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The life cycle of chondrocytes in the developing skeleton

Lillian Shum and Glen Nuckolls*

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Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, USA

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Arthritis Res 2002, 4:94-106  doi:10.1186/ar396


The electronic version of this article is the complete one and can be found online at:


Received:13 August 2001
Revisions received:14 September 2001
Accepted:19 September 2001
Published:8 November 2001

© 2002 BioMed Central Ltd

Abstract

Cartilage serves multiple functions in the developing embryo and in postnatal life. Genetic mutations affecting cartilage development are relatively common and lead to skeletal malformations, dysfunction or increased susceptibility to disease or injury. Characterization of these mutations and investigation of the molecular pathways in which these genes function have contributed to an understanding of the mechanisms regulating skeletal patterning, chondrogenesis, endochondral ossification and joint formation. Extracellular growth and differentiation factors including bone morphogenetic proteins, fibroblast growth factors, parathyroid hormone-related peptide, extracellular matrix components, and members of the hedgehog and Wnt families provide important signals for the regulation of cell proliferation, differentiation and apoptosis. Transduction of these signals within the developing mesenchymal cells and chondrocytes results in changes in gene expression mediated by transcription factors including Smads, Msx2, Sox9, signal transducer and activator of transcription (STAT), and core-binding factor alpha 1. Further investigation of the interactions of these signaling pathways will contribute to an understanding of cartilage growth and development, and will allow for the development of strategies for the early detection, prevention and treatment of diseases and disorders affecting the skeleton.

Keywords:
cartilage; chondrogenesis; endochondral ossification; limb bud; neural crest cells

Introduction

Cartilage is a connective tissue of diverse embryonic origin, that serves multiple prenatal and postnatal functions. Cartilage provides structural support for the early embryo, forms a template for developing endochondral bones, provides for rapid postnatal growth of the skeleton, cushions the joints, allows for flexible facial structure, and repairs fractured bones. Chondrocytes exhibit a life cycle of proliferation, differentiation, maturation, and apoptosis; the rate of each of these processes is dependent on temporal and spatial cues within the body. Identifying and characterizing these cues will reveal the molecular basis of cartilage form and function. Mutations or deregulation of these determinants of chondrogenesis and cartilage development can lead to skeletal malformation, limited skeletal function, or predisposition to injury. Congenital skeletal malformations are common and can be caused by a number of factors: inherited individual or multiple gene mutations; or acquired gene–environment interactions. Recent progress in linkage analysis and positional cloning has identified many genetic mutations associated with human skeletal syndromes or predispositions to certain skeletal diseases (Table 1). Sequencing the human genome also allows for the identification of the genetic loci of many bone- and cartilage-associated genes that serve as candidate links with additional skeletal disorders. Because of genetic background or gene–environment interactions, single gene mutations can cause different disorders. Furthermore, the same phenotype can be the result of mutations in different genes [1]. Complex diseases that are subject to multifactorial influences, such as osteoarthritis [2,3] provide the greatest challenges ahead. An understanding of the mechanisms that regulate chondrogenesis and cartilage development will, therefore, contribute to early gene-based detection of diseases and disorders that affect cartilage, and will provide the necessary foundation for novel prevention and treatment strategies, such as gene therapy and tissue engineering.

Table 1. Selected genes causal to skeletal diseases and disorders

Genetic and biomechanical determinants of chondrogenesis

Although chondrocytes appear to be a uniform cell type comprising the majority of the cells in cartilage, the origins and elaborations of the cartilage lineage are diverse. Chondrocytes arise from cranial neural crest cells (CNCCs) of the neural ectoderm, cephalic mesoderm, sclerotome of the paraxial mesoderm, or somato-pleure of the lateral plate mesoderm. Terminal differentiation of chondrocytes results in different types of cartilage: hyaline; elastic; and fibrous. Chondrocyte differentiation, therefore, provides unique opportunities for the study of 'what, when and how' a repertoire of morphogenetic signals are integrated into the developmental program. A number of molecules have been shown to function in cartilage formation. These include classes of extracellular ligands and their cognate receptors and cytoplasmic transducers [4], nuclear receptors [5], transcription factors or DNA-binding proteins [6], matrix proteins [7], matrix modifiers including matrix metalloproteinases [8], adhesion molecules [9] and the cytoskeleton [10]. The functions of these molecules have been reviewed in the literature cited and references therein. Although much is known about the gene products that characterize the cartilage phenotype, very little is known about the combinations of gene products that reflect the genesis of the cartilage cell lineage. Furthermore, the growth and development of the skeleton are particularly susceptible to the influence of biomechanical forces [11]. Mechanical loading regulates the shape, repair, regeneration, and senescence of the skeleton. Mechanical signals are transduced through the extracellular matrices, modify cell–matrix and cell–cell interactions, and impact on transcriptional responses. The interplay, therefore, between genetic and biomechanical determinants controls the integrity of cartilage produced both in vivo and in vitro [12].

Mesenchymal cell condensation

Chondroprogenitor mesenchymal cells aggregate into chondrogenic nodules as a necessary step in chondrocyte differentiation. This condensation process is dependent on signals initiated by cell–matrix and cell–cell adhesion, and these signals are modified by the cell's response to growth and differentiation factors in the extracellular environment. Condensation is hallmarked by changes in cell adhesion and cytoskeletal architecture [9,13]. The roles of N-cadherin, fibronectin, syndecans, tenascins, thrombospondins, neural cell adhesion molecule, focal adhesion kinase and paxillin in chondrogenic condensation have been reported. These molecules are expressed in restricted temporal and spatial patterns that correlate with chondroprogenitor cell condensation. Perturbations of the functions of these molecules leads to disruption in cell aggregation and inhibition of normal cartilage formation. Cell–cell and cell–matrix interactions activate cytoplasmic kinases, phosphatases and GTPases that can, in turn, be modulated by signaling from growth and differentiation factors such as the bone morphogenetic proteins (BMPs) and Wnts [14,15,16]. Although chondrogenesis is regulated by combinatorial signaling of a large number of factors, cell condensation can be regarded as the major event of the cell's commitment to the cartilage lineage, after which tissue-specific transcription factors and structural proteins begin to accumulate.

Bone morphogenetic proteins

BMPs are a pleiotropic group of extracellular ligands, first coined due to the ability of demineralized bone matrix (containing BMPs) to induce bone formation when injected into muscular compartments of animals [17]. Since then, a large number of molecules of the BMP family, and its superfamily, the transforming growth factor-betas (TGF-β s) have been cloned and identified, with mammalian BMP2 and BMP4 being the prototypes of the Drosophila homologue, decapentaplegic [18,19]. BMPs signal through transmembrane serine threonine kinase receptors [20]. There are two types of receptors, type I and type II, each having a number of subtypes and varying affinities to the different BMPs. Downstream from the receptors are various cytoplasmic and nuclear transducers, both positive and negative [21]. Over the past three decades since the discovery of BMPs, their ability to induce ectopic bone and cartilage formation and the mechanism of induction have been meticulously dissected [22]. BMPs, however, have been demonstrated to function in multiple systems and stages of development [19]. The diversity and specificity, therefore, of cellular competence and response towards BMPs remain to be topics of intense investigation. The pleiotropic actions of BMPs can be concentration-dependent, and BMP signaling can be regulated by positive and negative cellular feedback events [20]. Concomitantly, BMP signals are modulated by BMP binding proteins and other growth and differentiation factors, resulting in combinatorial signaling and divergent outcomes dependent on the modifiers, which can be either genetic or environmental [23].

