RESUMO
Persistent symptoms following SARS-CoV-2 infection are increasingly reported, although the drivers of post-acute sequelae (PASC) of COVID-19 are unclear. Here we assessed 214 individuals infected with SARS-CoV-2, with varying disease severity, for one year from COVID-19 symptom onset to determine the early correlates of PASC. A multivariate signature detected beyond two weeks of disease, encompassing unresolving inflammation, anemia, low serum iron, altered iron-homeostasis gene expression and emerging stress erythropoiesis; differentiated those who reported PASC months later, irrespective of COVID-19 severity. A whole-blood heme-metabolism signature, enriched in hospitalized patients at month 1-3 post onset, coincided with pronounced iron-deficient reticulocytosis. Lymphopenia and low numbers of dendritic cells persisted in those with PASC, and single-cell analysis reported iron maldistribution, suggesting monocyte iron loading and increased iron demand in proliferating lymphocytes. Thus, defects in iron homeostasis, dysregulated erythropoiesis and immune dysfunction due to COVID-19 possibly contribute to inefficient oxygen transport, inflammatory disequilibrium and persisting symptomatology, and may be therapeutically tractable.
Assuntos
COVID-19 , Ferro , Humanos , Eritropoese , SARS-CoV-2 , Pesquisadores , Progressão da DoençaRESUMO
Aged hematopoietic stem cells (HSCs) display diminished self-renewal and a myeloid differentiation bias. However, the drivers and mechanisms that underpin this fundamental switch are not understood. HSCs produce genotoxic formaldehyde that requires protection by the detoxification enzymes ALDH2 and ADH5 and the Fanconi anemia (FA) DNA repair pathway. We find that the HSCs in young Aldh2-/-Fancd2-/- mice harbor a transcriptomic signature equivalent to aged wild-type HSCs, along with increased epigenetic age, telomere attrition, and myeloid-biased differentiation quantified by single HSC transplantation. In addition, the p53 response is vigorously activated in Aldh2-/-Fancd2-/- HSCs, while p53 deletion rescued this aged HSC phenotype. To further define the origins of the myeloid differentiation bias, we use a GFP genetic reporter to find a striking enrichment of Vwf+ myeloid and megakaryocyte-lineage-biased HSCs. These results indicate that metabolism-derived formaldehyde-DNA damage stimulates the p53 response in HSCs to drive accelerated aging.
Assuntos
Envelhecimento , Aldeídos , Dano ao DNA , Hematopoese , Proteína Supressora de Tumor p53 , Animais , Camundongos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Aldeídos/metabolismo , Transcriptoma , Análise da Expressão Gênica de Célula Única , Células-Tronco Hematopoéticas/citologia , Células Mieloides/citologia , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologiaRESUMO
How cells respond to myriad stimuli with finite signaling machinery is central to immunology. In naive T cells, the inherent effect of ligand strength on activation pathways and endpoints has remained controversial, confounded by environmental fluctuations and intercellular variability within populations. Here we studied how ligand potency affected the activation of CD8+ T cells in vitro, through the use of genome-wide RNA, multi-dimensional protein and functional measurements in single cells. Our data revealed that strong ligands drove more efficient and uniform activation than did weak ligands, but all activated cells were fully cytolytic. Notably, activation followed the same transcriptional pathways regardless of ligand potency. Thus, stimulation strength did not intrinsically dictate the T cell-activation route or phenotype; instead, it controlled how rapidly and simultaneously the cells initiated activation, allowing limited machinery to elicit wide-ranging responses.
Assuntos
Linfócitos T CD8-Positivos/fisiologia , Citotoxicidade Imunológica , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Animais , Linhagem Celular , Genoma , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , RNA/genética , Transdução de Sinais , Análise de Célula ÚnicaRESUMO
The hierarchy of human hemopoietic progenitor cells that produce lymphoid and granulocytic-monocytic (myeloid) lineages is unclear. Multiple progenitor populations produce lymphoid and myeloid cells, but they remain incompletely characterized. Here we demonstrated that lympho-myeloid progenitor populations in cord blood - lymphoid-primed multi-potential progenitors (LMPPs), granulocyte-macrophage progenitors (GMPs) and multi-lymphoid progenitors (MLPs) - were functionally and transcriptionally distinct and heterogeneous at the clonal level, with progenitors of many different functional potentials present. Although most progenitors had the potential to develop into only one mature cell type ('uni-lineage potential'), bi- and rarer multi-lineage progenitors were present among LMPPs, GMPs and MLPs. Those findings, coupled with single-cell expression analyses, suggest that a continuum of progenitors execute lymphoid and myeloid differentiation, rather than only uni-lineage progenitors' being present downstream of stem cells.
