Chondrocytes and osteoblasts are two primary cell types in the skeletal system that are differentiated from common mesenchymal progenitors. It is believed that osteoblast differentiation is controlled by distinct mechanisms in intramembranous and endochondral ossification. We have found that ectopic canonical Wnt signaling leads to enhanced ossification and suppression of chondrocyte formation. Conversely, genetic inactivation of beta-catenin, an essential component transducing the canonical Wnt signaling, causes ectopic formation of chondrocytes at the expense of osteoblast differentiation during both intramembranous and endochondral ossification. Moreover, inactivation of beta-catenin in mesenchymal progenitor cells in vitro causes chondrocyte differentiation under conditions allowing only osteoblasts to form. Our results demonstrate that beta-catenin is essential in determining whether mesenchymal progenitors will become osteoblasts or chondrocytes regardless of regional locations or ossification mechanisms. Controlling Wnt/beta-catenin signaling is a common molecular mechanism underlying chondrocyte and osteoblast differentiation and specification of intramembranous and endochondral ossification.

A critical step in skeletal morphogenesis is the formation of synovial joints, which define the relative size of discrete skeletal elements and are required for the mobility of vertebrates. We have found that several Wnt genes, including Wnt4, Wnt14, and Wnt16, were expressed in overlapping and complementary patterns in the developing synovial joints, where ␤-catenin protein levels and transcription activity were up-regulated. Removal of ␤-catenin early in mesenchymal progenitor cells promoted chondrocyte differentiation and blocked the activity of Wnt14 in joint formation. Ectopic expression of an activated form of ␤-catenin or Wnt14 in early differentiating chondrocytes induced ectopic joint formation both morphologically and molecularly. In contrast, genetic removal of ␤-catenin in chondrocytes led to joint fusion. These results demonstrate that the Wnt/␤-catenin signaling pathway is necessary and sufficient to induce early steps of synovial joint formation. Wnt4, Wnt14, and Wnt16 may play redundant roles in synovial joint induction by signaling through the ␤-catenin-mediated canonical Wnt pathway.[Keywords: Wnt; ␤-catenin; joint formation; skeletal development] Supplemental material is available at http://www.genesdev.org. Formation of synovial joints between different skeletal elements is essential for the mobility of vertebrates. The number and position of joints also determine characteristic skeletal patterns in each vertebrate species by defining the size and shape of skeletal elements. As alterations of early patterning signals often lead to changes in the position and number of joints in the developing limb (Dahn and Fallon 2000;Suzuki et al. 2004), understanding the regulation of joint formation in the limb will also provide critical insights into how early-limb patterning is linked to later skeletal morphogenesis at the molecular level.In the developing limb, studies of descriptive embryology have shown that skeletal elements form through temporally and spatially regulated processes that include mesenchymal condensation, elongation, branching, and/ or segmentation (Shubin and Alberch 1986). Most of the synovial joints in the limb form through segmentation of a pre-existing cartilage rod. For instance, in the developing forelimb, the initial de novo mesenchymal condensation forms the cartilage anlagen of the humerus, the growth and branching of which then produce a Y-shaped bifurcation. It is the segmentation of this Y-shaped cartilage primordium that forms the elbow joint that separates the radius and ulna from the humerus (Shubin and Alberch 1986).Synovial joint formation starts from the differentiation of newly differentiated chondrocytes into flattened and densely packed interzone cells (for review, see Archer et al. 2003), which express joint-specific markers such as Gdf5 and lose the expression of chondrocytespecific markers such as ColII (Craig et al. 1987;Nalin et al. 1995;Morrison et al. 1996;Storm and Kingsley 1996). Later in development, the interzone cells differentiate and form three laye...

Chondrocyte fate determination and maintenance requires Sox9, an intrinsic transcription factor, but is inhibited by Wnt/ ␤-catenin signaling activated by extrinsic Wnt ligands. Here we explored the underlying molecular mechanism by which Sox9 antagonizes the Wnt/␤-catenin signaling in chondrocyte differentiation. We found that Sox9 employed two distinct mechanisms to inhibit Wnt/␤-catenin signaling: the Sox9 N terminus is necessary and sufficient to promote ␤-catenin degradation, whereas the C terminus is required to inhibit ␤-catenin transcriptional activity without affecting its stability. Sox9 binds to ␤-catenin and components of the ␤-catenin "destruction complex," glycogen synthase kinase 3 and ␤-transducin repeat containing protein, to promote their nuclear localization. Independent of its DNA binding ability, nuclear localization of Sox9 is both necessary and sufficient to enhance ␤-catenin phosphorylation and its subsequent degradation. Thus, one mechanism whereby Sox9 regulates chondrogenesis is to promote efficient ␤-catenin phosphorylation in the nucleus. This mechanism may be broadly employed by other intrinsic cell fate determining transcription factors to promptly turn off extrinsic inhibitory Wnt signaling mediated by ␤-catenin.