Sox9

The Sry-type, high-mobility group (HMG)-box containing transcription factor SOX9 comes closest to serving the function of a master regulator of the chondrocyte lineage of any molecule yet characterized. Sox9 expression is directly induced by BMP signaling [24,25,26]. In humans, SOX9 haploinsufficiency (Online Mendelian Inheritance in Man [OMIM] number 114290) results in campomelic dysplasia (a lethal skeletal malformation syndrome) with XY sex reversal [27]. During embryogenesis, Sox9 is expressed in all chondroprogenitors, coincident with the expression of type II collagen. Sox9 regulates chondrogenesis through binding to essential DNA sequence motifs in chondrocyte-specific enhancer elements of the type II and type XI collagen genes and the cartilage-derived retinoic-acid-sensitive protein. Sox9 can even bind to, and activate, these DNA enhancer sequences in cartilage genes that have been transfected into non chondrocytes [28,29,30]. Mouse embryonic stem cells with null mutations of Sox9 do not form cartilage in teratomas [31]. Animals that are heterozygous null for Sox9 exhibit defects in all cartilage primordia and present a phenotype similar to human campomelic dysplasia [32]. The phosphorylation of Sox9 by cAMP-dependent protein kinase A in response to parathyroid hormone-related peptide (PTHrP) signaling regulates the binding of Sox9 to responsive elements in the collagen promoters [33,34]. Furthermore, Sox9 is known to form complexes with L-Sox5 and Sox6, and may also interact with other chondrocyte-associated transcription factors [35]. The regulation of this key player in chondrogenesis, therefore, is at the level of expression, protein modification, complex formation, and transcriptional activation.

Patterning and cell fate determination

Chondrogenesis can be divided into two interdependent processes: patterning; and cell fate determination. Pattern formation is the process during which number, size, and shape of the cartilaginous template is delineated and established. Cell fate determination is the process by which the combinatorial interactions of genetic and environmental factors serve to direct the developmental progression of a cell lineage. Cell fate is progressively restricted, and tissue specificity is gradually committed. The actions of these determinants are dependent upon concentration, time, and position. Patterning and cell fate determination are governed by a series of tissue interactions, which include interactions between adjacent components of segmental structures, or between juxtaposed epithelium and mesenchyme. Chondrogenesis during craniofacial and limb development best illustrates the complexity and hierarchy of regulatory mechanisms underlying the developmental program. During vertebrate morphogenesis, CNCCs, as well as limb bud mesenchymal cells, respond to BMP4 [36]. Depending on the timing of exposure to BMP4, these mesenchymal cells may undergo apoptosis or chondrogenesis. The orchestration of the apoptotic and chondrogenic response results in the formation and delineation of cartilaginous structures in the developing face and limbs. Studies have shown that the regulation of BMP4-mediated divergent morphogenetic outcome is dependent on both positive and negative modulators, at the level of ligands, cytoplasmic signals, and transcription factors.

Craniofacial development

Cartilages of the craniofacial region are largely derived from CNCCs [37]. CNCCs are a specialized population of ectodermal cells in origin, and arise from the lateral margin of the developing hindbrain. At the early somite stage of the embryo, the hindbrain is segmented into compartments called rhombomeres. CNCCs that are generated from the hindbrain are thus segment-specific, and these cells undergo epithelial to mesenchymal transformation, leave the crest and migrate towards the forming face. The cells populate the branchial arches, expand, and eventually differentiate into cartilage and several other cell types. A series of experiments showed that CNCCs are responsive to BMP4 and, depending on the stage of development, there can be different outcomes. Instrumental to the differential responses is a homeodomain transcription factor, Msx2, which mediates craniofacial and limb morphogenesis [38,39]. A gain of function mutation in the human MSX2 gene (OMIM 123101) causes Boston-type craniosynostosis, while loss of function mutations in this gene cause parietal foramina type I [40]. Msx2 is present in all stages of CNCC development: formation; migration; and differentiation. In the developing rhombomeres, BMP4 and Msx2 are co expressed in rhombomeres 3 and 5 and correlate with extensive CNCC apoptosis observed in these rhombomeres, which results in limited contribution of CNCC to the craniofacial skeleton [41]. Apoptosis is the mechanism of eliminating CNCCs, which are not necessary for differentiation, and the occurrence of apoptosis in alternate segments of the hindbrain serves to pattern the migrating CNCCs into three major streams. In these early premigratory crest cells, BMP4 induces Msx2 expression and causes apoptosis [41]. Over expression of Msx2 along the cephalic neural tube results in increased apoptosis, suggesting that Msx2 is the mediator of BMP4 action [42]. In rhombomeres that produce a significant number of CNCCs, the action of BMP is restricted by a Wnt antagonist, cSFRP2 [43]. Taken together, the patterning of CNCC formation within the neural tube is regulated by both positive and negative signals modulated by BMP. At later stages, Msx2 functions as a repressor of chondrogenesis without inducing cell death [44]. During migration, Msx2 and Sox9 are co expressed in migrating CNCCs. Msx2 serves to repress the function of Sox9 such that these cells are allowed to migrate and arrive at their target site before overt differentiation occurs. A dominant negative form of Msx2 accelerates the rate and extent of chondrogenesis in CNCCs in cultures, demonstrating that, when the function of Msx2 is inhibited, cells are de-repressed, and allowed to differentiate [44]. Msx2 also functions as a repressor of chondrogenesis during the formation of Meckel's cartilage in the mandibular division of the first branchial arch [26]. Over expression of Msx2 in the developing mandible disrupts the formation of Meckel's cartilage. Interestingly, Msx2 expression closely borders areas of cartilage differentiation and is tightly juxtaposed to the expression of Sox9. This suggests that Msx2 normally functions to delineate and define the boundaries for cartilage formation. Implanting BMP4-soaked beads in the developing mandible induces the expression of both Sox9 and Msx2 [26]. The relative level of expression of these antagonistic molecules, however, is dependent on positional cues within the mandible. These positional cues may be genes that are locally expressed in a specific region of the developing mandible. These genes may modify cellular competence to respond to BMP4, and consequently the expression pattern and profile of Sox9 and Msx2 induced by BMP4. It is this relative expression of Sox9 and Msx2 that determines whether ectopic cartilage will form around the bead. The regional molecular differences in the mandible that account for differential expression of Sox9 and Msx2 remain to be explored.

Limb development

During skeletogenesis in the developing limb bud, chondroprogenitor cells initiate their differentiation while neighboring cells undergo apoptosis, thus defining the boundaries of the developing skeletal elements. Mesenchymal condensations followed by chondrocyte differentiation and maturation occur in digital zones, whereas mesenchymal cells undergo apoptotic elimination in interdigital web zones to give rise to the delineation of the digits [45]. Failure of one of these processes results in limb malformations such as polydactyly or syndactyly of soft or hard tissues. BMPs regulate not only the chondrogenic and the apoptotic responses of the mesenchymal cells, but also specify digit identity, as well as participate in the generation, maintenance, and regression of the apical ectodermal ridge (a structure that governs the proximal-distal patterning of the limb bud) [46,47,48]. BMPs, however, do not pattern each region of the limb bud individually. Rather, evidence supports the hypothesis that BMPs participate in communicating cell fate decisions interactively between adjacent regions of the limb bud. Interdigital mesenchyme destined to undergo apoptosis in vivo produces cartilage when it is isolated away from the digits and developed in vitro [49,50]. Furthermore, digit identity is specified by the correspondingly more posterior interdigital tissue [48]. Interestingly, similar to the early patterning of the CNCC, Msx2 is also a mediator of BMP-regulated apoptosis in the interdigital mesenchyme [51]. Recent data suggest that the specificity of BMP for multiple actions during limb morphogenesis reflects different activities of the receptor subtypes transducing the BMP signal [52]. BMP receptor type IB appears to be the necessary mediator of BMP-induced chondrogenesis [53,54,55], although overexpression of the receptor, or constitutive activation of the receptor can also cause excessive apoptosis [56,57]. A significant challenge of future research is to distinguish between the downstream signaling pathways from the BMP receptor subtypes. These differences may provide a molecular basis for the specific and often antithetic responses elicited by BMPs within developing limb buds and other tissues.

