RESUMEN
It is estimated that only 0.02% of disseminated tumour cells are able to seed overt metastases1. While this suggests the presence of environmental constraints to metastatic seeding, the landscape of host factors controlling this process remains largely unclear. Here, combining transposon technology2 and fluorescence niche labelling3, we developed an in vivo CRISPR activation screen to systematically investigate the interactions between hepatocytes and metastatic cells. We identify plexin B2 as a critical host-derived regulator of liver colonization in colorectal and pancreatic cancer and melanoma syngeneic mouse models. We dissect a mechanism through which plexin B2 interacts with class IV semaphorins on tumour cells, leading to KLF4 upregulation and thereby promoting the acquisition of epithelial traits. Our results highlight the essential role of signals from the liver parenchyma for the seeding of disseminated tumour cells before the establishment of a growth-promoting niche. Our findings further suggest that epithelialization is required for the adaptation of CRC metastases to their new tissue environment. Blocking the plexin-B2-semaphorin axis abolishes metastatic colonization of the liver and therefore represents a therapeutic strategy for the prevention of hepatic metastases. Finally, our screening approach, which evaluates host-derived extrinsic signals rather than tumour-intrinsic factors for their ability to promote metastatic seeding, is broadly applicable and lays a framework for the screening of environmental constraints to metastasis in other organs and cancer types.
Asunto(s)
Sistemas CRISPR-Cas , Hepatocitos , Neoplasias Hepáticas , Hígado , Metástasis de la Neoplasia , Proteínas del Tejido Nervioso , Animales , Femenino , Humanos , Masculino , Ratones , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Sistemas CRISPR-Cas/genética , Modelos Animales de Enfermedad , Elementos Transponibles de ADN , Fluorescencia , Hepatocitos/metabolismo , Hepatocitos/citología , Hepatocitos/patología , Factor 4 Similar a Kruppel/metabolismo , Hígado/citología , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/prevención & control , Neoplasias Hepáticas/secundario , Melanoma/metabolismo , Melanoma/patología , Metástasis de la Neoplasia/tratamiento farmacológico , Metástasis de la Neoplasia/patología , Metástasis de la Neoplasia/prevención & control , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Semaforinas/antagonistas & inhibidores , Semaforinas/metabolismoRESUMEN
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.
Asunto(s)
Colitis , Eosinófilos , Inmunidad , Intestinos , Animales , Humanos , Ratones , Colitis/inmunología , Colitis/patología , Eosinófilos/clasificación , Eosinófilos/citología , Eosinófilos/inmunología , Eosinófilos/metabolismo , Enfermedades Inflamatorias del Intestino/inmunología , Análisis de Expresión Génica de una Sola Célula , Transcriptoma , Proteoma , Interleucina-33 , Interferón gamma , Linfocitos T , Antígeno B7-1/metabolismo , Intestinos/inmunología , Intestinos/patologíaRESUMEN
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.
Asunto(s)
Cardiopatías Congénitas/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Factores de Transcripción/genética , Vía de Señalización Wnt , Proteínas de Pez Cebra/genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Corazón/embriología , Ratones , Mutación , Miocardio/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , beta Catenina/metabolismoRESUMEN
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.
Asunto(s)
Enterocitos , Mucosa Intestinal , Enterocitos/metabolismo , Ligandos , Mucosa Intestinal/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación CelularRESUMEN
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.
Asunto(s)
Colon/citología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Nicho de Células Madre/fisiología , Células Madre/metabolismo , Proteínas Wnt/metabolismo , Proteína con Dedos de Zinc GLI1/metabolismo , Animales , Autorrenovación de las Células , Femenino , Intestino Delgado/citología , Intestino Delgado/metabolismo , Masculino , Ratones , Células Madre/citología , Vía de Señalización WntRESUMEN
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.