Both the Wnt/␤-catenin and Ihh signaling pathways play essential roles in crucial aspects of endochondral ossification: osteoblast differentiation, chondrocyte proliferation and hypertrophy. To understand the genetic interaction between these two signaling pathways, we have inactivated the ␤-catenin gene and upregulated Ihh signaling simultaneously in the same cells during endochondral skeletal development using ␤-catenin and patched 1 floxed alleles. We uncovered previously unexpected roles of Ihh signaling in synovial joint formation and the essential function of Wnt/␤-catenin signaling in regulating chondrocyte survival. More importantly, we found that Wnt and Ihh signaling interact with each other in distinct ways to control osteoblast differentiation, chondrocyte proliferation, hypertrophy, survival and synovial joint formation in the developing endochondral bone. ␤-catenin is required downstream of Ihh signaling and osterix expression for osteoblast differentiation. But in chondrocyte survival, ␤-catenin is required upstream of Ihh signaling to inhibit chondrocyte apoptosis. In addition, Ihh signaling can inhibit chondrocyte hypertrophy and synovial joint formation independently of ␤-catenin. However, there is a strong synergistic interaction between Wnt/␤-catenin and Ihh signaling in regulating synovial joint formation.

Cell-cell signaling is a major strategy that vertebrate embryos employ to coordinately control cell proliferation, differentiation, and survival in many developmental processes. Similar cell signaling pathways also control adult tissue regeneration and repair. We demonstrated in the developing skeletal system that the Wnt/beta-catenin signaling controls the differentiation of progenitor cells into either osteoblasts or chondrocytes. Genetic ablation of beta-catenin in the developing mouse embryo resulted in ectopic formation of chondrocytes at the expense of osteoblast differentiation during both intramembranous and endochondral ossification. Conversely, ectopic upregulation of the canonical Wnt signaling led to suppression of chondrocyte formation and enhanced ossification. As other signaling pathways also play critical roles in controlling skeletal development, to gain a full picture of the molecular regulatory network of skeletal development, we investigated how the Wnt/beta-catenin signaling is integrated with Indian hedgehog (Ihh) signaling in controlling various aspects of skeletal development. We found that Wnt signaling acts downstream of Ihh signaling and is required in osteoblasts after Osterix expression to promote osteoblast maturation during endochondral bone formation. Since similar controlling mechanisms of osteoblast proliferation and differentiation may be employed by adult mesenchymal progenitor cells during fracture repair, these studies suggest that, to enhance fracture repair or bone formation, Ihh signaling needs to be enhanced at early stages, whereas Wnt signaling should be upregulated slightly later in differentiated osteoblasts.

Vertebrate limb development is controlled by three signaling centers that regulate limb patterning and growth along the proximodistal (PD),anteroposterior (AP) and dorsoventral (DV) limb axes. Coordination of limb development along these three axes is achieved by interactions and feedback loops involving the secreted signaling molecules that mediate the activities of these signaling centers. However, it is unknown how these signaling interactions are processed in the responding cells. We have found that distinct LIM homeodomain transcription factors, encoded by the LIM homeobox(LIM-HD) genes Lhx2, Lhx9 and Lmx1b integrate the signaling events that link limb patterning and outgrowth along all three axes. Simultaneous loss of Lhx2 and Lhx9 function resulted in patterning and growth defects along the AP and the PD limb axes. Similar, but more severe, phenotypes were observed when the activities of all three factors, Lmx1b, Lhx2 and Lhx9, were significantly reduced by removing their obligatory co-factor Ldb1. This reveals that the dorsal limb-specific factor Lmx1b can partially compensate for the function of Lhx2 and Lhx9 in regulating AP and PD limb patterning and outgrowth. We further showed that Lhx2and Lhx9 can fully substitute for each other, and that Lmx1bis partially redundant, in controlling the production of output signals in mesenchymal cells in response to Fgf8 and Shh signaling. Our results indicate that several distinct LIM-HD transcription factors in conjunction with their Ldb1 co-factor serve as common central integrators of distinct signaling interactions and feedback loops to coordinate limb patterning and outgrowth along the PD, AP and DV axes after limb bud formation.