Chondrocyte maturation

Embryonic cartilage is destined for one of several fates; it can remain as permanent cartilage, such as on the articular surfaces of bones, or it can provide a template for the formation of bones through the process of endochondral ossification (EO) [58,59]. Most of the bones of the axial and appendicular skeleton, and some of the bones of the craniofacial skeleton, develop through this process. The following two sections describe recent advances in understanding the molecular regulation of chondrocyte maturation during EO and joint formation.

Endochondral ossification

The anlagen of long bones develop as relatively homogeneous elongated masses of cartilage tissue surrounded by a perichondrium. Signaling between the perichondrium and the chondrocytes (discussed below) causes cells in the center of the anlagen to initiate progression in their maturation program to prepare a site of ossification. These chondrocytes undergo several rounds of more rapid proliferation, and then arrest in their cell cycle. The postmitotic cells change their morphology, alter their gene expression, and remodel their extracellular matrix to become hypertrophic chondrocytes (Fig. 1). Whereas proliferating and articular chondrocytes synthesize a cartilage matrix composed mostly of type II collagen, hypertrophic chondrocytes cease expressing type II and express type X collagen, which is recognized as a marker of hypertrophic cells in the chondrocyte lineage. The cartilage matrix also becomes mineralized, and the hypertrophic chondrocytes undergo apoptosis. Prior to their death, they deposit the angiogenic factor, vascular endothelial growth factor (VEGF), into their extracellular matrix, which promotes the invasion of blood vessels into the cartilage tissue [60]. The blood vessels bring chondroclasts, osteoblasts and osteoclasts into the new ossification center, which begin removing the mineralized cartilage matrix and forming bone tissue.

thumbnailFigure 1. A section through the growth plate of a fetal mouse metatarsal, 17 days post fertilization. Blue staining indicates endogenous alkaline phosphatase activity in the hypertrophic chondrocytes, diaphyseal perichondrium and bone collar, and primary spongiosa. Safranin O staining of chondromucin in the cartilage matrix is red. The regions populated by each stage of the chondrocyte lineage are shown.

The process of chondrocyte maturation expands from this initial central site toward the ends of the forming bones, with the zones of chondrocyte proliferation, cell cycle arrest, hypertrophy, and apoptosis arranged sequentially (Fig. 1). The perichondrium along the shaft differentiates into a collar of bone that expands toward the ends of the developing bone in pace with the advance of hypertrophic chondrocytes. During postnatal development, these cartilage structures, called growth plates, are 'sandwiched' between the bony metaphysis and epiphysis, and serve as factories for the rapid production of new bone. Regulation and coordination of the rates of chondrocyte proliferation, hypertrophic maturation, apoptosis, and bone collar formation are essential to normal bone morphogenesis. Human genetic disorders affecting EO, such as achondroplasia and chondrodysplasias, are relatively common. Positional cloning in affected pedigrees has contributed to the identification of genes that regulate bone development [59,61,62,63]. Further analysis of the regulatory mechanisms in animal models has provided an understanding of the interactions of these genes. Recent advances in understanding the regulation of EO have resulted from the studies of fibroblast growth factor receptors (FGFRs), Indian hedgehog (Ihh), PTHrP, BMPs and core-binding factor alpha 1 (Cbfa1). Although retinoids, nitric oxide, hypoxia, vitamin D, estrogens, and other small molecules, as well as extracellular matrix molecules and biomechanical signals contribute to the regulation of chondrocyte differentiation and maturation, this review will focus primarily on peptide and glycoprotein growth factor signaling pathways.

The PTHrP/Ihh pathway

Chondrocyte proliferation and maturation in the growth plate is regulated by a negative feedback loop of intercellular communication, mediated by the secreted signaling molecules PTHrP and Ihh [64,65]. PTHrP, a peptide hormone with homology to parathyroid hormone, is synthesized and secreted by periarticular perichondrial cells, and by chondrocytes later in development. It functions as a patterning molecule, inhibiting chondrocyte hypertrophy near the articular ends of the developing bone, thus maintaining a pool of proliferating cells [66]. Mutations in the PTH/PTHrP receptor that result in constitutive activation cause Jansen's chondrodysplasia [67]. These patients have decreased skeletal growth, abnormal metaphases and other skeletal malformations (OMIM 156400). Ihh, a member of the hedgehog family of cell-surface-associated ligands is expressed in the postmitotic, prehypertrophic cells, and provides the signal to maintain PTHrP expression at the ends of the developing bone [65]. By inhibiting chondrocyte maturation, PTHrP downregulates Ihh in the cells near the ends of the bone. Ihh promotes chondrocyte proliferation and specifies growth in the long axis through PTHrP-dependent and PTHrP-independent mechanisms [68]. Loss of Ihh function by gene targeting in mice results in decreased chondrocyte proliferation, loss of PTHrP expression, and abnormal positioning of hypertrophic chondrocytes close to the articular surface [66]. Ihh is also necessary for the signaling between the postmitotic chondrocytes and the perichondrium to establish and advance the bone collar [66,69]. Point mutations in Ihh that may inhibit binding to its receptor causes shortening of the digits (brachydactyly type A-1; OMIM 112500), consistent with its role in chondrocyte proliferation and bone growth [70].

The BMP pathways

BMP6 may serve a direct role in regulating chondrocyte maturation, while other BMPs may contribute to signaling between the chondrocytes and the perichondrium. BMP6 is expressed in prehypertrophic and hypertrophic chondrocytes, and several other BMPs are expressed in the perichondrium [71,72,73,74]. Treatment of chondrogenic cultures with BMP6 promotes the expression of type X collagen and alkaline phosphatase [75]. Misexpression of constitutively active BMP receptor type IA in developing limbs, however, delays chondrocyte maturation, and like Ihh overexpression, upregulates PTHrP [56,65]. This suggests that BMP stimulation of its receptor in the diaphyseal perichondrium mediates the signaling between Ihh from the prehypertrophic chondrocytes and PTHrP expression in the periarticular perichondrium.

The fibroblast growth factor pathways

Of the high affinity receptors for fibroblast growth factors (FGFs), three of the five family members FGFR1, FGFR2 and FGFR3 regulate skeletal development. The importance of FGFR3 in regulating chondrocyte proliferation and maturation has been revealed by analysis of patients with activating mutations in this gene, which causes achondroplasia (OMIM100800), hypochondroplasia (OMIM146000), thanatophoric dysplasias (OMIM187600), and other skeletal and soft tissue disorders, depending on the mutation (OMIM134934)[63]. Su et al. [76] demonstrated that chondrocytes from a fetus with thanatophoric dysplasia type II exhibited increased activation of the transcription factor STAT1, and increased expression of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1), a STAT-regulated gene. This suggests that mutations in FGFR cause defects in EO by inhibiting chondrocyte proliferation. Subsequent studies in mice and tissue culture cells have supported the hypothesis that increased FGFR activity disrupts the normal pattern of cartilage growth and maturation, at least in part by signaling through Stat molecules and increasing cyclin-dependent kinase inhibitor expression [77,78,79,80,81,82]. Furthermore, increased FGFR signaling causes premature apoptosis of growth plate chondrocytes in a Stat1-dependent manner [82,83]. Conversely, the FGFR3 null mutant mice exhibit increased chondrocyte proliferation and increased bone growth [84]. Interestingly, Ihh and PTHrP expression is decreased in mice expressing activated mutant FGFR3 or in wild type metatarsals grown in culture in the presence of FGF2 [85,86]. This suggests interactions between the FGF signaling pathway and the PTHrP/Ihh pathways in regulating chondrocyte proliferation and maturation, although the precise mechanisms of these interactions is not clear.

