RESUMO
30 years after the identification of WNTs, their signal transduction has become increasingly complex, with the discovery of more than 15 receptors and co-receptors in seven protein families. The recent discovery of three receptor classes for the R-spondin family of WNT agonists further adds to this complexity. What emerges is an intricate network of receptors that form higher-order ligand-receptor complexes routing downstream signalling. These are regulated both extracellularly by agonists such as R-spondin and intracellularly by post-translational modifications such as phosphorylation, proteolytic processing and endocytosis.
Assuntos
Receptores Wnt/fisiologia , Via de Sinalização Wnt , Animais , Drosophila melanogaster/metabolismo , Endocitose , Feminino , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Masculino , Camundongos , Processamento de Proteína Pós-Traducional , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Trombospondinas/metabolismo , Proteínas Wnt/fisiologia , beta Catenina/metabolismoRESUMO
Male germ cells are induced to form from the epiblast of the mouse embryo by a combination of WNT and bone morphogenetic protein signals. Ohinata et al. (2009) now clarify the steps of mouse germ cell formation and use this genetic insight to direct the specification and differentiation of germline progenitor cells in vitro.
Assuntos
Diferenciação Celular/fisiologia , Células Germinativas/citologia , Mesoderma/citologia , Células-Tronco/citologia , Animais , Proteínas Morfogenéticas Ósseas/fisiologia , Linhagem da Célula/fisiologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/fisiologia , Células Germinativas/fisiologia , Masculino , Mesoderma/fisiologia , Camundongos , Transdução de Sinais/fisiologia , Células-Tronco/fisiologia , Proteínas Wnt/fisiologiaRESUMO
Specification of the germ cell lineage is vital to development and heredity. In mice, the germ cell fate is induced in pluripotent epiblast cells by signaling molecules, yet the underlying mechanism remains unknown. Here we demonstrate that germ cell fate in the epiblast is a direct consequence of Bmp4 signaling from the extraembryonic ectoderm (ExE), which is antagonized by the anterior visceral endoderm (AVE). Strikingly, Bmp8b from the ExE restricts AVE development, thereby contributing to Bmp4 signaling. Furthermore, Wnt3 in the epiblast ensures its responsiveness to Bmp4. Serum-free, defined cultures revealed that, in response to Bmp4, competent epiblast cells uniformly expressed key transcriptional regulators Blimp1 and Prdm14 and acquired germ-cell properties, including genome-wide epigenetic reprogramming, in an orderly fashion. Notably, the induced cells contributed to both spermatogenesis and fertility of offspring. By identifying a signaling principle in germ cell specification, our study establishes a robust strategy for reconstituting the mammalian germ cell lineage in vitro.
Assuntos
Proteína Morfogenética Óssea 4/fisiologia , Linhagem da Célula/fisiologia , Células Germinativas , Transdução de Sinais/fisiologia , Animais , Proteínas Morfogenéticas Ósseas/fisiologia , Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/fisiologia , Células Germinativas/citologia , Células Germinativas/fisiologia , Masculino , Mesoderma/citologia , Mesoderma/fisiologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteínas de Ligação a RNA , Células-Tronco/citologia , Células-Tronco/fisiologia , Testículo/citologia , Testículo/fisiologia , Fatores de Transcrição/fisiologia , Proteínas Wnt/fisiologiaRESUMO
Wnt/ß-catenin signalling has been implicated in the terminal asymmetric divisions of neuronal progenitors in vertebrates and invertebrates. However, the role of Wnt ligands in this process remains poorly characterized. Here, we used the terminal divisions of the embryonic neuronal progenitors in C. elegans to characterize the role of Wnt ligands during this process, focusing on a lineage that produces the cholinergic interneuron AIY. We observed that, during interphase, the neuronal progenitor is elongated along the anteroposterior axis, then divides along its major axis, generating an anterior and a posterior daughter with different fates. Using time-controlled perturbations, we show that three Wnt ligands, which are transcribed at higher levels at the posterior of the embryo, regulate the orientation of the neuronal progenitor and its asymmetric division. We also identify a role for a Wnt receptor (MOM-5) and a cortical transducer APC (APR-1), which are, respectively, enriched at the posterior and anterior poles of the neuronal progenitor. Our study establishes a role for Wnt ligands in the regulation of the shape and terminal asymmetric divisions of neuronal progenitors, and identifies downstream components.