Asunto(s)
Adhesión Celular/fisiología , Neoplasias Mamarias Animales/metabolismo , Transducción de Señal/fisiología , Proteína Wnt3A/metabolismo , beta Catenina/metabolismo , Animales , Apoptosis , Ciclo Celular , Movimiento Celular , Transición Epitelial-Mesenquimal/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Mamarias Animales/genética , Ratones , Ratones Transgénicos , Invasividad Neoplásica , Metástasis de la Neoplasia , Transcriptoma , Factor de Crecimiento Transformador beta/farmacología , Proteína Wnt3A/genética , beta Catenina/genéticaRESUMEN
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.
Asunto(s)
Colon/metabolismo , Fibroblastos/clasificación , Fibroblastos/metabolismo , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Colon/fisiología , Células Epiteliales/metabolismo , Femenino , Perfilación de la Expresión Génica/métodos , Homeostasis , Mucosa Intestinal/metabolismo , Intestinos/fisiología , Mesodermo/citología , Mesodermo/fisiología , Ratones , Ratones Endogámicos C57BL , Transducción de Señal , Análisis de la Célula Individual/métodos , Células Madre/citología , Transcriptoma/genéticaRESUMEN
Cannabis sativa L., a plant historically utilized for textile fibers, oil, and animal feed, is progressively being recognized as a potential food source. This review elucidates the nutritional and functional attributes of hemp and cannabidiol (CBD) within the context of food science. Hemp is characterized by the presence of approximately 545 secondary metabolites, among which around 144 are bioactive cannabinoids of primary importance. The study looks in detail at the nutritional components of cannabis and the potential health benefits of CBD, encompassing anti-inflammatory, anxiolytic, and antipsychotic effects. The review deals with the legislation and potential applications of hemp in the food industry and with the future directions of cannabis applications as well. The paper emphasizes the need for more scientific investigation to validate the safety and efficacy of hemp components in food products, as current research suggests that CBD may have great benefits for a wide range of consumers.
Asunto(s)
Cannabidiol , Cannabinoides , Cannabis , Alucinógenos , Animales , Cannabidiol/farmacología , Cannabinoides/farmacología , Suplementos DietéticosRESUMEN
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.
Asunto(s)
Proteínas del Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Cristalino/embriología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo , Animales , Células Cultivadas , Proteínas del Ojo/genética , Técnicas de Sustitución del Gen , Proteínas de Homeodominio/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Estructura Terciaria de Proteína/genética , Proteínas Represoras/genética , Transducción de Señal , Factores de Transcripción/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
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.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , beta Catenina/genética , beta Catenina/metabolismo , Uniones Adherentes/genética , Animales , Células Epiteliales/citología , Células Epiteliales/patología , Gastrulación/genética , Ratones , Ratones Endogámicos , Mutación , Transducción de Señal/genética , Médula Espinal/citología , Médula Espinal/embriología , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/genéticaRESUMEN
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.
Asunto(s)
Proteína Morfogenética Ósea 2/metabolismo , Cojinetes Endocárdicos/metabolismo , Miocardio/metabolismo , Organogénesis , Transducción de Señal , beta Catenina/metabolismo , Animales , Proliferación Celular , Cojinetes Endocárdicos/embriología , Endocardio/metabolismo , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Modelos Biológicos , Comunicación Paracrina , Ratas , Vía de Señalización WntRESUMEN
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.
