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1.
PLoS One ; 15(4): e0232025, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32353019

RESUMO

The actin cytoskeleton plays a central role in establishing cell polarity and shape during embryonic morphogenesis. Daam1, a member of the Formin family of actin cytoskeleton regulators, is a Dvl2-binding protein that functions in the Wnt/Planar Cell Polarity (PCP) pathway. To examine the role of the Daam proteins in mammalian development, we generated Daam-deficient mice by gene targeting and found that Daam1, but not Daam2, is necessary for fetal survival. Embryonic development of Daam1 mutants was delayed most likely due to functional defects in the labyrinthine layer of the placenta. Examination of Daam2 and Daam1/2 double mutants revealed that Daam1 and Daam2 are functionally redundant during placental development. Of note, neural tube closure defects (NTD), which are observed in several mammalian PCP mutants, are not observed in Wnt5a or Daam1 single mutants, but arise in Daam1;Wnt5a double mutants. These findings demonstrate a unique function for Daam genes in placental development and are consistent with a role for Daam1 in the Wnt/PCP pathway in mammals.


Assuntos
Proteínas dos Microfilamentos/genética , Placentação/genética , Proteínas rho de Ligação ao GTP/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Transporte/metabolismo , Polaridade Celular , Citoesqueleto/metabolismo , Desenvolvimento Embrionário , Feminino , Forminas/genética , Forminas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Camundongos/embriologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Placenta/embriologia , Gravidez , Via de Sinalização Wnt , Proteínas rho de Ligação ao GTP/metabolismo
2.
Development ; 147(8)2020 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-32156757

RESUMO

Despite the importance of Wnt signaling for adult intestinal stem cell homeostasis and colorectal cancer, relatively little is known about its role in colon formation during embryogenesis. The development of the colon starts with the formation and extension of the hindgut. We show that Wnt3a is expressed in the caudal embryo in a dorsal-ventral (DV) gradient across all three germ layers, including the hindgut. Using genetic and lineage-tracing approaches, we describe novel dorsal and ventral hindgut domains, and show that ventrolateral hindgut cells populate the majority of the colonic epithelium. A Wnt3a-ß-catenin-Sp5/8 pathway, which is active in the dorsal hindgut endoderm, is required for hindgut extension and colon formation. Interestingly, the absence of Wnt activity in the ventral hindgut is crucial for proper hindgut morphogenesis, as ectopic stabilization of ß-catenin in the ventral hindgut via gain- or loss-of-function mutations in Ctnnb1 or Apc, respectively, leads to severe colonic hyperplasia. Thus, the DV Wnt gradient is required to coordinate growth between dorsal and ventral hindgut domains to regulate the extension of the hindgut that leads to colon formation.

3.
EMBO Rep ; 21(1): e48469, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31789450

RESUMO

Amino acids are essential for cellular metabolism, and it is important to understand how nutrient supply is coordinated with changing energy requirements during embryogenesis. Here, we show that the amino acid transporter Slc7a5/Lat1 is highly expressed in tissues undergoing morphogenesis and that Slc7a5-null mouse embryos have profound neural and limb bud outgrowth defects. Slc7a5-null neural tissue exhibited aberrant mTORC1 activity and cell proliferation; transcriptomics, protein phosphorylation and apoptosis analyses further indicated induction of the integrated stress response as a potential cause of observed defects. The pattern of stress response gene expression induced in Slc7a5-null embryos was also detected at low level in wild-type embryos and identified stress vulnerability specifically in tissues undergoing morphogenesis. The Slc7a5-null phenotype is reminiscent of Wnt pathway mutants, and we show that Wnt/ß-catenin loss inhibits Slc7a5 expression and induces this stress response. Wnt signalling therefore normally supports the metabolic demands of morphogenesis and constrains cellular stress. Moreover, operation in the embryo of the integrated stress response, which is triggered by pathogen-mediated as well as metabolic stress, may provide a mechanistic explanation for a range of developmental defects.

