Chondrogenesis is a multistep process that is essential for endochondral bone formation. Previous results have indicated a role for ␤-catenin and Wnt signaling in this pathway. Here we show the existence of physical and functional interactions between ␤-catenin and Sox9, a transcription factor that is required in successive steps of chondrogenesis. In vivo, either overexpression of Sox9 or inactivation of ␤-catenin in chondrocytes of mouse embryos produces a similar phenotype of dwarfism with decreased chondrocyte proliferation, delayed hypertrophic chondrocyte differentiation, and endochondral bone formation. Furthermore, either inactivation of Sox9 or stabilization of ␤-catenin in chondrocytes also produces a similar phenotype of severe chondrodysplasia. Sox9 markedly inhibits activation of ␤-catenin-dependent promoters and stimulates degradation of ␤-catenin by the ubiquitination/proteasome pathway. Likewise, Sox9 inhibits ␤-catenin-mediated secondary axis induction in Xenopus embryos. ␤-Catenin physically interacts through its Armadillo repeats with the C-terminal transactivation domain of Sox9. We hypothesize that the inhibitory activity of Sox9 is caused by its ability to compete with Tcf/Lef for binding to ␤-catenin, followed by degradation of ␤-catenin. Our results strongly suggest that chondrogenesis is controlled by interactions between Sox9 and the Wnt/␤-catenin signaling pathway. Chondrogenesis, an obligatory process in endochondral bone formation, starts with the recruitment of chondrogenic mesenchymal cells into condensations. This is followed by the differentiation of these cells into chondrocytes, which produce cartilage-specific extracellular matrix (ECM) proteins including type II collagen and the proteoglycan aggrecan. Chondrocytes then undergo a unidirectional proliferation to form orderly parallel columns, exit the cell cycle, become prehypertrophic, and then hypertrophic. Sox9, a high-mobility-group (HMGbox) transcription factor, is required at sequential steps in this pathway (Bi et al. 1999(Bi et al. , 2001Akiyama et al. 2002).Both the human disease campomelic dysplasia, which is caused by heterozygous mutations in the Sox9 gene and is due to Sox9 haploinsufficiency, as well as Sox9 heterozygous mutant mice are characterized by a general hypoplasia of endochondral bones (Foster et al. 1994;Wagner et al. 1994). Inactivation of Sox9 in limb buds using the Cre recombinase/loxP recombination system before chondrogenic mesenchymal condensations results in the complete absence of mesenchymal condensations and of subsequent cartilage and bone formation, indicating that Sox9 is needed for an early step in chondrogenesis, that of mesenchymal condensations (Akiyama et al. 2002). A similar conclusion was also reached by analysis of mouse embryo chimeras derived from homozygous Sox9 mutant embryonic stem (ES) cells (Bi et al. 1999). That Sox9 is needed at sequential steps is shown by the severe generalized chondrodysplasia of mouse embryos in which Sox9 is deleted after chondrogenic mesenchymal conde...

Beta-catenin-dependent or canonical Wnt signals are fundamental in animal development and tumor progression. Using Xenopus laevis, we report that the BTB/POZ zinc finger family member Kaiso directly represses canonical Wnt gene targets (Siamois, c-Fos, Cyclin-D1, and c-Myc) in conjunction with TCF/LEF (TCF). Analogous to beta-catenin relief of TCF repressive activity, we show that p120-catenin relieves Kaiso-mediated repression of Siamois. Furthermore, Kaiso and TCF coassociate, and combined Kaiso and TCF derepression results in pronounced Siamois expression and increased beta-catenin coprecipitation with the Siamois promoter. The functional interdependency is underlined by Kaiso suppression of beta-catenin-induced axis duplication and by TCF-3 rescue of Kaiso depletion phenotypes. These studies point to convergence of parallel p120-catenin/Kaiso and beta-catenin/TCF signaling pathways to regulate gene expression in vertebrate development and possibly carcinogenesis.

The recent identification of the genes responsible for several human genetic diseases affecting bone homeostasis and the characterization of mouse models for these diseases indicated that canonical Wnt signaling plays a critical role in the control of bone mass. Here, we report that the osteoblast-specific transcription factor Osterix (Osx), which is required for osteoblast differentiation, inhibits Wnt pathway activity. First, in calvarial cells of embryonic day (E)18.5 Osx-null embryos, expression of the Wnt antagonist Dkk1 was abolished, and that of Wnt target genes c-Myc and cyclin D1 was increased. Moreover, our studies demonstrated that Osx bound to and activated the Dkk1 promoter. In addition, Osx inhibited ␤-catenin-induced Topflash reporter activity and ␤-catenin-induced secondary axis formation in Xenopus embryos. Importantly, in calvaria of E18.5 Osx-null embryos harboring the TOPGAL reporter transgene, ␤-galactosidase activity was increased, suggesting that Osx inhibited the Wnt pathway in osteoblasts in vivo. Our data further showed that Osx disrupted binding of Tcf to DNA, providing a likely mechanism for the inhibition by Osx of ␤-catenin transcriptional activity. We also showed that Osx decreased osteoblast proliferation. Indeed, E18.5 Osx-null calvaria showed greater BrdU incorporation than wildtype calvaria and that Osx overexpression in C2C12 mesenchymal cells inhibited cell growth. Because Wnt signaling has a major role in stimulating osteoblast proliferation, we speculate that Osxmediated inhibition of osteoblast proliferation is a consequence of the Osx-mediated control of Wnt/␤-catenin activity. Our results add a layer of control to Wnt/␤-catenin signaling in bone.

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The pronephric kidney controls water and electrolyte balance during early fish and amphibian embryogenesis. Many Wnt signaling components have been implicated in kidney development. Specifically, in Xenopus pronephric development as well as the murine metanephroi, the secreted glycoprotein Wnt-4 has been shown to be essential for renal tubule formation. Despite the importance of Wnt signals in kidney organogenesis, little is known of the definitive downstream signaling pathway(s) that mediate their effects. Here we report that inhibition of Wnt/β-catenin signaling within the pronephric field of Xenopus results in significant losses to kidney epithelial tubulogenesis with little or no effect on adjoining axis or somite development. We find that the requirement for Wnt/β-catenin signaling extends throughout the pronephric primordium and is essential for the development of proximal and distal tubules of the pronephros as well as for the development of the duct and glomus. Although less pronounced than effects upon later pronephric tubule differentiation, inhibition of the Wnt/β-catenin pathway decreased expression of early pronephric mesenchymal markers indicating it is also needed in early pronephric patterning. We find that upstream inhibition of Wnt/β-catenin signals in zebrafish likewise reduces pronephric epithelial tubulogenesis. We also find that exogenous activation of Wnt/β-catenin signaling within the Xenopus pronephric field results in significant tubulogenic losses. Together, we propose Wnt/β-catenin signaling is required for pronephric tubule, duct and glomus formation in Xenopus laevis, and this requirement is conserved in zebrafish pronephric tubule formation.