BMP-2-enhanced chondrogenesis involves p38 MAPK-mediated down-regulation of Wnt-7a pathway

The bone morphogenetic protein (BMP) family has been implicated in control of cartilage development. Here, we demonstrate that BMP-2 promotes chondrogenesis by activating p38 mitogen-activated protein kinase (MAPK), which in turn downregulates Wnt-7a/b-catenin signaling responsible for proteasomal degradation of Sox9. Exposure of mesenchymal cells to BMP-2 resulted in upregulation of Sox9 protein and a concomitant decrease in the level of b-catenin protein and Wnt-7a signaling. In agreement with this, the interaction of Sox9 with b-catenin was inhibited in the presence of BMP-2. Inhibition of the p38 MAPK pathway using a dominant negative mutant led to sustained Wnt-7a signaling and decreased Sox9 expression, with consequent inhibition of precartilage condensation and chondrogenic differentiation. Moreover, overexpression of b-catenin caused degradation of Sox9 via the ubiquitin/26S proteasome pathway. Our results collectively indicate that the increase in Sox9 protein resulting from downregulation of b-catenin/Wnt-7a signaling is mediated by p38 MAPK during BMP-2 induced chondrogenesis in chick wing bud mesenchymal cells.     

Wnt signaling during BMP-2 stimulation of mesenchymal chondrogenesis

Members of both the Wnt and bone morphogenetic protein (BMP) families of signaling molecules have been implicated in the regulation of cartilage development. A key component of the Wnt signaling pathway is the cytosolic protein, beta-catenin. We have recently shown that the chondrogenic activity of BMP-2 in vitro involves the action of the cell-cell adhesion protein, N-cadherin, which functionally complexes with beta-catenin. The aim of this study is to test the hypothesis that Wnts may be involved in BMP-2 induced chondrogenesis, using an in vitro model of high-density micromass cultures of the murine multipotent mesenchymal cell line, C3H10T1/2. Expression of a number of Wnt members was detected in these cultures, including Wnt-3A and Wnt-7A, whose levels were up- and downregulated, respectively, by BMP-2. To assess the functional involvement of Wnt signaling in BMP-2 induced chondrogenesis, cultures were treated with lithium chloride, a Wnt-7A mimetic that acts by inhibiting the serine/threonine phosphorylation activity of glycogen synthase kinase-3beta (GSK-3beta). Lithium treatment significantly inhibited BMP-2 stimulation of chondrogenesis as well as GSK-3beta enzymatic activity, and decreased the levels of N-cadherin protein and mRNA. Furthermore, lithium decreased BMP-2 upregulation of total and nuclear levels of LEF-1 and beta-catenin as well as their interaction during later chondrogenesis; similarly, the interaction of beta-catenin with N-cadherin was also decreased. Interestingly, lithium treatment did not affect the ability of BMP-2 to decrease ubiquitination of beta-catenin, although it did reduce the interaction of beta-catenin with GSK-3beta during late chondrogenesis (days 9-13). We suggest that the chondro-inhibitory effect of lithium on BMP-2 induced chondrogenesis indicates antagonism between lithium-like Wnts and BMP-2 during mesenchymal condensation.     

Functional role of growth/differentiation factor 5 in chondrogenesis of limb mesenchymal cells

Growth/Differentiation Factor 5 (GDF5) plays an important role in limb mesenchymal cell condensation and chondrogenesis. Here we demonstrate, using high density cultures of chick embryonic limb mesenchyme, that GDF5 misexpression increased condensation of chondroprogenitor cells and enhanced chondrogenic differentiation. These effects were observed in the absence of altered cellular viability or biosynthetic activity, suggesting that GDF5 action might be directed at the level of cellular adhesion or cell-cell communication. GDF5- enhanced condensation occurred independent of cell density or N-cadherin mediated adhesion and signaling, but was inhibited upon interference of gap junction mediated communication. p38 MAP kinase signaling was required for the GDF5 effect on chondrocyte differentiation, but not for mesenchymal condensation. These findings suggest gap junction involvement in the action of GDF5 in developmental chondrogenesis.     