Assuntos
Divisão Celular Assimétrica/genética , Caenorhabditis elegans/embriologia , Células-Tronco Neurais/citologia , Proteínas Wnt/fisiologia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Divisão Celular/genética , Polaridade Celular , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Ligantes , Células-Tronco Neurais/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismoRESUMO
A Wnt signaling network governs early anterior-posterior (AP) specification and patterning of the deuterostome sea urchin embryo. We have previously shown that non-canonical Fzl1/2/7 signaling antagonizes the progressive posterior-to-anterior downregulation of the anterior neuroectoderm (ANE) gene regulatory network (GRN) by canonical Wnt/ß-catenin and non-canonical Wnt1/Wnt8-Fzl5/8-JNK signaling. This study focuses on the non-canonical function of the Wnt16 ligand during early AP specification and patterning. Maternally supplied wnt16 is expressed ubiquitously during cleavage and zygotic wnt16 expression is concentrated in the endoderm/mesoderm beginning at mid-blastula stage. Wnt16 antagonizes the ANE restriction mechanism and this activity depends on a functional Fzl1/2/7 receptor. Our results also show that zygotic wnt16 expression depends on both Fzl5/8 and Wnt/ß-catenin signaling. Furthermore, Wnt16 is necessary for the activation and/or maintenance of key regulatory endoderm/mesoderm genes and is essential for gastrulation. Together, our data show that Wnt16 has two functions during early AP specification and patterning: (1) an initial role activating the Fzl1/2/7 pathway that antagonizes the ANE restriction mechanism; and (2) a subsequent function in activating key endoderm GRN factors and the morphogenetic movements of gastrulation.
Assuntos
Padronização Corporal/genética , Morfogênese/genética , Ouriços-do-Mar , Proteínas Wnt/fisiologia , Animais , Embrião não Mamífero , Receptores Frizzled/genética , Receptores Frizzled/fisiologia , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/embriologia , Mesoderma/metabolismo , Placa Neural/embriologia , Placa Neural/metabolismo , Ouriços-do-Mar/embriologia , Ouriços-do-Mar/genética , Proteínas Wnt/genética , Via de Sinalização Wnt/fisiologiaRESUMO
The central regulator of the Wnt/ß-catenin pathway is the Axin/APC/GSK3ß destruction complex (DC), which, under unstimulated conditions, targets cytoplasmic ß-catenin for degradation. How Wnt activation inhibits the DC to permit ß-catenin-dependent signaling remains controversial, in part because the DC and its regulation have never been observed in vivo Using bimolecular fluorescence complementation (BiFC) methods, we have now analyzed the activity of the DC under near-physiological conditions in Drosophila By focusing on well-established patterns of Wnt/Wg signaling in the developing Drosophila wing, we have defined the sequence of events by which activated Wnt receptors induce a conformational change within the DC, resulting in modified Axin-GSK3ß interactions that prevent ß-catenin degradation. Surprisingly, the nucleus is surrounded by active DCs, which principally control the degradation of ß-catenin and thereby nuclear access. These DCs are inactivated and removed upon Wnt signal transduction. These results suggest a novel mechanistic model for dynamic Wnt signal transduction in vivo.