Assuntos
Diferenciação Celular/genética , Perfilação da Expressão Gênica/métodos , Células Progenitoras Linfoides/metabolismo , Células Progenitoras Mieloides/metabolismo , Análise de Célula Única/métodos , Animais , Linhagem da Célula/genética , Separação Celular/métodos , Células Cultivadas , Hematopoese/genética , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Camundongos , Transplante HeterólogoRESUMO
The kinetics of the immune changes in COVID-19 across severity groups have not been rigorously assessed. Using immunophenotyping, RNA sequencing, and serum cytokine analysis, we analyzed serial samples from 207 SARS-CoV2-infected individuals with a range of disease severities over 12 weeks from symptom onset. An early robust bystander CD8+ T cell immune response, without systemic inflammation, characterized asymptomatic or mild disease. Hospitalized individuals had delayed bystander responses and systemic inflammation that was already evident near symptom onset, indicating that immunopathology may be inevitable in some individuals. Viral load did not correlate with this early pathological response but did correlate with subsequent disease severity. Immune recovery is complex, with profound persistent cellular abnormalities in severe disease correlating with altered inflammatory responses, with signatures associated with increased oxidative phosphorylation replacing those driven by cytokines tumor necrosis factor (TNF) and interleukin (IL)-6. These late immunometabolic and immune defects may have clinical implications.
Assuntos
Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , COVID-19/virologia , Interações Hospedeiro-Patógeno/imunologia , Ativação Linfocitária/imunologia , SARS-CoV-2/imunologia , Biomarcadores , Linfócitos T CD8-Positivos/metabolismo , COVID-19/diagnóstico , COVID-19/genética , Citocinas/metabolismo , Suscetibilidade a Doenças , Perfilação da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Estudos Longitudinais , Ativação Linfocitária/genética , Fosforilação Oxidativa , Fenótipo , Prognóstico , Espécies Reativas de Oxigênio/metabolismo , Índice de Gravidade de Doença , TranscriptomaRESUMO
During mammalian embryonic development, a single fertilized egg cell will proliferate and differentiate into all the cell lineages and cell types that eventually form the adult organism. Cell lineage diversification involves repeated cell fate choices that ultimately occur at the level of the individual cell rather than at the cell-population level. Improvements in single-cell technologies are transforming our understanding of mammalian development, not only by overcoming the limitations presented by the extremely low cell numbers of early embryos but also by enabling the study of cell fate specification in greater detail. In this Review, we first discuss recent advances in single-cell transcriptomics and imaging and provide a brief outline of current bioinformatics methods available to analyse the resulting data. We then discuss how these techniques have contributed to our understanding of pre-implantation and early post-implantation development and of in vitro pluripotency. Finally, we overview the current challenges facing single-cell research and highlight the latest advances and potential future avenues.
Assuntos
Embrião de Mamíferos/fisiologia , Transcrição Gênica/genética , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Humanos , Transcrição Gênica/fisiologia , Transcriptoma/genética , Transcriptoma/fisiologiaRESUMO
It is not fully understood why COVID-19 is typically milder in children1-3. Here, to examine the differences between children and adults in their response to SARS-CoV-2 infection, we analysed paediatric and adult patients with COVID-19 as well as healthy control individuals (total n = 93) using single-cell multi-omic profiling of matched nasal, tracheal, bronchial and blood samples. In the airways of healthy paediatric individuals, we observed cells that were already in an interferon-activated state, which after SARS-CoV-2 infection was further induced especially in airway immune cells. We postulate that higher paediatric innate interferon responses restrict viral replication and disease progression. The systemic response in children was characterized by increases in naive lymphocytes and a depletion of natural killer cells, whereas, in adults, cytotoxic T cells and interferon-stimulated subpopulations were significantly increased. We provide evidence that dendritic cells initiate interferon signalling in early infection, and identify epithelial cell states associated with COVID-19 and age. Our matching nasal and blood data show a strong interferon response in the airways with the induction of systemic interferon-stimulated populations, which were substantially reduced in paediatric patients. Together, we provide several mechanisms that explain the milder clinical syndrome observed in children.
