Coordinated production and remodeling of the extracellular matrix is essential during development. It is of particular importance for skeletogenesis, as the ability of cartilage and bone to provide structural support is determined by the composition and organization of the extracellular matrix. Connective tissue growth factor (CTGF, CCN2) is a secreted protein containing several domains that mediate interactions with growth factors,integrins and extracellular matrix components. A role for CTGF in extracellular matrix production is suggested by its ability to mediate collagen deposition during wound healing. CTGF also induces neovascularization in vitro, suggesting a role in angiogenesis in vivo. To test whether CTGF is required for extracellular matrix remodeling and/or angiogenesis during development, we examined the pattern of Ctgf expression and generated Ctgf-deficient mice. Ctgf is expressed in a variety of tissues in midgestation embryos, with highest levels in vascular tissues and maturing chondrocytes. We confirmed that CTGF is a crucial regulator of cartilage extracellular matrix remodeling by generating Ctgf-/- mice. Ctgf deficiency leads to skeletal dysmorphisms as a result of impaired chondrocyte proliferation and extracellular matrix composition within the hypertrophic zone. Decreased expression of specific extracellular matrix components and matrix metalloproteinases suggests that matrix remodeling within the hypertrophic zones in Ctgf mutants is defective. The mutant phenotype also revealed a role for Ctgf in growth plate angiogenesis. Hypertrophic zones of Ctgf mutant growth plates are expanded, and endochondral ossification is impaired. These defects are linked to decreased expression of vascular endothelial growth factor (VEGF) in the hypertrophic zones of Ctgf mutants. These results demonstrate that CTGF is important for cell proliferation and matrix remodeling during chondrogenesis, and is a key regulator coupling extracellular matrix remodeling to angiogenesis at the growth plate.

Previous studies have demonstrated the ability of bone morphogenetic proteins (BMPs) to promote chondrogenic differentiation in vitro. However, the in vivo role of BMP signaling during chondrogenesis has been unclear. We report here that BMP signaling is essential for multiple aspects of early chondrogenesis. Whereas mice deficient in type 1 receptors Bmpr1a or Bmpr1b in cartilage are able to form intact cartilaginous elements, double mutants develop a severe generalized chondrodysplasia. The majority of skeletal elements that form through endochondral ossification are absent, and the ones that form are rudimentary. The few cartilage condensations that form in double mutants are delayed in the prechondrocytic state and never form an organized growth plate. The reduced size of mutant condensations results from increased apoptosis and decreased proliferation. Moreover, the expression of cartilage-specific extracellular matrix proteins is severely reduced in mutant elements. We demonstrate that this defect in chondrocytic differentiation can be attributed to lack of Sox9, L-Sox5, and Sox6 expression in precartilaginous condensations in double mutants. In summary, our study demonstrates that BMPR1A and BMPR1B are functionally redundant during early chondrogenesis and that BMP signaling is required for chondrocyte proliferation, survival, and differentiation in vivo.bone morphogenetic protein ͉ cartilage ͉ endochondral ossification ͉ Sox proteins ͉ skeletal development D uring endochondral ossification, mesenchymal cells condense and differentiate into chondrocytes. The chondrocytes undergo a highly organized differentiation program, forming the template for bone formation (1, 2). Bone morphogenetic proteins (BMPs) were identified by their ability to promote ectopic cartilage and bone formation (3). BMPs are members of the TGF␤ superfamily and transduce signals by binding to heteromeric complexes of type 1 and type 2 serine͞threonine kinase receptors. The binding of BMPs to the receptor complex results in the phosphorylation of intracellular Smads, which then translocate to the nucleus, where they regulate transcription (4). The differential affinities of distinct BMP ligands for the three type 1 receptors, BMPR1A (BMP receptor type 1A), BMPR1B (BMP receptor type 1B), and ActR1 (activin receptor type 1), are thought to contribute to the diversity of actions during development. Several lines of evidence suggest that Bmpr1a, Bmpr1b, and ActR1 have distinct roles during chondrogenesis. In the chick, Bmpr1a is expressed at low levels throughout the limb bud mesenchyme, whereas Bmpr1b is expressed in precartilaginous condensations, suggesting that these receptors have different roles in chondrogenesis. Furthermore, whereas constitutively active forms of either BMPR1A or BMPR1B promote chondrogenesis, only the overexpression of dominant negative (DN)-BMPR1B, and not DN-BMPR1A or DN-ActR1, blocks these events, suggesting that BMPR1B is the major transducer of BMP signals in limb condensations (5-8). However, Bmpr1b null mic...