4.
Development ; 145(8)2018 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-29615464

RESUMO

Embryonic morphogenesis of a complex organism requires proper regulation of patterning and directional growth. Planar cell polarity (PCP) signaling is emerging as a crucial evolutionarily conserved mechanism whereby directional information is conveyed. PCP is thought to be established by global cues, and recent studies have revealed an instructive role of a Wnt signaling gradient in epithelial tissues of both invertebrates and vertebrates. However, it remains unclear whether Wnt/PCP signaling is regulated in a coordinated manner with embryonic patterning during morphogenesis. Here, in mouse developing limbs, we find that apical ectoderm ridge-derived Fgfs required for limb patterning regulate PCP along the proximal-distal axis in a Wnt5a-dependent manner. We demonstrate with genetic evidence that the Wnt5a gradient acts as a global cue that is instructive in establishing PCP in the limb mesenchyme, and that Wnt5a also plays a permissive role to allow Fgf signaling to orient PCP. Our results indicate that limb morphogenesis is regulated by coordination of directional growth and patterning through integration of Wnt5a and Fgf signaling.


Assuntos
Padronização Corporal/fisiologia , Polaridade Celular/fisiologia , Fator 4 de Crescimento de Fibroblastos/fisiologia , Fator 8 de Crescimento de Fibroblasto/fisiologia , Proteína Wnt-5a/fisiologia , Animais , Padronização Corporal/genética , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Extremidades/embriologia , Fator 4 de Crescimento de Fibroblastos/deficiência , Fator 4 de Crescimento de Fibroblastos/genética , Fator 8 de Crescimento de Fibroblasto/deficiência , Fator 8 de Crescimento de Fibroblasto/genética , Mesoderma/embriologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Transdução de Sinais , Proteína Wnt-5a/deficiência , Proteína Wnt-5a/genética
5.
PLoS One ; 12(8): e0181549, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28846685

RESUMO

Resident tissue myeloid cells play a role in many aspects of physiology including development of the vascular systems. In the blood vasculature, myeloid cells use VEGFC to promote angiogenesis and can use Wnt ligands to control vascular branching and to promote vascular regression. Here we show that myeloid cells also regulate development of the dermal lymphatic vasculature using Wnt ligands. Using myeloid-specific deletion of the WNT transporter Wntless we show that myeloid Wnt ligands are active at two distinct stages of development of the dermal lymphatics. As lymphatic progenitors are emigrating from the cardinal vein and intersomitic vessels, myeloid Wnt ligands regulate both their numbers and migration distance. Later in lymphatic development, myeloid Wnt ligands regulate proliferation of lymphatic endothelial cells (LEC) and thus control lymphatic vessel caliber. Myeloid-specific deletion of WNT co-receptor Lrp5 or Wnt5a gain-of-function also produce elevated caliber in dermal lymphatic capillaries. These data thus suggest that myeloid cells produce Wnt ligands to regulate lymphatic development and use Wnt pathway co-receptors to regulate the balance of Wnt ligand activity during the macrophage-LEC interaction.


Assuntos
Derme/metabolismo , Células Endoteliais/metabolismo , Linfangiogênese/fisiologia , Vasos Linfáticos/metabolismo , Células Mieloides/metabolismo , Proteínas Wnt/metabolismo , Animais , Proliferação de Células/fisiologia , Camundongos , Via de Sinalização Wnt
6.
Dev Cell ; 40(5): 439-452.e4, 2017 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-28292423

RESUMO

Polarization of node cells along the anterior-posterior axis of mouse embryos is responsible for left-right symmetry breaking. How node cells become polarized has remained unknown, however. Wnt5a and Wnt5b are expressed posteriorly relative to the node, whereas genes for Sfrp inhibitors of Wnt signaling are expressed anteriorly. Here we show that polarization of node cells is impaired in Wnt5a-/-Wnt5b-/- and Sfrp mutant embryos, and also in the presence of a uniform distribution of Wnt5a or Sfrp1, suggesting that Wnt5 and Sfrp proteins act as instructive signals in this process. The absence of planar cell polarity (PCP) core proteins Prickle1 and Prickle2 in individual cells or local forced expression of Wnt5a perturbed polarization of neighboring wild-type cells. Our results suggest that opposing gradients of Wnt5a and Wnt5b and of their Sfrp inhibitors, together with intercellular signaling via PCP proteins, polarize node cells along the anterior-posterior axis for breaking of left-right symmetry.


