stratum intermedium. Indeed, early bell stage bovine tooth germs already displayed an obvious stratum intermedium with a typical multilayered organization and flanking the enamel knot. Strikingly, with further development, the cuspally located stratum intermedium underwent thinning and involution, whereas a multilayered stratum intermedium formed at successive sites along the cusp‐to‐cervix axis of odontogenesis. In situ hybridization and immunohistochemistry showed that stratum intermedium produces the signaling molecule Sonic hedgehog (Shh). Maximal Shh expression was invariably seen in its thickest multilayered portions. Shh was also produced by inner dental epithelium; expression was not constant but varied with development and cytodifferentiation of ameloblasts along the cusp‐to‐cervix axis. Interestingly, maximal Shh expression in inner dental epithelium did not coincide with that in stratum intermedium. Both stratum intermedium and inner dental epithelium expressed the Shh receptor Patched2 (Ptch2), an indication of autocrine signaling loops. Shh protein, but not RNA, was present in underlying dental mesenchyme, probably resulting from gradual diffusion from epithelial layers and reflecting paracrine loops of action. To analyze the regulation of Shh expression, epithelial and mesenchymal layers were separated and maintained in organ culture. Shh expression decreased over time, but was maintained in unoperated specimens. Our data show for the first time that stratum intermedium is a highly regulated and Shh‐expressing structure. Given its dynamic and apparently interactive properties, stratum intermedium may help orchestrate progression of odontogenesis from cusp to cervix.

Expression of Indian Hedgehog, BMP-4 and noggin in craniosynostosis induced by fetal constraint

Indian Hedgehog (Ihh), bone morphogenetic protein (BMP), and its antagonist Noggin play an important regulatory role in bone formation. We used an animal model to study the role of these molecules in craniosynostosis induced by fetal constraint. C57Bl/6 mice underwent cervical cerclage on the 18th day of gestation, and their pups were harvested 48 and 72 hours beyond the normal gestational period. Constrained and control calvariae were examined for expression of BMP-4, Noggin, Histone H4C, Ihh, Sonic Hedgehog (Shh), and Patched 1 (Ptch1), one of the Hh transcriptional target molecules/Hh receptors. Constraint-induced suture fusion was associated with decreased expression of Ihh and Noggin, whereas BMP-4 was expressed in both control and constrained sutures. Ptch1 colocalized with Ihh-positive osteogenic cells at the osteogenic fronts, but not with Shh transcripts, suggesting that Ihh, but not Shh, regulates Ptch1 expression in cranial suture development. Histone H4C was preferentially expressed in Ihh-positive cells, indicating that Ihh may regulate osteogenic cell proliferation at the osteogenic fronts. These results suggest a role for Ihh and Noggin signaling in constraint-induced craniosynostosis.

Retinoid signaling regulates CTGF expression in hypertrophic chondrocytes with differential involvement of MAP kinases

Retinoids are important for growth plate chondrocyte maturation, but their downstream effectors remain unclear. Recently, CTGF (CCN2) was found to regulate chondrocyte function, particularly in the hypertrophic zone. The goal of the study was to determine whether CTGF is a retinoid signaling effector molecule, how it is regulated, and how it acts.

Sonic hedgehog functions as a mitogen during bell stage of odontogenesis

Epithelial-mesenchymal interactions are required for tissue growth and gene expression patterns during odontogenesis. We showed previously that Sonic hedgehog (SHH) is detectable in both dental epithelium and mesenchyme, while Shh transcripts are present in dental epithelium only, suggesting that SHH functions as an autocrine signal in epithelium and a paracrine signal in mesenchyme. This hypothesis was tested here. We found by in situ hybridization that the SHH autocrine receptor Ptch-2 is indeed expressed in dental epithelium whereas the paracrine receptor Ptc is expressed in mesenchyme. Bovine bell stage tooth germs were microsurgically separated into epithelial and mesenchymal portions and the resulting tissue fragments were organ-cultured. In epithelium fragments cultured by themselves, gene expression of Shh and Gli-1 (a putative transcriptional mediator of hedgehog signaling) was significantly decreased in both inner dental epithelium and stratum intermedium layers; this was accompanied by a sharp drop in epithelial cell proliferation. However, in companion control tissue fragments containing both epithelium and mesenchyme, Shh and Gli-1 expression as well as cell proliferation were maintained. Treatment of dental epithelial or mesenchymal cell populations in monolayer cultures with exogenous recombinant SHH stimulated cell proliferation. Together, the data provide clear evidence that Shh is synthesized by dental epithelium, reaches the underlying mesenchyme, and appears to act as an autocrine mitogen for epithelial cells and a paracrine mitogen for mesenchymal cells, thus exerting crucial functions in tooth germ growth, morphogenesis, and tissue-tissue interactions of bell stage of odontogenesis.

Chick limbs with mouse teeth: An effective in vivo culture system for tooth germ development and analysis

Mouse tooth germ development is currently studied by three main approaches: in wild‐type and mutant mouse lines, after transplantation of tooth germs to ectopic sites, and in organ culture. The in vivo approaches are the most physiological but do not provide accessibility to tooth germs for further experimental manipulation. Organ cultures, although readily accessible, do not sustain full tooth germ development and are appropriate for short‐term analysis. Thus, we sought to establish a new approach that would combine experimental accessibility with sustained development. We implanted fragments of embryonic day 12 mouse embryo first branchial arch containing early bud stage tooth germs into the lateral mesenchyme of day 4–5 chick embryo wing buds in ovo. Eggs were reincubated, and implanted tissues were examined by histochemistry and in situ hybridization over time. The tooth germs underwent seemingly normal growth, differentiation, and morphogenesis. They reached the cap, bell, and crown stages in approximately 3, 6, and 10 days, respectively, mimicking in a striking manner native temporal patterns. To examine mechanisms regulating tooth germ development, we first implanted tooth germ fragments, microinjected them with neutralizing antibodies to the key signaling molecule Sonic hedgehog (Shh), and examined them over time. Tooth germ development was markedly delayed, as revealed by poor morphogenesis and lack of mature ameloblasts and odontoblasts displaying characteristic traits such as an elongated cell shape, nuclear relocalization, and amelogenin gene expression. These phenotypic changes began to be reversed upon further incubation. The data show that the limb bud represents an effective, experimentally accessible as well as economical system for growth and analysis of developing tooth germs. The inhibitory effects of Shh neutralizing antibody treatment are discussed in relation to roles of this signaling pathway proposed by this and other groups previously.