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Keywords:

  • connective tissue growth factor;
  • Meckel's cartilage;
  • chondrogenesis;
  • cell–matrix interactions;
  • TGF-β

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Meckel's 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 Meckel's cartilage. Indeed, CTGF was strongly expressed in anterior, central, and posterior regions of embryonic day (E) 12 condensing Meckel's 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 Meckel's 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. © 2004 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The development of cartilaginous elements involves several steps, including cell migration, cell–cell interactions, cell differentiation, and morphogenetic processes. Meckel's cartilage arises from neural crest- and mesoderm-derived progenitor cells (Nichols, 1981; Lumsden et al., 1991; Serbedzija et al., 1992; Couly et al., 1993; Imai et al., 1996; Chai et al., 2000; Mina, 2001) and is present transiently in the developing mandible, starting from mid-embryonic stages to early postnatal stages. As in other skeletal elements, one of the first signs of Meckel's cartilage formation is the appearance of condensed prechondrogenic cells at prescribed sites and times in the developing mandible. The cells then differentiate into chondrocytes and give rise to the characteristic rod-shaped cartilage, which is usually distinguished into anterior and central regions present within the developing mandible and a posterior region located near the developing condyle and ear. The cartilage is surrounded by a perichondrium composed of fibrous mesenchymal cells that separates it from neighboring nonchondrogenic cells. Recent studies have confirmed and provided additional evidence that, with further development, chondrocytes located in the anterior region and the most-rostral zone of central portion of Meckel's cartilage undergo a process of maturation resulting in marked changes in cell size and gene expression. These chondrocytes closely resemble hypertrophic chondrocytes present in other developing skeletal elements and express characteristic genes such as collagen X (Bhaskar et al., 1953; Frommer and Margolies, 1971; Richman and Diewert, 1988; Chung et al., 1995; Ishizeki et al., 1996; Harada and Ishizeki, 1998). At similar stages, intramembranous bone begins to form in close proximity to the hypertrophic cartilage, then grows and spreads anteriorly and posteriorly along the cartilaginous rod, and will ultimately produce the ossified jaw. Much of Meckel's cartilage eventually disappears, but its far-posterior end (close to the base of the skull) gives rise to the malleus and incus (Richany et al., 1956).

Connective tissue growth factor (CTGF/CCN2) is a cysteine-rich protein of approximately 38 kDa and a member of the CCN family of secreted signaling proteins. The family currently includes Cyr61/Cef10, Nov, WISP-1, WISP-2, and WISP-3 in addition to CTGF, and these proteins are involved in a variety of developmental processes, including cell determination and differentiation, morphogenesis, and cell migration and proliferation (Moussad and Brigstock, 2000; Perbal, 2001). CTGF was first identified as an immediate early gene induced by serum and transforming growth factor-β1 (TGF-β1) in cultured cells (Almendral et al., 1988; Brunner et al., 1991). Recent studies revealed that CTGF is expressed in several progenitor and differentiating cells, including vascular endothelial cells, osteoblasts, and chondrocytes (Surveyor and Brigstock, 1999, and references therein). Functional analyses suggested that CTGF stimulates proliferation of immature chondrocytes and favor terminal differentiation of mature chondrocytes (Nakanishi et al., 1997, 2000; Eguchi et al., 2001). These and other data have led to the proposal that CTGF has important roles in development of cartilage and bone during skeletogenesis, and disturbances in CTGF expression and action may be part of cartilage pathological conditions (Takigawa et al., 2003). Based on these interesting recent findings, we asked whether CTGF is expressed during Meckel's cartilage formation, and then tested possible roles it may have. Indeed, CTGF is expressed in a highly dynamic and very prominent manner during Meckel's cartilage development. Our data suggest that one of its roles is to favor cell interactions amongst first branchial arch mesenchymal cells and promote differentiation into Meckel's chondrocytes.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Phenotypic Expression

In the first set of experiments, we determined the patterns of CTGF gene expression at successive stages and different regions of Meckel's cartilage (Fig. 1) during mouse embryogenesis. Whole-mount in situ hybridization on embryonic day (E) 12 embryos with digoxigenin-labeled antisense riboprobes revealed that CTGF transcripts were already conspicuous throughout the condensed Meckel's prechondrogenic mesenchyme, extending from the anterior portion to posterior presumptive auricular portion (Fig. 2A, arrowheads). As to be expected, cartilaginous structures such as ribs and vertebrae were positive as well (Fig. 2B, single and double arrowheads, respectively), whereas no signal was detected in companion specimens hybridized with sense riboprobe (Fig. 2C).

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Figure 1. Schematic of Meckel's cartilage. The rod-like tissue is anatomically subdivided into three regions: anterior (orange), central (yellow), and posterior (purple). The central region is further subdivided into a rostral zone 1 and posterior zone 2. The dotted area encompassing a portion of the anterior region and zone 1 of the central region indicates the initiation site of ossification and represents the main focus of the present study.

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Figure 2. Gene expression patterns in developing Meckel's cartilage. Whole embryonic day (E) 12 embryos and sections from E12 (A–G), E15 (H–K), and E18 (L–O) embryos were analyzed by in situ hybridization for expression of connective tissue growth factor (CTGF), fibronectin (FN), Sox-9, and type II collagen (II). A,B: Abundant CTGF transcripts throughout E12 Meckel's cartilage primordia (A, arrowheads) and developing ribs and vertebral columns (B, single and double arrowheads). C: No hybridization signal in specimen hybridized with sense CTGF riboprobe. D–G: Meckel's condensing mesenchymal cells displaying strong expression of CTGF, FN, and Sox-9 (E–G, arrowheads) and transforming growth factor-β1 (TGF-β1; E, inset). H–K: E15 Meckel's immature cartilage exhibiting CTGF and FN only in peripheral chondrocytes and perichondrium (H–J, arrowheads and arrows, respectively) but uniform type II collagen expression (K). L–O: E18 Meckel's mature cartilage now displaying more widespread CTGF and FN expression in chondrocytes (L–N, arrowheads) but lower type II collagen and CTGF expression in perichondrium (L–N, arrows). Scale bars = 100 μm in D (applies to D–G), 50 μm in H (applies to H–O).