Assuntos
Padronização Corporal , Polaridade Celular , Transdução de Sinais , Proteínas Wnt/metabolismo , Proteína Wnt-5a/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Comunicação Celular , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas com Domínio LIM/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Modelos Biológicos , Proteínas/metabolismo
7.
Proc Natl Acad Sci U S A ; 113(50): E8079-E8088, 2016 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-27911818

RESUMO

Blastocyst implantation is a complex process requiring coordination of a dynamic sequence of embryo-uterine interactions. Blood vessels enter the uterus from the mesometrium, demarcating the uterus into mesometrial (M) and antimesometrial (AM) domains. Implantation occurs along the uterine longitudinal axis within specialized implantation chambers (crypts) that originate within the evaginations directed from the primary lumen toward the AM domain. The morphological orientation of crypts in rodent uteri was recognized more than a century ago, but the mechanism remained unknown. Here we provide evidence that planar cell polarity (PCP) signaling orchestrates directed epithelial evaginations to form crypts for implantation in mice. Uterine deletion of Vang-like protein 2, but not Vang-like protein 1, conferred aberrant PCP signaling, misdirected epithelial evaginations, defective crypt formation, and blastocyst attachment, leading to severely compromised pregnancy outcomes. The study reveals a previously unrecognized role for PCP in executing spatial cues for crypt formation and implantation. Because PCP is an evolutionarily conserved phenomenon, our study is likely to inspire implantation studies of this signaling pathway in humans and other species.


Assuntos
Polaridade Celular/fisiologia , Implantação do Embrião/fisiologia , Útero/fisiologia , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Comunicação Celular/fisiologia , Proteínas Desgrenhadas/fisiologia , Epitélio/anatomia & histologia , Epitélio/fisiologia , Feminino , Masculino , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Gravidez , Resultado da Gravidez , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/deficiência , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/fisiologia , Transdução de Sinais/fisiologia , Útero/anatomia & histologia , Proteína Wnt-5a/deficiência , Proteína Wnt-5a/genética , Proteína Wnt-5a/fisiologia
8.
Genesis ; 54(9): 497-502, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27411055

RESUMO

Wnt/ß-catenin signals are important regulators of embryonic and adult stem cell self-renewal and differentiation and play causative roles in tumorigenesis. Purified recombinant Wnt3a protein, or Wnt3a-conditioned culture medium, has been widely used to study canonical Wnt signaling in vitro or ex vivo. To study the role of Wnt3a in embryogenesis and cancer models, we developed a Cre recombinase activatable Rosa26(Wnt3a) allele, in which a Wnt3a cDNA was inserted into the Rosa26 locus to allow for conditional, spatiotemporally defined expression of Wnt3a ligand for gain-of-function (GOF) studies in mice. To validate this reagent, we ectopically overexpressed Wnt3a in early embryonic progenitors using the T-Cre transgene. This resulted in up-regulated expression of a ß-catenin/Tcf-Lef reporter and of the universal Wnt/ß-catenin pathway target genes, Axin2 and Sp5. Importantly, T-Cre; Rosa26(Wnt3a) mutants have expanded presomitic mesoderm (PSM) and compromised somitogenesis and closely resemble previously studied T-Cre; Ctnnb1(ex3) (ß-catenin(GOF) ) mutants. These data indicate that the exogenously expressed Wnt3a stimulates the Wnt/ß-catenin signaling pathway, as expected. The Rosa26(Wnt3a) mouse line should prove to be an invaluable tool to study the function of Wnt3a in vivo.


