RESUMO
Endogenous retroviruses (ERVs) are an integral part of the mammalian genome. The role of immune control of ERVs in general is poorly defined as is their function as anti-cancer immune targets or drivers of autoimmune disease. Here, we generate mouse-strains where Moloney-Murine Leukemia Virus tagged with GFP (ERV-GFP) infected the mouse germline. This enables us to analyze the role of genetic, epigenetic and cell intrinsic restriction factors in ERV activation and control. We identify an autoreactive B cell response against the neo-self/ERV antigen GFP as a key mechanism of ERV control. Hallmarks of this response are spontaneous ERV-GFP+ germinal center formation, elevated serum IFN-γ levels and a dependency on Age-associated B cells (ABCs) a subclass of T-bet+ memory B cells. Impairment of IgM B cell receptor-signal in nucleic-acid sensing TLR-deficient mice contributes to defective ERV control. Although ERVs are a part of the genome they break immune tolerance, induce immune surveillance against ERV-derived self-antigens and shape the host immune response.
Assuntos
Linfócitos B , Retrovirus Endógenos , Animais , Camundongos , Doenças Autoimunes/genética , Linfócitos B/imunologia , Retrovirus Endógenos/genética , Mamíferos/genéticaRESUMO
It is generally accepted that epiblast cells ingress into the primitive streak by epithelial-to-mesenchymal transition (EMT) to give rise to the mesoderm; however, it is less clear how the endoderm acquires an epithelial fate. Here, we used embryonic stem cell and mouse embryo knock-in reporter systems to combine time-resolved lineage labelling with high-resolution single-cell transcriptomics. This allowed us to resolve the morphogenetic programs that segregate the mesoderm from the endoderm germ layer. Strikingly, while the mesoderm is formed by classical EMT, the endoderm is formed independent of the key EMT transcription factor Snail1 by mechanisms of epithelial cell plasticity. Importantly, forkhead box transcription factor A2 (Foxa2) acts as an epithelial gatekeeper and EMT suppressor to shield the endoderm from undergoing a mesenchymal transition. Altogether, these results not only establish the morphogenetic details of germ layer formation, but also have broader implications for stem cell differentiation and cancer metastasis.
Assuntos
Blastocisto/fisiologia , Plasticidade Celular , Endoderma/fisiologia , Células Epiteliais/fisiologia , Transição Epitelial-Mesenquimal , Gastrulação , Células-Tronco Embrionárias Murinas/fisiologia , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Diferenciação Celular , Linhagem Celular , Endoderma/citologia , Endoderma/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Fator 3-beta Nuclear de Hepatócito/genética , Fator 3-beta Nuclear de Hepatócito/metabolismo , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/metabolismo , Fenótipo , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Fatores de TempoRESUMO
The INK4 locus is considered as a hot-spot region for the complex genetic disorders, including cancer, type 2 diabetes (T2D) and coronary artery disease (CAD). By CRISPR/Cas9 gene editing, we generated a human induced pluripotent stem cell (hiPSC) line (HMGUi001-A-5) deleting an 8 kb genomic DNA encompassing six T2D-associated SNPs at the INK4 locus. The resulting hiPSC line revealed a normal karyotype, preserved pluripotency and was able to differentiate towards germ layers, endoderm, mesoderm and ectoderm. Thus, the HMGUi001-A-5 line could provide a valuable cellular model to explore the molecular mechanisms linking these SNPs to T2D and other genetic disorders.
RESUMO
The field of gene therapy is striving more than ever to define a path to the clinic and the market. Twenty gene therapy products have already been approved and over two thousand human gene therapy clinical trials have been reported worldwide. These advances raise great hope to treat devastating rare and inherited diseases as well as incurable illnesses. Understanding of the precise pathomechanisms of diseases as well as the development of efficient and specific gene targeting and delivery tools are revolutionizing the global market. Currently, human cancers and monogenic disorders are indications number one. The elevated prevalence of genetic disorders and cancers, clear gene manipulation guidelines and increasing financial support for gene therapy in clinical trials are major trends. Gene therapy is presently starting to become commercially profitable as a number of gene and cell-based gene therapy products have entered the market and the clinic. This article reviews the history and development of twenty approved human gene and cell-based gene therapy products that have been approved up-to-now in clinic and markets of mainly North America, Europe and Asia.
