RESUMO
The gene mutated in colorectal cancer (MCC) encodes a coiled-coil protein implicated, as its name suggests, in the pathogenesis of hereditary human colon cancer. To date, however, the contributions of MCC to intestinal homeostasis and disease remain unclear. Here, we examine the subcellular localization of MCC, both at the mRNA and protein levels, in the adult intestinal epithelium. Our findings reveal that Mcc transcripts are restricted to proliferating crypt cells, including Lgr5+ stem cells, where the Mcc protein is distinctly associated with the centrosome. Upon intestinal cellular differentiation, Mcc is redeployed to the apical domain of polarized villus cells where non-centrosomal microtubule organizing centers (ncMTOCs) are positioned. Using intestinal organoids, we show that the shuttling of the Mcc protein depends on phosphorylation by casein kinases 1δ and ε, which are critical modulators of WNT signaling. Together, our findings support a role for MCC in establishing and maintaining the cellular architecture of the intestinal epithelium as a component of both the centrosome and ncMTOC.
Assuntos
Centrossomo , Centro Organizador dos Microtúbulos , Humanos , Centro Organizador dos Microtúbulos/metabolismo , Centrossomo/metabolismo , Intestinos , Diferenciação Celular , Proteínas/metabolismo , Mucosa Intestinal/metabolismoRESUMO
BACKGROUND: Polypoidal choroidal vasculopathy (PCV), a subtype of age-related macular degeneration (AMD), is a global leading cause of vision loss in older populations. Distinct from typical AMD, PCV is characterized by polyp-like dilatation of blood vessels and turbulent blood flow in the choroid of the eye. Gold standard anti-vascular endothelial growth factor (anti-VEGF) therapy often fails to regress polypoidal lesions in patients. Current animal models have also been hampered by their inability to recapitulate such vascular lesions. These underscore the need to identify VEGF-independent pathways in PCV pathogenesis. RESULTS: We cultivated blood outgrowth endothelial cells (BOECs) from PCV patients and normal controls to serve as our experimental disease models. When BOECs were exposed to heterogeneous flow, single-cell transcriptomic analysis revealed that PCV BOECs preferentially adopted migratory-angiogenic cell state, while normal BOECs undertook proinflammatory cell state. PCV BOECs also had a repressed protective response to flow stress by demonstrating lower mitochondrial functions. We uncovered that elevated hyaluronidase-1 in PCV BOECs led to increased degradation of hyaluronan, a major component of glycocalyx that interfaces between flow stress and vascular endothelium. Notably, knockdown of hyaluronidase-1 in PCV BOEC improved mechanosensitivity, as demonstrated by a significant 1.5-fold upregulation of Krüppel-like factor 2 (KLF2) expression, a flow-responsive transcription factor. Activation of KLF2 might in turn modulate PCV BOEC migration. Barrier permeability due to glycocalyx impairment in PCV BOECs was also reversed by hyaluronidase-1 knockdown. Correspondingly, hyaluronidase-1 was detected in PCV patient vitreous humor and plasma samples. CONCLUSIONS: Hyaluronidase-1 inhibition could be a potential therapeutic modality in preserving glycocalyx integrity and endothelial stability in ocular diseases with vascular origin.
Assuntos
Hialuronoglucosaminidase , Degeneração Macular , Idoso , Corioide/irrigação sanguínea , Corioide/patologia , Células Endoteliais , Angiofluoresceinografia , Glicocálix/patologia , Humanos , Hialuronoglucosaminidase/genética , Hialuronoglucosaminidase/uso terapêutico , Degeneração Macular/tratamento farmacológico , Degeneração Macular/patologiaRESUMO
Basal cells are multipotent stem cells responsible for the repair and regeneration of all the epithelial cell types present in the proximal lung. In mice, the elusive origins of basal cells and their contribution to lung development were recently revealed by high-resolution, lineage tracing studies. It however remains unclear if human basal cells originate and participate in lung development in a similar fashion, particularly with mounting evidence for significant species-specific differences in this process. To address this outstanding question, in the last several years differentiation protocols incorporating human pluripotent stem cells (hPSC) have been developed to produce human basal cells in vitro with varying efficiencies. To facilitate this endeavour, we introduced tdTomato into the human TP63 gene, whose expression specifically labels basal cells, in the background of a previously described hPSC line harbouring an NKX2-1GFP reporter allele. The functionality and specificity of the NKX2-1GFP;TP63tdTomato hPSC line was validated by directed differentiation into lung progenitors as well as more specialised lung epithelial subtypes using an organoid platform. This dual fluorescent reporter hPSC line will be useful for tracking, isolating and expanding basal cells from heterogenous differentiation cultures for further study.