Assuntos
COVID-19/sangue , COVID-19/imunologia , Células Dendríticas/imunologia , Interferons/imunologia , Células Matadoras Naturais/imunologia , SARS-CoV-2/imunologia , Linfócitos T Citotóxicos/imunologia , Adulto , Brônquios/imunologia , Brônquios/virologia , COVID-19/patologia , Chicago , Estudos de Coortes , Progressão da Doença , Células Epiteliais/citologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Feminino , Humanos , Imunidade Inata , Londres , Masculino , Mucosa Nasal/imunologia , Mucosa Nasal/virologia , SARS-CoV-2/crescimento & desenvolvimento , Análise de Célula Única , Traqueia/virologia , Adulto JovemRESUMO
Reactive aldehydes arise as by-products of metabolism and are normally cleared by multiple families of enzymes. We find that mice lacking two aldehyde detoxifying enzymes, mitochondrial ALDH2 and cytoplasmic ADH5, have greatly shortened lifespans and develop leukemia. Hematopoiesis is disrupted profoundly, with a reduction of hematopoietic stem cells and common lymphoid progenitors causing a severely depleted acquired immune system. We show that formaldehyde is a common substrate of ALDH2 and ADH5 and establish methods to quantify elevated blood formaldehyde and formaldehyde-DNA adducts in tissues. Bone-marrow-derived progenitors actively engage DNA repair but also imprint a formaldehyde-driven mutation signature similar to aging-associated human cancer mutation signatures. Furthermore, we identify analogous genetic defects in children causing a previously uncharacterized inherited bone marrow failure and pre-leukemic syndrome. Endogenous formaldehyde clearance alone is therefore critical for hematopoiesis and in limiting mutagenesis in somatic tissues.
Assuntos
Álcool Desidrogenase/genética , Aldeído-Desidrogenase Mitocondrial/genética , Formaldeído/sangue , Leucemia/genética , Adolescente , Aldeídos/sangue , Animais , Criança , Pré-Escolar , Adutos de DNA/genética , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Feminino , Formaldeído/toxicidade , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Lactente , Leucemia/sangue , Leucemia/patologia , Masculino , Camundongos , Mutação/genética , Especificidade por SubstratoRESUMO
Knowledge of locations and activities of cis-regulatory elements (CREs) is needed to decipher basic mechanisms of gene regulation and to understand the impact of genetic variants on complex traits. Previous studies identified candidate CREs (cCREs) using epigenetic features in one species, making comparisons difficult between species. In contrast, we conducted an interspecies study defining epigenetic states and identifying cCREs in blood cell types to generate regulatory maps that are comparable between species, using integrative modeling of eight epigenetic features jointly in human and mouse in our Validated Systematic Integration (VISION) Project. The resulting catalogs of cCREs are useful resources for further studies of gene regulation in blood cells, indicated by high overlap with known functional elements and strong enrichment for human genetic variants associated with blood cell phenotypes. The contribution of each epigenetic state in cCREs to gene regulation, inferred from a multivariate regression, was used to estimate epigenetic state regulatory potential (esRP) scores for each cCRE in each cell type, which were used to categorize dynamic changes in cCREs. Groups of cCREs displaying similar patterns of regulatory activity in human and mouse cell types, obtained by joint clustering on esRP scores, harbor distinctive transcription factor binding motifs that are similar between species. An interspecies comparison of cCREs revealed both conserved and species-specific patterns of epigenetic evolution. Finally, we show that comparisons of the epigenetic landscape between species can reveal elements with similar roles in regulation, even in the absence of genomic sequence alignment.
