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1.
Nature ; 615(7950): 151-157, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36509106

RESUMO

In the past decade, single-cell transcriptomics has helped to uncover new cell types and states and led to the construction of a cellular compendium of health and disease. Despite this progress, some difficult-to-sequence cells remain absent from tissue atlases. Eosinophils-elusive granulocytes that are implicated in a plethora of human pathologies1-5-are among these uncharted cell types. The heterogeneity of eosinophils and the gene programs that underpin their pleiotropic functions remain poorly understood. Here we provide a comprehensive single-cell transcriptomic profiling of mouse eosinophils. We identify an active and a basal population of intestinal eosinophils, which differ in their transcriptome, surface proteome and spatial localization. By means of a genome-wide CRISPR inhibition screen and functional assays, we reveal a mechanism by which interleukin-33 (IL-33) and interferon-γ (IFNγ) induce the accumulation of active eosinophils in the inflamed colon. Active eosinophils are endowed with bactericidal and T cell regulatory activity, and express the co-stimulatory molecules CD80 and PD-L1. Notably, active eosinophils are enriched in the lamina propria of a small cohort of patients with inflammatory bowel disease, and are closely associated with CD4+ T cells. Our findings provide insights into the biology of eosinophils and highlight the crucial contribution of this cell type to intestinal homeostasis, immune regulation and host defence. Furthermore, we lay a framework for the characterization of eosinophils in human gastrointestinal diseases.


Assuntos
Colite , Eosinófilos , Imunidade , Intestinos , Animais , Humanos , Camundongos , Colite/imunologia , Colite/patologia , Eosinófilos/classificação , Eosinófilos/citologia , Eosinófilos/imunologia , Eosinófilos/metabolismo , Doenças Inflamatórias Intestinais/imunologia , Análise da Expressão Gênica de Célula Única , Transcriptoma , Proteoma , Interleucina-33 , Interferon gama , Linfócitos T , Antígeno B7-1/metabolismo , Intestinos/imunologia , Intestinos/patologia
2.
Genes Dev ; 32(21-22): 1443-1458, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30366904

RESUMO

Bcl9 and Pygopus (Pygo) are obligate Wnt/ß-catenin cofactors in Drosophila, yet their contribution to Wnt signaling during vertebrate development remains unresolved. Combining zebrafish and mouse genetics, we document a conserved, ß-catenin-associated function for BCL9 and Pygo proteins during vertebrate heart development. Disrupting the ß-catenin-BCL9-Pygo complex results in a broadly maintained canonical Wnt response yet perturbs heart development and proper expression of key cardiac regulators. Our work highlights BCL9 and Pygo as selective ß-catenin cofactors in a subset of canonical Wnt responses during vertebrate development. Moreover, our results implicate alterations in BCL9 and BCL9L in human congenital heart defects.


Assuntos
Cardiopatias Congênitas/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fatores de Transcrição/genética , Via de Sinalização Wnt , Proteínas de Peixe-Zebra/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Coração/embriologia , Camundongos , Mutação , Miocárdio/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , beta Catenina/metabolismo
3.
EMBO Rep ; 24(9): e56454, 2023 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-37493498

RESUMO

The protective and absorptive functions of the intestinal epithelium rely on differentiated enterocytes in the villi. The differentiation of enterocytes is orchestrated by sub-epithelial mesenchymal cells producing distinct ligands along the villus axis, in particular Bmps and Tgfß. Here, we show that individual Bmp ligands and Tgfß drive distinct enterocytic programs specific to villus zonation. Bmp4 is expressed from the centre to the upper part of the villus and activates preferentially genes connected to lipid uptake and metabolism. In contrast, Bmp2 is produced by villus tip mesenchymal cells and it influences the adhesive properties of villus tip epithelial cells and the expression of immunomodulators. Additionally, Tgfß induces epithelial gene expression programs similar to those triggered by Bmp2. Bmp2-driven villus tip program is activated by a canonical Bmp receptor type I/Smad-dependent mechanism. Finally, we establish an organoid cultivation system that enriches villus tip enterocytes and thereby better mimics the cellular composition of the intestinal epithelium. Our data suggest that not only a Bmp gradient but also the activity of individual Bmp drives specific enterocytic programs.