Assuntos
Proteínas de Fluorescência Verde/análise , Proteínas Luminescentes/análise , Pulmão/citologia , Células-Tronco Pluripotentes/citologia , Fator Nuclear 1 de Tireoide/análise , Fatores de Transcrição/análise , Proteínas Supressoras de Tumor/análise , Linhagem Celular , Proteínas de Fluorescência Verde/genética , Humanos , Proteínas Luminescentes/genética , Pulmão/metabolismo , Organoides/citologia , Organoides/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fator Nuclear 1 de Tireoide/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Proteína Vermelha FluorescenteRESUMO
A population of more than six million people worldwide at high risk of Alzheimer's disease (AD) are those with Down Syndrome (DS, caused by trisomy 21 (T21)), 70% of whom develop dementia during lifetime, caused by an extra copy of ß-amyloid-(Aß)-precursor-protein gene. We report AD-like pathology in cerebral organoids grown in vitro from non-invasively sampled strands of hair from 71% of DS donors. The pathology consisted of extracellular diffuse and fibrillar Aß deposits, hyperphosphorylated/pathologically conformed Tau, and premature neuronal loss. Presence/absence of AD-like pathology was donor-specific (reproducible between individual organoids/iPSC lines/experiments). Pathology could be triggered in pathology-negative T21 organoids by CRISPR/Cas9-mediated elimination of the third copy of chromosome 21 gene BACE2, but prevented by combined chemical ß and γ-secretase inhibition. We found that T21 organoids secrete increased proportions of Aß-preventing (Aß1-19) and Aß-degradation products (Aß1-20 and Aß1-34). We show these profiles mirror in cerebrospinal fluid of people with DS. We demonstrate that this protective mechanism is mediated by BACE2-trisomy and cross-inhibited by clinically trialled BACE1 inhibitors. Combined, our data prove the physiological role of BACE2 as a dose-sensitive AD-suppressor gene, potentially explaining the dementia delay in ~30% of people with DS. We also show that DS cerebral organoids could be explored as pre-morbid AD-risk population detector and a system for hypothesis-free drug screens as well as identification of natural suppressor genes for neurodegenerative diseases.
Assuntos
Doença de Alzheimer , Síndrome de Down , Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Encéfalo/metabolismo , Síndrome de Down/genética , Genes Supressores , Humanos , Organoides/metabolismo , TrissomiaRESUMO
Mitchell-Riley syndrome (MRS) is caused by recessive mutations in the regulatory factor X6 gene (RFX6) and is characterised by pancreatic hypoplasia and neonatal diabetes. To determine why individuals with MRS specifically lack pancreatic endocrine cells, we micro-CT imaged a 12-week-old foetus homozygous for the nonsense mutation RFX6 c.1129C>T, which revealed loss of the pancreas body and tail. From this foetus, we derived iPSCs and show that differentiation of these cells in vitro proceeds normally until generation of pancreatic endoderm, which is significantly reduced. We additionally generated an RFX6HA reporter allele by gene targeting in wild-type H9 cells to precisely define RFX6 expression and in parallel performed in situ hybridisation for RFX6 in the dorsal pancreatic bud of a Carnegie stage 14 human embryo. Both in vitro and in vivo, we find that RFX6 specifically labels a subset of PDX1-expressing pancreatic endoderm. In summary, RFX6 is essential for efficient differentiation of pancreatic endoderm, and its absence in individuals with MRS specifically impairs formation of endocrine cells of the pancreas head and tail.