Assuntos
Marcação de Genes/métodos , Transgenes , Proteína Wnt3A/genética , Animais , Genes Reporter , Vetores Genéticos/genética , Integrases/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Regulação para Cima , Proteína Wnt3A/metabolismo
9.
Mol Cell Biol ; 36(12): 1793-802, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27090637

RESUMO

The Wnt/ß-catenin signaling pathway controls embryonic development and adult stem cell maintenance through the regulation of transcription. Failure to downregulate Wnt signaling can result in embryonic malformations and cancer, highlighting the important role of negative regulators of the pathway. The Wnt pathway activates several negative feedback targets, including axin2 and Dkk1, that function at different levels of the signaling cascade; however, none have been identified that directly target active ß-catenin/Tcf1 transcriptional complexes. We show that Zfp703 is a Wnt target gene that inhibits Wnt/ß-catenin activity in Wnt reporter assays and in Wnt-dependent mesoderm differentiation in embryonic stem cells. Zfp703 binds directly to Tcf1 to inhibit ß-catenin/Tcf1 complex formation and does so independently of the Groucho/Tle transcriptional corepressor. We propose that Zfp703 is a novel feedback suppressor of Wnt/ß-catenin signaling that functions by inhibiting the association of ß-catenin with Tcf1 on Wnt response elements in target gene enhancers.


Assuntos
Células-Tronco Embrionárias/citologia , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Proteínas Repressoras/metabolismo , beta Catenina/metabolismo , Animais , Diferenciação Celular , Desenvolvimento Embrionário , Células-Tronco Embrionárias/metabolismo , Humanos , Mesoderma/citologia , Camundongos , Proteínas Wnt/metabolismo , Via de Sinalização Wnt
10.
Proc Natl Acad Sci U S A ; 113(13): 3545-50, 2016 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-26969725

RESUMO

The ancient, highly conserved, Wnt signaling pathway regulates cell fate in all metazoans. We have previously shown that combined null mutations of the specificity protein (Sp) 1/Klf-like zinc-finger transcription factors Sp5 and Sp8 (i.e., Sp5/8) result in an embryonic phenotype identical to that observed when core components of the Wnt/ß-catenin pathway are mutated; however, their role in Wnt signal transduction is unknown. Here, we show in mouse embryos and differentiating embryonic stem cells that Sp5/8 are gene-specific transcriptional coactivators in the Wnt/ß-catenin pathway. Sp5/8 bind directly to GC boxes in Wnt target gene enhancers and to adjacent, or distally positioned, chromatin-bound T-cell factor (Tcf) 1/lymphoid enhancer factor (Lef) 1 to facilitate recruitment of ß-catenin to target gene enhancers. Because Sp5 is itself directly activated by Wnt signals, we propose that Sp5 is a Wnt/ß-catenin pathway-specific transcript on factor that functions in a feed-forward loop to robustly activate select Wnt target genes.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt/genética , beta Catenina/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos , Feminino , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1 de Ligação ao Facilitador Linfoide/genética , Camundongos , Camundongos Transgênicos , Gravidez , Fatores de Transcrição/genética , Ativação Transcricional , beta Catenina/genética
11.
Elife ; 5: e07727, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26845523

RESUMO

Endothelial cells respond to molecular and physical forces in development and vascular homeostasis. Deregulation of endothelial responses to flow-induced shear is believed to contribute to many aspects of cardiovascular diseases including atherosclerosis. However, how molecular signals and shear-mediated physical forces integrate to regulate vascular patterning is poorly understood. Here we show that endothelial non-canonical Wnt signalling regulates endothelial sensitivity to shear forces. Loss of Wnt5a/Wnt11 renders endothelial cells more sensitive to shear, resulting in axial polarization and migration against flow at lower shear levels. Integration of flow modelling and polarity analysis in entire vascular networks demonstrates that polarization against flow is achieved differentially in artery, vein, capillaries and the primitive sprouting front. Collectively our data suggest that non-canonical Wnt signalling stabilizes forming vascular networks by reducing endothelial shear sensitivity, thus keeping vessels open under low flow conditions that prevail in the primitive plexus.