Assuntos
Enterócitos , Mucosa Intestinal , Enterócitos/metabolismo , Ligantes , Mucosa Intestinal/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular
4.
Nature ; 558(7710): 449-453, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29875413

RESUMO

Wnt-ß-catenin signalling plays a pivotal role in the homeostasis of the intestinal epithelium by promoting stem cell renewal1,2. In the small intestine, epithelial Paneth cells secrete Wnt ligands and thus adopt the function of the stem cell niche to maintain epithelial homeostasis3,4. It is unclear which cells comprise the stem cell niche in the colon. Here we show that subepithelial mesenchymal GLI1-expressing cells form this essential niche. Blocking Wnt secretion from GLI1-expressing cells prevents colonic stem cell renewal in mice: the stem cells are lost and, as a consequence, the integrity of the colonic epithelium is corrupted, leading to death. GLI1-expressing cells also play an important role in the maintenance of the small intestine, where they serve as a reserve Wnt source that becomes critical when Wnt secretion from epithelial cells is prevented. Our data suggest a mechanism by which the stem cell niche is adjusted to meet the needs of the intestine via adaptive changes in the number of mesenchymal GLI1-expressing cells.


Assuntos
Colo/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Nicho de Células-Tronco/fisiologia , Células-Tronco/metabolismo , Proteínas Wnt/metabolismo , Proteína GLI1 em Dedos de Zinco/metabolismo , Animais , Autorrenovação Celular , Feminino , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Masculino , Camundongos , Células-Tronco/citologia , Via de Sinalização Wnt
5.
Proc Natl Acad Sci U S A ; 118(34)2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34408016

RESUMO

During malignant progression, epithelial cancer cells dissolve their cell-cell adhesion and gain invasive features. By virtue of its dual function, ß-catenin contributes to cadherin-mediated cell-cell adhesion, and it determines the transcriptional output of Wnt signaling: via its N terminus, it recruits the signaling coactivators Bcl9 and Pygopus, and via the C terminus, it interacts with the general transcriptional machinery. This duality confounds the simple loss-of-function analysis of Wnt signaling in cancer progression. In many cancer types including breast cancer, the functional contribution of ß-catenin's transcriptional activities, as compared to its adhesion functions, to tumor progression has remained elusive. Employing the mouse mammary tumor virus (MMTV)-PyMT mouse model of metastatic breast cancer, we compared the complete elimination of ß-catenin with the specific ablation of its signaling outputs in mammary tumor cells. Notably, the complete lack of ß-catenin resulted in massive apoptosis of mammary tumor cells. In contrast, the loss of ß-catenin's transcriptional activity resulted in a reduction of primary tumor growth, tumor invasion, and metastasis formation in vivo. These phenotypic changes were reflected by stalled cell cycle progression and diminished epithelial-mesenchymal transition (EMT) and cell migration of breast cancer cells in vitro. Transcriptome analysis revealed subsets of genes which were specifically regulated by ß-catenin's transcriptional activities upon stimulation with Wnt3a or during TGF-ß-induced EMT. Our results uncouple the signaling from the adhesion function of ß-catenin and underline the importance of Wnt/ß-catenin-dependent transcription in malignant tumor progression of breast cancer.


Assuntos
Adesão Celular/fisiologia , Neoplasias Mamárias Animais/metabolismo , Transdução de Sinais/fisiologia , Proteína Wnt3A/metabolismo , beta Catenina/metabolismo , Animais , Apoptose , Ciclo Celular , Movimento Celular , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Mamárias Animais/genética , Camundongos , Camundongos Transgênicos , Invasividade Neoplásica , Metástase Neoplásica , Transcriptoma , Fator de Crescimento Transformador beta/farmacologia , Proteína Wnt3A/genética , beta Catenina/genética
6.
EMBO J ; 38(2)2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30425074

RESUMO

During canonical Wnt signalling, the activity of nuclear ß-catenin is largely mediated by the TCF/LEF family of transcription factors. To challenge this view, we used the CRISPR/Cas9 genome editing approach to generate HEK 293T cell clones lacking all four TCF/LEF genes. By performing unbiased whole transcriptome sequencing analysis, we found that a subset of ß-catenin transcriptional targets did not require TCF/LEF factors for their regulation. Consistent with this finding, we observed in a genome-wide analysis that ß-catenin occupied specific genomic regions in the absence of TCF/LEF Finally, we revealed the existence of a transcriptional activity of ß-catenin that specifically appears when TCF/LEF factors are absent, and refer to this as ß-catenin-GHOST response. Collectively, this study uncovers a previously neglected modus operandi of ß-catenin that bypasses the TCF/LEF transcription factors.


