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1.
Nat Commun ; 11(1): 5701, 2020 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-33177522

RESUMO

Protein knockdown using the auxin-inducible degron (AID) technology is useful to study protein function in living cells because it induces rapid depletion, which makes it possible to observe an immediate phenotype. However, the current AID system has two major drawbacks: leaky degradation and the requirement for a high dose of auxin. These negative features make it difficult to control precisely the expression level of a protein of interest in living cells and to apply this method to mice. Here, we overcome these problems by taking advantage of a bump-and-hole approach to establish the AID version 2 (AID2) system. AID2, which employs an OsTIR1(F74G) mutant and a ligand, 5-Ph-IAA, shows no detectable leaky degradation, requires a 670-times lower ligand concentration, and achieves even quicker degradation than the conventional AID. We demonstrate successful generation of human cell mutants for genes that were previously difficult to deal with, and show that AID2 achieves rapid target depletion not only in yeast and mammalian cells, but also in mice.

2.
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
3.
Proc Natl Acad Sci U S A ; 116(47): 23636-23642, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31685615

RESUMO

Sonic hedgehog (SHH) signaling plays a pivotal role in 2 different phases during brain development. Early SHH signaling derived from the prechordal plate (PrCP) triggers secondary Shh induction in the forebrain, which overlies the PrCP, and the induced SHH signaling, in turn, directs late neuronal differentiation of the forebrain. Consequently, Shh regulation in the PrCP is crucial for initiation of forebrain development. However, no enhancer that regulates prechordal Shh expression has yet been found. Here, we identified a prechordal enhancer, named SBE7, in the vicinity of a cluster of known forebrain enhancers for Shh This enhancer also directs Shh expression in the ventral midline of the forebrain, which receives the prechordal SHH signal. Thus, the identified enhancer acts not only for the initiation of Shh regulation in the PrCP but also for subsequent Shh induction in the forebrain. Indeed, removal of the enhancer from the mouse genome markedly down-regulated the expression of Shh in the rostral domains of the axial mesoderm and in the ventral midline of the forebrain and hypothalamus in the mouse embryo, and caused a craniofacial abnormality similar to human holoprosencephaly (HPE). These findings demonstrate that SHH signaling mediated by the newly identified enhancer is essential for development and growth of the ventral midline of the forebrain and hypothalamus. Understanding of the Shh regulation governed by this prechordal and brain enhancer provides an insight into the mechanism underlying craniofacial morphogenesis and the etiology of HPE.


Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Prosencéfalo/embriologia , Animais , Sistemas CRISPR-Cas , Proteínas do Olho/fisiologia , Técnicas de Inativação de Genes , Genes Reporter , Proteínas Hedgehog/biossíntese , Proteínas Hedgehog/genética , Holoprosencefalia/genética , Proteínas de Homeodomínio/fisiologia , Hipotálamo/anormalidades , Hipotálamo/embriologia , Hipotálamo/metabolismo , Óperon Lac , Mesencéfalo/embriologia , Mesencéfalo/metabolismo , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Prosencéfalo/anormalidades , Prosencéfalo/metabolismo , Transdução de Sinais , Transgenes
4.
Elife ; 72018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29761784

RESUMO

The metameric structure in vertebrates is based on the periodic formation of somites from the anterior end of the presomitic mesoderm (PSM). The segmentation boundary is defined by the Tbx6 expression domain, whose anterior limit is determined by Tbx6 protein destabilization via Ripply2. However, the molecular mechanism of this process is poorly understood. Here, we show that Ripply2 directly binds to Tbx6 in cultured cells without changing the stability of Tbx6, indicating an unknown mechanism for Tbx6 degradation in vivo. We succeeded in reproducing in vivo events using a mouse ES induction system, in which Tbx6 degradation occurred via Ripply2. Mass spectrometry analysis of the PSM-fated ES cells revealed that proteasomes are major components of the Ripply2-binding complex, suggesting that recruitment of a protein-degradation-complex is a pivotal function of Ripply2. Finally, we identified a motif in the T-box, which is required for Tbx6 degradation independent of binding with Ripply2 in vivo.