RESUMO
Cilia play a major role in the regulation of numerous signaling pathways and are essential for embryonic development. Mutations in genes affecting ciliary function can cause a variety of diseases in humans summarized as ciliopathies. To facilitate the detection and visualization of cilia in a temporal and spatial manner in mouse tissues, we generated a Cre-inducible cilium-specific reporter mouse line expressing an ARL13B-tRFP fusion protein driven by a CMV enhancer/chicken ß actin promotor (pCAG) from the Hprt locus. We detected bright and specific ciliary signals by immunostainings of various mono- and multiciliated tissues and by time-lapse live-cell analysis of cultured embryos and organ explant cultures. Additionally, we monitored cilium assembly and disassembly in embryonic fibroblast cells using live-cell imaging. Thus, the ARL13B-tRFP reporter mouse strain is a valuable tool for the investigation of ciliary structure and function in a tissue-specific manner to understand processes, such as ciliary protein trafficking or cilium-dependent signaling in vitro and in vivo.
Assuntos
Fatores de Ribosilação do ADP/genética , Células Epiteliais/metabolismo , Técnicas de Introdução de Genes/métodos , Genes Reporter , Proteínas Luminescentes/genética , Fatores de Ribosilação do ADP/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Cílios/genética , Cílios/metabolismo , Células Epiteliais/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Integrases/genética , Integrases/metabolismo , Proteínas Luminescentes/metabolismo , Camundongos , Proteína Vermelha FluorescenteRESUMO
The mechanisms underlying haematopoietic lineage decisions remain disputed. Lineage-affiliated transcription factors with the capacity for lineage reprogramming, positive auto-regulation and mutual inhibition have been described as being expressed in uncommitted cell populations. This led to the assumption that lineage choice is cell-intrinsically initiated and determined by stochastic switches of randomly fluctuating cross-antagonistic transcription factors. However, this hypothesis was developed on the basis of RNA expression data from snapshot and/or population-averaged analyses. Alternative models of lineage choice therefore cannot be excluded. Here we use novel reporter mouse lines and live imaging for continuous single-cell long-term quantification of the transcription factors GATA1 and PU.1 (also known as SPI1). We analyse individual haematopoietic stem cells throughout differentiation into megakaryocytic-erythroid and granulocytic-monocytic lineages. The observed expression dynamics are incompatible with the assumption that stochastic switching between PU.1 and GATA1 precedes and initiates megakaryocytic-erythroid versus granulocytic-monocytic lineage decision-making. Rather, our findings suggest that these transcription factors are only executing and reinforcing lineage choice once made. These results challenge the current prevailing model of early myeloid lineage choice.
Assuntos
Diferenciação Celular , Linhagem da Célula , Fator de Transcrição GATA1/metabolismo , Células Mieloides/citologia , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Animais , Eritrócitos/citologia , Retroalimentação Fisiológica , Feminino , Genes Reporter , Granulócitos/citologia , Hematopoese , Células-Tronco Hematopoéticas/citologia , Masculino , Megacariócitos/citologia , Camundongos , Modelos Biológicos , Monócitos/citologia , Reprodutibilidade dos Testes , Análise de Célula Única , Processos EstocásticosRESUMO
The HMG-box transcription factor Sox17 is essential for endoderm formation, vascular development, and definitive hematopoiesis. To investigate the fate of distinct Sox17-expressing progenitor cells in a spatiotemporal manner, we generated a hormone-inducible CreERT2 knock-in mouse line. By homologous recombination we fused a codon improved, ligand-dependent estrogen receptor Cre recombinase by an intervening viral T2A sequence for co-translational cleavage to the 3' coding region of Sox17. Induction of Cre activity by administration of tamoxifen at defined time points of early mouse development and subsequent genetic lineage tracing confirmed the inducibility and tissue specificity of Cre recombination. Furthermore, Cre activity could be selectively induced in extra-embryonic and embryonic endoderm lineages, the primitive gut tube, and in endothelial cells of the vascular system as well as in the hemogenic endothelium of the dorsal aorta. The Sox17CreERT2 mouse line therefore represents a new tool for genetic lineage tracing in a tissue-specific manner and in addition enables lineage-restricted functional analysis.