The Cbfa1 pathway

Cbfa1 (also called Runx2, PEBP2A or Osf2; OMIM600211) is a critical gene in the regulation of skeletal development as it is necessary for endochondral and intramembranous bone formation, and it is sufficient to induce premature and ectopic chondrocyte hypertrophy [87,88,89,90]. Cbfa1 encodes a transcription factor containing a conserved runt domain, that is expressed in mesenchymal condensations, chondrocytes, and cells of the osteoblast lineage [90,91,92,93]. Heterozygous loss of function mutations in Cbfa1 cause cleidocranial dysplasia (OMIM119600), a syndrome that includes clavicle hypoplasia or aplasia, failure in closure of the anterior fontanel, and other skeletal and dental malformations [94]. In addition to the essential role that Cbfa1 plays in osteoblast differentiation, it also regulates chondrocyte maturation. Loss of Cbfa1 by gene targeting in mice results in a complete lack of bone formation and a lack of chondrocyte hypertrophy in most of the skeleton [87,88,92,93]. Ectopic expression of Cbfa1 in non hypertrophic chondrocytes of transgenic mice promotes their hypertrophic differentiation and disrupts joint formation [90,95]. Cbfa1 is a direct regulator of osteocalcin and other genes in osteoblasts, and may also directly regulate hypertrophic-chondrocyte-specific genes [89]. VEGF, which is normally expressed in hypertrophic chondrocytes, is not expressed in the chondrocytes of Cbfa1 null mutant mice. Furthermore, VEGF expression is upregulated by Cbfa1 in fibroblasts in tissue culture [96]. These data suggest that Cbfa1 is an important regulator of EO, controlling chondrocyte maturation, osteoblast differentiation, and angiogenesis in the developing bone.

The regulation of Cbfa1 expression and activity serves as a point of convergence of multiple signaling pathways. Cbfa1 expression is upregulated by BMP2, BMP4 or BMP7 treatment of multipotential, skeletal, or myogenic cell lines [91,97,98,99]. The regulation of Cbfa1 by BMP may be mediated by Msx2, since Cbfa1 expression is decreased in Msx2 null mutant mice [39]. Negative regulators of Cbfa1 expression in osteogenic cells include glu cocorticoid, 1,25(OH)2D3, and TGF-β [91,100,101]. The function of Cbfa1 is repressed by its association with Smad3 in TGF-β stimulated cells [101]. Cbfa1 expression is upregulated in transgenic mice carrying an activated mutant FGFR1, and upregulated in a mesenchymal cell line treated with FGF2 or FGF8 [102]. Ihh may be both a regulator of Cbfa1 expression and a target of Cbfa1 transcriptional activity. Cbfa1 expression and bone collar formation is dependent on Ihh expression in prehypertrophic chondrocytes [66,69]. Expression of Cbfa1 in non hypertrophic chondrocytes induces Ihh expression in these cells and eventual hypertrophy [90,95]. Further study of the regulation of Cbfa1, its protein interactions, and the targets of its transcriptional activity will contribute to the detailed characterization of the molecular mechanisms regulating EO.

Joint formation

Another fate for embryonic skeletal cartilage is the formation of joints. The cartilage template for the developing limb skeleton forms as a continuous, branched cartilage element from the humerus/femur to the digit rays, with only a few skeletal elements formed from independent condensations [103,104]. These cartilage structures are then segmented through the differentiation and apoptosis of chondrocytes to form joint cavities, through a process called cavitation. Concurrently, adjacent chondrocytes and perichondrial cells differentiate to form articular cartilage and other joint-associated tissues [105,106].

Cartilage-derived morphogenetic protein 1 (CDMP1) and its mouse homologue, Gdf5, are members of the TGF-β superfamily, related to BMPs. Null mutation of Gdf5 causes shortening of the digits and defects in joint formation in the limbs as seen in the classical mouse mutant line brachypodism (bp) [105,107]. Mutations in CDMP1 cause the human skeletal disorders Grebe type chondrodysplasia (OMIM200700), Hunter-Thompson type acromesomelic dysplasia (OMIM201250) and brachydactyly type C (OMIM113100), which all include shortened or missing phalanges. Gdf5 is normally expressed in developing joints. It is one of the earliest markers of joint formation and is strongly expressed throughout cavitation [108,109,110]. Gli3, a transcription factor that functions in the hedgehog signaling pathway, is also expressed in developing joints, and its expression pattern is expanded in bp mice [110]. Focal application of exogenous Gdf5 protein to the cartilage digit rays of bp mouse limb buds in culture inhibits the expanded expression of Gli3 [110]. This suggests that CDMP1/Gdf5 provides an important signal for the sites of joint formation, and this signal regulates the expression of genes that control chondrocyte differentiation.

Recent studies by Hartmann and Tabin [111] demonstrated the importance of Wnt-14 in initiating joint formation and in the spacing of joints within the cartilage condensation. Wnt-14 is a member of the large Wnt family of secreted glycoproteins that bind to receptors of the frizzled family. Wnt-14 is expressed in the early joint-forming regions of the developing chicken limb in a pattern similar to Gdf5. In fully developed joints, however, Wnt-14 is expressed in the joint capsule and synovial membrane, while Gdf5 is restricted to the joint fibroarticular cartilage. Ectopic expression of Wnt-14 in developing digits induces morphological and molecular changes indicative of ectopic joint formation, including inhibition of cartilage differentiation, and upregulation of Gli3, Gdf5, autotaxin, chordin, Wnt-4 and CD44rel expression in relative patterns similar to those seen in normal joint development [111]. Furthermore, ectopic Wnt-14 expression represses adjacent endogenous joint development in the same cartilage condensation. This suggests that joint initiation by Wnt-14 activates a signal that regulates the positioning of the next joint in the patterning of the digits.

Study of the Wnt/frizzled pathway will contribute to an understanding of skeletal patterning and joint formation and may also provide molecular characterization of cellular changes in rheumatoid arthritis. Wnt-5a and frizzled 5 are both over expressed in the synovial tissues of rheumatoid arthritis patients when compared to normal joint tissue or tissue from osteoarthritic joints [112]. This may suggest a change in the state of differentiation of synoviocytes that contributes to progression of the disease.

Future directions

Skeletal morphogenesis depends greatly on the patterning and formation of cartilage, and the subsequent remodeling of cartilage into bones or joints. Molecules such as BMPs regulate important steps at different times in the life cycle of cartilage. This may suggest that BMPs instruct chondrocytes when to change their patterns of gene expression and behavior rather than providing specific instructions of which genes to express. The set of genes ready for expression may be determined by the history of signals to which the cell has been exposed, and the collection of receptors, signaling molecules and transcription factors accumulated in response to those signals. During chondrogenesis and endochondral ossification, chondrocyte proliferation is regulated by FGFs, BMPs, PTHrP, Ihh, cell–cell and cell–matrix adhesion, and biomechanical signals. These multiple concurrent signals converge on the regulation of cell cycle progression and cell differentiation. Further study of the interaction of cartilage-associated transcription factors such as Msx2 and Cbfa1 with cell–cycle regulators will contribute to an understanding of the important connection between proliferation and differentiation. Tissue engineering for the treatment of skeletal diseases and disorders will depend on effective tools for expanding populations of chondrocytes while maintaining or restoring their state of differentiation.

Abbreviations

BMP = bone morphogenetic protein; bp = brachypodism; Cbfa1 = core-binding factor alpha 1; CDMP1 = cartilage-derived morphogenetic protein1; CNCC = cranial neural crest cell; EO = endochondral ossification; FGF = fibroblast growth factor; FGFR = fibroblast growth factor receptor; HMG = high-mobility group; Ihh = Indian hedgehog; PTHrP = parathyroid hormone-related peptide; VEGF = vascular endothelial growth factor.