Assuntos
Perfilação da Expressão Gênica/métodos , Fatores de Transcrição TCF/genética , Transcrição Gênica , beta Catenina/metabolismo , Sistemas CRISPR-Cas , Edição de Genes , Regulação da Expressão Gênica , Células HEK293 , Humanos , Fatores de Transcrição TCF/metabolismo , Sequenciamento do Exoma/métodos , Via de Sinalização Wnt
7.
PLoS Biol ; 18(12): e3001032, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33306673

RESUMO

Despite recent progress in recognizing the importance of mesenchymal cells for the homeostasis of the intestinal system, the current picture of how these cells communicate with the associated epithelial layer remains unclear. To describe the relevant cell populations in an unbiased manner, we carried out a single-cell transcriptome analysis of the adult murine colon, producing a high-quality atlas of matched colonic epithelium and mesenchyme. We identify two crypt-associated colonic fibroblast populations that are demarcated by different strengths of platelet-derived growth factor receptor A (Pdgfra) expression. Crypt-bottom fibroblasts (CBFs), close to the intestinal stem cells, express low levels of Pdgfra and secrete canonical Wnt ligands, Wnt potentiators, and bone morphogenetic protein (Bmp) inhibitors. Crypt-top fibroblasts (CTFs) exhibit high Pdgfra levels and secrete noncanonical Wnts and Bmp ligands. While the Pdgfralow cells maintain intestinal stem cell proliferation, the Pdgfrahigh cells induce differentiation of the epithelial cells. Our findings enhance our understanding of the crosstalk between various colonic epithelial cells and their associated mesenchymal signaling hubs along the crypt axis-placing differential Pdgfra expression levels in the spotlight of intestinal fibroblast identity.


Assuntos
Colo/metabolismo , Fibroblastos/classificação , Fibroblastos/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Colo/fisiologia , Células Epiteliais/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Homeostase , Mucosa Intestinal/metabolismo , Intestinos/fisiologia , Mesoderma/citologia , Mesoderma/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Análise de Célula Única/métodos , Células-Tronco/citologia , Transcriptoma/genética
8.
Nat Rev Mol Cell Biol ; 11(11): 789-801, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20944666

RESUMO

Proteomes, the ensembles of all proteins expressed by cells or tissues, are typically analysed by mass spectrometry. Recent technical and computational advances have greatly increased the fraction of a proteome that can be identified and quantified in a single study. Current mass spectrometry-based proteomic strategies have the potential to reproducibly, accurately, quantitatively and comprehensively measure any protein or whole proteomes from cells and tissues at different states. Achieving these goals will require complete proteome maps and analytical strategies that use these maps as prior information and will greatly enhance the impact of proteomics on biological and clinical research.


Assuntos
Espectrometria de Massas/métodos , Proteínas/análise , Proteoma/análise , Proteômica/métodos , Animais , Humanos , Reprodutibilidade dos Testes
9.
Genes Dev ; 28(17): 1879-84, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25184676

RESUMO

Bcl9 and Bcl9l (Bcl9/9l) encode Wnt signaling components that mediate the interaction between ß-catenin and Pygopus (Pygo) via two evolutionarily conserved domains, HD1 and HD2, respectively. We generated mouse strains lacking these domains to probe the ß-catenin-dependent and ß-catenin-independent roles of Bcl9/9l and Pygo during mouse development. While lens development is critically dependent on the presence of the HD1 domain, it is not affected by the lack of the HD2 domain, indicating that Bcl9/9l act in this context in a ß-catenin-independent manner. Furthermore, we uncover a new regulatory circuit in which Pax6, the master regulator of eye development, directly activates Bcl9/9l transcription.


Assuntos
Proteínas do Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Cristalino/embriologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Repressoras/metabolismo , Animais , Células Cultivadas , Proteínas do Olho/genética , Técnicas de Introdução de Genes , Proteínas de Homeodomínio/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Estrutura Terciária de Proteína/genética , Proteínas Repressoras/genética , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
10.
Nat Rev Mol Cell Biol ; 10(4): 276-86, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19305417

RESUMO

The canonical Wnt pathway has gathered much attention in recent years owing to its fundamental contribution to metazoan development, tissue homeostasis and human malignancies. Wnt target gene transcription is regulated by nuclear beta-catenin, and genetic assays have revealed various collaborating protein cofactors. Their daunting number and diverse nature, however, make it difficult to arrange an orderly picture of the nuclear Wnt transduction events. Yet, these findings emphasize that beta-catenin-mediated transcription affects chromatin. How does beta-catenin cope with chromatin regulation to turn on Wnt target genes?