Assuntos
Células Endoteliais/fisiologia , Estresse Mecânico , Remodelação Vascular , Via de Sinalização Wnt , Animais , Linhagem Celular , Movimento Celular , Polaridade Celular , Regulação da Expressão Gênica , Camundongos
12.
Dev Biol ; 412(1): 18-31, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26916252

RESUMO

Wnt5a, a non-canonical Wnt ligand critical for outflow tract (OFT) morphogenesis, is expressed specifically in second heart field (SHF) progenitors in the caudal splanchnic mesoderm (SpM) near the inflow tract (IFT). Using a conditional Wnt5a gain of function (GOF) allele and Islet1-Cre, we broadly over-expressed Wnt5a throughout the SHF lineage, including the entire SpM between the IFT and OFT. Wnt5a over-expression in Wnt5a null mutants can rescue the cell polarity and actin polymerization defects as well as severe SpM shortening, but fails to rescue OFT shortening. Moreover, Wnt5a over-expression in wild-type background is able to cause OFT shortening. We find that Wnt5a over-expression does not perturb SHF cell proliferation, apoptosis or differentiation, but affects the deployment of SHF cells by causing them to accumulate into a large bulge at the rostral SpM and fail to enter the OFT. Our immunostaining analyses suggest an inverse correlation between cell cohesion and Wnt5a level in the wild-type SpM. Ectopic Wnt5a expression in the rostral SpM of Wn5a-GOF mutants diminishes the upregulation of adherens junction; whereas loss of Wnt5a in Wnt5a null mutants causes premature increase in adherens junction level in the caudal SpM. Over-expression of mouse Wnt5a in Xenopus animal cap cells also reduces C-cadherin distribution on the plasma membrane without affecting its overall protein level, suggesting that Wnt5a may play an evolutionarily conserved role in controlling the cell surface level of cadherin to modulate cell cohesion during tissue morphogenesis. Collectively, our data indicate that restricted expression of Wnt5a in the caudal SpM is essential for normal OFT morphogenesis, and uncover a novel function of spatially regulated cell cohesion by Wnt5a in driving the deployment of SHF cells from the SpM into the OFT.


Assuntos
Miocárdio/citologia , Células-Tronco/citologia , Proteínas Wnt/fisiologia , Animais , Camundongos , Transdução de Sinais , Proteína Wnt-5a
14.
Dev Biol ; 408(1): 126-39, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26526197

RESUMO

Wnt ligands regulate heart morphogenesis but the underlying mechanisms remain unclear. Two Formin-related proteins, DAAM1 and 2, were previously found to bind the Wnt effector Disheveled. Here, since DAAM1 and 2 nucleate actin and mediate Wnt-induced cytoskeletal changes, a floxed-allele of Daam1 was used to disrupt its function specifically in the myocardium and investigate Wnt-associated pathways. Homozygous Daam1 conditional knockout (CKO) mice were viable but had misshapen hearts and poor cardiac function. The defects in Daam1 CKO mice were observed by mid-gestation and were associated with a loss of protrusions from cardiomyocytes invading the outflow tract. Further, these mice exhibited noncompaction cardiomyopathy (NCM) and deranged cardiomyocyte polarity. Interestingly, Daam1 CKO mice that were also homozygous for an insertion disrupting Daam2 (DKO) had stronger NCM, severely reduced cardiac function, disrupted sarcomere structure, and increased myocardial proliferation, suggesting that DAAM1 and DAAM2 have redundant functions. While RhoA was unaffected in the hearts of Daam1/2 DKO mice, AKT activity was lower than in controls, raising the issue of whether DAAM1/2 are only mediating Wnt signaling. Daam1-floxed mice were thus bred to Wnt5a null mice to identify genetic interactions. The hearts of Daam1 CKO mice that were also heterozygous for the null allele of Wnt5a had stronger NCM and more severe loss of cardiac function than Daam1 CKO mice, consistent with DAAM1 and Wnt5a acting in a common pathway. However, deleting Daam1 further disrupted Wnt5a homozygous-null hearts, suggesting that DAAM1 also has Wnt5a-independent roles in cardiac development.