Assuntos
Proteína Axina/metabolismo , Complexo de Sinalização da Axina/fisiologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/fisiologia , Animais , Animais Geneticamente Modificados , Proteína Axina/química , Complexo de Sinalização da Axina/química , Complexo de Sinalização da Axina/metabolismo , Padronização Corporal/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Embrião não Mamífero , Teste de Complementação Genética , Glicogênio Sintase Quinase 3 beta/química , Imagem Óptica , Fosforilação/genética , Ligação Proteica/genética , Conformação Proteica , Dobramento de Proteína , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Proteínas Wnt/metabolismo , Proteínas Wnt/fisiologia , Via de Sinalização Wnt/genética , beta Catenina/metabolismoRESUMO
The link between single-cell variation and population-level fate choices lacks a mechanistic explanation despite extensive observations of gene expression and epigenetic variation among individual cells. Here, we found that single human embryonic stem cells (hESCs) have different and biased differentiation potentials toward either neuroectoderm or mesendoderm depending on their G1 lengths before the onset of differentiation. Single-cell variation in G1 length operates in a dynamic equilibrium that establishes a G1 length probability distribution for a population of hESCs and predicts differentiation outcome toward neuroectoderm or mesendoderm lineages. Although sister stem cells generally share G1 lengths, a variable proportion of cells have asymmetric G1 lengths, which maintains the population dispersion. Environmental Wingless-INT (WNT) levels can control the G1 length distribution, apparently as a means of priming the fate of hESC populations once they undergo differentiation. As a downstream mechanism, global 5-hydroxymethylcytosine levels are regulated by G1 length and thereby link G1 length to differentiation outcomes of hESCs. Overall, our findings suggest that intrapopulation heterogeneity in G1 length underlies the pluripotent differentiation potential of stem cell populations.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/fisiologia , Fase G1 , Proteínas Wnt/fisiologia , Linhagem Celular , HumanosRESUMO
Stem cells reside in a niche, a local environment whose cellular and molecular complexity is still being elucidated. In Drosophila ovaries, germline stem cells depend on cap cells for self-renewing signals and physical attachment. Germline stem cells also contact the anterior escort cells, and here we report that anterior escort cells are absolutely required for germline stem cell maintenance. When escort cells die from impaired Wnt signaling or hid expression, the loss of anterior escort cells causes loss of germline stem cells. Anterior escort cells function as an integral niche component by promoting DE-cadherin anchorage and by transiently expressing the Dpp ligand to promote full-strength BMP signaling in germline stem cells. Anterior escort cells are maintained by Wnt6 ligands produced by cap cells; without Wnt6 signaling, anterior escort cells die leaving vacancies in the niche, leading to loss of germline stem cells. Our data identify anterior escort cells as constituents of the germline stem cell niche, maintained by a cap cell-produced Wnt6 survival signal.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/citologia , Drosophila melanogaster/fisiologia , Nicho de Células-Tronco/fisiologia , Proteínas Wnt/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas Morfogenéticas Ósseas/metabolismo , Caderinas/metabolismo , Contagem de Células , Diferenciação Celular , Linhagem da Célula , Sobrevivência Celular , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Feminino , Células Germinativas/citologia , Células Germinativas/fisiologia , Ligantes , Modelos Biológicos , Ovário/citologia , Ovário/metabolismo , Transdução de Sinais , Nicho de Células-Tronco/genética , Proteínas Wnt/genéticaRESUMO
During embryogenesis, morphogens form a concentration gradient in responsive tissue, which is then translated into a spatial cellular pattern. The mechanisms by which morphogens spread through a tissue to establish such a morphogenetic field remain elusive. Here, we investigate by mutually complementary simulations and in vivo experiments how Wnt morphogen transport by cytonemes differs from typically assumed diffusion-based transport for patterning of highly dynamic tissue such as the neural plate in zebrafish. Stochasticity strongly influences fate acquisition at the single cell level and results in fluctuating boundaries between pattern regions. Stable patterning can be achieved by sorting through concentration dependent cell migration and apoptosis, independent of the morphogen transport mechanism. We show that Wnt transport by cytonemes achieves distinct Wnt thresholds for the brain primordia earlier compared with diffusion-based transport. We conclude that a cytoneme-mediated morphogen transport together with directed cell sorting is a potentially favored mechanism to establish morphogen gradients in rapidly expanding developmental systems.
Assuntos
Padronização Corporal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Vertebrados/embriologia , Proteínas Wnt/fisiologia , Animais , Apoptose , Encéfalo/embriologia , Linhagem da Célula , Movimento Celular , Biologia Computacional , Simulação por Computador , Desenvolvimento Embrionário , Crista Neural/embriologia , Placa Neural/embriologia , Transporte Proteico , Transdução de Sinais , Software , Processos Estocásticos , Peixe-Zebra/embriologia , beta Catenina/fisiologiaRESUMO
Germline stem cell (GSC) self-renewal and differentiation into gametes is regulated by both intrinsic factors in the germ line as well as extrinsic factors from the surrounding somatic niche. dWnt4, in the escort cells of the adult somatic niche promotes GSC differentiation using the canonical ß-catenin-dependent transcriptional pathway to regulate escort cell survival, adhesion to the germ line and downregulation of self-renewal signaling. Here, we show that in addition to the ß-catenin-dependent canonical pathway, dWnt4 also uses downstream components of the Wnt non-canonical pathway to promote escort cell function earlier in development. We find that the downstream non-canonical components, RhoA, Rac1 and cdc42, are expressed at high levels and are active in escort cell precursors of the female larval gonad compared to the adult somatic niche. Consistent with this expression pattern, we find that the non-canonical pathway components function in the larval stages but not in adults to regulate GSC differentiation. In the larval gonad, dWnt4, RhoA, Rac1 and cdc42 are required to promote intermingling of escort cell precursors, a function that then promotes proper escort cell function in the adults. We find that dWnt4 acts by modulating the activity of RhoA, Rac1 and cdc42, but not their protein levels. Together, our results indicate that at different points of development, dWnt4 switches from using the non-canonical pathway components to using a ß-catenin-dependent canonical pathway in the escort cells to facilitate the proper differentiation of GSCs.