Assuntos
Linhagem da Célula , Células-Tronco Embrionárias/metabolismo , Técnicas de Introdução de Genes , Integrases/metabolismo , Fatores de Transcrição SOXF/genética , Animais , Aorta/metabolismo , Diferenciação Celular , Embrião de Mamíferos , Endoderma/metabolismo , Células Endoteliais/metabolismo , Gástrula/metabolismo , Genótipo , Células-Tronco Hematopoéticas/metabolismo , Integrases/genética , Camundongos , Camundongos Transgênicos , Modelos Animais , Especificidade de Órgãos , Tamoxifeno/farmacologiaRESUMO
A variety of developmental disorders have been associated with ciliary defects, yet the controls that govern cilia disassembly are largely unknown. Here we report a mouse embryonic node gene, which we named Pitchfork (Pifo). Pifo associates with ciliary targeting complexes and accumulates at the basal body during cilia disassembly. Haploinsufficiency causes a unique node cilia duplication phenotype, left-right asymmetry defects, and heart failure. This phenotype is likely relevant in humans, because we identified a heterozygous R80K PIFO mutation in a fetus with situs inversus and cystic liver and kidneys, and in patient with double-outflow right ventricle. We show that PIFO, but not R80K PIFO, is sufficient to activate Aurora A, a protooncogenic kinase that induces cilia retraction, and that Pifo/PIFO mutation causes cilia retraction, basal body liberation, and overreplication defects. Thus, the observation of a disassembly phenotype in vivo provides an entry point to understand and categorize ciliary disease. AUTHOR AUDIO:
Assuntos
Padronização Corporal/genética , Padronização Corporal/fisiologia , Cílios/genética , Cílios/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Sequência de Aminoácidos , Animais , Aurora Quinase A , Aurora Quinases , Centrossomo/ultraestrutura , Cílios/ultraestrutura , Dupla Via de Saída do Ventrículo Direito/genética , Feminino , Genes Homeobox , Heterozigoto , Humanos , Doenças Renais Císticas/embriologia , Doenças Renais Císticas/genética , Fígado/anormalidades , Masculino , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Mutação , Fenótipo , Gravidez , Proteínas Serina-Treonina Quinases/fisiologia , Homologia de Sequência de Aminoácidos , Situs Inversus/embriologia , Situs Inversus/genéticaRESUMO
Sox17 encodes an SRY-related high-mobility group (HMG) box transcription factor that is essential for endoderm formation and fetal hematopoietic stem cell maintenance. In the mouse, expression of Sox17 is first observed in the extraembryonic endoderm and is subsequently seen in the definitive endoderm as well as in blood and the endothelial cells of the developing vasculature. To conditionally inactivate genes in these domains, we have targeted the Sox17 locus to generate a bicistronic mRNA linking Sox17 to a codon improved Cre recombinase (iCre) via a viral 2A sequence. Here we report a new Cre knock-in mouse line, Sox17-2A-iCre, with activity in the developing endoderm, the vascular endothelial cells of the cardiovascular system and the hematopoietic system. Our results indicate that the Sox17-2A-iCre is active in an early endoderm progenitor and recombination of the Rosa26 reporter was observed in all previously reported expression domains of Sox17. The Sox17-2A-iCre line will be an excellent tool to conditionally inactivate genes in the definitive endoderm as well as in the vasculature and hematopoietic system.