Assuntos
Diferenciação Celular , Diabetes Mellitus/genética , Diabetes Mellitus/patologia , Endoderma/embriologia , Doenças da Vesícula Biliar/genética , Doenças da Vesícula Biliar/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Atresia Intestinal/genética , Atresia Intestinal/patologia , Mutação/genética , Pâncreas/embriologia , Fatores de Transcrição de Fator Regulador X/genética , Alelos , Sequência de Bases , Diferenciação Celular/genética , Cromatina/metabolismo , Consanguinidade , Diabetes Mellitus/diagnóstico por imagem , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Família , Feminino , Doenças da Vesícula Biliar/diagnóstico por imagem , Genoma Humano , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Atresia Intestinal/diagnóstico por imagem , Masculino , Linhagem , Transcrição Gênica , Transcriptoma/genética , Microtomografia por Raio-XRESUMO
Microfibril-associated glycoprotein 4 (MFAP4) is an extracellular matrix protein belonging to the fibrinogen-related protein superfamily. MFAP4 is produced by vascular smooth muscle cells and is highly enriched in the blood vessels of the heart and lung, where it is thought to contribute to the structure and function of elastic fibers. Genetic studies in humans have implicated MFAP4 in the pathogenesis of Smith-Magenis syndrome, in which patients present with multiple congenital abnormalities and mental retardation, as well as in the severe cardiac malformation left-sided congenital heart disease. Comprehensive genetic analysis of the role of MFAP4 orthologues in model organisms during development and tissue homeostasis is however lacking. Here, we demonstrate that zebrafish mfap4 transcripts are detected embryonically, resolving to the macrophage lineage by 24 h post fertilization. mfap4 null mutant zebrafish are unexpectedly viable and fertile, without ostensible phenotypes. However, tail fin amputation assays reveal that mfap4 mutants have reduced numbers of macrophages, with a concomitant increase in neutrophilic granulocytes, although recruitment of both cell types to the site of injury was unaffected. Molecular analyses suggest that loss of Mfap4 alters the balance between myeloid and lymphoid lineages during both primitive and definitive haematopoiesis, which could significantly impact the downstream function of the immune system.
Assuntos
Proteínas da Matriz Extracelular/genética , Hematopoese/genética , Peixe-Zebra/genética , Animais , Proteínas de Transporte , Desenvolvimento Embrionário/genética , Proteínas da Matriz Extracelular/metabolismo , Deleção de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Glicoproteínas , Humanos , Contagem de Leucócitos , Microfibrilas/metabolismo , Fenótipo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismoRESUMO
Holoprosencephaly (HPE) is a congenital forebrain defect often associated with embryonic lethality and lifelong disabilities. Currently, therapeutic and diagnostic options are limited by lack of knowledge of potential disease-causing mutations. We have identified a new mutation in the PRDM15 gene (C844Y) associated with a syndromic form of HPE in multiple families. We demonstrate that C844Y is a loss-of-function mutation impairing PRDM15 transcriptional activity. Genetic deletion of murine Prdm15 causes anterior/posterior (A/P) patterning defects and recapitulates the brain malformations observed in patients. Mechanistically, PRDM15 regulates the transcription of key effectors of the NOTCH and WNT/PCP pathways to preserve early midline structures in the developing embryo. Analysis of a large cohort of patients with HPE revealed potentially damaging mutations in several regulators of both pathways. Our findings uncover an unexpected link between NOTCH and WNT/PCP signaling and A/P patterning and set the stage for the identification of new HPE candidate genes.