Assuntos
Diferenciação Celular/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster , Células Germinativas/fisiologia , Glicoproteínas/fisiologia , Nicho de Células-Tronco , Células-Tronco/fisiologia , Proteínas Wnt/fisiologia , Via de Sinalização Wnt/genética , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero , Feminino , Genes de Troca/fisiologia , Glicoproteínas/genética , Gônadas/citologia , Gônadas/fisiologia , Masculino , Nicho de Células-Tronco/genética , Proteínas Wnt/genéticaRESUMO
ß-Catenin signaling controls the development and maintenance of the blood-brain barrier (BBB) and the blood-retina barrier (BRB), but the division of labor and degree of redundancy between the two principal ligand-receptor systems-the Norrin and Wnt7a/Wnt7b systems-are incompletely defined. Here, we present a loss-of-function genetic analysis of postnatal BBB and BRB maintenance in mice that shows striking threshold and partial redundancy effects. In particular, the combined loss of Wnt7a and Norrin or Wnt7a and Frizzled4 (Fz4) leads to anatomically localized BBB defects that are far more severe than observed with loss of Wnt7a, Norrin, or Fz4 alone. In the cerebellum, selective loss of Wnt7a in glia combined with ubiquitous loss of Norrin recapitulates the phenotype observed with ubiquitous loss of both Wnt7a and Norrin, implying that glia are the source of Wnt7a in the cerebellum. Tspan12, a coactivator of Norrin signaling in the retina, is also active in BBB maintenance but is less potent than Norrin, consistent with a model in which Tspan12 enhances the amplitude of the Norrin signal in vascular endothelial cells. Finally, in the context of a partially impaired Norrin system, the retina reveals a small contribution to BRB development from the Wnt7a/Wnt7b system. Taken together, these experiments define the extent of CNS region-specific cooperation for several components of the Norrin and Wnt7a/Wnt7b systems, and they reveal substantial regional heterogeneity in the extent to which partially redundant ligands, receptors, and coactivators maintain the BBB and BRB.
Assuntos
Barreira Hematoencefálica/crescimento & desenvolvimento , Barreira Hematoencefálica/fisiologia , Barreira Hematorretiniana/crescimento & desenvolvimento , Barreira Hematorretiniana/fisiologia , Proteínas do Olho/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Wnt/fisiologia , Animais , Barreira Hematoencefálica/citologia , Barreira Hematorretiniana/citologia , Técnicas de Cultura de Células , Proteínas do Olho/genética , Receptores Frizzled/deficiência , Receptores Frizzled/genética , Receptores Frizzled/fisiologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Modelos Neurológicos , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Tetraspaninas/deficiência , Tetraspaninas/genética , Tetraspaninas/fisiologia , Proteínas Wnt/deficiência , Proteínas Wnt/genética , beta Catenina/fisiologiaRESUMO
Wnt/beta-catenin signaling may play an essential role in endometrial decidualization, placentation, and the establishment of pregnancy. We investigate here the possible roles, immunolocalizations, and synthesis of the Wnt3, Wnt7a, and beta-catenin proteins in the rat endometrium during the estrous cycle and early postimplantation period. Wnt3 and Wnt7a had a similar localization and dynamic expression relative to the endometrial stages. Wnt7a immunostaining was not limited only to the luminal epithelial cells, but also to strong stainings in the stromal and endothelial cells. Wnt3, Wnt7a, and beta-catenin were highly synthesized and colocalized at the trophoblast-decidual interface; and were more obvious in the primary decidual zone, the GTCs, and the ectoplacental cone. Beta-catenin was strongly localized at the borders of the mature decidual cells; however, Wnt3 and Wnt7a immunolocalizations were decreased in those cells. As such, the immunolocalization of Wnt3, Wnt7a, and beta-catenin shifted with decidualization and placentation. The expression level of Wnt3, Wnt7a, and beta-catenin messenger RNAs increased in early pregnancy, and especially between Days 8.5 and 9.5. The dramatic changes in the expression of Wnt3, Wnt7a, and beta-catenin observed during the early days of pregnancy and the estrous cycle may indicate their roles in decidualization, stromal cell proliferation, and trophoblast invasion.