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To define more precisely the spatiotemporal patterns of CTGF expression, we processed continuous coronal sections of developing Meckel's cartilages from E12 to E18 mouse embryos by in situ hybridization, focusing in particular on the intramandibular region. As seen above, E12 condensed Meckel's mesenchymal cells (Fig. 2D, arrowhead) exhibited very strong CTGF expression (Fig. 2E, arrowhead) as well as strong expression of the condensation-characteristic matrix protein fibronectin (Fig. 2F, arrowhead) and the chondrogenic factor TGF-β1 (Fig. 2E, inset), regardless of location along the presumptive rod. Of interest, the condensed cells also exhibited strong expression of the chondrogenic master gene Sox-9 (Fig. 2G, arrowhead). Differentiation of condensed mesenchymal cells into chondrocytes by E15 was accompanied by a significant reduction in CTGF expression (Fig. 2H,I), but abundant transcripts persisted in peripheral chondrocytes and adjacent perichondrium (Fig. 2H,I, arrowhead and arrow, respectively). Peripheral chondrocytes and perichondrium also contained significant levels of fibronectin RNA (Fig. 2J, arrowhead and arrow, respectively), while cartilage-characteristic type II collagen transcripts were present throughout the cartilage (Fig. 2K).

By E18, the anterior region and rostral half of the central region (Fig. 1, zone 1) contained mature hypertrophic-like chondrocytes surrounded by a thin perichondrial tissue (Fig. 2L, arrowhead and arrow, respectively) and incipient intramembranous bone (Fig. 2L, double arrowhead). These developmental events were accompanied by a reversal in gene expression patterns. CTGF was now expressed throughout the mature cartilage (Fig. 2M, arrowhead) as was fibronectin (Fig. 2N, arrowhead); expression of these two genes was very low in perichondrial tissue (Fig. 2L–N, arrows). As to be expected, type II collagen expression had decreased in the mature hypertrophic-like chondrocytes (Fig. 2O) compared with the strong expression in younger immature chondrocytes (Fig. 2K).

There continues to be much interest in the behavior, role, and fate of Meckel's chondrocytes and possible relationships to bone formation (Bhaskar et al., 1953; Frommer and Margolies, 1971; Ishizeki et al., 1999). Thus, we carried out a further and more detailed analysis of organization and gene expression patterns of chondrocytes in the anterior region and zone 1 of central region in E18 Meckel's cartilage. Longitudinal sections parallel to the anterior–posterior axis of Meckel's cartilage showed that the cells were organized in growth plate-like structures similar to those present in developing long bones (Fig. 3). This finding was particularly in the anterior region that displayed small-sized immature chondrocytes at its rostral end (Fig. 3A, arrowhead) and large hypertrophic cells at its opposite end (Fig. 3A, arrow). Zone 1 of the central region was not as well organized but did display hypertrophic-like chondrocytes and less-mature chondrocytes along its rostral-to-posterior axis (Fig. 3B, arrow and arrowhead, respectively). Of interest, the two growth plate-like structures flanked a long ossified area that stained positively with the bone stain alizarin red and resembled endochondrally derived bone (Fig. 3A–D, double arrows). The latter was also suggested by dual histological staining; safranin O–positive cartilage remnants were intimately surrounded by fast green–positive bone tissue (Fig. 3D, inset), an arrangement common during the transition from calcified hypertrophic cartilage to endochondral bone in embryonic and postnatal long bone growth plates (Poole et al., 1982).

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Figure 3. Histological organization and gene expression in embryonic day (E) 18 Meckel's cartilage. A–R: Longitudinal sections from the anterior (left panels) and zone 1 (right panels) portions of intramandibular E18 Meckel's cartilage were stained with hematoxylin–eosin (A,B), alizarin red (C,D), or safranin-O/fast green (D, inset) or were processed for in situ hybridization with indicated genes (E–R). Note in A and B the organization of chondrocytes in growth plate-resembling structures, with zones of immature cells (A,B, arrowheads), zones of hypertrophy (A,B, arrows), and an ossifying alizarin red-staining central area (C,D, double arrows). See text for further details. TGF-β1, transforming growth factor-β1; VEGF, vascular endothelial growth factor; OP, osteopontin; X, collagen X; Ihh, Indian hedgehog; CTGF, connective tissue growth factor. Scale bar =100 μm in A (applies to A–R).

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These histological observations were reaffirmed and expanded by gene expression analyses by in situ hybridization. As seen above, CTGF expression was not uniform but was evident in chondrocytes scattered in several sites, particularly the hypertrophic zones (Fig. 3E,F, arrows). The prehypertrophic marker Ihh was expressed in the intermediate zones of the growth plate-like structure (Fig. 3G,H, arrows), and collagen X transcripts were abundant in mature hypertrophic zones (Fig. 3I,J). Gene products associated with transition from hypertrophic cartilage to endochondral bone, specifically osteopontin, MMP-9, and vascular endothelial growth factor (VEGF), were all expressed in posthypertrophic chondrocytes and bone cells (Fig. 3K–P, arrows). In addition, TGF-β1 transcripts were particularly evident in posthypertrophic zone, periosteal tissues and bone cells (Fig. 3Q,R).

Chondrocyte maturation in other skeletal structures involves significant changes in proliferation (Koyama et al., 1995, 1999). To determine whether the same applies to Meckel's development, E14 and E18 embryos were pulse-labeled with 5′-bromodeoxyuridine (BrdU) for 2 hr and the resulting histological sections were processed for immunohistochemical detection of proliferating cells. BrdU-positive cells were scattered throughout Meckel's cartilage in E14 embryos that largely contained immature chondrocytes (Fig. 4A, arrowheads), while very few were seen in anterior and central subregions of E18 specimens containing mature hypertrophic chondrocytes (Fig. 4B). Proliferating cells were present in perichondrium at both stages (Fig. 4A,B, arrows), and no signal was seen in control sections that received no primary antibody (Fig. 4C).

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Figure 4. Cell proliferation patterns. Mandibular explants from embryonic day (E) 14 and E18 mouse embryos were pulse-labeled with bromodeoxyuridine (BrdU) for 2 hr and incorporated nucleotide was revealed by immunohistochemical detection. A,B: Note the presence of proliferating chondrocytes in immature E14 cartilage (A, arrowheads) and absence in mature E18 cartilage (B). Proliferating cells are seen in perichondrial tissue at each stage (A,B, arrows). C: Control tissue that was not pulse-labeled with BrdU failed to stain. Scale bar = 100 μm in C (applies to A–C).