Assuntos
Células-Tronco Embrionárias Murinas/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Repressoras/metabolismo , Somitos/embriologia , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Espectrometria de Massas , Camundongos , Ligação Proteica , Proteólise , Proteínas com Domínio T
5.
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
6.
PLoS One ; 12(11): e0187248, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29095923

RESUMO

The segmental pattern of the vertebrate body is established via the periodic formation of somites from the presomitic mesoderm (PSM). This periodical process is controlled by the cyclic and synchronized activation of Notch signaling in the PSM. Protein O-fucosyltransferase1 (Pofut1), which transfers O-fucose to the EGF domains of the Notch1 receptor, is indispensable for Notch signaling activation. The Drosophila homologue Ofut1 was reported to control Notch localization via two different mechanisms, working as a chaperone for Notch or as a regulator of Notch endocytosis. However, these were found to be independent of O-fucosyltransferase activity because the phenotypes were rescued by Ofut1 mutants lacking O-fucosyltransferase activity. Pofut1 may also be involved in the Notch receptor localization in mice. However, the contribution of enzymatic activity of Pofut1 to the Notch receptor dynamics remains to be elucidated. In order to clarify the importance of the O-fucosyltransferase activity of Pofut1 for Notch signaling activation and the protein localization in the PSM, we established mice carrying point mutations at the 245th a.a. or 370-372th a.a., highly conserved amino-acid sequences whose mutations disrupt the O-fucosyltransferase activity of both Drosophila Ofut1 and mammalian Pofut1, with the CRISPR/Cas9 mediated genome-engineering technique. Both mutants displayed the same severely perturbed somite formation and Notch1 subcellular localization defects as the Pofut1 null mutants. In the mutants, Pofut1 protein, but not RNA, became undetectable by E9.5. Furthermore, both wild-type and mutant Pofut1 proteins were degraded through lysosome dependent machinery. Pofut1 protein loss in the point mutant embryos caused the same phenotypes as those observed in Pofut1 null embryos.


Assuntos
Fucosiltransferases/metabolismo , Mutação Puntual , Receptor Notch1/metabolismo , Transdução de Sinais , Somitos/crescimento & desenvolvimento , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Regulação para Baixo , Fucosiltransferases/genética , Camundongos , Processamento Pós-Transcricional do RNA
7.
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
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.
Genes Cells ; 21(7): 728-39, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27353389

RESUMO

Lrrc6 encodes a cytoplasmic protein that is expressed specifically in cells with motile cilia including the node, trachea and testes of the mice. A mutation of Lrrc6 has been identified in human patients with primary ciliary dyskinesia (PCD). Mutant mice lacking Lrrc6 show typical PCD defects such as hydrocephalus and laterality defects. We found that in the absence of Lrrc6, the morphology of motile cilia remained normal, but their motility was completely lost. The 9 + 2 arrangement of microtubules remained normal in Lrrc6(-/-) mice, but the outer dynein arms (ODAs), the structures essential for the ciliary beating, were absent from the cilia. In the absence of Lrrc6, ODA proteins such as DNAH5, DNAH9 and IC2, which are assembled in the cytoplasm and transported to the ciliary axoneme, remained in the cytoplasm and were not transported to the ciliary axoneme. The IC2-IC1 interaction, which is the first step of ODA assembly, was normal in Lrrc6(-/-) mice testes. Our results suggest that ODA proteins may be transported from the cytoplasm to the cilia by an Lrrc6-dependent mechanism.


Assuntos
Cílios/genética , Síndrome de Kartagener/genética , Proteínas/genética , Animais , Dineínas do Axonema/genética , Axonema/genética , Axonema/patologia , Cílios/patologia , Citoplasma/genética , Citoplasma/metabolismo , Proteínas do Citoesqueleto , Modelos Animais de Doenças , Dineínas/genética , Humanos , Síndrome de Kartagener/patologia , Camundongos , Camundongos Transgênicos , Mutação
10.
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
11.
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
12.
Dev Biol ; 400(1): 105-17, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25641698

RESUMO

The precise border of somites formed during mouse somitogenesis is defined by a Tbx6 expression domain, which is established by Mesp2-mediated Tbx6 suppression in the anterior part of the presomitic mesoderm (PSM). Ripply2, a target of Mesp2, is proposed to be involved in this down-regulation because Ripply2 deficiency causes an anterior expansion of the Tbx6 domain, resembling the Mesp2-null phenotype. However, it is unclear whether Ripply2 acts on Tbx6 independently or in association with Mesp2. To address this question, we generated three sets of transgenic mice with the following Ripply2 expression patterns: (1) overexpression in the endogenous expression domain, (2) expression instead of Mesp2 (Ripply2-knockin), and (3) ectopic expression in the entire PSM. We found accelerated Tbx6 degradation in the embryos showing Ripply2 overexpression. In the Ripply2-knockin embryos, the anterior limit of Tbx6 domain was generated by Ripply2 even in the absence of Mesp2. Ectopic Ripply2 expression along the entire PSM suppressed Tbx6 and induced Sox2-positive neural tube formation at the bilateral domain, resembling the Tbx6-null phenotype. This phenotype resulted from Tbx6 protein and not mRNA elimination, suggesting the post-translational down-regulation of Tbx6 by Ripply2. Taken together, our results demonstrate that Ripply2 represses Tbx6 in a Mesp2-independent manner, which contributes to the accurate segmental border formation.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Padronização Corporal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Repressoras/metabolismo , Somitos/embriologia , Fatores de Transcrição/metabolismo , Animais , Western Blotting , Primers do DNA/genética , Técnicas de Introdução de Genes , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/genética , Somitos/metabolismo , Proteínas com Domínio T
13.
Hum Mol Genet ; 23(25): 6807-14, 2014 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-25082826

