RESUMO
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/beta-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/beta-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/beta-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/beta-catenin signals in zebrafish likewise reduces pronephric epithelial tubulogenesis. We also find that exogenous activation of Wnt/beta-catenin signaling within the Xenopus pronephric field results in significant tubulogenic losses. Together, we propose Wnt/beta-catenin signaling is required for pronephric tubule, duct and glomus formation in Xenopus laevis, and this requirement is conserved in zebrafish pronephric tubule formation.
Assuntos
Rim/embriologia , Rim/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , Xenopus/embriologia , beta Catenina/metabolismo , Animais , Biomarcadores/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Túbulos Renais Coletores/embriologia , Túbulos Renais Coletores/metabolismo , Larva/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mesoderma/metabolismo , Mutação/genética , Organogênese , Fenótipo , Proteínas Recombinantes de Fusão/metabolismo , Somitos/embriologia , Xenopus/genética , Proteínas de Xenopus/metabolismo , Peixe-Zebra/embriologiaRESUMO
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 beta-catenin-induced Topflash reporter activity and beta-catenin-induced secondary axis formation in Xenopus embryos. Importantly, in calvaria of E18.5 Osx-null embryos harboring the TOPGAL reporter transgene, beta-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 beta-catenin transcriptional activity. We also showed that Osx decreased osteoblast proliferation. Indeed, E18.5 Osx-null calvaria showed greater BrdU incorporation than wild-type 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 Osx-mediated inhibition of osteoblast proliferation is a consequence of the Osx-mediated control of Wnt/beta-catenin activity. Our results add a layer of control to Wnt/beta-catenin signaling in bone.
Assuntos
Osteoblastos/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Regulação para Baixo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Modelos Biológicos , Especificidade de Órgãos , Osteoblastos/citologia , Peptídeos/metabolismo , Domínios Proteicos Ricos em Prolina , Regiões Promotoras Genéticas/genética , Ligação Proteica , Fator de Transcrição Sp7 , Fatores de Transcrição/química , Transcrição Gênica , Transfecção , Proteínas Wnt/genética , Xenopus , beta Catenina/genéticaRESUMO
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.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Transcrição Gênica/fisiologia , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/fisiologia , Animais , Western Blotting/métodos , Padronização Corporal/genética , Padronização Corporal/fisiologia , Imunoprecipitação da Cromatina/métodos , Clonagem Molecular/métodos , Relação Dose-Resposta a Droga , Interações Medicamentosas , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Imunofluorescência/métodos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Imunoprecipitação/métodos , Hibridização In Situ/métodos , Microinjeções/métodos , Mutação/fisiologia , Oligodesoxirribonucleotídeos Antissenso/farmacologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transcrição Gênica/efeitos dos fármacos , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis , beta CateninaRESUMO
The Wnt signaling pathway is central to the development of all animals and to cancer progression, yet largely unknown are the pairings of secreted Wnt ligands to their respective Frizzled transmembrane receptors or, in many cases, the relative contributions of canonical (beta-catenin/LEF/TCF) versus noncanonical Wnt signals. Specifically, in the kidney where Wnt-4 is essential for the mesenchymal to epithelial transition that generates the tissue's collecting tubules, the corresponding Frizzled receptor(s) and downstream signaling mechanism(s) are unclear. In this report, we addressed these issues using Madin-Darby Canine Kidney (MDCK) cells, which are competent to form tubules in vitro. Employing established reporter constructs of canonical Wnt/beta-catenin pathway activity, we have determined that MDCK cells are highly responsive to Wnt-4, -1, and -3A, but not to Wnt-5A and control conditions, precisely reflecting functional findings from Wnt-4 null kidney mesenchyme ex vivo rescue studies. We have confirmed that Wnt-4's canonical signaling activity in MDCK cells is mediated by downstream effectors of the Wnt/beta-catenin pathway using beta-Engrailed and dnTCF-4 constructs that suppress this pathway. We have further found that MDCK cells express the Frizzled-6 receptor and that Wnt-4 forms a biochemical complex with the Frizzled-6 CRD. Since Frizzled-6 did not appear to transduce Wnt-4's canonical signal, data supported recently by Golan et al., there presumably exists another as yet unknown Frizzled receptor(s) mediating Wnt-4 activation of beta-catenin/LEF/TCF. Finally, we report that canonical Wnt/beta-catenin signals cells help maintain cell growth and survival in MDCK cells but do not contribute to standard HGF-induced (nonphysiologic) tubule formation. Our results in combination with work from Xenopus laevis (not shown) lead us to believe that Wnt-4 binds both canonical and noncanonical Frizzled receptors, thereby activating Wnt signaling pathways that may each contribute to kidney tubulogenesis.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/metabolismo , Rim/embriologia , Organogênese/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transativadores/metabolismo , Sequência de Aminoácidos/genética , Animais , Sequência de Bases/genética , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Cães , Indução Embrionária/fisiologia , Células Epiteliais/citologia , Receptores Frizzled , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Rim/citologia , Rim/metabolismo , Túbulos Renais/citologia , Túbulos Renais/embriologia , Túbulos Renais/metabolismo , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Ligação Proteica/fisiologia , Proteínas Proto-Oncogênicas/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/isolamento & purificação , Receptores Acoplados a Proteínas G/genética , Alinhamento de Sequência , Transdução de Sinais/fisiologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteínas Wnt , Proteína Wnt4 , Proteínas de Xenopus , beta CateninaRESUMO
Chondrogenesis is a multistep process that is essential for endochondral bone formation. Previous results have indicated a role for beta-catenin and Wnt signaling in this pathway. Here we show the existence of physical and functional interactions between beta-catenin and Sox9, a transcription factor that is required in successive steps of chondrogenesis. In vivo, either overexpression of Sox9 or inactivation of beta-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 beta-catenin in chondrocytes also produces a similar phenotype of severe chondrodysplasia. Sox9 markedly inhibits activation of beta-catenin-dependent promoters and stimulates degradation of beta-catenin by the ubiquitination/proteasome pathway. Likewise, Sox9 inhibits beta-catenin-mediated secondary axis induction in Xenopus embryos. Beta-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 beta-catenin, followed by degradation of beta-catenin. Our results strongly suggest that chondrogenesis is controlled by interactions between Sox9 and the Wnt/beta-catenin signaling pathway.