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Cell Interactions and Aggregation

CTGF mediates cell adhesion and interactions in other systems (Kireeva et al., 1997; Shimo et al., 1999) and is expressed in condensing Meckel's mesenchyme (see Fig. 1 above). To test whether the factor interacts with the cell surface and may mediate cell–cell interactions and condensation, we isolated mesenchymal cells from E10 mouse embryo first branchial arches and tested them for interaction with recombinant CTGF (rCTGF). Freshly isolated cells were seeded onto microwell dishes precoated with different concentrations of rCTGF and, for comparison, onto wells coated with fibronectin or bovine serum albumin (BSA). Cells readily attached to rCTGF or fibronectin-coated dishes (Fig. 5A–D) but not to BSA-coated dishes (Fig. 5E,F). Adhesion increased in a dose-dependent manner (Fig. 5G) and, interestingly, was maximal at 5.0 μg/ml fibronectin and 10 μg/ml rCTGF, indicating slight differences in cellular avidity for each protein (Fig. 5G). Adhesion to rCTGF was prevented by incubation with CTGF antibodies, suggesting specificity of cell–rCTGF interactions (Fig. 5H).

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Figure 5. Cell adhesion assays. A–F: Progenitor cells freshly isolated from embryonic day (E) 10 first branchial arches were tested for adhesion over a 1-hr period at room temperature to wells coated with 5 or 10 μg/ml of recombinant connective tissue growth factor (rCTGF; A,B), fibronectin (FN; C,D), or bovine serum albumin (BSA; E,F). G,H: In parallel experiments, cells were tested for adhesion to wells coated with increasing amounts of rCTGF or fibronectin (G; 0–10 μg/ml), and wells coated with 5 μg/ml rCTGF in the presence of CTGF antibodies or control immunoglobulins (IgGs; H; 0–180 μg/ml). The number of adherent cells was determined by counting random microscopic fields. Data represent mean values ± standard deviation. Scale bar = 50 μm in F (applies to A–F).

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To analyze cell aggregation, we used the single-cell suspension test previously used to analyze Wnt protein function on cell aggregation; this assay measures aggregation as loss of single cells during aggregation test periods (Bradley et al., 1993). Freshly isolated single cell suspensions prepared from E10 first branchial arches were maintained in suspension for 2 hr in the absence or presence of rCTGF. After incubation, the number of single cells in each sample was determined microscopically (Fig. 6). Treatment with rCTGF increased aggregation of mesenchymal cells as revealed by decreases in single cell number over control values (Fig. 6). When cultures were treated with neutralizing CTGF antibodies, aggregation was markedly inhibited and single cell number went up, indicating interference with endogenous CTGF (Fig. 6). When the CTGF antibodies were premixed with peptide used to raise them, the mixture had no effects on aggregation, attesting to specificity of antibody action.

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Figure 6. Cell aggregation assays. Embryonic day (E) 10 first branchial arch cells were incubated at room temperature in serum-free medium containing no additives (control), 50 ng/ml recombinant connective tissue growth factor (rCTGF), 50 μg/ml CTGF antibodies, or 50 μg/ml CTGF antibodies plus an excess amount of immunizing peptide. After a 2 hr incubation, the number of single cells was determined in multiple randomly chosen microscopic fields. Data are the average of quadruplicate determinations and are presented relative to control set at 100% ± standard deviation. Asterisks indicate significance (P < 0.05).

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CTGF Effects on Chondrogenic Cell Differentiation

Because CTGF appears to favor cell–cell interactions and aggregation (Figs. 5, 6), we asked next whether it would favor chondrogenic cell differentiation as well. For these experiments, first branchial arch mesenchymal cells were isolated from E10 mouse embryos (that is, well before the initiation of overt chondrogenesis in vivo). Cells were reared in micromass cultures in the presence of increasing amounts of rCTGF (0–100 ng/ml); micromass cultures are widely used to study chondrogenesis in other systems (Daniels et al., 1996). Several cartilage nodules were present in control untreated cultures by day 4 to 6, but their number had increased in rCTGF-treated cultures (Fig. 7C). To estimate more accurately the degree of chondrogenesis, cultures were stained with Alcian blue and the amount of stained matrix material was quantified by spectrophotometry. This analysis showed that chondrogenesis had increased by approximately 25% at 100 ng/ml rCTGF over control values (Fig. 7D). In reciprocal loss-of-function experiments, cultures were reared for 4 to 6 days in the presence of 50 μg/ml of CTGF neutralizing antibodies or a mixture of antibodies and immunizing peptide. Clearly, treatment with CTGF antibodies inhibited chondrogenesis, while cultures treated with antibodies plus peptide had levels of chondrogenesis comparable to control (Fig. 7E,F). Immunostaining of control day 6 cultures showed that the cartilaginous nodules were very rich in endogenous CTGF (Fig. 7A), and no staining was seen with cultures stained with preimmune IgGs (Fig. 7B).

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Figure 7. Connective tissue growth factor (CTGF) distribution and effects of rCTGF treatment on chondrogenesis. E10 first branchial arch cells were reared in micromass cultures for 4–6 days in control medium or medium containing recombinant CTGF (0–100 ng/ml) or CTGF antibodies plus immunizing peptide. Cultures were then processed for immunostaining or Alcian blue staining. A,B: Immunodetection of endogenous CTGF in cartilage nodules (A) and lack of staining in companion cultures reacted with preimmune IgGs (B). C,D: Alcian blue staining and quantification showing that nodules formation was increased by rCTGF treatment in a dose-dependent manner. E,F: Alcian blue staining and quantification showing that nodule formation was decreased by treatment with CTGF antibodies (50 μg/ml) but left unchanged by treatment with a mixture of CTGF antibodies plus immunizing peptide. Data represent mean values ± standard deviation. Asterisks indicate significance (P < 0.05). Scale bar = 50 μm in B (applies to A,B).