Assuntos
Endométrio/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Wnt/fisiologia , Via de Sinalização Wnt , Proteína Wnt3/fisiologia , beta Catenina/fisiologia , Animais , Decídua/citologia , Embrião de Mamíferos/metabolismo , Ciclo Estral/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Gravidez , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Células Estromais/metabolismo , Trofoblastos/citologia , Proteínas Wnt/biossíntese , Proteínas Wnt/genética , Proteína Wnt3/biossíntese , Proteína Wnt3/genética , beta Catenina/biossíntese , beta Catenina/genéticaRESUMO
Wingless/integrated (Wnt) signaling has emerged as a major mechanism for promoting bone formation and a target pathway for developing bone anabolic agents against osteoporosis. However, the downstream events mediating the potential therapeutic effect of Wnt proteins are not fully understood. Previous studies have indicated that increased glycolysis is associated with osteoblast differentiation in response to Wnt signaling, but direct genetic evidence for the importance of glucose metabolism in Wnt-induced bone formation is lacking. Here, we have generated compound transgenic mice to overexpress Wnt family member 7B (Wnt7b) transiently in the osteoblast lineage of postnatal mice, with or without concurrent deletion of the glucose transporter 1 (Glut1), also known as solute carrier family 2, facilitated glucose transporter member 1. Overexpression of Wnt7b in 1-mo-old mice for 1 wk markedly stimulated bone formation, but the effect was essentially abolished without Glut1, even though transient deletion of Glut1 itself did not affect normal bone accrual. Consistent with the in vivo results, Wnt7b increased Glut1 expression and glucose consumption in the primary culture of osteoblast lineage cells, and deletion of Glut1 diminished osteoblast differentiation in vitro. Thus, Wnt7b promotes bone formation in part through stimulating glucose metabolism in osteoblast lineage cells.-Chen, H., Ji, X., Lee, W.-C., Shi, Y., Li, B., Abel, E. D., Jiang, D., Huang, W., Long, F. Increased glycolysis mediates Wnt7b-induced bone formation.
Assuntos
Transportador de Glucose Tipo 1/fisiologia , Glucose/metabolismo , Glicólise , Osteoblastos/metabolismo , Osteogênese/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Wnt/fisiologia , Animais , Linhagem da Célula , Células Cultivadas , Fêmur/crescimento & desenvolvimento , Fêmur/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Genes Reporter , Transportador de Glucose Tipo 1/deficiência , Transportador de Glucose Tipo 1/genética , Camundongos , Camundongos Transgênicos , Osteogênese/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Proteínas Recombinantes/metabolismo , Tamoxifeno/farmacologia , Tíbia/crescimento & desenvolvimento , Tíbia/ultraestrutura , Proteínas Wnt/genéticaRESUMO
Gastrulation was arguably the key evolutionary innovation that enabled metazoan diversification, leading to the formation of distinct germ layers and specialized tissues. Differential gene expression specifying cell fate is governed by the inputs of intracellular and/or extracellular signals. Beta-catenin/Tcf and the TGF-beta bone morphogenetic protein (BMP) provide critical molecular signaling inputs during germ layer specification in bilaterian metazoans, but there has been no direct experimental evidence for a specific role for BMP signaling during endomesoderm specification in the early branching metazoan Nematostella vectensis (an anthozoan cnidarian). Using forward transcriptomics, we show that beta-catenin/Tcf signaling and BMP2/4 signaling provide differential inputs into the cnidarian endomesodermal gene regulatory network (GRN) at the onset of gastrulation (24 h postfertilization) in N. vectensis Surprisingly, beta-catenin/Tcf signaling and BMP2/4 signaling regulate a subset of common downstream target genes in the GRN in opposite ways, leading to the spatial and temporal differentiation of fields of cells in the developing embryo. Thus, we show that regulatory interactions between beta-catenin/Tcf signaling and BMP2/4 signaling are required for the specification and determination of different embryonic regions and the patterning of the oral-aboral axis in Nematostella We also show functionally that the conserved "kernel" of the bilaterian heart mesoderm GRN is operational in N. vectensis, which reinforces the hypothesis that the endoderm and mesoderm in triploblastic bilaterians evolved from the bifunctional endomesoderm (gastrodermis) of a diploblastic ancestor, and that slow rhythmic contractions might have been one of the earliest functions of mesodermal tissue.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Cnidários/embriologia , Mesoderma/embriologia , Transdução de Sinais , Proteínas Wnt/fisiologia , Actinas/metabolismo , Animais , Sistemas CRISPR-Cas , Linhagem da Célula , Endoderma/embriologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Masculino , Oligonucleotídeos Antissenso/genéticaRESUMO