Assuntos
Proteínas dos Microfilamentos/metabolismo , Miocárdio/metabolismo , Sarcômeros/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Adesão Celular , Proliferação de Células , Citoesqueleto/metabolismo , Embrião de Mamíferos/metabolismo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Quinase 3 da Glicogênio Sintase/metabolismo , Testes de Função Cardíaca , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Heterozigoto , Camundongos Knockout , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Morfogênese , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Especificidade de Órgãos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Proteínas Wnt , Proteína Wnt-5a , Proteínas rho de Ligação ao GTP/deficiência , Proteínas rho de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
15.
Am J Pathol ; 185(8): 2194-205, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26100214

RESUMO

Activation of Wnt/ß-catenin signaling during liver regeneration (LR) after partial hepatectomy (PH) is observed in several species. However, how this pathway is turned off when hepatocyte proliferation is no longer required is unknown. We assessed LR in liver-specific knockouts of Wntless (Wls-LKO), a protein required for Wnt secretion from a cell. When subjected to PH, Wls-LKO showed prolongation of hepatocyte proliferation for up to 4 days compared with littermate controls. This coincided with increased ß-catenin-T-cell factor 4 interaction and cyclin-D1 expression. Wls-LKO showed decreased expression and secretion of inhibitory Wnt5a during LR. Wnt5a expression increased between 24 and 48 hours, and Frizzled-2 between 24 and 72 hours, after PH in normal mice. Treatment of primary mouse hepatocytes and liver tumor cells with Wnt5a led to a notable decrease in ß-catenin-T-cell factor activity, cyclin-D1 expression, and cell proliferation. Intriguingly, Wnt5a-LKO did not display any prolongation of LR because of compensation by other cells. In addition, Wnt5a-LKO hepatocytes failed to respond to exogenous Wnt5a treatment in culture because of a compensatory decrease in Frizzled-2 expression. In conclusion, we demonstrate Wnt5a to be, by default, a negative regulator of ß-catenin signaling and hepatocyte proliferation, both in vitro and in vivo. We also provide evidence that the Wnt5a/Frizzled-2 axis suppresses ß-catenin signaling in hepatocytes in an autocrine manner, thereby contributing to timely conclusion of the LR process.


Assuntos
Proliferação de Células/fisiologia , Hepatócitos/metabolismo , Regeneração Hepática/fisiologia , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Animais , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Regeneração Hepática/efeitos dos fármacos , Camundongos , Camundongos Knockout , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Proteína Wnt-5a
16.
Development ; 142(9): 1628-38, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25922526

RESUMO

In the development of the vertebrate body plan, Wnt3a is thought to promote the formation of paraxial mesodermal progenitors (PMPs) of the trunk region while suppressing neural specification. Recent lineage-tracing experiments have demonstrated that these trunk neural progenitors and PMPs derive from a common multipotent progenitor called the neuromesodermal progenitor (NMP). NMPs are known to reside in the anterior primitive streak (PS) region; however, the extent to which NMPs populate the PS and contribute to the vertebrate body plan, and the precise role that Wnt3a plays in regulating NMP self-renewal and differentiation are unclear. To address this, we used cell-specific markers (Sox2 and T) and tamoxifen-induced Cre recombinase-based lineage tracing to locate putative NMPs in vivo. We provide functional evidence for NMP location primarily in the epithelial PS, and to a lesser degree in the ingressed PS. Lineage-tracing studies in Wnt3a/ß-catenin signaling pathway mutants provide genetic evidence that trunk progenitors normally fated to enter the mesodermal germ layer can be redirected towards the neural lineage. These data, combined with previous PS lineage-tracing studies, support a model that epithelial anterior PS cells are Sox2(+)T(+) multipotent NMPs and form the bulk of neural progenitors and PMPs of the posterior trunk region. Finally, we find that Wnt3a/ß-catenin signaling directs trunk progenitors towards PMP fates; however, our data also suggest that Wnt3a positively supports a progenitor state for both mesodermal and neural progenitors.