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CTGF Effects on Matrix Molecule Expression

To obtain insights into how CTGF may regulate chondrogenesis, we determined whether it affects expression of fibronectin and tenascin-C, matrix molecules involved in mesenchymal cell condensation (Mackie et al., 1987; Gluhak et al., 1996). The E10 first branchial arch cells were reared in micromass cultures for 3 days and then treated with 100 ng/ml rCTGF during the following 6 and 24 hr in serum-free conditions. Total RNAs isolated simultaneously from control and treated cultures at the 24-hr time point were processed for Northern blot analysis. Expression of fibronectin was markedly stimulated by 6 hr of rCTGF treatment and remained so by 24 hr (Fig. 8A). Tenascin-C expression was increased as well but to a lesser extent (Fig. 8A). Because TGF-β1 is a known stimulator of chondrogenesis (Seyedin et al., 1987; Leonard et al., 1991; Chai et al., 1994), we asked whether it would induce similar changes in gene expression. Indeed, treatment of day 3 cultures with 5 ng/ml TGF-β1 markedly boosted fibronectin and tenascin-C gene expression (Fig. 8B). Strikingly, it also boosted CTGF gene expression (Fig. 8B).

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Figure 8. Gene expression analysis. A,B: Embryonic day (E) 10 first branchial arch cells in micromass cultures were grown for 3 days and then treated during the following 6 and 24 hr with 100 ng/ml recombinant connective tissue growth factor (rCTGF; A) or 5 ng/ml transforming growth factor-β1 (TGF-β1; B). Total cellular RNAs were used to determine expression of fibronectin (FN), tenascin-C (TN), and CTGF by Northern blot. Blots were stained with methylene blue to reveal ribosomal 18S RNA and equal loading among lanes.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The study provides evidence that CTGF participates in Meckel's cartilage development. The factor exhibits highly dynamic patterns of expression that are restricted temporally and spatially during embryogenesis. These expression patterns and CTGF functional activities revealed by our in vitro data together suggest that the factor is engaged in and regulates events spanning the genesis and eventual demise of Meckel's cartilage.

CTGF and Meckel's Cartilage Roles

Our in vivo expression data show that CTGF is first strongly expressed in the condensing mesenchyme of incipient Meckel's cartilage from its most anterior tip to its posterior end. Differentiation of the mesenchymal cells into chondrocytes is accompanied by a significant reduction in expression, while strong expression persists along the chondroperichondrial border. Maturation and hypertrophy of chondrocytes located within anterior and central (zone 1) regions are accompanied by strong re-expression of the factor. While prominent CTGF expression in prechondrogenic condensed mesenchyme has never been observed previously, the expression patterns of CTGF in maturing Meckel's chondrocytes are clearly reminiscent of those seen during chondrocyte maturation in other developing skeletal elements (Takigawa, 2000; Ivkovic et al., 2003). It is conceivable then that Meckel-associated CTGF has roles similar to those proposed in those elements. The relatively low levels of expression in immature chondrocytes may be linked to cell proliferation, while high levels in mature cells may be linked to, and actually favor, terminal maturation, angiogenesis, and ossification. Although this possibility awaits direct testing, it correlates well with our observation that immature Meckel's chondrocytes are engaged in active cell proliferation, whereas mature cells are not, precisely as it is observed in chondrocytes undergoing maturation in developing long bones or other elements (Koyama et al., 1995). Our interpretation also correlates well with, and is sustained by, the recent report describing the consequences of CTGF gene ablation on mouse skeletal development (Ivkovic et al., 2003). The authors found that the CTGF-null mice have severe skeletal defects in the limbs, trunk, and craniofacial area, including Meckel's cartilage. Lack of CTGF appears to have particularly strong negative effects on chondrocyte hypertrophy and the transition from cartilage to bone.

In this regard, it is interesting that Meckel's chondrocytes share additional striking similarities with maturing chondrocytes at other skeletal sites. As our histological and molecular analyses make evident, chondrocytes present in the anterior region and zone 1 of the central region are organized in growth plate-like structures, undergo hypertrophy, and are replaced by endochondral-like bone. The cells express typical growth plate regulators, including Ihh in prehypertrophic cells and collagen X, VEGF, and MMP-9 in hypertrophic cells. These are all key regulators of skeletogenesis whose roles have been studied and carefully assessed in developing long bones. For example, Ihh regulates intramembranous bone collar formation (Koyama et al., 1996; Nakamura et al., 1997) and lack of Ihh causes arrest of chondrocyte maturation and absence of intramembranous and endochondral bone formation in mouse embryo limbs (St-Jacques et al., 1999). Defects in VEGF expression and angiogenesis or lack of MMP-9 also lead to impairments in chondrocyte maturation, cartilage invasion, and intramembranous and endochondral ossification (Vu et al., 1998; Zelzer et al., 2002; Yin et al., 2002). Thus, it appears that Meckel's chondrocytes in anterior and zone 1 regions express several key regulators and behave and function as chondrocytes in growth plates of developing long bones. The Meckel's chondrocytes undergo terminal maturation and replacement by endochondral bone, while the adjacent and surrounding mesenchyme undergoes intramembranous ossification. Because CTGF is strongly expressed in both hypertrophic chondrocytes and adjacent perichondrial tissues, the factor may have roles in both chondrocyte terminal differentiation and ossification processes. These interpretations are in line with older studies by Bhaskar et al. (1953) and Frommer and Margolies (1971), suggesting that the anterior portion of Meckel's cartilage undergoes maturation, hypertrophy, and endochondral ossification. They also agree with more recent studies indicating that the same occurs in chondrocytes in the rostral (zone 1) half of the central region; terminal hypertrophy, calcification, and vascularization in the cartilaginous tissue would thus contribute an endochondral component to overall mandible formation (Ishizeki et al., 1999). Based on these conclusions, it will be of interest to re-examine the phenotypes of CTGF-null mice (Ivkovic et al., 2003) and determine more precisely how Meckel's cartilage and overall mandible development are affected. It will be of equal interest to examine and compare Meckel's development in mice lacking other chondrocyte regulators such as Ihh or MMP-9.