Intestinal stem cells (ISCs) require well-defined signals from their environment in order to carry out their specific functions. Most of these signals are provided by neighboring cells that form a stem cell niche, whose shape and cellular composition self-organize. Major features of this self-organization can be studied in ISC-derived organoid culture. In this system, manipulation of essential pathways of stem cell maintenance and differentiation results in well-described growth phenotypes. We here provide an individual cell-based model of intestinal organoids that enables a mechanistic explanation of the observed growth phenotypes. In simulation studies of the 3D structure of expanding organoids, we investigate interdependences between Wnt- and Notch-signaling which control the shape of the stem cell niche and, thus, the growth pattern of the organoids. Similar to in vitro experiments, changes of pathway activities alter the cellular composition of the organoids and, thereby, affect their shape. Exogenous Wnt enforces transitions from branched into a cyst-like growth pattern; known to occur spontaneously during long term organoid expansion. Based on our simulation results, we predict that the cyst-like pattern is associated with biomechanical changes of the cells which assign them a growth advantage. The results suggest ongoing stem cell adaptation to in vitro conditions during long term expansion by stabilizing Wnt-activity. Our study exemplifies the potential of individual cell-based modeling in unraveling links between molecular stem cell regulation and 3D growth of tissues. This kind of modeling combines experimental results in the fields of stem cell biology and cell biomechanics constituting a prerequisite for a better understanding of tissue regeneration as well as developmental processes.
Assuntos
Simulação por Computador , Intestinos/citologia , Modelos Biológicos , Organoides/crescimento & desenvolvimento , Células-Tronco/fisiologia , Animais , Apoptose , Fenômenos Biomecânicos , Adesão Celular , Forma Celular , Camundongos , Camundongos Endogâmicos C57BL , Polímeros , Receptores Notch/fisiologia , Regeneração/fisiologia , Nicho de Células-Tronco , Técnicas de Cultura de Tecidos , Proteínas Wnt/fisiologia , Via de Sinalização WntRESUMO
Once urinary bladder cancer (UBC) develops into muscle-invasive bladder cancer, its mortality rate increases dramatically. However, the molecular mechanisms of UBC invasion and metastasis remain largely unknown. Herein, using 5637 UBC cells, we generated two sublines with low (5637 NMI) and high (5637 HMI) invasive capabilities. Mass spectrum analyses revealed that the Wnt family protein Wnt7a is more highly expressed in 5637 HMI cells than in 5637 NMI cells. We also found that increased Wnt7a expression is associated with UBC metastasis and predicted worse clinical outcome in UBC patients. Wnt7a depletion in 5637 HMI and T24 cells reduced UBC cell invasion and decreased levels of active ß-catenin and its downstream target genes involved in the epithelial-to-mesenchymal transition (EMT) and extracellular matrix (ECM) degradation. Consistently, treating 5637 NMI and J82 cells with recombinant Wnt7a induced cell invasion, EMT, and expression of ECM degradation-associated genes. Moreover, TOP/FOPflash luciferase assays indicated that Wnt7a activated canonical ß-catenin signaling in UBC cells, and increased Wnt7a expression was associated with nuclear ß-catenin in UBC samples. Wnt7a ablation suppressed matrix metalloproteinase 10 (MMP10) expression, and Wnt7a overexpression increased MMP10 promoter activity through two TCF/LEF promoter sites, confirming that Wnt7a-mediated MMP10 activation is mediated by the canonical Wnt/ß-catenin pathway. Of note, the microRNA miR-370-3p directly repressed Wnt7a expression and thereby suppressed UBC cell invasion, which was partially restored by Wnt7a overexpression. Our results have identified an miR-370-3p/Wnt7a axis that controls UBC invasion through canonical Wnt/ß-catenin signaling, which may offer prognostic and therapeutic opportunities.