Long-term in vitro analysis of limb cartilage development: involvement of Wnt signaling

Endochondral skeletal development involves the condensation of mesenchymal cells, their differentiation into chondrocytes, followed by chondrocyte maturation, hypertrophy, and matrix mineralization, and replacement by osteoblasts. The Wnt family of secreted proteins have been shown to play important roles in vertebrate limb formation. To examine the role(s) of Wnt members and their transmembrane-spanning receptor(s), Frizzled (fz), we retrovirally misexpressed Wnt-5a, Wnt-7a, chicken frizzled-1 (Chfz-1), and frizzled-7 (Chfz-7) in long-term (21 day) high density, micromass cultures of stage 23/24 chick embryonic limb mesenchyme. This culture system recapitulates in vitro the entire differentiation (days 1-10), growth (days 5-12), and maturation and hypertrophy (from day 12 on) program of cartilage development. Wnt-7a misexpression severely inhibited chondrogenesis from day 7 onward. Wnt-5a misexpression resulted in a poor hypertrophic phenotype by day 14. Chfz-7 misexpression caused a slight delay of chondrocyte maturation based on histology, whereas Chfz-1 misexpression did not affect the chondrogenic phenotype. Misexpression of all Wnt members decreased collagen type X expression and alkaline phosphatase activity at day 21. Our findings implicate functional role(s) for Wnt signaling throughout embryonic cartilage development, and show the utility of the long-term in vitro limb mesenchyme culture system for such studies.     

Wnt-5a is involved in TGF-beta3-stimulated chondrogenic differentiation of chick wing bud mesenchymal cells

In this study we investigated whether signalling by TGF-beta3 and Wnt-5a cross-talk during chondrogenic differentiation of chick wing mesenchyme. Using differential display polymerase chain reaction screening, we found the expression of Wnt-5a to be significantly increased during transforming growth factor-beta3 (TGF-beta3)-induced precartilage condensation in mesenchyme micromass cultures. Transfection of cells with a Wnt-5a expression construct promoted precartilage condensation and chondrogenesis in micromass cultures, similar to that observed when chondrogenic-competent cells were exposed to TGF-beta3. Overexpression of Wnt-5a or treatment with TGF-beta3 stimulated the activation of protein kinase C-alpha (PKC-alpha) and p38 mitogen-activated protein kinase (MAPK), both positive regulators of chondrogenic differentiation. Inactivation of PKC-alpha and p38 MAPK by specific inhibitors abrogated chondrogenesis stimulated by both TGF-beta3 and Wnt-5a. Similarly, partial reduction in TGF-beta3-induced Wnt-5a expression by small interfering RNA resulted in decreased activities of PKC-alpha and p38 MAPK, and abolished the chondro-stimulatory effect of TGF-beta3. Collectively, these findings indicate that Wnt-5a, a non-canonical Wnt, can mediate the chondro-stimulatory effect of TGF-beta3 through upregulation of PKC-alpha and p38MAPK signaling.     

MicroRNA-34a regulates migration of chondroblast and IL-1β-induced degeneration of chondrocytes by targeting EphA5

MicroRNAs function as an endogenous mode of fine gene regulation and have been implicated in multiple differentiation and developmental processes. In the present study, we investigated the role of miRNA-34 during chondrogenic differentiation of chick limb mesenchymal cells. We found that the expression of miR-34a increased upon chondrogenic inhibition. Blockade of miR-34a via PNA-based antisense oligonucleotides (ASOs) recovered the chondro-inhibitory actions of JNK inhibitor on migration of chondrogenic progenitors and the formation of precartilage condensation. Furthermore, we determined that EphA5 is a relevant target of miR-34a during chondrogenesis. MiR-34a was necessary and sufficient to down-regulate EphA5 expression, and up-modulation of EphA5 is sufficient to overcome inhibitory actions of miR-34 inhibition on cell migration and condensation of chick limb mesenchymal cells on collagen substrate. Taken together, our data suggest that miR-34a is a negative modulator of chondrogenesis, particularly in migration of chondroblasts, by targeting EphA5 and resulting inhibition of cellular condensation during chondrogenesis of chick limb mesenchymal cells.     