CTGF and Chondrogenic Cell Differentiation

In addition to a very strong association between condensed Meckel's mesenchyme and CTGF gene expression, we provide evidence here that the factor plays an important role in differentiation of the cells. We find that prechondrogenic and chondrogenic nodules in micromass cultures of E10 first brachial arch-derived cells contain high levels of immunodetectable CTGF. The prechondrogenic cells readily interact with substrate-associated rCTGF, and their aggregation in suspension is stimulated by rCTGF and inhibited by CTGF neutralizing antibodies. We find also that rCTGF stimulates chondrogenesis in the micromass cultures, whereas antibody treatment inhibits it. It appears safe to conclude that CTGF has the ability to promote interactions and aggregation of first branchial arch cells and, in so doing, stimulates their differentiation into chondrocytes. This finding is in line with the well-established fact in systems such as the developing limb that mesenchymal cell aggregation and condensation stimulate, and are required for, chondrogenesis (Daniels et al., 1996). It remains to be established whether CTGF acts on aggregation and condensation by direct interaction with cell surface receptors or mediates these events by interaction with surface-associated macromolecules. Recent studies have indicated that integrin αvβ3 and low density lipoprotein receptor-related protein represent potential CTGF cell surface receptors (Babic et al., 1999; Moussad and Brigstock, 2000; Segarini et al., 2001). If these receptors turn out to be expressed by Meckel's prechondrogenic cells, they could obviously exert a similar function and mediate cell/CTGF interactions. An alternative possibility is that CTGF stimulates synthesis of cell-associated macromolecules that mediate cell aggregation. Indeed, we show here that the factor stimulates gene expression of fibronectin and tenascin-C, which are known to participate in the early cell aggregation phase of chondrogenesis at other embryonic sites (Mackie et al., 1987; Frenz et al., 1989; Downie and Newman, 1995).

It has been established that TGF-β and the transcription factor Sox-9 regulate chondrogenesis in limbs, trunk, and craniofacial region, including Meckel's cartilage (Seyedin et al., 1987; Leonard et al., 1991; Chai et al., 1994; Bi et al., 1999). Our data indicate that TGF-β1 rapidly and strongly stimulates expression of fibronectin and tenascin-C; of interest, it also boosts expression of CTGF itself. Similar stimulatory effects of TGF-β on fibronectin and tenascin-C expression were reported in other cell types and tissues (Roark and Greer, 1994; Merino et al., 1998). Given that TGF-β1 is coexpressed along with CTGF and fibronectin in Meckel's mesenchymal condensations (Fig. 2), it is possible that it may act as an upstream factor in Meckel's chondrogenesis and induce expression of cell aggregation-related molecules and CTGF itself. In doing so, TGF-β is likely to act by means of signaling mechanisms involving Smads, as indicated by recent studies (Ito et al., 2002). What about Sox-9? Our data indicate that Sox-9 expression overlaps CTGF expression at Meckel's condensation stage in young embryos. However, while Sox-9 expression persists in newly emerged chondrocytes, CTGF expression is drastically reduced. Thus, it is unlikely that CTGF may be a direct target of Sox-9 action and one of the effector molecules through which Sox-9 exerts its required roles in chondrogenesis. We should mention previous studies showing that another CCN family member, Cyr61, is expressed in developing skeletal elements (O'Brien and Lau, 1992; Igarashi et al., 1993) and that exogenous Cyr61 stimulates chondrogenesis in limb mesenchymal micromass cultures and synthesis of collagens and other extracellular matrix macromolecules (Wong et al., 1997). Thus, Cyr61 may also be expressed in Meckel's development and could represent a target of Sox-9 and/or TGF-β action. Ongoing studies are addressing these lingering and interesting questions.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

In Situ Hybridization

This procedure was carried out as described (Koyama et al., 1995, 1999). Paraffin-embedded serial tissue sections were pretreated with 1 μg/ml proteinase K (Sigma, St. Louis, MO) in 50 mM Tris-HCl, 5 mM ethylenediaminetetraacetic acid pH 7.5 for 1 min at room temperature, immediately post-fixed in 4% paraformaldehyde buffer for 10 min, and then washed twice in 1× phosphate buffered saline (PBS) containing 2 mg/ml glycine for 10 min/wash. Sections were treated for 15 min with a freshly prepared solution of 0.25% acetic anhydride in triethanolamine buffer. Sections were hybridized with antisense or sense 35S-labeled probes (approximately 1 × 106 DPM/section) at 50°C for 16 hr. After hybridization, slides were washed three times with 2× standard saline citrate (SSC) containing 50% formamide at 50°C for 20 min/wash, treated with 20 μg/ml RNase A for 30 min at 37°C, and washed three times with 0.1× SSC at 50°C for 10 min/wash. Sections were dehydrated by immersion in 70, 90, and 100% ethanol for 5 min/step, coated with Kodak NTB-3 emulsion diluted 1:1 with water, and exposed for 10 to 14 days. Slides were developed with Kodak D-19 at 20°C for 3 min, stained with hematoxylin and eosin, and analyzed and photographed with a Nikon microscope equipped for brightfield and darkfield optics. A full-length cDNA and a 680- to 840-bp fragment of mouse CTGF, a 1243- to 1990-bp fragment of mouse type II collagen, a 1376- to 1915-bp fragment of mouse TGF-β1, a 960- to 1380-bp fragment of mouse Ihh, a 121- to 867-bp fragment of mouse Sox-9, a 1937- to 2258-bp fragment of mouse MMP-9, and a 115- to 539-bp fragment of mouse VEGF were cloned by reverse transcriptase-polymerase chain reaction. The cDNA clones were sequenced to confirm identity based on published sequences.

Cell Proliferation

Tissue explants containing developing Meckel's cartilage and associated mandibular tissues were microdissected from E14 and E18 mouse embryos under sterile conditions and maintained in organ-culture for 2 hr. Explants were placed on filter paper on top of a steel grid at the air-liquid interface, using DMEM medium supplemented with 2.5% fetal calf serum (Gibco, BRL). Explants were pulse-labeled with 10 μM BrdU (Amersham Pharmacia Biotec) during last 2 hr of culture, fixed with 75% ethanol, and processed for immunohistochemical detection of incorporated BrdU (Vector Laboratories).