Assuntos
Cromatina/metabolismo , Ativação Transcricional , Proteínas Wnt/genética , beta Catenina/metabolismo , Animais , Humanos
11.
Nature ; 522(7557): 482-6, 2015 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-25874673

RESUMO

Disruption of epithelial polarity is a key event in the acquisition of neoplastic growth. JNK signalling is known to play an important part in driving the malignant progression of many epithelial tumours, although the link between loss of polarity and JNK signalling remains elusive. In a Drosophila genome-wide genetic screen designed to identify molecules implicated in neoplastic growth, we identified grindelwald (grnd), a gene encoding a transmembrane protein with homology to members of the tumour necrosis factor receptor (TNFR) superfamily. Here we show that Grnd mediates the pro-apoptotic functions of Eiger (Egr), the unique Drosophila TNF, and that overexpression of an active form of Grnd lacking the extracellular domain is sufficient to activate JNK signalling in vivo. Grnd also promotes the invasiveness of Ras(V12)/scrib(-/-) tumours through Egr-dependent Matrix metalloprotease-1 (Mmp1) expression. Grnd localizes to the subapical membrane domain with the cell polarity determinant Crumbs (Crb) and couples Crb-induced loss of polarity with JNK activation and neoplastic growth through physical interaction with Veli (also known as Lin-7). Therefore, Grnd represents the first example of a TNFR that integrates signals from both Egr and apical polarity determinants to induce JNK-dependent cell death or tumour growth.


Assuntos
Polaridade Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Receptores do Fator de Necrose Tumoral/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/genética , Moléculas de Adesão Celular/metabolismo , Divisão Celular/genética , Polaridade Celular/genética , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Proteínas de Drosophila/química , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Feminino , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Metaloproteinase 1 da Matriz/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Dados de Sequência Molecular , Invasividade Neoplásica/genética , Neoplasias/enzimologia , Neoplasias/genética , Receptores do Fator de Necrose Tumoral/química , Receptores do Fator de Necrose Tumoral/genética , Proteínas ras/genética , Proteínas ras/metabolismo
12.
PLoS Genet ; 13(4): e1006700, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28369070

RESUMO

The hallmark of canonical Wnt signaling is the transcriptional induction of Wnt target genes by the beta-catenin/TCF complex. Several studies have proposed alternative interaction partners for beta-catenin or TCF, but the relevance of potential bifurcations in the distal Wnt pathway remains unclear. Here we study on a genome-wide scale the requirement for Armadillo (Arm, Drosophila beta-catenin) and Pangolin (Pan, Drosophila TCF) in the Wnt/Wingless(Wg)-induced transcriptional response of Drosophila Kc cells. Using somatic genetics, we demonstrate that both Arm and Pan are absolutely required for mediating activation and repression of target genes. Furthermore, by means of STARR-sequencing we identified Wnt/Wg-responsive enhancer elements and found that all responsive enhancers depend on Pan. Together, our results confirm the dogma of canonical Wnt/Wg signaling and argue against the existence of distal pathway branches in this system.


Assuntos
Proteínas do Domínio Armadillo/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt , Proteína Wnt1/metabolismo , Animais , Proteínas do Domínio Armadillo/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Genoma de Inseto , Proteínas Repressoras/genética , Análise de Sequência de RNA , Transdução de Sinais , Fatores de Transcrição/genética , Proteína Wnt1/genética
13.
Genes Dev ; 25(24): 2631-43, 2011 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-22190459