Asunto(s)
Enfermedades Autoinmunes/etiología , Miocarditis/etiología , Miocardio/patología , Factor de Crecimiento Transformador beta/fisiología , Proteínas Wnt/metabolismo , Animales , Bencenoacetamidas/farmacología , Diferenciación Celular/fisiología , Progresión de la Enfermedad , Fibrosis/fisiopatología , Humanos , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/fisiología , Proteínas de la Membrana/metabolismo , Ratones Endogámicos BALB C , Miofibroblastos/fisiología , Piridinas/farmacología , Transducción de Señal/fisiología , Células Madre/fisiología , Factores de Transcripción TCF/metabolismo , Disfunción Ventricular/fisiopatología , Proteína Wnt-5a/metabolismo , Proteína Wnt1/metabolismo , beta Catenina/metabolismoRESUMEN
RATIONALE: Proper patterning of the atrioventricular canal (AVC) is essential for delay of electrical impulses between atria and ventricles, and defects in AVC maturation can result in congenital heart disease. OBJECTIVE: To determine the role of canonical Wnt signaling in the myocardium during AVC development. METHODS AND RESULTS: We used a novel allele of ß-catenin that preserves ß-catenin's cell adhesive functions but disrupts canonical Wnt signaling, allowing us to probe the effects of Wnt loss of function independently. We show that the loss of canonical Wnt signaling in the myocardium results in tricuspid atresia with hypoplastic right ventricle associated with the loss of AVC myocardium. In contrast, ectopic activation of Wnt signaling was sufficient to induce formation of ectopic AV junction-like tissue as assessed by morphology, gene expression, and electrophysiological criteria. Aberrant AVC development can lead to ventricular pre-excitation, a characteristic feature of Wolff-Parkinson-White syndrome. We demonstrate that postnatal activation of Notch signaling downregulates canonical Wnt targets within the AV junction. Stabilization of ß-catenin protein levels can rescue Notch-mediated ventricular pre-excitation and dysregulated ion channel gene expression. CONCLUSIONS: Our data demonstrate that myocardial canonical Wnt signaling is an important regulator of AVC maturation and electric programming upstream of Tbx3. Our data further suggest that ventricular pre-excitation may require both morphological patterning defects, as well as myocardial lineage reprogramming, to allow robust conduction across accessory pathway tissue.
Asunto(s)
Atrios Cardíacos/metabolismo , Sistema de Conducción Cardíaco/metabolismo , Ventrículos Cardíacos/metabolismo , Atresia Tricúspide/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo , Animales , Atrios Cardíacos/embriología , Atrios Cardíacos/fisiopatología , Sistema de Conducción Cardíaco/embriología , Sistema de Conducción Cardíaco/fisiopatología , Ventrículos Cardíacos/embriología , Ventrículos Cardíacos/fisiopatología , Ratones , Miocardio/metabolismo , Receptores Notch/metabolismo , Proteínas de Dominio T Box/metabolismo , Atresia Tricúspide/genética , Atresia Tricúspide/fisiopatología , beta Catenina/genéticaRESUMEN
ß-Catenin (Armadillo in Drosophila) is a multitasking and evolutionary conserved molecule that in metazoans exerts a crucial role in a multitude of developmental and homeostatic processes. More specifically, ß-catenin is an integral structural component of cadherin-based adherens junctions, and the key nuclear effector of canonical Wnt signalling in the nucleus. Imbalance in the structural and signalling properties of ß-catenin often results in disease and deregulated growth connected to cancer and metastasis. Intense research into the life of ß-catenin has revealed a complex picture. Here, we try to capture the state of the art: we try to summarize and make some sense of the processes that regulate ß-catenin, as well as the plethora of ß-catenin binding partners. One focus will be the interaction of ß-catenin with different transcription factors and the potential implications of these interactions for direct cross-talk between ß-catenin and non-Wnt signalling pathways.
Asunto(s)
Adhesión Celular , Eucariontes/fisiología , Vía de Señalización Wnt , beta Catenina/metabolismo , Regulación de la Expresión GénicaRESUMEN
Pygopus has been discovered as a fundamental Wnt signaling component in Drosophila. The mouse genome encodes two Pygopus homologs, Pygo1 and Pygo2. They serve as context-dependent ß-catenin coactivators, with Pygo2 playing the more important role. All Pygo proteins share a highly conserved plant homology domain (PHD) that allows them to bind di- and trimethylated lysine 4 of histone H3 (H3K4me2/3). Despite the structural conservation of this domain, the relevance of histone binding for the role of Pygo2 as a Wnt signaling component and as a reader of chromatin modifications remains speculative. Here we generate a knock-in mouse line, homozygous for a Pygo2 mutant defective in chromatin binding. We show that even in the absence of the potentially redundant Pygo1, Pygo2 does not require the H3K4me2/3 binding activity to sustain its function during mouse development. Indeed, during tissue homeostasis, Wnt/ß-catenin-dependent transcription is largely unaffected. However, the Pygo2-chromatin interaction is relevant in testes, where, importantly, Pygo2 binds in vivo to the chromatin in a PHD-dependent manner. Its presence on regulatory regions does not affect the transcription of nearby genes; rather, it is important for the recruitment of the histone acetyltransferase Gcn5 to chromatin, consistent with a testis-specific and Wnt-unrelated role for Pygo2 as a chromatin remodeler.