RESUMO

Congenital anomalies of the kidney and urinary tract (CAKUT) affect about 1 in 500 births and are a major cause of morbidity in infants. Duplex collecting systems rank among the most common abnormalities of CAKUT, but the molecular basis for this defect is poorly understood. In mice, conditional deletion of Wnt5a in mesoderm results in bilateral duplex kidney and ureter formation. The ureteric buds (UBs) in mutants emerge as doublets from the intermediate mesoderm (IM)-derived nephric duct (ND) without anterior expansion of the glial cell line-derived neurotrophic factor (Gdnf) expression domain in the surrounding mesenchyme. Wnt5a is normally expressed in a graded manner at the posterior end of the IM, but its expression is down-regulated prior to UB outgrowth at E10.5. Furthermore, ablation of Wnt5a in the mesoderm with an inducible Cre at E7.5 results in duplex UBs, whereas ablation at E8.5 yields normal UB outgrowth, demonstrating that Wnt5a functions in IM development well before the formation of the metanephros. In mutants, the posterior ND is duplicated and surrounding Pax2-positive mesenchymal cells persist in the nephric cord, suggesting that disruption of normal ND patterning prompts the formation of duplex ureters and kidneys. Ror2 homozygous mutants, which infrequently yield duplex collecting systems, show a dramatic increase in incidence with the additional deletion of one copy of Wnt5a, implicating this receptor in non-canonical Wnt5a signaling during IM development. This work provides the first evidence of a role of Wnt5a/Ror2 signaling in IM extension and offers new insights into the etiology of CAKUT and possible involvement of Wnt5a/Ror2 mutations.


Assuntos
Rim/metabolismo , Mesoderma/metabolismo , Morfogênese/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Transdução de Sinais/genética , Proteínas Wnt/genética , Animais , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Homozigoto , Integrases/genética , Integrases/metabolismo , Rim/crescimento & desenvolvimento , Rim/patologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Mesoderma/crescimento & desenvolvimento , Mesoderma/patologia , Camundongos , Camundongos Transgênicos , Fator de Transcrição PAX2/genética , Fator de Transcrição PAX2/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Fatores de Tempo , Ureter/crescimento & desenvolvimento , Ureter/metabolismo , Ureter/patologia , Proteínas Wnt/deficiência , Proteína Wnt-5a , Ductos Mesonéfricos/crescimento & desenvolvimento , Ductos Mesonéfricos/metabolismo , Ductos Mesonéfricos/patologia
14.
Cell Rep ; 8(2): 382-92, 2014 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-25043182

RESUMO

Embryo homing and implantation occur within a crypt (implantation chamber) at the antimesometrial (AM) pole along the uterus. The mechanism by which this is achieved is not known. Here, we show that villi-like epithelial projections from the main uterine lumen toward the AM pole at regularly spaced intervals that form crypts for embryo implantation were disrupted in mice with uterine loss or gain of function of Wnt5a, or loss of function of both Ror1 and Ror2. This disruption of Wnt5a-ROR signaling resulted in disorderly epithelial projections, crypt formation, embryo spacing, and impaired implantation. These early disturbances under abnormal Wnt5a-ROR signaling were reflected in adverse late pregnancy events, including defective decidualization and placentation, ultimately leading to compromised pregnancy outcomes. This study presents deeper insight regarding the formation of organized epithelial projections for crypt formation and embryo implantation for pregnancy success.