RESUMO

ß-Catenin, apart from playing a cell-adhesive role, is a key nuclear effector of Wnt signaling. Based on activity assays in Drosophila, we generated mouse strains where the endogenous ß-catenin protein is replaced by mutant forms, which retain the cell adhesion function but lack either or both of the N- and the C-terminal transcriptional outputs. The C-terminal activity is essential for mesoderm formation and proper gastrulation, whereas N-terminal outputs are required later during embryonic development. By combining the double-mutant ß-catenin with a conditional null allele and a Wnt1-Cre driver, we probed the role of Wnt/ß-catenin signaling in dorsal neural tube development. While loss of ß-catenin protein in the neural tube results in severe cell adhesion defects, the morphology of cells and tissues expressing the double-mutant form is normal. Surprisingly, Wnt/ß-catenin signaling activity only moderately regulates cell proliferation, but is crucial for maintaining neural progenitor identity and for neuronal differentiation in the dorsal spinal cord. Our model animals thus allow dissecting signaling and structural functions of ß-catenin in vivo and provide the first genetic tool to generate cells and tissues that entirely and exclusively lack canonical Wnt pathway activity.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , beta Catenina/genética , beta Catenina/metabolismo , Junções Aderentes/genética , Animais , Células Epiteliais/citologia , Células Epiteliais/patologia , Gastrulação/genética , Camundongos , Camundongos Endogâmicos , Mutação , Transdução de Sinais/genética , Medula Espinal/citologia , Medula Espinal/embriologia , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/genética
14.
J Mol Cell Cardiol ; 123: 150-158, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30201295

RESUMO

Abnormal endocardial cushion formation is a major cause of congenital heart valve disease, which is a common birth defect with significant morbidity and mortality. Although ß-catenin and BMP2 are two well-known regulators of endocardial cushion formation, their interaction in this process is largely unknown. Here, we report that deletion of ß-catenin in myocardium results in formation of hypoplastic endocardial cushions accompanying a decrease of mesenchymal cell proliferation. Loss of ß-catenin reduced Bmp2 expression in myocardium and SMAD signaling in cushion mesenchyme. Exogenous BMP2 recombinant proteins fully rescued the proliferation defect of mesenchymal cells in cultured heart explants from myocardial ß-catenin knockout embryos. Using a canonical WNT signaling reporter mouse line, we showed that cushion myocardium exhibited high WNT/ß-catenin activities during endocardial cushion growth. Selective disruption of the signaling function of ß-catenin resulted in a cushion growth defect similar to that caused by the complete loss of ß-catenin. Together, these observations demonstrate that myocardial ß-catenin signaling function promotes mesenchymal cell proliferation and endocardial cushion expansion through inducing BMP signaling.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Coxins Endocárdicos/metabolismo , Miocárdio/metabolismo , Organogênese , Transdução de Sinais , beta Catenina/metabolismo , Animais , Proliferação de Células , Coxins Endocárdicos/embriologia , Endocárdio/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Modelos Biológicos , Comunicação Parácrina , Ratos , Via de Sinalização Wnt
15.
Genome Res ; 25(4): 514-23, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25568052

RESUMO

Transcription factors (TFs) are key regulators of cell fate. The estimated 755 genes that encode DNA binding domain-containing proteins comprise ∼ 5% of all Drosophila genes. However, the majority has remained uncharacterized so far due to the lack of proper genetic tools. We generated 594 site-directed transgenic Drosophila lines that contain integrations of individual UAS-TF constructs to facilitate spatiotemporally controlled misexpression in vivo. All transgenes were expressed in the developing wing, and two-thirds induced specific phenotypic defects. In vivo knockdown of the same genes yielded a phenotype for 50%, with both methods indicating a great potential for misexpression to characterize novel functions in wing growth, patterning, and development. Thus, our UAS-TF library provides an important addition to the genetic toolbox of Drosophila research, enabling the identification of several novel wing development-related TFs. In parallel, we established the chromatin landscape of wing imaginal discs by ChIP-seq analyses of five chromatin marks and RNA Pol II. Subsequent clustering revealed six distinct chromatin states, with two clusters showing enrichment for both active and repressive marks. TFs that carry such "bivalent" chromatin are highly enriched for causing misexpression phenotypes in the wing, and analysis of existing expression data shows that these TFs tend to be differentially expressed across the wing disc. Thus, bivalently marked chromatin can be used as a marker for spatially regulated TFs that are functionally relevant in a developing tissue.