Assuntos
Polaridade Celular , Proteínas de Ligação a DNA/genética , Holoprosencefalia/genética , Mutação com Perda de Função/genética , Receptores Notch/metabolismo , Fatores de Transcrição/genética , Via de Sinalização Wnt , Animais , Padronização Corporal/genética , Encéfalo/anormalidades , Encéfalo/embriologia , Polaridade Celular/genética , Estudos de Coortes , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/metabolismo , Feminino , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Placa Neural/metabolismo , Gravidez , Transcrição Gênica , Dedos de ZincoRESUMO
Heterozygous de novo mutations in GATA6 are the most frequent cause of pancreatic agenesis in humans. In mice, however, a similar phenotype requires the biallelic loss of Gata6 and its paralog Gata4. To elaborate the human-specific requirements for GATA6, we chose to model GATA6 loss in vitro by combining both gene-edited and patient-derived pluripotent stem cells (hPSCs) and directed differentiation toward ß-like cells. We find that GATA6 heterozygous hPSCs show a modest reduction in definitive endoderm (DE) formation, while GATA6-null hPSCs fail to enter the DE lineage. Consistent with these results, genome-wide studies show that GATA6 binds and cooperates with EOMES/SMAD2/3 to regulate the expression of cardinal endoderm genes. The early deficit in DE is accompanied by a significant reduction in PDX1+ pancreatic progenitors and C-PEPTIDE+ ß-like cells. Taken together, our data position GATA6 as a gatekeeper to early human, but not murine, pancreatic ontogeny.
Assuntos
Diferenciação Celular , Endoderma/metabolismo , Fator de Transcrição GATA6/genética , Redes Reguladoras de Genes , Células Secretoras de Insulina/metabolismo , Pâncreas/anormalidades , Pancreatopatias/congênito , Células-Tronco Pluripotentes/metabolismo , Linhagem da Célula , Células Cultivadas , Endoderma/citologia , Fator de Transcrição GATA6/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células Secretoras de Insulina/citologia , Pâncreas/metabolismo , Pancreatopatias/genética , Pancreatopatias/metabolismo , Células-Tronco Pluripotentes/citologia , Ligação Proteica , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
The historic town of Taos, New Mexico, with its rich multicultural history of art and craft, was the site of the second Keystone Symposium on 'Endoderm Development and Disease', which was held in February 2018. The theme of the meeting was 'Cross-Organ Comparison and Interplay', emphasizing an integrative and multisystem approach to the broad topics of organ physiology, homeostasis, repair, regeneration and disease. As we review here, participants shared their recent discoveries and discussed how new technologies developed in one organ system might be applied to answer crucial questions in another. Other integrative themes were how agents such as parasites, microbes, immune cells, physical forces and innervation can affect tissue organization and progenitor cell dynamics, and how defects in the development of an organ can impact its adult function. Participants came away with a broader vision of their field and a renewed sense of collective energy empowered by novel tools and fresh ideas.
Assuntos
Endoderma , Animais , Congressos como Assunto , Humanos , New MexicoRESUMO
Corneal tissue is the most transplanted of all body tissues. Currently, cadaveric donor tissues are used for transplantation. However, a global shortage of transplant grade material has prompted development of alternative, cell-based therapies for corneal diseases. Pluripotent stem cells are attractive sources of cells for regenerative medicine, because large numbers of therapeutically useful cells can be generated. However, a detailed understanding of how to differentiate clinically relevant cell types from stem cells is fundamentally required. Periocular mesenchyme (POM), a subtype of cranial neural crest, is vital for development of multiple cell types in the cornea, including clinically relevant cells such as corneal endothelium and stromal keratocytes. Herein, we describe protocols for differentiation of POM from pluripotent stem cells. Using defined media containing inhibitors of TGFß and WNT signalling, we generated neural crest cells that express high levels of the POM transcription factors PITX2 and FOXC1. Furthermore, we identified cells resembling POM in the adult cornea, located in a niche between the trabecular meshwork and peripheral endothelium. The generation and expansion of POM is an important step in the generation of a number of cells types that could prove to be clinically useful for a number of diseases of the cornea.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias Humanas/citologia , Crista Neural/citologia , Células-Tronco Pluripotentes/citologia , Células Cultivadas , Córnea/citologia , Humanos , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Preeclampsia (PE) is a gestational hypertensive syndrome affecting between 5 and 8% of all pregnancies. Although PE is the leading cause of fetal and maternal morbidity and mortality, its molecular etiology is still unclear. Here, we show that ELABELA (ELA), an endogenous ligand of the apelin receptor (APLNR, or APJ), is a circulating hormone secreted by the placenta. Elabela but not Apelin knockout pregnant mice exhibit PE-like symptoms, including proteinuria and elevated blood pressure due to defective placental angiogenesis. In mice, infusion of exogenous ELA normalizes hypertension, proteinuria, and birth weight. ELA, which is abundant in human placentas, increases the invasiveness of trophoblast-like cells, suggesting that it enhances placental development to prevent PE. The ELA-APLNR signaling axis may offer a new paradigm for the treatment of common pregnancy-related complications, including PE.