RESUMO
Early organogenesis represents a key step in animal development, during which pluripotent cells diversify to initiate organ formation. Here, we sampled 300,000 single-cell transcriptomes from mouse embryos between E8.5 and E9.5 in 6-h intervals and combined this new dataset with our previous atlas (E6.5-E8.5) to produce a densely sampled timecourse of >400,000 cells from early gastrulation to organogenesis. Computational lineage reconstruction identified complex waves of blood and endothelial development, including a new programme for somite-derived endothelium. We also dissected the E7.5 primitive streak into four adjacent regions, performed scRNA-seq and predicted cell fates computationally. Finally, we defined developmental state/fate relationships by combining orthotopic grafting, microscopic analysis and scRNA-seq to transcriptionally determine cell fates of grafted primitive streak regions after 24â h of in vitro embryo culture. Experimentally determined fate outcomes were in good agreement with computationally predicted fates, demonstrating how classical grafting experiments can be revisited to establish high-resolution cell state/fate relationships. Such interdisciplinary approaches will benefit future studies in developmental biology and guide the in vitro production of cells for organ regeneration and repair.
Assuntos
Gastrulação , Organogênese , Camundongos , Animais , Diferenciação Celular , Organogênese/genética , Linha Primitiva , Endotélio , Embrião de Mamíferos , MamíferosRESUMO
The Human Developmental Cell Atlas (HDCA) initiative, which is part of the Human Cell Atlas, aims to create a comprehensive reference map of cells during development. This will be critical to understanding normal organogenesis, the effect of mutations, environmental factors and infectious agents on human development, congenital and childhood disorders, and the cellular basis of ageing, cancer and regenerative medicine. Here we outline the HDCA initiative and the challenges of mapping and modelling human development using state-of-the-art technologies to create a reference atlas across gestation. Similar to the Human Genome Project, the HDCA will integrate the output from a growing community of scientists who are mapping human development into a unified atlas. We describe the early milestones that have been achieved and the use of human stem-cell-derived cultures, organoids and animal models to inform the HDCA, especially for prenatal tissues that are hard to acquire. Finally, we provide a roadmap towards a complete atlas of human development.
Assuntos
Movimento Celular , Rastreamento de Células , Células/citologia , Biologia do Desenvolvimento/métodos , Embrião de Mamíferos/citologia , Feto/citologia , Disseminação de Informação , Organogênese , Adulto , Animais , Atlas como Assunto , Técnicas de Cultura de Células , Sobrevivência Celular , Visualização de Dados , Feminino , Humanos , Imageamento Tridimensional , Masculino , Modelos Animais , Organogênese/genética , Organoides/citologia , Células-Tronco/citologiaRESUMO
ABSTRACT: Loss of long-term hematopoietic stem cell (LT-HSC) function ex vivo hampers the success of clinical protocols that rely on culture. However, the kinetics and mechanisms through which this occurs remain incompletely characterized. In this study, through time-resolved single-cell RNA sequencing, matched in vivo functional analysis, and the use of a reversible in vitro system of early G1 arrest, we defined the sequence of transcriptional and functional events that occur during the first ex vivo division of human LT-HSCs. We demonstrated that the sharpest loss in LT-HSC repopulation capacity happens early on, between 6 and 24 hours of culture, before LT-HSCs commit to cell cycle progression. During this time window, LT-HSCs adapt to the culture environment, limit the global variability in gene expression, and transiently upregulate gene networks involved in signaling and stress responses. From 24 hours, LT-HSC progression past early G1 contributes to the establishment of differentiation programs in culture. However, contrary to the current assumptions, we demonstrated that the loss of HSC function ex vivo is independent of cell cycle progression. Finally, we showed that targeting LT-HSC adaptation to culture by inhibiting the early activation of JAK/STAT signaling improves HSC long-term repopulating function ex vivo. Collectively, our study demonstrated that controlling early LT-HSC adaptation to ex vivo culture, for example, via JAK inhibition, is critically important to improve HSC gene therapy and expansion protocols.