Assuntos
Padronização Corporal/fisiologia , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Mesoderma/embriologia , Células-Tronco Neurais/fisiologia , Linha Primitiva/citologia , Transdução de Sinais/fisiologia , Animais , Técnicas Histológicas , Imuno-Histoquímica , Hibridização In Situ , Mesoderma/citologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Proteína Wnt3A/metabolismo
17.
Mol Cell Biol ; 35(11): 2007-23, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25825523

RESUMO

Wnt signaling pathways are tightly regulated by ubiquitination, and dysregulation of these pathways promotes tumorigenesis. It has been reported that the ubiquitin ligase RNF43 plays an important role in frizzled-dependent regulation of the Wnt/ß-catenin pathway. Here, we show that RNF43 suppresses both Wnt/ß-catenin signaling and noncanonical Wnt signaling by distinct mechanisms. The suppression of Wnt/ß-catenin signaling requires interaction between the extracellular protease-associated (PA) domain and the cysteine-rich domain (CRD) of frizzled and the intracellular RING finger domain of RNF43. In contrast, these N-terminal domains of RNF43 are not required for inhibition of noncanonical Wnt signaling, but interaction between the C-terminal cytoplasmic region of RNF43 and the PDZ domain of dishevelled is essential for this suppression. We further show the mechanism by which missense mutations in the extracellular portion of RNF43 identified in patients with tumors activate Wnt/ß-catenin signaling. Missense mutations of RNF43 change their localization from the endosome to the endoplasmic reticulum (ER), resulting in the failure of frizzled-dependent suppression of Wnt/ß-catenin signaling. However, these mutants retain the ability to suppress noncanonical Wnt signaling, probably due to interaction with dishevelled. RNF43 is also one of the potential target genes of Wnt/ß-catenin signaling. Our results reveal the molecular role of RNF43 and provide an insight into tumorigenesis.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas Oncogênicas/genética , Transdução de Sinais/genética , Proteínas Wnt/genética , Via de Sinalização Wnt/genética , Linhagem Celular , Linhagem Celular Tumoral , Citoplasma/genética , Proteínas do Citoesqueleto/genética , Retículo Endoplasmático/genética , Endossomos/genética , Receptores Frizzled/genética , Células HCT116 , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Células MCF-7 , Mutação de Sentido Incorreto/genética , Domínios RING Finger/genética , Transativadores/genética , Ubiquitina-Proteína Ligases , beta Catenina/genética
18.
Dev Dyn ; 244(6): 797-807, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25809880

RESUMO

BACKGROUND: Vertebrate body axis extension occurs in a head-to-tail direction from a caudal progenitor zone that responds to interacting signals. Wnt/ß-catenin signaling is critical for generation of paraxial mesoderm, somite formation, and maintenance of the axial stem cell pool. Body axis extension requires Wnt8a in lower vertebrates, but in mammals Wnt3a is required, although the anterior trunk develops in the absence of Wnt3a. RESULTS: We examined mouse Wnt8a(-/-) and Wnt3a(-/-) single and double mutants to explore whether mammalian Wnt8a contributes to body axis extension and to determine whether a posterior growth function for Wnt8a is conserved throughout the vertebrate lineage. We find that caudal Wnt8a is expressed only during early somite stages and is required for normal development of the anterior trunk in the absence of Wnt3a. During this time, we show that Wnt8a and Wnt3a cooperate to maintain Fgf8 expression and prevent premature Sox2 up-regulation in the axial stem cell niche, critical for posterior growth. Similar to Fgf8, Wnt8a requires retinoic acid (RA) signaling to restrict its caudal expression boundary and possesses an upstream RA response element that binds RA receptors. CONCLUSIONS: These findings provide new insight into interaction of caudal Wnt-FGF-RA signals required for body axis extension.