Assuntos
Anormalidades Cardiovasculares/genética , Proteínas de Transporte/genética , Hormônios Placentários/genética , Placentação/genética , Pré-Eclâmpsia/genética , Animais , Apelina/genética , Apelina/metabolismo , Peso ao Nascer , Proteínas de Transporte/administração & dosagem , Proteínas de Transporte/metabolismo , Proteínas de Transporte/farmacologia , Feminino , Camundongos , Camundongos Knockout , Neovascularização Fisiológica/genética , Hormônios Peptídicos , Placenta/irrigação sanguínea , Placenta/metabolismo , Gravidez , Proteinúria , Transdução de SinaisRESUMO
The transcriptional network acting downstream of LIF, WNT and MAPK-ERK to stabilize mouse embryonic stem cells (ESCs) in their naive state has been extensively characterized. However, the upstream factors regulating these three signaling pathways remain largely uncharted. PR-domain-containing proteins (PRDMs) are zinc-finger sequence-specific chromatin factors that have essential roles in embryonic development and cell fate decisions. Here we characterize the transcriptional regulator PRDM15, which acts independently of PRDM14 to regulate the naive state of mouse ESCs. Mechanistically, PRDM15 modulates WNT and MAPK-ERK signaling by directly promoting the expression of Rspo1 (R-spondin1) and Spry1 (Sprouty1). Consistent with these findings, CRISPR-Cas9-mediated disruption of PRDM15-binding sites in the Rspo1 and Spry1 promoters recapitulates PRDM15 depletion, both in terms of local chromatin organization and the transcriptional modulation of these genes. Collectively, our findings uncover an essential role for PRDM15 as a chromatin factor that modulates the transcription of upstream regulators of WNT and MAPK-ERK signaling to safeguard naive pluripotency.
Assuntos
Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Sistema de Sinalização das MAP Quinases/genética , Fatores de Transcrição/genética , Via de Sinalização Wnt/genética , Animais , Western Blotting , Linhagem Celular , Autorrenovação Celular/genética , Células Cultivadas , Reprogramação Celular/genética , Proteínas de Ligação a DNA/metabolismo , Imunofluorescência , Perfilação da Expressão Gênica/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos Knockout , Camundongos Nus , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismoRESUMO
Pluripotent stem cells have been proposed as an unlimited source of pancreatic ß cells for studying and treating diabetes. However, the long, multi-step differentiation protocols used to generate functional ß cells inevitably exhibit considerable variability, particularly when applied to pluripotent cells from diverse genetic backgrounds. We have developed culture conditions that support long-term self-renewal of human multipotent pancreatic progenitors, which are developmentally more proximal to the specialized cells of the adult pancreas. These cultured pancreatic progenitor (cPP) cells express key pancreatic transcription factors, including PDX1 and SOX9, and exhibit transcriptomes closely related to their in vivo counterparts. Upon exposure to differentiation cues, cPP cells give rise to pancreatic endocrine, acinar, and ductal lineages, indicating multilineage potency. Furthermore, cPP cells generate insulin+ ß-like cells in vitro and in vivo, suggesting that they offer a convenient alternative to pluripotent cells as a source of adult cell types for modeling pancreatic development and diabetes.