Assuntos
MicroRNAs/fisiologia , Neoplasias da Bexiga Urinária/patologia , Proteínas Wnt/fisiologia , Via de Sinalização Wnt , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , Espectrometria de Massas , Metaloproteinase 10 da Matriz/biossíntese , Invasividade Neoplásica , Metástase Neoplásica , Oncogenes , Prognóstico , Neoplasias da Bexiga Urinária/metabolismo , Proteínas Wnt/genética , beta Catenina/metabolismoRESUMO
BACKGROUND: WNT signaling is implicated in embryonic development, and in adult tissue homeostasis, while its deregulation is evident in disease. This study investigates the unique roles of canonical WNT10B in both normal prostate development and prostate cancer (PCa) progression. METHODS: Organ culture and rat ventral prostates (VPs) were used to study Wnt10b ontogeny and growth effect of WNT10B protein. PB-SV40 LTag rat VPs were utilized for Wnt expression polymerase chain reaction (PCR) array and immunohistochemistry. Human localized PCa tissue microarrays (TMAs) were investigated for differential WNT10B expression. Human RNA-seq data sets were queried for differential expression of WNT10B in metastatic and localized PCa. Knockdown of WNT10B in PC3 cells was utilized to study its effects on proliferation, stemness, epithelial to mesenchymal transition (EMT), and xenograft propagation. RESULTS: Wnt10b expression was highest at birth and rapidly declined in the postnatal rat VP. Exogenous WNT10B addition to culture developing VPs decreased growth suggesting an antiproliferative role. VPs from PB-SV40 LTag rats with localized PCa showed a 25-fold reduction in Wnt10b messenger RNA (mRNA) expession, confirmed at the protein level. Human PCa TMAs revealed elevated WNT10B protein in prostate intraepithelial neoplasia compared with normal prostates but reduced levels in localized PCa specimens. In contrast, RNA-seq data set of annotated human PCa metastasis found a significant increase in WNT10B mRNA expression compared with localized tumors suggesting stage-specific functions of WNT10B. Similarly, WNT10B mRNA levels were increased in metastatic cell lines PC3, PC3M, as well as in HuSLC, a PCa stem-like cell line, as compared with disease-free primary prostate epithelial cells. WNT10B knockdown in PC3 cells reduced expression of EMT genes, MMP9 and stemness genes NANOG and SOX2 and markedly reduced the stem cell-like side population. Furthermore, loss of WNT10B abrogated the ability of PC3 cells to propagate tumors via serial transplantation. CONCLUSIONS: Taken together, these results suggest a dual role for WNT10B in normal development and in PCa progression with opposing functions depending on disease stage. We propose that decreased WNT10B levels in localized cancer allow for a hyperproliferative state, whereas increased levels in advanced disease confer a stemness and malignant propensity which is mitigated by knocking down WNT10B levels. This raises the potential for WNT10B as a novel target for therapeutic intervention in metastatic PCa.
Assuntos
Próstata/crescimento & desenvolvimento , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Wnt/fisiologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Transição Epitelial-Mesenquimal , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Nus , Metástase Neoplásica/patologia , Transplante de Neoplasias , Técnicas de Cultura de Órgãos , Células PC-3 , Neoplasia Prostática Intraepitelial/patologia , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Proteínas Wnt/análise , Proteínas Wnt/genéticaRESUMO
A hydra has a simple structure consisting of a head, body column, and foot along a single axis called the oral-aboral axis. The tissue dynamics of a hydra consist of a steady state of production and loss of tissue involving the entire animal. Axis formation and its maintenance is controlled by the head organizer, which is located at the apex of the animal. The head organizer produces two signals, the head activator and head inhibitor, which are transmitted to, and are distributed in, descending gradients among the epithelial cells along the body column. The two gradients control axial patterning along the oral-aboral axis. In the context of the tissue dynamics of the adult hydra, these three elements controlling axis formation and axial patterning are in a steady state of production and loss. The canonical Wnt pathway plays a major role in setting up and maintaining the head organizer.