Assuntos
Decídua/metabolismo , Implantação do Embrião , Células Epiteliais/citologia , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Animais , Decídua/citologia , Decídua/fisiologia , Células Epiteliais/metabolismo , Feminino , Camundongos , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Proteínas Wnt/genética , Proteína Wnt-5a
15.
Science ; 338(6103): 108-13, 2012 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-22956684

RESUMO

Reestablishing homeostasis after tissue damage depends on the proper organization of stem cells and their progeny, though the repair mechanisms are unclear. The mammalian intestinal epithelium is well suited to approach this problem, as it is composed of well-delineated units called crypts of Lieberkühn. We found that Wnt5a, a noncanonical Wnt ligand, was required for crypt regeneration after injury in mice. Unlike controls, Wnt5a-deficient mice maintained an expanded population of proliferative epithelial cells in the wound. We used an in vitro system to enrich for intestinal epithelial stem cells to discover that Wnt5a inhibited proliferation of these cells. Surprisingly, the effects of Wnt5a were mediated by activation of transforming growth factor-ß (TGF-ß) signaling. These findings suggest a Wnt5a-dependent mechanism for forming new crypt units to reestablish homeostasis.


Assuntos
Colo/lesões , Colo/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Proteínas Wnt/fisiologia , Cicatrização/fisiologia , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colo/embriologia , Meios de Cultivo Condicionados/farmacologia , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Mucosa Intestinal/embriologia , Mucosa Intestinal/lesões , Mucosa Intestinal/fisiologia , Ligantes , Mesoderma/citologia , Mesoderma/embriologia , Camundongos , Camundongos Knockout , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/fisiologia , Tamoxifeno/farmacologia , Proteínas Wnt/genética , Proteínas Wnt/farmacologia , Proteína Wnt-5a , Cicatrização/efeitos dos fármacos
16.
Nat Commun ; 2: 390, 2011 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-21750544

RESUMO

Segmentation is an organizing principle of body plans. The segmentation clock, a molecular oscillator best illustrated by the cyclic expression of Notch signalling genes, controls the periodic cleavage of somites from unsegmented presomitic mesoderm during vertebrate segmentation. Wnt3a controls the spatiotemporal expression of cyclic Notch genes; however, the underlying mechanisms remain obscure. Here we show by transcriptional profiling of Wnt3a (-/-) embryos that the bHLH transcription factor, Mesogenin1 (Msgn1), is a direct target gene of Wnt3a. To identify Msgn1 targets, we conducted genome-wide studies of Msgn1 activity in embryonic stem cells. We show that Msgn1 is a major transcriptional activator of a Notch signalling program and synergizes with Notch to trigger clock gene expression. Msgn1 also indirectly regulates cyclic genes in the Fgf and Wnt pathways. Thus, Msgn1 is a central component of a transcriptional cascade that translates a spatial Wnt3a gradient into a temporal pattern of clock gene expression.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Relógios Biológicos/fisiologia , Padronização Corporal/fisiologia , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Animais , Diferenciação Celular , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Células-Tronco Embrionárias , Perfilação da Expressão Gênica , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Wnt/genética , Proteína Wnt3 , Proteína Wnt3A , beta Catenina/metabolismo
17.
Nat Cell Biol ; 13(6): 636-7, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21633348

RESUMO

The primary cilium is proposed to restrain the level of canonical Wnt signalling, but it was unknown how the cilium achieves this. ß-catenin, a component of the canonical Wnt signalling pathway, is now shown to be sequestered to the cilium by the Wnt signalling modulator Jouberin (Jbn) to restrain Wnt responses.


Assuntos
Cílios/metabolismo , Proteínas Wnt/metabolismo , Animais , Fibroblastos/metabolismo , Humanos , Camundongos , Transdução de Sinais
18.
Nature ; 474(7352): 511-5, 2011 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-21623369

RESUMO

Myeloid cells are a feature of most tissues. Here we show that during development, retinal myeloid cells (RMCs) produce Wnt ligands to regulate blood vessel branching. In the mouse retina, where angiogenesis occurs postnatally, somatic deletion in RMCs of the Wnt ligand transporter Wntless results in increased angiogenesis in the deeper layers. We also show that mutation of Wnt5a and Wnt11 results in increased angiogenesis and that these ligands elicit RMC responses via a non-canonical Wnt pathway. Using cultured myeloid-like cells and RMC somatic deletion of Flt1, we show that an effector of Wnt-dependent suppression of angiogenesis by RMCs is Flt1, a naturally occurring inhibitor of vascular endothelial growth factor (VEGF). These findings indicate that resident myeloid cells can use a non-canonical, Wnt-Flt1 pathway to suppress angiogenic branching.