Assuntos
Autorrenovação Celular/fisiologia , Células-Tronco Pluripotentes/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Regulação para Baixo , Células Alimentadoras/citologia , Células Alimentadoras/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Insulina/farmacologia , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Rim/metabolismo , Rim/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pâncreas/citologia , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição SOX9/metabolismo , Células-Tronco/metabolismo , Transativadores/metabolismo , Transplante HeterólogoRESUMO
ELABELA (ELA) is a peptide hormone required for heart development that signals via the Apelin Receptor (APLNR, APJ). ELA is also abundantly secreted by human embryonic stem cells (hESCs), which do not express APLNR. Here we show that ELA signals in a paracrine fashion in hESCs to maintain self-renewal. ELA inhibition by CRISPR/Cas9-mediated deletion, shRNA, or neutralizing antibodies causes reduced hESC growth, cell death, and loss of pluripotency. Global phosphoproteomic and transcriptomic analyses of ELA-pulsed hESCs show that it activates PI3K/AKT/mTORC1 signaling required for cell survival. ELA promotes hESC cell-cycle progression and protein translation and blocks stress-induced apoptosis. INSULIN and ELA have partially overlapping functions in hESC medium, but only ELA can potentiate the TGFß pathway to prime hESCs toward the endoderm lineage. We propose that ELA, acting through an alternate cell-surface receptor, is an endogenous secreted growth factor in human embryos and hESCs that promotes growth and pluripotency.
Assuntos
Células-Tronco Embrionárias Humanas/metabolismo , Hormônios Peptídicos/metabolismo , Transdução de Sinais , Anticorpos Neutralizantes , Receptores de Apelina , Diferenciação Celular , Linhagem Celular , Autorrenovação Celular , Endoderma/citologia , Endoderma/metabolismo , Perfilação da Expressão Gênica , Células-Tronco Embrionárias Humanas/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Comunicação Parácrina , Fosfatidilinositol 3-Quinases/metabolismo , Proteômica , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Inactivation of the Pancreatic and Duodenal Homeobox 1 (PDX1) gene causes pancreatic agenesis, which places PDX1 high atop the regulatory network controlling development of this indispensable organ. However, little is known about the identity of PDX1 transcriptional targets. We simulated pancreatic development by differentiating human embryonic stem cells (hESCs) into early pancreatic progenitors and subjected this cell population to PDX1 chromatin immunoprecipitation sequencing (ChIP-seq). We identified more than 350 genes bound by PDX1, whose expression was upregulated on day 17 of differentiation. This group included known PDX1 targets and many genes not previously linked to pancreatic development. ChIP-seq also revealed PDX1 occupancy at hepatic genes. We hypothesized that simultaneous PDX1-driven activation of pancreatic and repression of hepatic programs underlie early divergence between pancreas and liver. In HepG2 cells and differentiating hESCs, we found that PDX1 binds and suppresses expression of endogenous liver genes. These findings rebrand PDX1 as a context-dependent transcriptional repressor and activator within the same cell type.
Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Pâncreas/citologia , Pâncreas/metabolismo , Transativadores/metabolismo , Sítios de Ligação , Biomarcadores , Linhagem Celular , Análise por Conglomerados , Biologia Computacional , Perfilação da Expressão Gênica , Humanos , Fígado/metabolismo , Motivos de Nucleotídeos , Especificidade de Órgãos/genética , Organogênese/genética , Matrizes de Pontuação de Posição Específica , Ligação Proteica , Elementos de Resposta , Transcrição GênicaRESUMO
During vertebrate gastrulation, a complex set of mass cellular rearrangements shapes the embryonic body plan and appropriately positions the organ primordia. In zebrafish and Xenopus, convergence and extension (CE) movements simultaneously narrow the body axis mediolaterally and elongate it from head to tail. This process is governed by polarized cell behaviors that are coordinated by components of the non-canonical, ß-catenin-independent Wnt signaling pathway, including Wnt5b and the transmembrane planar cell polarity (PCP) protein Vangl2. However, the intracellular events downstream of Wnt/PCP signals are not fully understood. Here, we show that zebrafish mutated in colorectal cancer (mcc), which encodes an evolutionarily conserved PDZ domain-containing putative tumor suppressor, is required for Wnt5b/Vangl2 signaling during gastrulation. Knockdown of mcc results in CE phenotypes similar to loss of vangl2 and wnt5b, whereas overexpression of mcc robustly rescues the depletion of wnt5b, vangl2 and the Wnt5b tyrosine kinase receptor ror2. Biochemical experiments establish a direct physical interaction between Mcc and the Vangl2 cytoplasmic tail. Lastly, CE defects in mcc morphants are suppressed by downstream activation of RhoA and JNK. Taken together, our results identify Mcc as a novel intracellular effector of non-canonical Wnt5b/Vangl2/Ror2 signaling during vertebrate gastrulation.