Assuntos
Padronização Corporal , Cabeça/fisiologia , Hydra/fisiologia , Via de Sinalização Wnt , Animais , Diferenciação Celular , Linhagem da Célula , Endoderma/fisiologia , Células Epiteliais/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Hydra/genética , Regeneração , Fatores de Transcrição TCF/fisiologia , Proteínas Wnt/genética , Proteínas Wnt/fisiologia , beta Catenina/fisiologiaRESUMO
OBJECTIVES: Wnt16 is implicated in bone fracture and bone mass accrual both in animals and humans. However, its functional roles and molecular mechanism in chondrocyte differentiation and osteoarthritis (OA) pathophysiology remain largely undefined. In this study, we analysed its mechanistic association and functional relationship in OA progression in chondrocyte lineage. METHODS: The role of Wnt16 during skeletal development was examined by Col2a1-Wnt16 transgenic mice and Wnt16fl/fl;Col2a1-Cre (Wnt16-cKO) mice. OA progression was assessed by micro-CT analysis and Osteoarthritis Research Society International score after anterior cruciate ligament transection (ACLT) surgery with Wnt16 manipulation by adenovirus intra-articular injection. The molecular mechanism was investigated in vitro using 3D chondrocyte pellet culture and biochemical analyses. Histological analysis was performed in mouse joints and human cartilage specimens. RESULTS: Wnt16 overexpression in chondrocytes in mice significantly inhibited chondrocyte hypertrophy during skeletal development. Wnt16 deficiency exaggerated OA progression, whereas intra-articular injection of Ad-Wnt16 markedly attenuated ACLT-induced OA. Cellular and molecular analyses showed that, instead of ß-catenin and calcium pathways, Wnt16 activated the planar cell polarity (PCP) and JNK pathway by interacting mainly with AP2b1, and to a lesser extend Ror2 and CD146, and subsequently induced PTHrP expression through phosphor-Raptor mTORC1 pathway. CONCLUSIONS: Our findings indicate that Wnt16 activates PCP/JNK and crosstalks with mTORC1-PTHrP pathway to inhibit chondrocyte hypertrophy. Our preclinical study suggests that Wnt16 may be a potential therapeutic target for OA treatment.
Assuntos
Artrite Experimental/patologia , Osteoartrite/patologia , Proteínas Wnt/fisiologia , Animais , Artrite Experimental/metabolismo , Artrite Experimental/fisiopatologia , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Diferenciação Celular/fisiologia , Polaridade Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Condrócitos/patologia , Condrócitos/fisiologia , Progressão da Doença , Humanos , Hipertrofia/prevenção & controle , Sistema de Sinalização das MAP Quinases/fisiologia , Alvo Mecanístico do Complexo 1 de Rapamicina/fisiologia , Camundongos Transgênicos , Osteoartrite/metabolismo , Osteoartrite/fisiopatologia , Proteína Relacionada ao Hormônio Paratireóideo/fisiologia , Proteínas Wnt/deficiência , Proteínas Wnt/metabolismoRESUMO
ß-Catenin-dependent Wnt signaling controls numerous aspects of skeletal development and postnatal bone repair. Currently available transgenic Wnt reporter mice allow for visualization of global canonical Wnt signaling activity within skeletal tissues, without delineation of cell type. This is particularly important in a bone repair context, in which the inflammatory phase can obscure the visualization of mesenchymal cell types of interest. To tackle the issue of tissue-specific Wnt signaling, we have generated and characterized a transgenic mouse strain [termed paired related homeobox 1 (Prx1)-Wnt-green fluorescent protein (GFP), by crossing a previously validated Prx1-Cre strain with a nuclear fluorescent reporter driven by T-cell factor/lymphoid enhancer factor activity (Rosa26-Tcf/Lef-LSL-H2B-GFP)]. Prx1-Wnt-GFP animals were subject to three models of long bone and membranous bone repair (displaced forelimb fracture, tibial cortical defect, and frontal bone defect). Results showed that, irrespective of bone type, locoregional mesenchymal cell activation of Wnt signaling occurs in a defined temporospatial pattern among Prx1-Wnt-GFP mice. In summary, Prx1-Wnt-GFP reporter animals allow for improved visualization, spatial discrimination, and facile quantification of Wnt-activated mesenchymal cells within models of adult bone repair.