Assuntos
Células Mieloides/metabolismo , Neovascularização Fisiológica/fisiologia , Retina/citologia , Transdução de Sinais , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Wnt/metabolismo , Animais , Vasos Sanguíneos/crescimento & desenvolvimento , Células Endoteliais/metabolismo , Fibroblastos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas Relacionadas a Receptor de LDL/metabolismo , Ligantes , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Receptores Acoplados a Proteínas-G , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/deficiência , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Proteínas Wnt/deficiência , Proteínas Wnt/genética , Proteína Wnt-5a
19.
Dev Biol ; 352(1): 58-69, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21256838

RESUMO

Wnt4 and ß-catenin are both required for nephrogenesis, but studies using TCF-reporter mice suggest that canonical Wnt signaling is not activated in metanephric mesenchyme (MM) during its conversion to the epithelia of the nephron. To better define the role of Wnt signaling, we treated rat metanephric mesenchymal progenitors directly with recombinant Wnt proteins. These studies revealed that Wnt4 protein, which is required for nephron formation, induces tubule formation and differentiation markers Lim1 and E-cadherin in MM cells, but does not activate a TCF reporter or up regulate expression of canonical Wnt target gene Axin-2 and has little effect on the stabilization of ß-catenin or phosphorylation of disheveled-2. Furthermore, Wnt4 causes membrane localization of ZO-1 and occludin in tight junctions. To directly examine the role of ß-catenin/TCF-dependent transcription, we developed synthetic cell-permeable analogs of ß-catenin's helix C, which is required for transcriptional activation, in efforts to specifically inhibit canonical Wnt signaling. One inhibitor blocked TCF-dependent transcription and induced degradation of ß-catenin but did not affect tubule formation and stimulated the expression of Lim1 and E-cadherin. Since a canonical mechanism appears not to be operative in tubule formation, we assessed the involvement of the non-canonical Ca(2+)-dependent pathway. Treatment of MM cells with Wnt4 induced an influx of Ca(2+) and caused phosphorylation of CaMKII. Moreover, Ionomycin, a Ca(2+)-dependent pathway activator, stimulated tubule formation. These results demonstrate that the canonical Wnt pathway is not responsible for mesenchymal-epithelial transition (MET) in nephron formation and suggest that the non-canonical calcium/Wnt pathway mediates Wnt4-induced tubulogenesis in the kidney.


Assuntos
Mesoderma/efeitos dos fármacos , Mesoderma/embriologia , Modelos Biológicos , Néfrons/efeitos dos fármacos , Néfrons/embriologia , Proteínas Wnt/farmacologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Genes Reporter/genética , Humanos , Ionomicina/farmacologia , Túbulos Renais/citologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/embriologia , Túbulos Renais/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Morfogênese/efeitos dos fármacos , Néfrons/citologia , Néfrons/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição TCF/metabolismo , Transcrição Genética/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Proteína Wnt4 , beta Catenina/química , beta Catenina/metabolismo
20.
J Cell Sci ; 123(Pt 3): 472-83, 2010 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-20067994

RESUMO

The Wnt planar cell polarity (Wnt/PCP) pathway signals through small Rho-like GTPases to regulate the cytoskeleton. The core PCP proteins have been mapped to the Wnt/PCP pathway genetically, but the molecular mechanism of their action remains unknown. Here, we investigate the function of the mammalian PCP protein Vang-like protein 2 (Vangl2). RNAi knockdown of Vangl2 impaired cell-cell adhesion and cytoskeletal integrity in the epithelial cell lines HEK293T and MDCK. Similar effects were observed when Vangl2 was overexpressed in HEK293T, MDCK or C17.2 cells. The effects of Vangl2 overexpression could be blocked by knockdown of the small GTPase Rac1 or by dominant-negative Rac1. In itself, knockdown of Rac1 impaired cytoskeletal integrity and reduced cell-cell adhesion. We found that Vangl2 bound and re-distributed Rac1 within the cells but did not alter Rac1 activity. Moreover, both transgenic mouse embryos overexpressing Vangl2 in neural stem cells and loop-tail Vangl2 loss-of-function embryos displayed impaired adherens junctions, a cytoskeletal unit essential for neural tube rigidity and neural tube closure. In vivo, Rac1 was re-distributed within the cells in a similar way to that observed by us in vitro. We propose that Vangl2 affects cell adhesion and the cytoskeleton by recruiting Rac1 and targeting its activity in the cell to adherens junctions.


Assuntos
Junções Aderentes/metabolismo , Proteínas de Membrana/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Junções Aderentes/genética , Animais , Linhagem Celular , Cães , Humanos , Imuno-Histoquímica , Imunoprecipitação , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica/genética , Ligação Proteica/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas rac1 de Ligação ao GTP/genética
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