Acknowledgements

We are grateful to Harold C Slavkin for bringing our research together and for providing continued encouragement and guidance. We thank Alan Horner for critical reading of the manuscript and Sirinee Chiamvichitr for assisting in the preparation of the manuscript. Barbara Schmitt provided valuable assistance in compiling the information for Table 1. Lillian Shum and Glen Nuckolls are supported by NIH Z01-AR41114.

References

  1. Nuckolls GH, Shum L, Slavkin HC: Progress toward understanding craniofacial malformations.

    Cleft Palate Craniofac J 1999, 36:12-26. PubMed Abstract | Publisher Full Text OpenURL

  2. Felson DT, Lawrence RC, Dieppe PA, Hirsch R, Helmick CG, Jordan JM, Kington RS, Lane NE, Nevitt MC, Zhang Y, Sowers M, McAlindon T, Spector TD, Poole AR, Yanovski SZ, Ateshian G, Sharma L, Buckwalter JA, Brandt KD, Fries JF: Osteoarthritis: new insights. Part 1: the disease and its risk factors.

    Ann Intern Med 2000, 133:635-646. PubMed Abstract | Publisher Full Text OpenURL

  3. Loughlin J: Genetic epidemiology of primary osteoarthritis.

    Curr Opin Rheumatol 2001, 13:111-116. PubMed Abstract | Publisher Full Text OpenURL

  4. Hill DJ, Logan A: Peptide growth factors and their interactions during chondrogenesis.

    Prog Growth Factor Res 1992, 4:45-68. PubMed Abstract OpenURL

  5. Underhill TM, Sampaio AV, Weston AD: Retinoid signalling and skeletal development.

    Novartis Found Symp 2001, 232:171-185. PubMed Abstract | Publisher Full Text OpenURL

  6. Mundlos S, Olsen BR: Heritable diseases of the skeleton. Part I: Molecular insights into skeletal development-transcription factors and signaling pathways.

    Faseb J 1997, 11:125-132. PubMed Abstract OpenURL

  7. Mundlos S, Olsen BR: Heritable diseases of the skeleton. Part II: Molecular insights into skeletal development-matrix components and their homeostasis.

    Faseb J 1997, 11:227-233. PubMed Abstract OpenURL

  8. Wu W, Mwale F, Tchetina E, Kojima T, Yasuda T, Poole AR: Cartilage matrix resorption in skeletogenesis.

    Novartis Found Symp 2001, 232:158-166. PubMed Abstract | Publisher Full Text OpenURL

  9. DeLise AM, Fischer L, Tuan RS: Cellular interactions and signaling in cartilage development.

    Osteoarthritis Cartilage 2000, 8:309-334. PubMed Abstract | Publisher Full Text OpenURL

  10. Daniels K, Solursh M: Modulation of chondrogenesis by the cytoskeleton and extracellular matrix.

    J Cell Sci 1991, 100:249-254. PubMed Abstract OpenURL

  11. Hasler EM, Herzog W, Wu JZ, Muller W, Wyss U: Articular cartilage biomechanics: theoretical models, material properties, and biosynthetic response.

    Crit Rev Biomed Eng 1999, 27:415-488. PubMed Abstract OpenURL

  12. Reddi AH: Morphogenesis and tissue engineering of bone and cartilage: inductive signals, stem cells, and biomimetic biomaterials.

    Tissue Eng 2000, 6:351-359. PubMed Abstract | Publisher Full Text OpenURL

  13. Hall BK, Miyake T: All for one and one for all: condensations and the initiation of skeletal development.

    Bioessays 2000, 22:138-147. PubMed Abstract | Publisher Full Text OpenURL

  14. Haas AR, Tuan RS: Chondrogenic differentiation of murine C3H10T1/2 multipotential mesenchymal cells: II. Stimulation by bone morphogenetic protein-2 requires modulation of N-cadherin expression and function.

    Differentiation 1999, 64:77-89. PubMed Abstract | Publisher Full Text OpenURL

  15. Stott NS, Jiang TX, Chuong CM: Successive formative stages of precartilaginous mesenchymal condensations in vitro: modulation of cell adhesion by Wnt-7A and BMP-2.

    J Cell Physiol 1999, 180:314-324. PubMed Abstract | Publisher Full Text OpenURL

  16. Oh CD, Chang SH, Yoon YM, Lee SJ, Lee YS, Kang SS, Chun JS: Opposing role of mitogen-activated protein kinase subtypes, erk-1/2 and p38, in the regulation of chondrogenesis of mesenchymes.

    J Biol Chem 2000, 275:5613-5619. PubMed Abstract | Publisher Full Text OpenURL

  17. Urist MR: Bone: formation by autoinduction.

    Science 1965, 150:893-899. PubMed Abstract OpenURL

  18. Ducy P, Karsenty G: The family of bone morphogenetic proteins.

    Kidney Int 2000, 57:2207-2214. PubMed Abstract | Publisher Full Text OpenURL

  19. Wozney JM: The bone morphogenetic protein family: multi-functional cellular regulators in the embryo and adult.

    Eur J Oral Sci 1998, 106:160-166. PubMed Abstract OpenURL

  20. Miyazono K, Kusanagi K, Inoue H: Divergence and convergence of TGF-beta/BMP signaling.

    J Cell Physiol 2001, 187:265-276. PubMed Abstract | Publisher Full Text OpenURL

  21. Wrana JL: Regulation of Smad activity.

    Cell 2000, 100:189-192. PubMed Abstract | Publisher Full Text OpenURL

  22. Reddi AH: Cartilage morphogenesis: role of bone and cartilage morphogenetic proteins, homeobox genes and extracel-lular matrix.

    Matrix Biol 1995, 14:599-606. PubMed Abstract OpenURL

  23. Reddi AH: Interplay between bone morphogenetic proteins and cognate binding proteins in bone and cartilage development: noggin, chordin and DAN.

    Arthritis Res 2001, 3:1-5. PubMed Abstract | BioMed Central Full Text OpenURL

  24. Healy C, Uwanogho D, Sharpe PT: Regulation and role of Sox9 in cartilage formation.

    Dev Dyn 1999, 215:69-78. PubMed Abstract | Publisher Full Text OpenURL

  25. Zehentner BK, Dony C, Burtscher H: The transcription factor Sox9 is involved in BMP-2 signaling.

    J Bone Miner Res 1999, 14:1734-1741. PubMed Abstract OpenURL

  26. Semba I, Nonaka K, Takahashi I, Takahashi K, Dashner R, Shum L, Nuckolls GH, Slavkin HC: Positionally-dependent chondrogenesis induced by BMP4 is co-regulated by Sox9 and Msx2.

    Dev Dyn 2000, 217:401-414. PubMed Abstract | Publisher Full Text OpenURL

  27. Wheatley S, Wright E, Jeske Y, McCormack A, Bowles J, Koopman P: Aetiology of the skeletal dysmorphology syndrome campomelic dysplasia: expression of the Sox9 gene during chondrogenesis in mouse embryos.

    Ann N Y Acad Sci 1996, 785:350-352. PubMed Abstract OpenURL

  28. Lefebvre V, de Crombrugghe B: Toward understanding SOX9 function in chondrocyte differentiation.

    Matrix Biol 1998, 16:529-540. PubMed Abstract | Publisher Full Text OpenURL

  29. Xie WF, Zhang X, Sakano S, Lefebvre V, Sandell LJ: Trans-activation of the mouse cartilage-derived retinoic acid-sensitive protein gene by Sox9.

    J Bone Miner Res 1999, 14:757-763. PubMed Abstract OpenURL

  30. de Crombrugghe B, Lefebvre V, Behringer RR, Bi W, Murakami S, Huang W: Transcriptional mechanisms of chondrocyte differentiation.