Assuntos
Padronização Corporal/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Nicho de Células-Tronco/fisiologia , Proteína Wnt3A/fisiologia , Anormalidades Múltiplas/embriologia , Anormalidades Múltiplas/genética , Oxirredutases do Álcool/deficiência , Oxirredutases do Álcool/genética , Animais , Padronização Corporal/genética , Sequência Conservada , Fator 8 de Crescimento de Fibroblasto/biossíntese , Fator 8 de Crescimento de Fibroblasto/genética , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Knockout , Fenótipo , Receptores do Ácido Retinoico/fisiologia , Elementos de Resposta/genética , Fatores de Transcrição SOXB1/biossíntese , Fatores de Transcrição SOXB1/genética , Transdução de Sinais/fisiologia , Somitos/crescimento & desenvolvimento , Somitos/metabolismo , Tretinoína/farmacologia , Vertebrados/embriologia , Proteínas Wnt , Proteína Wnt3A/deficiência , Proteína Wnt3A/genética
19.
Diabetes ; 64(4): 1235-48, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25352637

RESUMO

Adipose tissue dysfunction plays a pivotal role in the development of insulin resistance in obese individuals. Cell culture studies and gain-of-function mouse models suggest that canonical Wnt proteins modulate adipose tissue expansion. However, no genetic evidence supports a role for endogenous Wnt proteins in adipose tissue dysfunction, and the role of noncanonical Wnt signaling remains largely unexplored. Here we provide evidence from human, mouse, and cell culture studies showing that Wnt5a-mediated, noncanonical Wnt signaling contributes to obesity-associated metabolic dysfunction by increasing adipose tissue inflammation. Wnt5a expression is significantly upregulated in human visceral fat compared with subcutaneous fat in obese individuals. In obese mice, Wnt5a ablation ameliorates insulin resistance, in parallel with reductions in adipose tissue inflammation. Conversely, Wnt5a overexpression in myeloid cells augments adipose tissue inflammation and leads to greater impairments in glucose homeostasis. Wnt5a ablation or overexpression did not affect fat mass or adipocyte size. Mechanistically, Wnt5a promotes the expression of proinflammatory cytokines by macrophages in a Jun NH2-terminal kinase-dependent manner, leading to defective insulin signaling in adipocytes. Exogenous interleukin-6 administration restores insulin resistance in obese Wnt5a-deficient mice, suggesting a central role for this cytokine in Wnt5a-mediated metabolic dysfunction. Taken together, these results demonstrate that noncanonical Wnt signaling contributes to obesity-induced insulin resistance independent of adipose tissue expansion.


Assuntos
Gordura Intra-Abdominal/metabolismo , Obesidade/metabolismo , Gordura Subcutânea/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Glucose/metabolismo , Humanos , Inflamação/metabolismo , Resistência à Insulina , Macrófagos/metabolismo , Camundongos , Fosforilação
20.
Nat Med ; 20(12): 1464-71, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25362254

RESUMO

Peripheral artery disease (PAD) generates tissue ischemia through arterial occlusions and insufficient collateral vessel formation. Vascular insufficiency in PAD occurs despite higher circulating levels of vascular endothelial growth factor A (VEGF-A), a key regulator of angiogenesis. Here we show that clinical PAD is associated with elevated levels of an antiangiogenic VEGF-A splice isoform (VEGF-A165b) and a corresponding reduction in levels of the proangiogenic VEGF-A165a splice isoform. In mice, VEGF-A165b expression was upregulated by conditions associated with impaired limb revascularization, including leptin deficiency, diet-induced obesity, genetic ablation of the secreted frizzled-related protein 5 (Sfrp5) adipokine and transgenic overexpression of Wnt5a in myeloid cells. In a mouse model of PAD, delivery of VEGF-A165b inhibited revascularization of ischemic hind limbs, whereas treatment with an isoform-specific neutralizing antibody reversed impaired revascularization caused by metabolic dysfunction or perturbations in the Wnt5a-Sfrp5 regulatory system. These results indicate that inflammation-driven expression of the antiangiogenic VEGF-A isoform can contribute to impaired collateralization in ischemic cardiovascular disease.


Assuntos
Neovascularização Fisiológica/fisiologia , Doença Arterial Periférica/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Circulação Colateral , Modelos Animais de Doenças , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Isoformas de Proteínas , Proteínas Wnt/genética , Proteína Wnt-5a
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