Assuntos
Padronização Corporal/genética , Drosophila melanogaster/embriologia , Discos Imaginais/embriologia , Fatores de Transcrição/genética , Asas de Animais/embriologia , Animais , Animais Geneticamente Modificados , Cromatina/genética , Cromatina/metabolismo , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Histonas/genética , Fenótipo , Regiões Promotoras Genéticas/genética , Estrutura Terciária de Proteína/genética , Interferência de RNA , RNA Polimerase II/genética , RNA Interferente Pequeno
16.
Eur Heart J ; 38(18): 1413-1425, 2017 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-27099262

RESUMO

AIMS: Myocardial fibrosis critically contributes to cardiac dysfunction in inflammatory dilated cardiomyopathy (iDCM). Activation of transforming growth factor-ß (TGF-ß) signalling is a key-step in promoting tissue remodelling and fibrosis in iDCM. Downstream mechanisms controlling these processes, remain elusive. METHODS AND RESULTS: Experimental autoimmune myocarditis (EAM) was induced in BALB/c mice with heart-specific antigen and adjuvant. Using heart-inflammatory precursors, as well as mouse and human cardiac fibroblasts, we demonstrated rapid secretion of Wnt proteins and activation of Wnt/ß-catenin pathway in response to TGF-ß signalling. Inactivation of extracellular Wnt with secreted Frizzled-related protein 2 (sFRP2) or inhibition of Wnt secretion with Wnt-C59 prevented TGF-ß-mediated transformation of inflammatory precursors and cardiac fibroblasts into pathogenic myofibroblasts. Inhibition of T-cell factor (TCF)/ß-catenin-mediated transcription with ICG-001 or genetic loss of ß-catenin also prevented TGF-ß-induced myofibroblasts formation. Furthermore, blocking of Smad-independent TGF-ß-activated kinase 1 (TAK1) pathway completely abrogated TGF-ß-induced Wnt secretion. Activation of Wnt pathway in the absence of TGF-ß, however, failed to transform precursors into myofibroblasts. The critical role of Wnt axis for cardiac fibrosis in iDCM is also supported by elevated Wnt-1/Wnt-5a levels in human samples from hearts with myocarditis. Accordingly, and as an in vivo proof of principle, inhibition of Wnt secretion or TCF/ß-catenin-mediated transcription abrogated the development of post-inflammatory fibrosis in EAM. CONCLUSION: We identified TAK1-mediated rapid Wnt protein secretion as a novel downstream key mechanism of TGF-ß-mediated myofibroblast differentiation and myocardial fibrosis progression in human and mouse myocarditis. Thus, pharmacological targeting of Wnts might represent a promising therapeutic approach against iDCM in the future.


Assuntos
Doenças Autoimunes/etiologia , Miocardite/etiologia , Miocárdio/patologia , Fator de Crescimento Transformador beta/fisiologia , Proteínas Wnt/metabolismo , Animais , Benzenoacetamidas/farmacologia , Diferenciação Celular/fisiologia , Progressão da Doença , Fibrose/fisiopatologia , Humanos , MAP Quinase Quinase Quinases/metabolismo , MAP Quinase Quinase Quinases/fisiologia , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos BALB C , Miofibroblastos/fisiologia , Piridinas/farmacologia , Transdução de Sinais/fisiologia , Células-Tronco/fisiologia , Fatores de Transcrição TCF/metabolismo , Disfunção Ventricular/fisiopatologia , Proteína Wnt-5a/metabolismo , Proteína Wnt1/metabolismo , beta Catenina/metabolismo
17.
Genes Dev ; 24(9): 881-6, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20439429

RESUMO

Wingless (Wg) signaling regulates expression of its target genes via Pangolin and Armadillo, and their interacting cofactors. In the absence of Wg, Pangolin mediates transcriptional repression. In the presence of Wg, Pangolin, Armadillo, and a cohort of coactivators mediate transcriptional activation. Here we uncover Coop (corepressor of Pan) as a Pangolin-interacting protein. Coop and Pangolin form a complex on DNA containing a Pangolin/TCF-binding motif. Overexpression of Coop specifically represses Wg target genes, while loss of Coop function causes derepression. Finally, we show that Coop antagonizes the binding of Armadillo to Pangolin, providing a mechanism for Coop-mediated repression of Wg target gene transcription.


Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Regulação da Expressão Gênica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Proteína Wnt1/metabolismo , Animais , Proteínas do Domínio Armadillo/metabolismo , Proteínas Correpressoras/metabolismo , Drosophila melanogaster/genética , Fatores de Transcrição/metabolismo
18.
Anal Chem ; 89(18): 9664-9670, 2017 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-28727418

RESUMO

Using label-free ToF-SIMS imaging mass spectrometry, we generated a map of small molecules differentially expressed in the Drosophila wing imaginal disc. The distributions of these moieties were in line with gene expression patterns observed during wing imaginal disc development. Combining ToF-SIMS imaging and coherent anti-Stokes Raman spectroscopy (CARS) microspectroscopy allowed us to locally identify acylglycerols as the main constituents of the pattern differentiating the future body wall tissue from the wing blade tissue. The findings presented herein clearly demonstrate that lipid localization patterns are strongly correlated with a developmental gene expression. From this correlation, we hypothesize that lipids play a so far unrecognized role in organ development.


Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Perfilação da Expressão Gênica , Glicerídeos/análise , Discos Imaginais/crescimento & desenvolvimento , Espectrometria de Massa de Íon Secundário , Asas de Animais/crescimento & desenvolvimento , Animais , Drosophila melanogaster/anatomia & histologia , Glicerídeos/genética , Discos Imaginais/anatomia & histologia , Análise Espectral Raman , Fatores de Tempo , Asas de Animais/anatomia & histologia
19.
Development ; 141(12): 2483-93, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24917503

RESUMO

The potent activity of Wnt/Wingless (Wg) signals necessitates sophisticated mechanisms that spatially and temporally regulate their distribution and range of action. The two main receptor components for Wg - Arrow (Arr) and Frizzled 2 (Fz2) - are transcriptionally downregulated by Wg signaling, thus forming gradients that oppose that of Wg. Here, we analyze the relevance of this transcriptional regulation for the formation of the Wg gradient in the Drosophila wing disc by combining in vivo receptor overexpression with an in silico model of Wg receptor interactions. Our experiments show that ubiquitous upregulation of Arr and Fz2 has no significant effects on Wg output, whereas clonal overexpression of these receptors leads to signaling discontinuities that have detrimental phenotypic consequences. These findings are supported by our in silico model for Wg diffusion and signal transduction, which suggests that abrupt changes in receptor levels causes discontinuities in Wg signaling. Furthermore, we identify a 200 bp regulatory element in the arr locus that can account for the Arr gradient, and we show that this is indirectly negatively controlled by Wg activity. Finally, we analyze the role of Frizzled 3 (Fz3) in this system and find that its expression, which is induced by Wg, contributes to the establishment of the Arr and Fz2 gradients through counteracting canonical signaling. Taken together, our results provide a model in which the regulatory network of Wg and the three receptor components account for the range and shape of this prototypical morphogen system.


Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteína Wnt1/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Receptores Frizzled/genética , Ligantes , Modelos Biológicos , Fenótipo , Regiões Promotoras Genéticas , Transdução de Sinais , Transcrição Gênica , Regulação para Cima , Asas de Animais/embriologia , Proteína Wnt1/genética
20.
PLoS Biol ; 12(11): e1001988, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25369031

RESUMO

The Wingless (Wg/Wnt) signaling pathway is essential for metazoan development, where it is central to tissue growth and cellular differentiation. Deregulated Wg pathway activation underlies severe developmental abnormalities, as well as carcinogenesis. Armadillo/ß-Catenin plays a key role in the Wg transduction cascade; its cytoplasmic and nuclear levels directly determine the output activity of Wg signaling and are thus tightly controlled. In all current models, once Arm is targeted for degradation by the Arm/ß-Catenin destruction complex, its fate is viewed as set. We identified a novel Wg/Wnt pathway component, Armless (Als), which is required for Wg target gene expression in a cell-autonomous manner. We found by genetic and biochemical analyses that Als functions downstream of the destruction complex, at the level of the SCF/Slimb/ßTRCP E3 Ub ligase. In the absence of Als, Arm levels are severely reduced. We show by biochemical and in vivo studies that Als interacts directly with Ter94, an AAA ATPase known to associate with E3 ligases and to drive protein turnover. We suggest that Als antagonizes Ter94's positive effect on E3 ligase function and propose that Als promotes Wg signaling by rescuing Arm from proteolytic degradation, spotlighting an unexpected step where the Wg pathway signal is modulated.


Assuntos
Proteínas do Domínio Armadillo/metabolismo , Proteínas de Drosophila/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , Proteína Wnt1/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Animais , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Drosophila , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Proteínas Hedgehog/metabolismo , Humanos , Receptores Notch/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína com Valosina , Asas de Animais/crescimento & desenvolvimento
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