Epidermal growth factor negatively regulates chondrogenesis of mesenchymal cells by modulating the protein kinase C-alpha, Erk-1, and p38 MAPK signaling pathways

During limb development, epithelial cells in the apical ectodermal ridge keep the underlying mesenchymal cells in a proliferative state preventing differentiation by secreting signaling molecules such as epidermal growth factor (EGF). We investigated the molecular mechanism of the EGF effect on the regulation of micromass culture-induced chondrogenesis of chick limb bud mesenchymal cells as a model system. We found that expression and tyrosine phosphorylation of the EGF receptor was increased transiently during chondrogenesis. Exogenous EGF inhibited chondrogenic differentiation of mesenchymal cells, and this effect was reversed by the EGF receptor inhibitor AG1478. EGF treatment also inhibited the expression and activation of protein kinase C-alpha, whereas it activated Erk-1 and inhibited p38 mitogen-activated protein kinase, all of which appeared to be involved in the EGF-induced inhibition of chondrogenesis. Stimulation of the EGF receptor blocked precartilage condensation and altered the expression of cell adhesion molecules such as N-cadherin and integrins alpha(5) and beta(1). All these EGF effects were reversible by AG1478. The data indicate that EGF negatively regulate chondrogenesis of chick limb bud mesenchymal cells by inhibiting precartilage condensation and by modulating signaling pathways including those of protein kinase C-alpha, Erk-1, and p38 mitogen-activated protein kinase.     

Chondrogenesis induced by actin cytoskeleton disruption is regulated via protein kinase C-dependent p38 mitogen-activated protein kinase signaling

Disruption of the actin cytoskeleton in subconfluent mesenchymal cells induces chondrogenic differentiation via protein kinase C (PKC) alpha signaling. In this study, we investigated the role of p38 mitogen-activated protein (MAP) kinase in the chondrogenic differentiation of mesenchymal cells that is induced by depolymerization of the actin cytoskeleton. Treatment of mesenchymal cells derived from chick embryonic limb buds with cytochalasin D (CD) disrupted the actin cytoskeleton with concomitant chondrogenic differentiation. The chondrogenesis was accompanied by an increase in p38 MAP kinase activity and inhibition of p38 MAP kinase with SB203580 blocked chondrogenesis. Together these results suggest an essential role for p38 MAP kinase in chondrogenesis. In addition, inhibition of p38 MAP kinase did not alter CD-induced increased expression and activity of PKC alpha, whereas down-regulation of PKC by prolonged exposure of cells to phorbol ester inhibited CD-induced p38 MAP kinase activation. Our results therefore suggest that PKC is involved in the regulation of chondrogenesis induced by disruption of the actin cytoskeleton via a p38 MAP kinase signaling pathway.     

Successive formative stages of precartilaginous mesenchymal condensations in vitro: Modulation of cell adhesion by Wnt-7A and BMP-2

High-density chick limb bud cell culture is a useful model to study mesenchymal condensatifons and chondrogenesis. Most previous studies have focused on the effects of soluble reagents on terminal chondrogenic differentiation and have not defined the early cellular processes and signaling events. In this study, we defined five successive stages in the differentiation process: 1) dissociated cells, 2) small aggregates, 3) formation of cell clusters, 4) precartilaginous condensations, and 5) cartilage nodule. We used RCAS retrovirus-mediated Wnt-7a gene transduction to test the effect of Wnt-7a on the differentiation process. We found that Wnt-7a suppressed chondrogenic differentiation. Wnt-7a did not inhibit the initiation of condensation formation but blocked the progression of precartilaginous condensations to cartilage nodules. The Wnt-7a-transduced cultures showed characteristics of a less mature culture with persistent expression of NCAM, N-cadherin, wider distribution of integrin β1 and fibronectin, and suppression of tenascin-C. BMP-2 is known to enhance chondrogenic differentiation in these cultures by promoting cell clusters to form continuous sheet-like precartilaginous condensations. However, cultures exposed to both BMP-2 and Wnt-7a showed inhibition of chondrogenic differentiation. Different signaling molecules such as Wnt-7a and BMP-2 may have antagonistic effects on cartilage differentiation and the gradient of the two molecules may be involved in defining the boundaries of the initial precartilaginous condensation. We propose that the shape of the precartilaginous condensations may be modulated by local concentrations of signaling molecules, such as Wnt-7a and BMP-2, which act to alter cell-substrate and cell-cell adhesions. J. Cell. Physiol. 180:314–324, 1999. © 1999 Wiley-Liss, Inc.     