CTGF Antibodies and rCTGF Production

Preparation and characterization of CTGF antibodies were described previously (Shimo et al., 2002). Briefly, antisera were raised in rabbits against, and affinity-purified with, the synthetic peptides TVYRSGESFQSSC (residues 108–120) and TDGRCCTPHR (residues 288–297). The peptides derive from the N-terminal and C-terminal portions of human CTGF, respectively, and were chosen because of their predicted antigenicity and conservation among species.

rCTGF was produced in baculovirus (BV) as described (Shimo et al., 2002). Briefly, the open reading frame for full-length human CTGF encompassing residues 22–349 was cloned into the BamHI/EcoRI site of the pVT-Bac transfer vector, in frame with a vector-derived signal peptide. This construct targets the recombinant open reading frame to the polyhedron locus of the BV genome. To facilitate purification, six His residues were placed on the C-terminus of the protein. The construct was sequenced to verify accuracy and subsequently transfected, along with BV DNA (BaculoGold, Pharmingen) into insect cells. Recombinant virus was plaque purified and used for protein expression. By using this system, the expressed protein is secreted into the culture medium. For production of rCTGF, insect cells were infected with virus (4 pfu/cell) and grown in suspension culture in Sf-900 (serum-free insect cell culture medium) for 3 days (∼70% survival) and the cells are pelleted by centrifugation at 3,000 rpm for 30 min at 4°C. CTGF was purified from the medium under native conditions using a metal-chelate resin (Mr ∼ 38 kDa), resulting in a yield ∼4 mg of purified rCTGF per liter of culture medium.

Cell Cultures

Micromass cultures were prepared according to previous methods (Daniels et al., 1996). First branchial arches from E10 mouse embryos were digested with 0.1% trypsin and 0.1% collagenase (Gibco, BRL) in Hanks' balanced salt solution and dissociated by pipetting after neutralization of enzymatic activity with serum. Cells were filtered through 20-μm Nitex filter. Cells were plated in 24-well plates (Fisher) at densities of at 2 × 107 cells/ml, with one 10-μl drop per well, and allowed to attach for 2 hr at 37°C, 5% CO2, before addition of complete culture medium (1 ml/well). Complete medium contained DMEM high glucose with 10% fetal bovine serum and antibiotics in the presence of increasing amounts of rCTGF (0–100 ng/ml). At indicated times, cultures were fixed for 10 min with 4% paraformaldehyde, stained with 0.75% Alcian blue (SIGMA) in 0.1 N HCl, pH 1.0, overnight, and rinsed with distilled water. Amounts of stained extracellular matrix were quantified by measuring extractable dye (San Antonio and Tuan, 1986). Alcian blue-stained cultures were extracted with 6 M guanidine–HCl for 2 hr at room temperature; optical density of the extracted dye was measured at 650 nm in 96-well plate reader. Statistical significance was calculated by Student's t-test. When indicated, paraformaldehyde-fixed cultures were processed for immunostaining with CTGF primary antibodies (overnight incubation at 4°C) followed by fluorescein isothiocyanate–conjugated goat anti-rabbit antibodies for 1 hr at room temperature.

Cell Adhesion and Aggregation Assays

Single cell suspensions prepared from E10 mouse first branchial arches were incubated for 1 to 2 hr in serum-free DMEM to recover from the dissociation procedure. rCTGF and fibronectin (Gibco) were diluted in 0.1% BSA in 1× PBS to a final concentration of 0.1–10 μg/ml. Immunological 96-well plates were coated with each protein at 4°C for 48 hr, blocked with 6% BSA in 1× PBS for 1 hr at room temperature, and washed with 1× PBS three times. Cells were plated onto the precoated dishes at 3 × 104 cells/well in serum-free DMEM; after incubation for 1 hr, DMEM containing 10% FBS was added and plates were placed bottom up for 15 min at room temperature. After discarding the floating cells, attached cells were fixed with methanol and their numbers were determined by counting at least five fields microscopically. When indicated, adhesion tests were carried out in the presence of increasing amounts of CTGF antibodies (0 to 180 μg/ml) or equal amounts of control rabbit IgGs.

For aggregation assays, freshly isolated cells were diluted to a concentration of 1 × 106 cells/ml in serum-free medium and plated in quadruplicate in 24 multiwell plates in the presence of 50 ng/ml rCTGF, 50 μg/ml CTGF antibodies, or antibodies premixed with the excess amounts of immunizing peptide. After 2 hr incubation at room temperature, the number of single cells was determined in three randomly chosen microscopic fields per well and used to calculate average single numbers ± standard deviation. Statistical significance was calculated by Student's t-test.