Asunto(s)
Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Histonas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Vía de Señalización Wnt , Animales , Ensamble y Desensamble de Cromatina , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/fisiología , Femenino , Fertilidad , Técnicas de Sustitución del Gen , Genotipo , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Dominios y Motivos de Interacción de Proteínas , Testículo/metabolismo , Factores de Transcripción p300-CBP/metabolismoRESUMEN
The fate of neural progenitor cells (NPCs) is determined by a complex interplay of intrinsic programs and extrinsic signals, very few of which are known. ß-Catenin transduces extracellular Wnt signals, but also maintains adherens junctions integrity. Here, we identify for the first time the contribution of ß-catenin transcriptional activity as opposed to its adhesion role in the development of the cerebral cortex by combining a novel ß-catenin mutant allele with conditional inactivation approaches. Wnt/ß-catenin signaling ablation leads to premature NPC differentiation, but, in addition, to a change in progenitor cell cycle kinetics and an increase in basally dividing progenitors. Interestingly, Wnt/ß-catenin signaling affects the sequential fate switch of progenitors, leading to a shortened neurogenic period with decreased number of both deep and upper-layer neurons and later, to precocious astrogenesis. Indeed, a genome-wide analysis highlighted the premature activation of a corticogenesis differentiation program in the Wnt/ß-catenin signaling-ablated cortex. Thus, ß-catenin signaling controls the expression of a set of genes that appear to act downstream of canonical Wnt signaling to regulate the stage-specific production of appropriate progenitor numbers, neuronal subpopulations, and astroglia in the forebrain.
Asunto(s)
Corteza Cerebral/citología , Células-Madre Neurales/citología , Neuronas/citología , Vía de Señalización Wnt , beta Catenina/metabolismo , Animales , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Corteza Cerebral/metabolismo , Ratones , Ratones Endogámicos C57BL , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: ß-catenin plays a central role in multiple developmental processes. However, it has been difficult to study its pleiotropic effects, because of the dual capacity of ß-catenin to coordinate cadherin-dependent cell adhesion and to act as a component of Wnt signal transduction. To distinguish between the divergent functions of ß-catenin during peripheral nervous system development, we made use of a mutant allele of ß-catenin that can mediate adhesion but not Wnt-induced TCF transcriptional activation. This allele was combined with various conditional inactivation approaches. RESULTS: We show that of all peripheral nervous system structures, only sensory dorsal root ganglia require ß-catenin for proper formation and growth. Surprisingly, however, dorsal root ganglia development is independent of cadherin-mediated cell adhesion. Rather, both progenitor cell proliferation and fate specification are controlled by ß-catenin signaling. These can be divided into temporally sequential processes, each of which depends on a different function of ß-catenin. CONCLUSIONS: While early stage proliferation and specific Neurog2- and Krox20-dependent waves of neuronal subtype specification involve activation of TCF transcription, late stage progenitor proliferation and Neurog1-marked sensory neurogenesis are regulated by a function of ß-catenin independent of TCF activation and adhesion. Thus, switching modes of ß-catenin function are associated with consecutive cell fate specification and stage-specific progenitor proliferation.