    Matrix Biol 2000, 19:389-394. PubMed Abstract | Publisher Full Text OpenURL

  31. Bi W, Deng JM, Zhang Z, Behringer RR, de Crombrugghe B: Sox9 is required for cartilage formation.

    Nat Genet 1999, 22:85-89. PubMed Abstract | Publisher Full Text OpenURL

  32. Bi W, Huang W, Whitworth DJ, Deng JM, Zhang Z, Behringer RR, de Crombrugghe B: Haploinsufficiency of Sox9 results in defective cartilage primordia and premature skeletal mineralization.

    Proc Natl Acad Sci USA 2001, 98:6698-6703. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  33. Huang W, Chung UI, Kronenberg HM, de Crombrugghe B: The chondrogenic transcription factor Sox9 is a target of signaling by the parathyroid hormone-related peptide in the growth plate of endochondral bones.

    Proc Natl Acad Sci USA 2001, 98:160-165. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  34. Huang W, Zhou X, Lefebvre V, de Crombrugghe B: Phosphory-lation of SOX9 by cyclic AMP-dependent protein kinase A enhances SOX9's ability to transactivate a Col2a1 chondrocyte-specific enhancer.

    Mol Cell Biol 2000, 20:4149-4158. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  35. Lefebvre V, Li P, de Crombrugghe B: A new long form of Sox5 (L-Sox5), Sox6 and Sox9 are coexpressed in chondrogenesis and cooperatively activate the type II collagen gene.

    EMBO J 1998, 17:5718-5733. PubMed Abstract | Publisher Full Text OpenURL

  36. Nuckolls GH, Slavkin HC, Shum L: Bone morphogenetic protein signaling in limb and craniofacial development.

    In Proceedings of the Biological Mechanisms of Tooth Eruption, Resorption and Replacement by Implants. Edited by Davidowitch Z. Birmingham, AL: Harvard Society for the Advancement of Orthodontics, EBSCO media; 1998, 39-47. OpenURL

  37. Shum L, Takahashi K, Takahashi I, Nagata M, Tan DP, Semba I, Tanaka O, Bringas P, Nuckolls GH, Slavkin HC: Embryogenesis and the classification of craniofacial dysmorphogenesis.

    In Oral and Maxillofacial Surgery. Edited by Fonseca RJ. Philadelphia: WB Saunders; 2000, 149-194. OpenURL

  38. Bendall AJ, Abate-Shen C: Roles for Msx and Dlx homeoproteins in vertebrate development.

    Gene 2000, 247:17-31. PubMed Abstract | Publisher Full Text OpenURL

  39. Satokata I, Ma L, Ohshima H, Bei M, Woo I, Nishizawa K, Maeda T, Takano Y, Uchiyama M, Heaney S, Peters H, Tang Z, Maxson R, Maas R: Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation.

    Nat Genet 2000, 24:391-395. PubMed Abstract | Publisher Full Text OpenURL

  40. Wilkie AO, Oldridge M, Tang Z, Maxson RE Jr: Craniosynostosis and related limb anomalies.

    Novartis Found Symp 2001, 232:122-133. PubMed Abstract | Publisher Full Text OpenURL

  41. Graham A, Francis-West P, Brickell P, Lumsden A: The signalling molecule BMP4 mediates apoptosis in the rhomben-cephalic neural crest.

    Nature 1994, 372:684-686. PubMed Abstract | Publisher Full Text OpenURL

  42. Takahashi K, Nuckolls GH, Tanaka O, Semba I, Takahashi I, Dashner R, Shum L, Slavkin HC: Adenovirus-mediated ectopic expression of Msx2 in even-numbered rhombomeres induces apoptotic elimination of cranial neural crest cells in ovo.

    Development 1998, 125:1627-1635. PubMed Abstract | Publisher Full Text OpenURL

  43. Ellies DL, Church V, Francis-West P, Lumsden A: The WNT antagonist cSFRP2 modulates programmed cell death in the developing hindbrain.

    Development 2000, 127:5285-5295. PubMed Abstract | Publisher Full Text OpenURL

  44. Takahashi K, Nuckolls GH, Takahashi I, Nonaka K, Nagata M, Ikura T, Slavkin HC, Shum L: Msx2 is a repressor of chondrogenic differentiation in migratory cranial neural crest cells.

    Dev Dynamics 2001, 222:252-262. Publisher Full Text OpenURL

  45. Pizette S, Niswander L: Early steps in limb patterning and chondrogenesis.

    Novartis Found Symp 2001, 232:23-36. PubMed Abstract | Publisher Full Text OpenURL

  46. Dahn RD, Fallon JF: Limiting outgrowth: BMPs as negative regulators in limb development.

    Bioessays 1999, 21:721-725. PubMed Abstract | Publisher Full Text OpenURL

  47. Merino R, Ganan Y, Macias D, Rodriguez-Leon J, Hurle JM: Bone morphogenetic proteins regulate interdigital cell death in the avian embryo.

    Ann N Y Acad Sci 1999, 887:120-132. PubMed Abstract | Publisher Full Text OpenURL

  48. Dahn RD, Fallon JF: Interdigital regulation of digit identity and homeotic transformation by modulated BMP signaling.

    Science 2000, 289:438-441. PubMed Abstract | Publisher Full Text OpenURL

  49. Ros MA, Piedra ME, Fallon JF, Hurle JM: Morphogenetic potential of the chick leg interdigital mesoderm when diverted from the cell death program.

    Dev Dyn 1997, 208:406-419. PubMed Abstract | Publisher Full Text OpenURL

  50. Tang MK, Leung AK, Kwong WH, Chow PH, Chan JY, Ngo-Muller V, Li M, Lee KK: Bmp-4 requires the presence of the digits to initiate programmed cell death in limb interdigital tissues.

    Dev Biol 2000, 218:89-98. PubMed Abstract | Publisher Full Text OpenURL

  51. Ferrari D, Lichtler AC, Pan ZZ, Dealy CN, Upholt WB, Kosher RA: Ectopic expression of Msx-2 in posterior limb bud mesoderm impairs limb morphogenesis while inducing BMP-4 expression, inhibiting cell proliferation, and promoting apoptosis.

    Dev Biol 1998, 197:12-24. PubMed Abstract | Publisher Full Text OpenURL

  52. Cheifetz S: BMP receptors in limb and tooth formation.

    Crit Rev Oral Biol Med 1999, 10:182-198. PubMed Abstract OpenURL

  53. Nonaka K, Shum L, Takahashi I, Takahashi K, Ikura T, Dashner R, Nuckolls GH, Slavkin HC: Convergence of the BMP and EGF signaling pathways on Smad1 in the regulation of chondroge-nesis.

    Int J Dev Biol 1999, 43:795-807. PubMed Abstract OpenURL

  54. Baur ST, Mai JJ, Dymecki SM: Combinatorial signaling through BMP receptor IB and GDF5: shaping of the distal mouse limb and the genetics of distal limb diversity.

    Development 2000, 127:605-619. PubMed Abstract | Publisher Full Text OpenURL

  55. Yi SE, Daluiski A, Pederson R, Rosen V, Lyons KM: The type I BMP receptor BMPRIB is required for chondrogenesis in the mouse limb.

    Development 2000, 127:621-630. PubMed Abstract | Publisher Full Text OpenURL

  56. Zou H, Wieser R, Massague J, Niswander L: Distinct roles of type I bone morphogenetic protein receptors in the formation and differentiation of cartilage.

    Genes Dev 1997, 11:2191-2203. PubMed Abstract | Publisher Full Text OpenURL

  57. Zhang Z, Yu X, Zhang Y, Geronimo B, Lovlie A, Fromm SH, Chen Y: Targeted misexpression of constitutively active BMP receptor-IB causes bifurcation, duplication, and posterior transformation of digit in mouse limb.