Assuntos
Gastrulação/fisiologia , Genes MCC/genética , Morfogênese/fisiologia , Via de Sinalização Wnt/fisiologia , Peixe-Zebra/embriologia , Animais , Western Blotting , Polaridade Celular/fisiologia , Imunoprecipitação , Hibridização In Situ , Luciferases , Proteínas de Membrana/metabolismo , Microscopia Confocal , Domínios PDZ/genética , Reação em Cadeia da Polimerase , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Proteínas Wnt/metabolismo , Proteína Wnt-5a , Proteínas de Peixe-Zebra/metabolismoRESUMO
A core network of genes maintaining pluripotency has been at least partially defined. How the genetic switch is flipped to differentiation is the subject of a new study that reveals some unexpected players.
RESUMO
Activin/Nodal signaling via SMAD2/3 maintains human embryonic stem cell (hESC) pluripotency by direct transcriptional regulation of NANOG or, alternatively, induces mesoderm and definitive endoderm (DE) formation. In search of an explanation for these contrasting effects, we focused on SNON (SKIL), a potent SMAD2/3 corepressor that is expressed in hESCs but rapidly down-regulated upon differentiation. We show that SNON predominantly associates with SMAD2 at the promoters of primitive streak (PS) and early DE marker genes. Knockdown of SNON results in premature activation of PS and DE genes and loss of hESC morphology. In contrast, enforced SNON expression inhibits DE formation and diverts hESCs toward an extraembryonic fate. Thus, our findings provide novel mechanistic insight into how a single signaling pathway both regulates pluripotency and directs lineage commitment.
Assuntos
Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Mesoderma/metabolismo , Transdução de Sinais , Proteína Smad2/genética , Proteína Smad3/genéticaRESUMO
Human embryonic stem cells (hESCs) herald tremendous promise for the production of clinically useful cell types for the treatment of injury and disease. Numerous reports demonstrate their differentiation into definitive endoderm (DE) cells, the germ layer from which pancreatic ß cells and hepatocytes arise, solely from exposure to a high dose of recombinant Activin/Nodal. We show that combining a second related ligand, BMP4, in combination with Activin A yields 15%-20% more DE as compared with Activin A alone. The addition of recombinant BMP4 accelerates the downregulation of pluripotency genes, particularly SOX2, and results in upregulation of endogenous BMP2 and BMP4, which in turn leads to elevated levels of phospho-SMAD1/5/8. Combined Activin A and BMP4 treatment also leads to an increase in the expression of DE genes CXCR4, SOX17, and FOXA2 when compared with Activin A addition alone. Comparative microarray studies between DE cells harvested on day 3 of differentiation further reveal a novel set of genes upregulated in response to initial BMP4 exposure. Several of these, including APLNR, LRIG3, MCC, LEPREL1, ROR2, and LZTS1, are expressed in the mouse primitive streak, the site of DE formation. Thus, this synergism between Activin A and BMP4 during the in vitro differentiation of hESC into DE suggests a complex interplay between BMP and Activin/Nodal signaling during the in vivo allocation and expansion of the endoderm lineage.