Transforming growth factor-beta-mediated chondrogenesis of human mesenchymal progenitor cells involves N-cadherin and mitogen-activated protein kinase and Wnt signaling cross-talk

The multilineage differentiation potential of adult tissue-derived mesenchymal progenitor cells (MPCs), such as those from bone marrow and trabecular bone, makes them a useful model to investigate mechanisms regulating tissue development and regeneration, such as cartilage. Treatment with transforming growth factor-beta (TGF-beta) superfamily members is a key requirement for the in vitro chondrogenic differentiation of MPCs. Intracellular signaling cascades, particularly those involving the mitogen-activated protein (MAP) kinases, p38, ERK-1, and JNK, have been shown to be activated by TGF-betas in promoting cartilage-specific gene expression. MPC chondrogenesis in vitro also requires high cell seeding density, reminiscent of the cellular condensation requirements for embryonic mesenchymal chondrogenesis, suggesting common chondro-regulatory mechanisms. Prompted by recent findings of the crucial role of the cell adhesion protein, N-cadherin, and Wnt signaling in condensation and chondrogenesis, we have examined here their involvement, as well as MAP kinase signaling, in TGF-beta1-induced chondrogenesis of trabecular bone-derived MPCs. Our results showed that TGF-beta1 treatment initiates and maintains chondrogenesis of MPCs through the differential chondro-stimulatory activities of p38, ERK-1, and to a lesser extent, JNK. This regulation of MPC chondrogenic differentiation by the MAP kinases involves the modulation of N-cadherin expression levels, thereby likely controlling condensation-like cell-cell interaction and progression to chondrogenic differentiation, by the sequential up-regulation and progressive down-regulation of N-cadherin. TGF-beta1-mediated MAP kinase activation also controls WNT-7A gene expression and Wnt-mediated signaling through the intracellular beta-catenin-TCF pathway, which likely regulates N-cadherin expression and subsequent N-cadherin-mediated cell-adhesion complexes during the early steps of MPC chondrogenesis.     

N-Cadherin expression and signaling in limb mesenchymal chondrogenesis: stimulation by poly-L-lysine

Cellular condensation is a requisite step in the initiation of mesenchymal chondrogenesis in the embryonic limb bud. We have previously shown that cellular condensation of limb chondroprogenitor mesenchymal cells is accompanied by elevated expression of N-cadherin during chondrogenesis both in vivo and in vitro. N-Cadherin-mediated cell-cell interaction is also functionally required for proper mesenchymal chondrogenesis both in vivo and in vitro. In this report, we have further analyzed the functional importance of N-cadherin in the cellular condensation-chondrogenesis pathway by examining N-cadherin expression and related activities in high density micromass cultures of chick limb mesenchymal cells in which chondrogenesis is being stimulated with the cationic polymer, poly-L-lysine (PL). The chondrogenesis-promoting action of PL is thought to involve the clustering of cells via ionic cross-linking, perhaps mimicking the action of an endogenous matrix component. Immunohistochemistry, immunoblotting, and Northern blot analysis all show that PL treatment results in a time-dependent increase in N-cadherin expression at both the protein and mRNA levels. In addition, inhibition of N-cadherin function with a neutralizing monoclonal antibody directed to its extracellular domain inhibits the chondrogenesis-stimulating effect of PL. PL treatment also alters the tyrosine-phosphorylation state of the N-cadherin associated signaling protein, beta-catenin. These results suggest that N-cadherin-mediated cell adhesion is a requisite regulatory component of the limb mesenchymal chondrogenic differentiation program, involving at least in part beta-catenin tyrosine phosphorylation as a signaling step.     

MicroRNA-221 Regulates Chondrogenic Differentiation through Promoting Proteosomal Degradation of Slug by Targeting Mdm2

MicroRNAs (miRNAs) are small RNAs that fulfill diverse functions by negatively regulating gene expression. Here, we investigated the involvement of miRNAs in the chondrogenic differentiation of chick limb mesenchymal cells and found that the expression of miR-221 increased upon chondrogenic inhibition. Blockade of miR-221 via peanut agglutinin-based antisense oligonucleotides reversed the chondro-inhibitory actions of a JNK inhibitor on the proliferation and migration of chondrogenic progenitors as well as the formation of precartilage condensations. We determined that mdm2 is a relevant target of miR-221 during chondrogenesis. miR-221 was necessary and sufficient to down-regulate Mdm2 expression, and this down-modulation of Mdm2 by miR-221 prevented the degradation of (and consequently up-regulated) the Slug protein, which negatively regulates the proliferation of chondroprogenitors. These results indicate that miR-221 contributes to the regulation of cell proliferation by negatively regulating Mdm2 and thereby inhibiting Slug degradation during the chondrogenesis of chick limb mesenchymal cells.     