    Dev Biol 2000, 220:154-167. PubMed Abstract | Publisher Full Text OpenURL

  58. Erlebacher A, Filvaroff EH, Gitelman SE, Derynck R: Toward a molecular understanding of skeletal development.

    Cell 1995, 80:371-378. PubMed Abstract | Publisher Full Text OpenURL

  59. Olsen BR, Reginato AM, Wang W: Bone Development.

    Annu Rev Cell Dev Biol 2000, 16:191-220. PubMed Abstract | Publisher Full Text OpenURL

  60. Gerber HP, Vu TH, Ryan AM, Kowalski J, Werb Z, Ferrara N: VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation.

    Nat Med 1999, 5:623-628. PubMed Abstract | Publisher Full Text OpenURL

  61. Francomano CA, McIntosh I, Wilkin DJ: Bone dysplasias in man: molecular insights.

    Curr Opin Genet Dev 1996, 6:301-308. PubMed Abstract | Publisher Full Text OpenURL

  62. Mundlos S, Olsen BR: Heritable diseases of the skeleton. Part II: Molecular insights into skeletal development-matrix components and their homeostasis.

    FASEB J 1997, 11:227-233. PubMed Abstract OpenURL

  63. Vajo Z, Francomano CA, Wilkin DJ: The molecular and genetic basis of fibroblast growth factor receptor 3 disorders: the achondroplasia family of skeletal dysplasias, Muenke cran-iosynostosis, and Crouzon syndrome with acanthosis nigri-cans.

    Endocr Rev 2000, 21:23-39. PubMed Abstract | Publisher Full Text OpenURL

  64. Lanske B, Karaplis AC, Lee K, Luz A, Vortkamp A, Pirro A, Karpe-rien M, Defize LH, Ho C, Mulligan RC, Abou-Samra AB, Juppner H, Segre GV, Kronenberg HM: PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth.

    Science 1996, 273:663-666. PubMed Abstract OpenURL

  65. Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ: Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein.

    Science 1996, 273:613-622. PubMed Abstract OpenURL

  66. St-Jacques B, Hammerschmidt M, McMahon AP: Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation.

    Genes Dev 1999, 13:2072-2086. PubMed Abstract | Publisher Full Text OpenURL

  67. Schipani E, Kruse K, Juppner H: A constitutively active mutant PTH-PTHrP receptor in Jansen-type metaphyseal chondrodysplasia.

    Science 1995, 268:98-100. PubMed Abstract OpenURL

  68. Karp SJ, Schipani E, St-Jacques B, Hunzelman J, Kronenberg H, McMahon AP: Indian hedgehog coordinates endochondral bone growth and morphogenesis via parathyroid hormone related-protein-dependent and -independent pathways.

    Development 2000, 127:543-548. PubMed Abstract | Publisher Full Text OpenURL

  69. Chung UI, Schipani E, McMahon AP, Kronenberg HM: Indian hedgehog couples chondrogenesis to osteogenesis in endo-chondral bone development.

    J Clin Invest 2001, 107:295-304. PubMed Abstract | Publisher Full Text OpenURL

  70. Gao B, Guo J, She C, Shu A, Yang M, Tan Z, Yang X, Guo S, Feng G, He L: Mutations in IHH, encoding Indian hedgehog, cause brachydactyly type A-1.

    Nat Genet 2001, 28:386-388. PubMed Abstract | Publisher Full Text OpenURL

  71. Lyons KM, Pelton RW, Hogan BL: Organogenesis and pattern formation in the mouse: RNA distribution patterns suggest a role for bone morphogenetic protein-2A (BMP-2A).

    Development 1990, 109:833-844. PubMed Abstract OpenURL

  72. Jones CM, Lyons KM, Hogan BL: Involvement of Bone Morpho-genetic Protein-4 (BMP-4) and Vgr-1 in morphogenesis and neurogenesis in the mouse.

    Development 1991, 111:531-542. PubMed Abstract OpenURL

  73. Kingsley DM: What do BMPs do in mammals? Clues from the mouse short-ear mutation.

    Trends Genet 1994, 10:16-21. PubMed Abstract | Publisher Full Text OpenURL

  74. Pathi S, Rutenberg JB, Johnson RL, Vortkamp A: Interaction of Ihh and BMP/Noggin signaling during cartilage differentiation.

    Dev Biol 1999, 209:239-253. PubMed Abstract | Publisher Full Text OpenURL

  75. Grimsrud CD, Romano PR, D'Souza M, Puzas JE, Reynolds PR, Rosier RN, O'Keefe RJ: BMP-6 is an autocrine stimulator of chondrocyte differentiation.

    J Bone Miner Res 1999, 14:475-482. PubMed Abstract OpenURL

  76. Su WC, Kitagawa M, Xue N, Xie B, Garofalo S, Cho J, Deng C, Horton WA, Fu XY: Activation of Stat1 by mutant fibroblast growth-factor receptor in thanatophoric dysplasia type II dwarfism.

    Nature 1997, 386:288-292. PubMed Abstract | Publisher Full Text OpenURL

  77. Chen L, Adar R, Yang X, Monsonego EO, Li C, Hauschka PV, Yayon A, Deng CX: Gly369Cys mutation in mouse FGFR3 causes achondroplasia by affecting both chondrogenesis and osteogenesis.

    J Clin Invest 1999, 104:1517-1525. PubMed Abstract | Publisher Full Text OpenURL

  78. Li C, Chen L, Iwata T, Kitagawa M, Fu XY, Deng CX: A Lys644Glu substitution in fibroblast growth factor receptor 3 (FGFR3) causes dwarfism in mice by activation of STATs and ink4 cell cycle inhibitors.

    Hum Mol Genet 1999, 8:35-44. PubMed Abstract | Publisher Full Text OpenURL

  79. Sahni M, Ambrosetti DC, Mansukhani A, Gertner R, Levy D, Basil-ico C: FGF signaling inhibits chondrocyte proliferation and regulates bone development through the STAT-1 pathway.

    Genes Dev 1999, 13:1361-1366. PubMed Abstract | Publisher Full Text OpenURL

  80. Hart KC, Robertson SC, Kanemitsu MY, Meyer AN, Tynan JA, Donoghue DJ: Transformation and Stat activation by derivatives of FGFR1, FGFR3, and FGFR4.

    Oncogene 2000, 19:3309-3320. PubMed Abstract | Publisher Full Text OpenURL

  81. Aikawa T, Segre GV, Lee K: Fibroblast growth factor inhibits chondrocytic growth through induction of p21 and subsequent inactivation of Cyclin E-Cdk2.

    J Biol Chem 2001, 276:29347-29352. PubMed Abstract | Publisher Full Text OpenURL

  82. Sahni M, Raz R, Coffin JD, Levy D, Basilico C: STAT1 mediates the increased apoptosis and reduced chondrocyte proliferation in mice overexpressing FGF2.

    Development 2001, 28:2119-2129. OpenURL

  83. Legeai-Mallet L, Benoist-Lasselin C, Delezoide AL, Munnich A, Bonaventure J: Fibroblast growth factor receptor 3 mutations promote apoptosis but do not alter chondrocyte proliferation in thanatophoric dysplasia.

    J Biol Chem 1998, 273:13007-13014. PubMed Abstract | Publisher Full Text OpenURL

  84. Deng C, Wynshaw-Boris A, Zhou F, Kuo A, Leder P: Fibroblast growth factor receptor 3 is a negative regulator of bone growth.

    Cell 1996, 84:911-921. PubMed Abstract | Publisher Full Text OpenURL

  85. Naski MC, Colvin JS, Coffin JD, Ornitz DM: Repression of hedgehog signaling and BMP4 expression in growth plate cartilage by fibroblast growth factor receptor 3.

    Development 1998, 125:4977-4988. PubMed Abstract | Publisher Full Text OpenURL

  86. Chen L, Li C, Qiao W, Xu X, Deng C: A Ser(365)->Cys mutation of fibroblast growth factor receptor 3 in mouse downregulates Ihh/PTHrP signals and causes severe achondroplasia.