Asunto(s)
Neurogénesis , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/metabolismo , beta Catenina/metabolismo , Animales , Cadherinas/metabolismo , Adhesión Celular , Linaje de la Célula/genética , Movimiento Celular , Proliferación Celular , Ganglios Espinales/citología , Regulación del Desarrollo de la Expresión Génica , Ratones , Modelos Biológicos , Mutación/genética , Cresta Neural/citología , Células-Madre Neurales/citología , Fenotipo , Transducción de Señal , Factores de Transcripción TCF/metabolismo , Factores de Tiempo , Proteínas Wnt/metabolismo , alfa Catenina/metabolismoRESUMEN
BACKGROUND: Precise spatiotemporal control of gene expression is essential for the establishment of correct cell numbers and identities during brain development. This process involves epigenetic control mechanisms, such as those mediated by the polycomb group protein Ezh2, which catalyzes trimethylation of histone H3K27 (H3K27me3) and thereby represses gene expression. RESULTS: Herein, we show that Ezh2 plays a crucial role in the development and maintenance of the midbrain. Conditional deletion of Ezh2 in the developing midbrain resulted in decreased neural progenitor proliferation, which is associated with derepression of cell cycle inhibitors and negative regulation of Wnt/ß-catenin signaling. Of note, Ezh2 ablation also promoted ectopic expression of a forebrain transcriptional program involving derepression of the forebrain determinants Foxg1 and Pax6. This was accompanied by reduced expression of midbrain markers, including Pax3 and Pax7, as a consequence of decreased Wnt/ß-catenin signaling. CONCLUSION: Ezh2 is required for appropriate brain growth and maintenance of regional identity by H3K27me3-mediated gene repression and control of canonical Wnt signaling.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Mesencéfalo/crecimiento & desarrollo , Complejo Represivo Polycomb 2/genética , Prosencéfalo/crecimiento & desarrollo , Vía de Señalización Wnt , Animales , Proteína Potenciadora del Homólogo Zeste 2 , Epigénesis Genética , Mesencéfalo/embriología , Ratones , Complejo Represivo Polycomb 2/metabolismo , Prosencéfalo/embriologíaRESUMEN
In the context of growing interest in plant-based food products for their potential health benefits and sustainability, this study investigates the effect of mono- and diglycerides of fatty acids application on physico-chemical properties of various plant-based cream products, compared to lecithin application in rice cream. Rheological and textural parameters, colour profile, and colloidal stability were analysed. The application of mono- and diglycerides modified the creams' viscoelastic behaviour, showing a decrease in viscoelasticity across the samples; although in oat-coconut cream resulted in a higher viscoelasticity, indicating the formation of a gel-like structure. Rice cream with lecithin emulsifier showed lower viscoelastic properties characterised by higher phase angle (tan δ). All samples behaved as pseudoplastic materials (with a flow behaviour index n < 1). For coconut and almond creams, the consistency coefficient increased and flow behaviour index decreased after emulsifier application. Interestingly, the emulsifier addition did not significantly affect the cream's colour profile, characterised by yellow hue angle (h*) as a dominant spectral component. The colloidal stability, indicated by a stability index (SI), was determined as well.
RESUMEN
The single-layer epithelium of the gastrointestinal tract is a dynamically renewing tissue that ensures nutrient absorption, secretory and barrier functions and is involved in immune responses. The basis for this homeostatic renewal is the Wnt signaling pathway. Blocking this pathway can lead to epithelial damage, while its abnormal activation can result in the development of intestinal tumors. In this study, we investigated the dynamics of intestinal epithelial cells and tumorigenesis using a conditional mouse model. Using single-cell and bulk RNA sequencing and histological analysis, we elucidated the cellular responses following the loss of specific cell types. We focused on the fate of cells in the lower parts of the intestinal crypts and the development of colon adenomas. By partially inactivating the transcription factor Tcf4, a key effector of the Wnt signaling pathway, we analyzed the regeneration of isolated hyperproliferative foci (crypts). Our results suggest that the damaged epithelium is not restored by a specific regeneration program associated with oncofetal gene production, but rather by a standard homeostatic renewal pathway. Moreover, disruption of Tcf4 in secretory progenitors resulted in a significant shift in the cell lineage from Paneth cells to goblet cells, characterized by morphological changes and loss of Paneth cell-specific genes. We also found that hyperactivation of the Wnt signaling pathway in colonic adenomas correlated with the upregulation of genes typical of Paneth cells in the intestine, followed by the emergence of secretory tumor cells producing the Wnt3 ligand. The absence of Tcf4 led to a phenotypic shift of the tumor cells towards goblet cells. Our study presents a new model of epithelial regeneration based on the genetically driven partial elimination of intestinal crypts. We highlight the critical role of Tcf4 in the control of cell lineage decisions in the intestinal epithelium and colon tumors.