Tumor necrosis factor-α-inducible protein 8 (TNFAIP8) is the first discovered oncogenic and an anti-apoptotic member of a conserved TNFAIP8 or TIPE family of proteins. TNFAIP8 mRNA is induced by NF-kB, and overexpression of TNFAIP8 has been correlated with poor prognosis in many cancers. Downregulation of TNFAIP8 expression has been associated with decreased pulmonary colonization of human tumor cells, and enhanced sensitivities of tumor xenografts to radiation and docetaxel. Here we have investigated the effects of depletion of TNFAIP8 on the mRNA, microRNA and protein expression profiles in prostate and breast cancers and melanoma. Depending on the tumor cell type, knockdown of TNFAIP8 was found to be associated with increased mRNA expression of several antiproliferative and apoptotic genes (e.g., IL-24, FAT3, LPHN2, EPHA3) and fatty acid oxidation gene ACADL, and decreased mRNA levels of oncogenes (e.g., NFAT5, MALAT1, MET, FOXA1, KRAS, S100P, OSTF1) and glutamate transporter gene SLC1A1. TNFAIP8 knockdown cells also exhibited decreased expression of multiple onco-proteins (e.g., PIK3CA, SRC, EGFR, IL5, ABL1, GAP43), and increased expression of the orphan nuclear receptor NR4A1 and alpha 1 adaptin subunit of the adaptor-related protein complex 2 AP2 critical to clathrin-mediated endocytosis. TNFAIP8-centric molecules were found to be predominately implicated in the hypoxia-inducible factor-1α (HIF-1α) signaling pathway, and cancer and development signaling networks. Thus TNFAIP8 seems to regulate the cell survival and cancer progression processes in a multifaceted manner. Future validation of the molecules identified in this study is likely to lead to new subset of molecules and functional determinants of cancer cell survival and progression.

Human ACTG1 mutations are associated with high-frequency hearing loss, and patients with mutations in this gene are good candidates for electric acoustic stimulation. To better understand the genetic etiology of hearing loss cases, massively parallel DNA sequencing was performed on 7,048 unrelated Japanese hearing loss probands. Among 1,336 autosomal dominant hearing loss patients, we identified 15 probands (1.1%) with 13 potentially pathogenic ACTG1 variants. Six variants were novel and seven were previously reported. We collected and analyzed the detailed clinical features of these patients. The average progression rate of hearing deterioration in pure-tone average for four frequencies was 1.7 dB/year from 0 to 50 years age, and all individuals over 60 years of age had severe hearing loss. To better understand the underlying disease-causing mechanism, intracellular localization of wild-type and mutant gamma-actins were examined using the NIH/3T3 fibroblast cell line. ACTG1 mutants p.I34M p.M82I, p.K118M and p.I165V formed small aggregates while p.R37H, p.G48R, p.E241K and p.H275Y mutant gamma-actins were distributed in a similar manner to the WT. From these results, we believe that some part of the pathogenesis of ACTG1 mutations may be driven by the inability of defective gamma-actin to be polymerized into F-actin. Autosomal dominant non-syndromic hearing loss (ADNSHL) occurs in about 20% of non-syndromic hereditary hearing loss (HL) cases 1 , and 38 genes have been reported to be associated with ADNSHL (Van Camp G, Smith RJH. Hereditary Hearing Loss Homepage: http://hereditaryhearingloss.org). The emergence of massively parallel DNA sequencing has allowed the rapid and cost-effective detection of disease-causing variants, and it is already available for particularly effective medical care based on the accurate diagnosis of Mendelian disorders 2. Among the 38 genes associated with ADNSHL, ACTG1(OMIM: *102560) has received special attention for several reasons. First, ACTG1-associated HL (DFNA20/26, OMIM: #604717) patients show high-frequency progressive HL 3 and are good candidates for electric acoustic stimulation (EAS) 4. Second, the ACTG1-encoding protein, γ (gamma)-actin, is a component of the well-studied stereocilia 5-9 , and the functional consequences of some ACTG1 mutations can be analyzed by molecular biology 5. Hair cell stereocilia are crucial for converting the mechanical forces of sound waves into electrical signals (i.e., mechanotransduction) 10. These specialized