    Hum Mol Genet 2001, 10:457-465. PubMed Abstract | Publisher Full Text OpenURL

  87. Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao YH, Inada M, Sato M, Okamoto R, Kitamura Y, Yoshiki S, Kishimoto T: Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts.

    Cell 1997, 89:755-764. PubMed Abstract | Publisher Full Text OpenURL

  88. Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC, Rosewell IR, Stamp GW, Beddington RS, Mundlos S, Olsen BR, Selby PB, Owen MJ: Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development.

    Cell 1997, 89:765-771. PubMed Abstract | Publisher Full Text OpenURL

  89. Ducy P: Cbfa1: a molecular switch in osteoblast biology.

    Dev Dyn 2000, 219:461-471. PubMed Abstract | Publisher Full Text OpenURL

  90. Takeda S, Bonnamy JP, Owen MJ, Ducy P, Karsenty G: Continuous expression of Cbfa1 in nonhypertrophic chondrocytes uncovers its ability to induce hypertrophic chondrocyte differentiation and partially rescues Cbfa1-deficient mice.

    Genes Dev 2001, 15:467-481. PubMed Abstract | Publisher Full Text OpenURL

  91. Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G: Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation.

    Cell 1997, 89:747-754. PubMed Abstract | Publisher Full Text OpenURL

  92. Inada M, Yasui T, Nomura S, Miyake S, Deguchi K, Himeno M, Sato M, Yamagiwa H, Kimura T, Yasui N, Ochi T, Endo N, Kita-mura Y, Kishimoto T, Komori T: Maturational disturbance of chondrocytes in Cbfa1-deficient mice.

    Dev Dyn 1999, 214:279-290. PubMed Abstract | Publisher Full Text OpenURL

  93. Kim IS, Otto F, Zabel B, Mundlos S: Regulation of chondrocyte differentiation by Cbfa1.

    Mech Dev 1999, 80:159-170. PubMed Abstract | Publisher Full Text OpenURL

  94. Mundlos S, Otto F, Mundlos C, Mulliken JB, Aylsworth AS, Albright S, Lindhout D, Cole WG, Henn W, Knoll JH, Owen MJ, Mertelsmann R, Zabel BU, Olsen BR: Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia.

    Cell 1997, 89:773-779. PubMed Abstract | Publisher Full Text OpenURL

  95. Ueta C, Iwamoto M, Kanatani N, Yoshida C, Liu Y, Enomoto-Iwamoto M, Ohmori T, Enomoto H, Nakata K, Takada K, Kurisu K, Komori T: Skeletal malformations caused by overexpression of Cbfa1 or its dominant negative form in chondrocytes.

    J Cell Biol 2001, 153:87-100. PubMed Abstract | Publisher Full Text OpenURL

  96. Zelzer E, Glotzer DJ, Hartmann C, Thomas D, Fukai N, Soker S, Olsen BR: Tissue specific regulation of VEGF expression during bone development requires Cbfa1/Runx2.

    Mech Dev 2001, 106:97-106. PubMed Abstract | Publisher Full Text OpenURL

  97. Tsuji K, Ito Y, Noda M: Expression of the PEBP2alphaA/AML3/CBFA1 gene is regulated by BMP4/7 heterodimer and its overexpression suppresses type I collagen and osteocalcin gene expression in osteoblastic and nonosteoblastic mes-enchymal cells.

    Bone 1998, 22:87-92. PubMed Abstract | Publisher Full Text OpenURL

  98. Gori F, Thomas T, Hicok KC, Spelsberg TC, Riggs BL: Differentiation of human marrow stromal precursor cells: bone morphogenetic protein-2 increases OSF2/CBFA1, enhances osteoblast commitment, and inhibits late adipocyte maturation.

    J Bone Miner Res 1999, 14:1522-1535. PubMed Abstract OpenURL

  99. Lee MH, Javed A, Kim HJ, Shin HI, Gutierrez S, Choi JY, Rosen V, Stein JL, van Wijnen AJ, Stein GS, Lian JB, Ryoo HM: Transient upregulation of CBFA1 in response to bone morphogenetic protein-2 and transforming growth factor beta 1 in C2C12 myogenic cells coincides with suppression of the myogenic phenotype but is not sufficient for osteoblast differentiation.

    J Cell Biochem 1999, 73:114-125. PubMed Abstract | Publisher Full Text OpenURL

  100. Chang DJ, Ji C, Kim KK, Casinghino S, McCarthy TL, Centrella M: Reduction in transforming growth factor beta receptor I expression and transcription factor CBFa1 on bone cells by glucocorticoid.

    J Biol Chem 1998, 273:4892-4896. PubMed Abstract | Publisher Full Text OpenURL

  101. Alliston T, Choy L, Ducy P, Karsenty G, Derynck R: TGF-beta-induced repression of CBFA1 by Smad3 decreases cbfa1 and osteocalcin expression and inhibits osteoblast differentiation.

    EMBO J 2001, 20:2254-2272. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  102. Zhou YX, Xu X, Chen L, Li C, Brodie SG, Deng CX: A Pro250Arg substitution in mouse Fgfr1 causes increased expression of Cbfa1 and premature fusion of calvarial sutures.

    Hum Mol Genet 2000, 9:2001-2008. PubMed Abstract | Publisher Full Text OpenURL

  103. Shubin NH, Alberch P: A morphogenetic approach to the origin and basic organization of the tetrapod limb.

    Evol Biol 1986, 20:319-387. OpenURL

  104. Oster GF, Shubin N, Murray JD, Alberch P: Evolution and morphogenetic rules: the shape of the vertebrate limb in ontogeny and phylogney.

    Evolution 1988, 42:862-884. OpenURL

  105. Kingsley DM: Genetic control of bone and joint formation.

    Novartis Found Symp 2001, 232:213-22. PubMed Abstract | Publisher Full Text OpenURL

  106. Pacifici M, Koyama E, Iwamoto M, Gentili C: Development of articular cartilage: what do we know about it and how may it occur?

    Connect Tissue Res 2000, 41:175-184. PubMed Abstract OpenURL

  107. Storm EE, Huynh TV, Copeland NG, Jenkins NA, Kingsley DM, Lee SJ: Limb alterations in brachypodism mice due to mutations in a new member of the TGF beta-superfamily.

    Nature 1994, 368:639-643. PubMed Abstract | Publisher Full Text OpenURL

  108. Storm EE, Kingsley DM: Joint patterning defects caused by single and double mutations in members of the bone morphogenetic protein (BMP) family.

    Development 1996, 122:3969-3979. PubMed Abstract | Publisher Full Text OpenURL

  109. Wolfman NM, Hattersley G, Cox K, Celeste AJ, Nelson R, Yamaji N, Dube JL, DiBlasio-Smith E, Nove J, Song JJ, Wozney JM, Rosen V: Ectopic induction of tendon and ligament in rats by growth and differentiation factors 5, 6, and 7, members of the TGF-beta gene family.

    J Clin Invest 1997, 100:321-330. PubMed Abstract | Publisher Full Text OpenURL

  110. Storm EE, Kingsley DM: GDF5 coordinates bone and joint formation during digit development.

    Dev Biol 1999, 209:11-27. PubMed Abstract | Publisher Full Text OpenURL

  111. Hartmann C, Tabin CJ: Wnt-14 plays a pivotal role in inducing synovial joint formation in the developing appendicular skeleton.

    Cell 2001, 104:341-351. PubMed Abstract | Publisher Full Text OpenURL

  112. Sen M, Lauterbach K, El-Gabalawy H, Firestein GS, Corr M, Carson DA: Expression and function of wingless and frizzled homologs in rheumatoid arthritis.

    Proc Natl Acad Sci USA 2000, 97:2791-2796. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  113. Online Mendelian Inheritance in Man. McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD); [http://www.ncbi.nlm.nih.gov/omim/] webcite

    2000.