Assuntos
Ciclo Celular , Células-Tronco Hematopoéticas , Humanos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Células Cultivadas , Transdução de Sinais , Diferenciação Celular , Técnicas de Cultura de Células/métodos , Adaptação FisiológicaRESUMO
In COVID-19, hyperinflammatory and dysregulated immune responses contribute to severity. Patients with pre-existing autoimmune conditions can therefore be at increased risk of severe COVID-19 and/or associated sequelae, yet SARS-CoV-2 infection in this group has been little studied. Here, we performed single-cell analysis of peripheral blood mononuclear cells from patients with three major autoimmune diseases (rheumatoid arthritis, psoriasis, or multiple sclerosis) during SARS-CoV-2 infection. We observed compositional differences between the autoimmune disease groups coupled with altered patterns of gene expression, transcription factor activity, and cell-cell communication that substantially shape the immune response under SARS-CoV-2 infection. While enrichment of HLA-DRlow CD14+ monocytes was observed in all three autoimmune disease groups, type-I interferon signaling as well as inflammatory T cell and monocyte responses varied widely between the three groups of patients. Our results reveal disturbed immune responses to SARS-CoV-2 in patients with pre-existing autoimmunity, highlighting important considerations for disease treatment and follow-up.
Assuntos
Doenças Autoimunes , COVID-19 , Humanos , SARS-CoV-2 , Leucócitos Mononucleares , Multiômica , Autoimunidade , Análise de Célula ÚnicaRESUMO
Hematopoietic stem and progenitor cells (HSPCs) rely on a complex interplay among transcription factors (TFs) to regulate differentiation into mature blood cells. A heptad of TFs (FLI1, ERG, GATA2, RUNX1, TAL1, LYL1, LMO2) bind regulatory elements in bulk CD34+ HSPCs. However, whether specific heptad-TF combinations have distinct roles in regulating hematopoietic differentiation remains unknown. We mapped genome-wide chromatin contacts (HiC, H3K27ac, HiChIP), chromatin modifications (H3K4me3, H3K27ac, H3K27me3) and 10 TF binding profiles (heptad, PU.1, CTCF, STAG2) in HSPC subsets (stem/multipotent progenitors plus common myeloid, granulocyte macrophage, and megakaryocyte erythrocyte progenitors) and found TF occupancy and enhancer-promoter interactions varied significantly across cell types and were associated with cell-type-specific gene expression. Distinct regulatory elements were enriched with specific heptad-TF combinations, including stem-cell-specific elements with ERG, and myeloid- and erythroid-specific elements with combinations of FLI1, RUNX1, GATA2, TAL1, LYL1, and LMO2. Furthermore, heptad-occupied regions in HSPCs were subsequently bound by lineage-defining TFs, including PU.1 and GATA1, suggesting that heptad factors may prime regulatory elements for use in mature cell types. We also found that enhancers with cell-type-specific heptad occupancy shared a common grammar with respect to TF binding motifs, suggesting that combinatorial binding of TF complexes was at least partially regulated by features encoded in DNA sequence motifs. Taken together, this study comprehensively characterizes the gene regulatory landscape in rare subpopulations of human HSPCs. The accompanying data sets should serve as a valuable resource for understanding adult hematopoiesis and a framework for analyzing aberrant regulatory networks in leukemic cells.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core , Células-Tronco Hematopoéticas , Humanos , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Regulação da Expressão Gênica , Hematopoese/genética , Cromatina/metabolismoRESUMO
Despite the overwhelming evidence that multiple sclerosis is an autoimmune disease, relatively little is known about the precise nature of the immune dysregulation underlying the development of the disease. Reasoning that the CSF from patients might be enriched for cells relevant in pathogenesis, we have completed a high-resolution single-cell analysis of 96 732 CSF cells collected from 33 patients with multiple sclerosis (n = 48 675) and 48 patients with other neurological diseases (n = 48 057). Completing comprehensive cell type annotation, we identified a rare population of CD8+ T cells, characterized by the upregulation of inhibitory receptors, increased in patients with multiple sclerosis. Applying a Multi-Omics Factor Analysis to these single-cell data further revealed that activity in pathways responsible for controlling inflammatory and type 1 interferon responses are altered in multiple sclerosis in both T cells and myeloid cells. We also undertook a systematic search for expression quantitative trait loci in the CSF cells. Of particular interest were two expression quantitative trait loci in CD8+ T cells that were fine mapped to multiple sclerosis susceptibility variants in the viral control genes ZC3HAV1 (rs10271373) and IFITM2 (rs1059091). Further analysis suggests that these associations likely reflect genetic effects on RNA splicing and cell-type specific gene expression respectively. Collectively, our study suggests that alterations in viral control mechanisms might be important in the development of multiple sclerosis.