RNA Analysis

Micromass cultures of E10 first branchial arch cells were switched to serum-free conditions and treated for 6 or 24 hr with 100 ng/ml rCTGF or 5 ng/ml TGF-β1. Treatments were carried out such that control and treated cultures were harvested simultaneously at the end of the 24-hr time periods and processed for isolation of total cellular RNAs using TRIZOL (Life Technologies, Inc.). For Northern blot analysis, RNAs were denatured by glyoxalation, size-fractionated by gel electrophoresis in 1% agarose gels at 10 μg/lane, and transferred to membranes by capillary blotting (Koyama et al., 1999). Blots were stained with 0.04% methylene blue to verify that each sample had been transferred efficiently. Blots were hybridized for 16 hr to 32P-labeled DNA probes at a concentration of 2.5 × 106 cpm/ml of hybridization solution containing 50% formamide, 6× SSC, 1% sodium dodecyl sulfate (SDS), 200 μg/ml sheared denatured salmon sperm DNA, and 10× Denhardt's reagent. Hybridization temperature was 42°C. After hybridization, blots were rinsed several times at room temperature with 2× SSC and 0.5% SDS; a final high-stringency rinse was with 0.1× SSC and 0.5% SDS at room temperature. Blots were exposed to Kodak (Rochester, N.Y.) X-ray films at −70°C.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We thank Dr. Hyun-Duck Nah, University of Pennsylvania, for valuable comments and suggestions. E.K. was funded by the NIH.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  • Almendral JM, Sommer D, MacDonald-Bravo H, Burckhardt J, Perera J, Bravo R. 1988. Complexity of the early genetic response to growth factors in mouse fibroblasts. Mol Cell Biol 8: 21402148.
  • Babic AM, Chen CC, Lau LF. 1999. Fisp12/mouse connective tissue growth factor mediates endothelial cell adhesion and migration through integrin alphavbeta3, promotes endothelial cell survival, and induces angiogenesis in vivo. Mol Cell Biol 19: 29582966.
  • Bhaskar SN, Weinmann JP, Schour I. 1953. Role of Meckel's cartilage in the development and growth of the rat mandible. J Dent Res 32: 398410.
  • Bi W, Deng JM, Zhang Z, Behringer RR, de Crombrugghe B. 1999. Sox9 is required for cartilage formation. Nat Genet 22: 8589.
  • Bradley RS, Cowin P, Brown AM. 1993. Expression of Wnt-1 in PC12 cells results in modulation of plakoglobin and E-cadherin and increased cellular adhesion. J Cell Biol 123: 18571865.
  • Brunner A, Chinn J, Neubauer M, Purchio AF. 1991. Identification of a gene family regulated by transforming growth factor-β. DNA Cell Biol 10: 293300.
  • Chai Y, Mah A, Crohin C, Groff S, Bringas P Jr, Le T, Santos V, Slavkin HC. 1994. Specific transforming growth factor-β subtypes regulate embryonic mouse Meckel's cartilage and tooth development. Dev Biol 162: 85103.
  • Chai Y, Jiang X, Ito Y, Bringas PJ, Han J, Rowitch DH, Soriano P, McMahon AP, Sucov HM. 2000. Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. Development 127: 16711679.
  • Chung KS, Park HH, Ting K, Takita H, Apte SS, Kuboki Y, Nishimura I. 1995. Modulated expression of type X collagen in Meckel's cartilage with different developmental fates. Dev Biol 170: 387396.
  • Couly GF, Coltey PM, Le Douarin NM. 1993. The triple origin of skull in higher vertebrates: a study in quail-chick chimeras. Development 117: 409429.
  • Daniels K, Reiter R, Solursh M. 1996. Micromass cultures of limb and other mesenchyme. Methods Cell Biol 51: 237247.
  • Downie SA, Newman SA. 1995. Different roles for fibronectin in the generation of fore and hind limb precartilage condensations. Dev Biol 172: 519530.
  • Eguchi T, Kubota S, Kondo S, Shimo T, Hattori T, Nakanishi T, Kuboki T, Yatani H, Takigawa M. 2001. Regulatory mechanism of human connective tissue growth factor (ctgf/hcs24) gene expression in a human chondrocytic cell line, hcs-2/8. J Biochem 130: 7987.
  • Frenz DA, Jaikaria NS, Newman SA. 1989. The mechanism of precartilage mesenchymal condensation: a major role for interaction of the cell surface with the amino-terminal heparin-binding domain of fibronectin. Dev Biol 136: 97103.
  • Frommer J, Margolies MR. 1971. Contribution of Meckel's cartilage to ossification of the mandible in mice. J Dent Res 50: 12601267.
  • Gluhak J, Mais A, Mina M. 1996. Tenascin-C is associated with early stages of chondrogenesis by chick mandibular ectomesenchymal cells in vivo and in vitro. Dev Dyn 205: 2440.
  • Harada Y, Ishizeki K. 1998. Evidence for transformation of chondrocytes and site-specific resorption during the degradation of Meckel's cartilage. Anat Embryol (Berl) 197: 439450.
  • Igarashi A, Okochi H, Bradham DM, Grotendorst GR. 1993. Regulation of connective tissue growth factor gene expression in human skin fibroblasts and during wound repair. Mol Biol Cell 4: 637645.
  • Imai H, Osumi-Yamashita N, Ninomiya Y, Eto K. 1996. Contribution of early-emigrating midbrain crest cells to the dental mesenchyme of mandibular molar teeth in rat embryos. Dev Biol 176: 151165.
  • Ishizeki K, Takigawa M, Harada Y, Suzuki F, Nawa T. 1996. Meckel's cartilage chondrocytes in organ culture synthesize bone-type proteins accompanying osteocytic phenotype expression. Anat Embryol (Berl) 193: 6171.
  • Ishizeki K, Saito H, Shinagawa T, Fujiwara N, Nawa T. 1999. Histochemical and immunohistochemical analysis of the mechanism of calcification of Meckel's cartilage during mandible development in rodents. J Anat 194: 265277.
  • Ito Y, Bringas P Jr, Mogharel A, Zhao J, Deng C, Chai Y. 2002. Receptor-regulated and inhibitory Smads are critical in regulating transforming growth factorβ-mediated Meckel's cartilage development. Dev Dyn 224: 6978.
  • Ivkovic S, Yoon BS, Popoff SN, Safadi FF, Libuda DE, Stephenson RC, Daluiski A, Lyons KM. 2003. Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development. Development 130: 27792791.
  • Kireeva ML, Latinkic BV, Kolesnikova TV, Chen CC, Yang GP, Abler AS, Lau LF. 1997. Cyr61 and Fisp12 are both ECM-associated signaling molecules: activities, metabolism, and localization during development. Exp Cell Res 233: 6377.
  • Koyama E, Leatherman JL, Shimazu A, Nah HD, Pacifici M. 1995. Syndecan-3, tenascin-C, and the development of cartilaginous skeletal elements and joints in chick limbs. Dev Dyn 203: 152162.