Assuntos
Endoderma/metabolismo , Células Endoteliais/metabolismo , Integrases/metabolismo , Modelos Animais , Fatores de Transcrição SOXF/metabolismo , Animais , Southern Blotting , Primers do DNA/genética , Células-Tronco Embrionárias/metabolismo , Vetores Genéticos/genética , Genótipo , Camundongos , Camundongos Transgênicos , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
In the mouse, one of the earliest events in the determination of cell fate is the segregation of cells into germ layers during gastrulation; however, the cellular and molecular details are not well defined due to intrauterine development. We were able to visualize a clear sequence of events occurring in the process of germ-layer formation, using immunohistochemistry and time-lapse confocal imaging. The T-box transcription factor brachyury (T) and the Forkhead transcription factor Foxa2 specify mesoderm and endoderm in the posterior epiblast. Fate-specified epiblast cells lose their polarity and undergo epithelial-mesenchymal transition to invade into the primitive streak region, where these cell populations quickly separate and differentiate into morphologically and molecularly distinct Foxa2-positive endoderm and T-positive mesoderm populations. The endoderm cells flatten and acquire apical-basal polarity during intercalation into the outside epithelium in order to establish proper intracellular junctions with pre-existing cells. By contrast, the mesodermal cells become spherical during migration and acquire a mesenchymal fate. Interestingly, axial mesodermal cells are descended from Foxa2-positive epiblast cells that upregulate T protein in the anterior primitive streak region. These cells, as well as Foxa2-positive endoderm cells, are highly polarized and epithelialized, suggesting that Foxa2 promotes an epithelial fate and suppresses a mesenchymal fate. This observation is supported by the fact that Foxa2 mutant endodermal cells fail to maintain polarity and do not establish proper cellular junctions, and are thus unable to functionally integrate into the endoderm epithelium. We propose that Foxa2 regulates a molecular program that induces an epithelial cellular phenotype.
Assuntos
Endoderma/embriologia , Endoderma/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Camadas Germinativas/embriologia , Camadas Germinativas/metabolismo , Fator 3-beta Nuclear de Hepatócito/metabolismo , Animais , Adesão Celular , Linhagem Celular , Polaridade Celular , Forma Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Endoderma/citologia , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/citologia , Fator 3-beta Nuclear de Hepatócito/genética , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Mutação/genética , Fatores de TempoRESUMO
Transient myeloid disorder is a unique self-regressing neoplasia specific to Down's syndrome. The transcription factor GATA1 is needed for normal growth and maturation of erythroid cells and megakaryocytes. Mutations in GATA1 have been reported in acute megakaryoblastic leukaemia in Down's syndrome. We aimed to investigate changes in GATA1 in patients with Down's syndrome and either transient myeloid disorder (n=10) or acute megakaryoblastic leukaemia (n=6). We recorded mutations eliminating exon 2 from GATA1 in all patients with transient myeloid disorder (age 0-24 days) and in all with acute megakaryoblastic leukaemia (age 14-38 months). The range of mutations did not differ between patients with each disorder. Patients with transient myeloid disorder with mutations in GATA1 can regress spontaneously to complete remission, and mutations do not necessarily predict later acute megakaryoblastic leukaemia.
Assuntos
Proteínas de Ligação a DNA/genética , Síndrome de Down/genética , Leucemia Mieloide/genética , Fatores de Transcrição/genética , Pré-Escolar , Síndrome de Down/complicações , Fatores de Ligação de DNA Eritroide Específicos , Éxons/genética , Feminino , Fator de Transcrição GATA1 , Humanos , Lactente , Recém-Nascido , Cariotipagem , Leucemia Mieloide/complicações , Masculino , MutaçãoRESUMO
Expression of insulin-like growth factors (IGFs) and their cognate receptor, the IGF-1 receptor, is frequently upregulated during the development of many types of cancer. Besides stimulating cell cycle progression and the transformation status of tumor cells, a wealth of recent experimental data suggests that IGF-mediated signaling exerts a central tumor-promoting function through the repression of tumor cell apoptosis. These functions are all conveyed by the IGF-1 receptor, thus making it an attractive target for therapeutic intervention. Notably, inhibition of IGF-mediated survival function appears to synergize with conventional chemotherapeutic ablation of tumor cells, raising the possibility of combinatorial cancer therapies with significantly reduced side-effects. Copyright 1999 Harcourt Publishers Ltd.