Wnt-3A enhances bone morphogenetic protein-2-mediated chondrogenesis of murine C3H10T1/2 mesenchymal cells

We have recently reported the chondrogenic effect of bone morphogenetic protein-2 (BMP-2) in high density cultures of the mouse multipotent mesenchymal C3H10T1/2 cell line and have shown the functional requirement of the cell-cell adhesion molecule N-cadherin in BMP-2-induced chondrogenesis in vitro (Denker, A. E., Nicoll, S. B., and Tuan, R. S. (1995) Differentiation 59, 25-34; Haas, A. R., and Tuan, R. S. (1999) Differentiation 64, 77-89). Furthermore, BMP-2 treatment also results in an increased protein level of beta-catenin, a known N-cadherin-associated Wnt signal transducer (Fischer, L., Haas, A., and Tuan, R. S. (2001) Signal Transduction 2, 66-78), suggesting functional cross-talk between the BMP-2 and Wnt signaling pathways. We have observed previously that BMP-2 treatment up-regulates expression of Wnt-3A in high density cultures of C3H10T1/2 cells. To assess the contribution of Wnt-3A to BMP-2-mediated chondrogenesis, we have generated C3H10T1/2 cell lines overexpressing Wnt-3A and various forms of glycogen synthase kinase-3beta (GSK-3beta), an immediate cytosolic component of the Wnt signaling pathway, and examined their response to BMP-2. We show that overexpression of either Wnt-3A or kinase-dead GSK-3beta enhances BMP-2-mediated chondrogenesis. Furthermore, Wnt-3A overexpression results in decreases in both N-cadherin and GSK-3beta protein levels, whereas Wnt-3A as well as kinase-dead GSK-3beta overexpression increase total and nuclear levels of both beta-catenin and LEF-1. Direct cross-talk between Wnts and BMP-2 was also indicated by the up-regulated interaction between beta-catenin and SMAD-4 in response to BMP-2. These results suggest that Wnt-3A acts in a manner opposite to that of other Wnts, such as Wnt-7A, which were previously identified as inhibitory to chondrogenesis, and is the first BMP-2-regulated, chondrogenesis-enhancing member of the Wnt family.     

Dual roles of Wnt signaling during chondrogenesis in the chicken limb

Long bones of the appendicular skeleton are formed from a cartilage template in a process known as endochondral bone development. Chondrocytes within this template undergo a progressive program of differentiation from proliferating to postmitotic prehypertrophic to hypertrophic chondrocytes, while mesenchymal cells immediately surrounding the early cartilage template form the perichondrium. Recently, members of the Wnt family of secreted signaling molecules have been implicated in regulating chondrocyte differentiation. We find that Wnt-5a, Wnt-5b and Wnt-4 genes are expressed in chondrogenic regions of the chicken limb: Wnt-5a is expressed in the perichondrium, Wnt-5b is expressed in a subpopulation of prehypertrophic chondrocytes and in the outermost cell layer of the perichondrium, and Wnt-4 is expressed in cells of the joint region. Misexpression experiments demonstrate that two of these Wnt molecules, Wnt-5a and Wnt-4, have opposing effects on the differentiation of chondrocytes and that these effects are mediated through divergent signaling pathways. Specifically, Wnt-5a misexpression delays the maturation of chondrocytes and the onset of bone collar formation, while Wnt-4 misexpression accelerates these two processes. Misexpression of a stabilized form of beta-catenin also results in accelerated chondrogenesis, suggesting that a beta-catenin/TCF-LEF complex is involved in mediating the positive regulatory effect of Wnt-4. A number of the genes involved in Wnt signal tranduction, including two members of the Frizzled gene family, which are believed to encode Wnt-receptors, show very dynamic and distinct expression patterns in cartilaginous elements of developing chicken limbs. Misexpression of putative dominant-negative forms of the two Frizzled proteins results in severe shortening of the infected cartilage elements due to a delay in chondrocyte maturation, indicating that an endogenous Wnt signal does indeed function to promote chondrogenic differentiation.     