  • Koyama E, Leatherman JL, Noji S, Pacifici M. 1996. Early chick limb cartilaginous elements possess polarizing activity and express hedgehog-related morphogenetic factors. Dev Dyn 207: 344354.
  • Koyama E, Golden EB, Kirsch T, Adams SL, Chandraratna RA, Michaille JJ, Pacifici M. 1999. Retinoid signaling is required for chondrocyte maturation and endochondral bone formation during limb skeletogenesis. Dev Biol 208: 375391.
  • Leonard CM, Fuld HM, Frenz DA, Downie SA, Massague J, Newman SA. 1991. Role of transforming growth factor-beta in chondrogenic pattern formation in the embryonic limb: stimulation of mesenchymal condensation and fibronectin gene expression by exogenenous TGF-beta and evidence for endogenous TGF-beta-like activity. Dev Biol 145: 99109.
  • Lumsden A, Sprawson N, Graham A. 1991. Segmental origin and migration of neural crest cells in the hindbrain region of the chick embryo. Development 113: 12811291.
  • Mackie EJ, Thesleff I, Chiquet-Ehrismann R. 1987. Tenascin is associated with chondrogenic and osteogenic differentiation in vivo and promotes chondrogenesis in vitro. J Cell Biol 105: 25692579.
  • Merino R, Ganan Y, Macias D, Economides AN, Sampath KT, Hurle JM. 1998. Morphogenesis of digits in the avian limb is controlled by FGFs, TGFbetas, and noggin through BMP signaling. Dev Biol 200: 3545.
  • Mina M. 2001. Regulation of mandibular growth and morphogenesis. Crit Rev Oral Biol Med 12: 276300.
  • Moussad EE, Brigstock DR. 2000. Connective tissue growth factor: what's in a name? Mol Genet Metab 71: 276292.
  • Nakamura T, Aikawa T, Iwamoto-Enomoto M, Iwamoto M, Higuchi Y, Pacifici M, Kinto N, Yamaguchi A, Noji S, Kurisu K, Matsuya T, Maurizio P. 1997. Induction of osteogenic differentiation by hedgehog proteins. Biochem Biophys Res Commun 237: 465469.
  • Nakanishi T, Kimura Y, Tamura T, Ichikawa H, Yamaai Y, Sugimoto T, Takigawa M. 1997. Cloning of a mRNA preferentially expressed in chondrocytes by differential display-PCR from a human chondrocytic cell line that is identical with connective tissue growth factor (CTGF) mRNA. Biochem Biophys Res Commun 8: 206210.
  • Nakanishi T, Nishida T, Shimo T, Kobayashi K, Kubo T, Tamatani T, Tezuka K, Takigawa M. 2000. Effects of CTGF/Hcs24, a product of a hypertrophic chondrocyte-specific gene, on the proliferation and differentiation of chondrocytes in culture. Endocrinology 141: 264273.
  • Nichols DH. 1981. Neural crest formation in the head of the mouse embryo as observed using a new histological technique. J Embryol Exp Morphol 64: 105120.
  • O'Brien TP, Lau LF. 1992. Expression of the growth factor-inducible immediate early gene cyr61 correlates with chondrogenesis during mouse embryonic development. Cell Growth Differ 3: 645654.
  • Perbal B. 2001. NOV (nephroblastoma overexpressed) and the CCN family of genes: structural and functional issues. Mol Pathol 54: 5779.
  • Poole AR, Pidoux I, Rosenberg L. 1982. Role of proteoglycans in endochondral ossification: immunofluorescent localization of link protein and proteoglycan monomer in bovine fetal epiphyseal growth plate. J Cell Biol 92: 249260.
  • Richany SF, Bast TH, Anson BJ. 1956. The development of the first branchial arch in man and the fate of Meckel's cartilage. Q Bull Northwest Univ Med Sch 30: 331355.
  • Richman JM, Diewert VM. 1988. The fate of Meckel's cartilage chondrocytes in ocular culture. Dev Biol 129: 4860.
  • Roark EF, Greer K. 1994. Transforming growth factor-beta and bone morphogenetic protein-2 act by distinct mechanisms to promote chick limb cartilage differentiation in vitro. Dev Dyn 200: 103116.
  • San Antonio JD, Tuan RS. 1986. Chondrogenesis of limb bud mesenchyme in vitro: stimulation by cations. Dev Biol 115: 313324.
  • Segarini PR, Nesbitt JE, Li D, Hays LG, Yates JR, Carmichael DF. 2001. The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor is a receptor for connective tissue growth factor. J Biol Chem 276: 4065940667.
  • Serbedzija GN, Bronner-Fraser M, Fraser SE. 1992. Vital dye analysis of cranial neural crest cell migration in the mouse embryo. Development 116: 297307.
  • Seyedin SM, Segarini PR, Rosen DM, Thompson AY, Bentz H, Graycar J. 1987. Cartilage-inducing factor-B is a unique protein structurally and functionally related to transforming growth factor-beta. J Biol Chem 262: 19461949.
  • Shimo T, Nakanishi T, Nishida T, Asano M, Kanyama M, Kuboki T, Tamatani T, Tezuka K, Takemura M, Matsumura T, Takigawa M. 1999. Connective tissue growth factor induces the proliferation, migration, and tube formation of vascular endothelial cells in vitro, and angiogenesis in vivo. J Biochem 126: 137145.
  • Shimo T, Wu C, Billings PC, Piddington R, Rosenbloom J, Pacifici M, Koyama E. 2002. Expression, gene regulation, and roles of Fisp12/CTGF in developing tooth germs. Dev Dyn 224: 267278.
  • St-Jacques B, Hammerschidt M, McMahon AP. 1999. Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev 1320762086.
  • Surveyor GA, Brigstock DR. 1999. Immunohistochemical localization of connective tissue growth factor (CTGF) in the mouse embryo between days 7.5 and 14.5 of gestation. Growth Factors 17: 115124.
  • Takigawa M. 2000. Physiological roles of connective tissue growth factor (CTGF/Hcs24): promotion of endochondral ossification, angiogenesis and tissue remodeling. In: IkadaY, ShimizuY, editors. Tissue engineering for therapeutic use. Amsterdam: Elsevier. p 113.
  • Takigawa M, Nakanishi T, Kubota S, Nishida T. 2003. Role of CTGF/HCS24/ecogenin in skeletal growth control. J Cell Physiol 194: 256266.
  • Vu TH, Shipley JM, Bergers G, Berger JE, Helms JA, hanahan D, Shapiro SD, Senior RM, Werb Z. 1998. MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 93: 411422.
  • Wong M, Kireeva ML, Kolesnikova TV, Lau LF. 1997. Cyr61, product of a growth factor-inducible immediate-early gene, regulates chondrogenesis in mouse limb bud mesenchymal cells. Dev Biol 192: 492508.
  • Yin M, Gentili C, Koyama E, Zasloff M, Pacifici M. 2002. Antiangiogenic treatment delays chondrocyte maturation and bone formation during limb skeletogenesis. J Bone Miner Res 17: 5665.
  • Zelzer E, McLean W, Ng YS, Fukai N, Reginato AM, Lovejoy S, D'Amore PA, Olsen BR. 2002. Skeletal defects in VEGF(120/120) mice reveal multiple roles for VEGF in skeletogenesis. Development 129: 18931904.