Bone morphogenetic protein (BMP) signaling is required for endochondral bone formation. However, whether or not the effects of BMPs are mediated via canonical Smad pathways or through noncanonical pathways is unknown. In this study we have determined the role of receptor Smads 1, 5 and 8 in chondrogenesis. Deletion Development 136, 1093Development 136, -1104Development 136, (2009 signaling. Finally, we provide evidence that linker phosphorylation of Smads represents a physiologically significant mechanism regulating BMP signaling in the growth plate, but that the inhibitory effects of FGFs are likely to be mediated through different mechanisms. ). MATERIALS AND METHODS Generation of HistologySkeletal preparations were generated as described (Ivkovic et al., 2003;Yoon et al., 2006). Alcian Blue/nuclear Fast Red staining was performed as described (Luna, 1992). Von Kossa staining was performed by incubation in 1% silver nitrate under UV light for 20 minutes and counterstaining with nuclear Fast Red. Safranin O staining was performed by staining in Weigert's iron hematoxylin solution for 10 minutes, followed by Fast Green (0.001%) and Safranin O (0.1%) for 5 minutes each.For immunofluorescence, sections were boiled for 15 minutes in citrate buffer (Ivkovic et al., 2003). Sections were blocked with 5% goat or donkey serum for 1 hour and incubated with primary antibody overnight at 4°C, followed by incubation with secondary antibody for 1 hour at room temperature, then with fluorophore for 30 minutes at room temperature. Primary antibodies were as follows: phospho-Smad1/5/8 and phospho-Smad1/5 (Cell Signaling Technology); type II collagen and Pth1r (Abcam); type I collagen (Southern Biotech); type X collagen (a kind gift from Robin Poole, Shriners Hospitals for Children, Montreal, Québec, Canada); aggrecan (Developmental Studies Hybridoma Bank, Iowa City, USA); Pcna (Zymed); Fgfr1 and Stat1 (Sigma); phosphoSmad1L (a kind gift from Eddy De Robertis, University of California, Los Angeles, CA, USA). Secondary antibodies were conjugated with AlexaFluor-555 and AlexaFluor-488. Sections were counterstained with DAPI (Vectashield). For TUNEL staining, the fluorescein In Situ Cell Death Detection Kit (Roche) was used according to the manufacturer's protocol. In situ hybridization was performed as described (Song et al., 2007). Limb cultureEmbryos were harvested at 16.5 days of gestation (E16.5). Forelimbs were isolated and cultured as described (Minina et al., 2001;Minina et al., 2002). The contralateral limb was cultured in the presence of recombinant human FGF18 (10 ng/ml; Invitrogen) or the FGFR inhibitor SU5402 (10 μM; Calbiochem). In all cases, the right forelimb served as the untreated control. A total of six limbs were examined for each condition, in two separate experiments. RT-PCR and western analysis of growth plate cartilageRNA was extracted from proximal humeri using the RNeasy Kit (Qiagen). Synthesis of cDNA was performed with Superscript III (Invitrogen). Reverse transcriptase (RT)-PCR reactions compris...

The ability of bone morphogenetic proteins (BMPs) to promote chondrogenesis has been investigated extensively over the past two decades. Although BMPs promote almost every aspect of chondrogenesis, from commitment to terminal differentiation is well known, the mechanisms of BMP action in discrete aspects of endochondral bone formation have only recently begun to be investigated. In this review, we focus on in vivo studies that have identified interactions between BMP signaling pathways and key downstream targets of BMP action in chondrogenesis. We also discuss evidence regarding the potential roles of BMP receptors in mediating distinct aspects of chondrogenesis, and studies investigating the intersection of BMP pathways with other pathways known to coordinate the progression of chondrocytes through the growth plate. These studies indicate that both Smad-dependent and -independent BMP pathways are required for chondrogenesis, and that BMPs exert essential roles via regulation of the Indian hedgehog (IHH)/parathyroid hormone-related protein (PTHrP) and fibroblast growth factor (FGF) pathways in the growth plate.

Bone morphogenetic protein (BMP) signaling pathways are essential regulators of chondrogenesis. However, the roles of these pathways in vivo are not well understood. Limb-culture studies have provided a number of essential insights, including the demonstration that BMP pathways are required for chondrocyte proliferation and differentiation. However, limb-culture studies have yielded contradictory results; some studies indicate that BMPs exert stimulatory effects on differentiation, whereas others support inhibitory effects. Therefore, we characterized the skeletal phenotypes of mice lacking Bmpr1a in chondrocytes (Bmpr1a ) in order to test the roles of BMP pathways in the growth plate in vivo. These mice reveal requirements for BMP signaling in multiple aspects of chondrogenesis. They also demonstrate that the balance between signaling outputs from BMP and fibroblast growth factor (FGF) pathways plays a crucial role in the growth plate. These studies indicate that BMP signaling is required to promote Ihh expression, and to inhibit activation of STAT and ERK1/2 MAPK, key effectors of FGF signaling. BMP pathways inhibit FGF signaling, at least in part, by inhibiting the expression of FGFR1. These results provide a genetic in vivo demonstration that the progression of chondrocytes through the growth plate is controlled by antagonistic BMP and FGF signaling pathways.

Maintenance of female reproductive competence depends on the actions of several hormones and signaling factors. Recent reports suggest roles for bone morphogenetic proteins (BMPs) in early stages of folliculogenesis. A role for the type I BMP receptor BmprIB as a regulator of ovulation rates in sheep has been described recently, but little is known about the roles of BMP signaling pathways in other aspects of reproductive function. We report here that BMPRIB is essential for multiple aspects of female fertility. Mice deficient in BmprIB exhibit irregular estrous cycles and an impaired pseudopregnancy response. BmprIB mutants produce oocytes that can be fertilized in vitro, but defects in cumulus expansion prevent fertilization in vivo. This defect is associated with decreased levels of aromatase production in granulosa cells. Unexpectedly, levels of mRNA for cyclooxygenase 2, an enzyme required for cumulus expansion, are increased. BmprIB mutants also exhibit a failure in endometrial gland formation. The expression of BmprIB in uterine linings suggests that these defects are a direct consequence of loss of BMP signaling in this tissue. In summary, these studies demonstrate the importance of BMP signaling pathways for estrus cyclicity, estradiol biosynthesis, and cumulus cell expansion in vivo and reveal sites of action for BMP signaling pathways in reproductive tissues.