Wnt-signaling and skeletogenesis

Members of the Wnt gene family, encoding secreted cystein-rich glycoproteins, have been isolated from a variety of organisms. They serve as important developmental signaling molecules and have been implicated to play crucial roles in such diverse processes as cancer, organogenesis and pattern formation. Experiments by Zakany and Duboule, and Rudnicki and Brown have suggested a role for Wnt molecules in negatively regulating chondrogenesis. However, neither of the two Wnt genes used in these studies is endogenously expressed in chondrogenic regions. We and others have found that in the chick limb at least four members of the Wnt gene family, Wnt-4, Wnt-5a, Wnt-5b, and Wnt-14, are expressed in defined regions of the developing chondrogenic elements. With the exception of Wnt-5b, which is expressed in perichondrial cells and prehypertrophic chondrocytes, the expression of the three other Wnt genes is restricted to the perichondrium surrounding the cartilage element. Viral misexpression studies in the chick suggested that Wnt-4 acts as a positive signal originating from the joint region and when misexpressed accelerates chondrocyte maturation, while Wnt-5a and Wnt-5b both negatively regulate chondrocyte maturation. We have further shown that they utilize different signaling pathways; while Wnt-4 signals through the canonical Wnt-pathway, Wnt-5a and Wnt-5b do not. Interestingly, the delay in chondrocyte maturation due to Wnt-5a misexpression is associated with an up regulation of Wnt-5b expression in the prehypertropic chondrocytes. Concomitantly, Wnt-5b misexpression also delays chondrocyte maturation. However, preliminary studies suggest that the two Wnt genes affect different steps in the maturation process. Wnt signaling, however, is not only regulating chondrogenesis but is also involved in the segmentation process of the appendicular skeleton. Localized misexpression of the fourth Wnt gene, Wnt-14, which is expressed early in the presumptive joint region, induces morphological and molecular changes indicative of an early joint interzone, suggesting that Wnt-14 plays a pivotal role in the induction of the joint interzone.     

Simulated microgravity using a rotary cell culture system promotes chondrogenesis of human adipose-derived mesenchymal stem cells via the p38 MAPK pathway

Mesenchymal stem cells (MSCs) are multi-potent, and the chondrogenesis of MSCs is affected by mechanical stimulation. The aim of this study was to investigate, using a rotary cell culture system (RCCS) bioreactor, the effects of microgravity on the chondrogenic differentiation of human adipose-derived MSCs (ADSCs), which were cultured in pellets with or without the chondrogenic growth factor TGF-β1. In addition, we evaluated the role of the p38 MAPK pathway in this process. The real-time PCR and histological results show that microgravity has a synergistic effect on chondrogenesis with TGF-β1. The p38 MAPK pathway was activated by TGF-β1 alone and was further stimulated by microgravity. Inhibition of p38 activity with SB203580 suppressed chondrocyte-specific gene expression and matrix production. These findings suggest that the p38 MAPK signal acts as an essential mediator in the microgravity-induced chondrogenesis of ADSCs.     

Alterations in the spatiotemporal expression pattern and function of N-cadherin inhibit cellular condensation and chondrogenesis of limb mesenchymal cells in vitro

Cartilage formation in the embryonic limb is presaged by a cellular condensation phase that is mediated by both cell-cell and cell-matrix interactions. N-Cadherin, a Ca(2+)-dependent cell-cell adhesion molecule, is expressed at higher levels in the condensing mesenchyme, followed by down-regulation upon chondrogenic differentiation, strongly suggesting a functional role in the cellular condensation process. To further examine the role of N-cadherin, we have generated expression constructs of wild type and two deletion mutants (extracellular and intracellular) of N-cadherin in the avian replication-competent, RCAS retrovirus, and transfected primary chick limb mesenchymal cell cultures with these constructs. The effects of altered, sustained expression of N-cadherin and its mutant forms on cellular condensation, on the basis of peanut agglutinin (DNA) staining, and chondrogenesis, based on expression of chondrocyte phenotypic markers, were characterized. Cellular condensation was relatively unchanged in cultures overexpressing wild type N-cadherin, compared to controls on all days in culture. However, expression of either of the deletion mutant forms of N-cadherin resulted in decreased condensation, with the extracellular deletion mutant demonstrating the most severe inhibition, suggesting a requirement for N-cadherin mediated cell-cell adhesion and signaling in cellular condensation. Subsequent chondrogenic differentiation was also affected in all cultures overexpressing the N-cadherin constructs, on the basis of metabolic sulfate incorporation, the presence of the cartilage matrix proteins collagen type II and cartilage proteoglycan link protein, and alcian blue staining of the matrix. The characteristics of the cultures suggest that the N-cadherin mutants disrupt proper cellular condensation and subsequent chondrogenesis, while the cultures overexpressing wild type N-cadherin appear to condense normally, but are unable to proceed toward differentiation, possibly due to the prolonged maintenance of increased cell-cell adhesiveness. Thus, spatiotemporally regulated N-cadherin expression and function, at the level of both homotypic binding and linkage to the cytoskeleton, is required for chondrogenesis of limb mesenchymal cells.