Assuntos
Esclerose Múltipla , Humanos , Linfócitos T CD8-Positivos , Regulação para Cima , Antivirais , Líquido Cefalorraquidiano/metabolismo , Proteínas de Membrana/genéticaRESUMO
Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time points ranging from 6.5 to 8.5 days post-fertilization. We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we use single-cell profiling to show that Tal1-/- chimeric embryos display defects in early mesoderm diversification, and we thus demonstrate how combining temporal and transcriptional information can illuminate gene function. Together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development, as well as a roadmap for the optimization of in vitro differentiation protocols for regenerative medicine.
Assuntos
Diferenciação Celular/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Gastrulação , Organogênese , Análise de Célula Única , Animais , Linhagem da Célula/genética , Quimera/embriologia , Quimera/genética , Quimera/metabolismo , Endoderma/citologia , Endoderma/embriologia , Endoderma/metabolismo , Endotélio/citologia , Endotélio/embriologia , Endotélio/metabolismo , Feminino , Gastrulação/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Hematopoese/genética , Masculino , Mesoderma/citologia , Mesoderma/embriologia , Camundongos , Mutação/genética , Células Mieloides/citologia , Organogênese/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Linha Primitiva/citologia , Linha Primitiva/embriologia , Proteína 1 de Leucemia Linfocítica Aguda de Células T/deficiência , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genéticaRESUMO
Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan and is associated with major transcriptional changes1-5. Global epigenetic reprogramming accompanies these changes6-8, but the role of the epigenome in regulating early cell-fate choice remains unresolved, and the coordination between different molecular layers is unclear. Here we describe a single-cell multi-omics map of chromatin accessibility, DNA methylation and RNA expression during the onset of gastrulation in mouse embryos. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements at enhancer marks, driven by ten-eleven translocation (TET)-mediated demethylation and a concomitant increase of accessibility. By contrast, the methylation and accessibility landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or remodelled before cell-fate decisions, providing the molecular framework for a hierarchical emergence of the primary germ layers.
Assuntos
Metilação de DNA , Epigênese Genética , Gástrula/citologia , Gástrula/metabolismo , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , RNA/genética , Análise de Célula Única , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Cromatina/genética , Cromatina/metabolismo , Desmetilação , Corpos Embrioides/citologia , Endoderma/citologia , Endoderma/embriologia , Endoderma/metabolismo , Elementos Facilitadores Genéticos/genética , Epigenoma/genética , Eritropoese , Análise Fatorial , Gástrula/embriologia , Gastrulação/fisiologia , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , RNA/análise , Fatores de Tempo , Dedos de ZincoRESUMO
Recent advances in molecular profiling provide descriptive datasets of complex differentiation landscapes including the haematopoietic system, but the molecular mechanisms defining progenitor states and lineage choice remain ill-defined. Here, we employed a cellular model of murine multipotent haematopoietic progenitors (Hoxb8-FL) to knock out 39 transcription factors (TFs) followed by RNA-Seq analysis, to functionally define a regulatory network of 16,992 regulator/target gene links. Focussed analysis of the subnetworks regulated by the B-lymphoid TF Ebf1 and T-lymphoid TF Gata3 revealed a surprising role in common activation of an early myeloid programme. Moreover, Gata3-mediated repression of Pax5 emerges as a mechanism to prevent precocious B-lymphoid differentiation, while Hox-mediated activation of Meis1 suppresses myeloid differentiation. To aid interpretation of large transcriptomics datasets, we also report a new method that visualises likely transitions that a progenitor will undergo following regulatory network perturbations. Taken together, this study reveals how molecular network wiring helps to establish a multipotent progenitor state, with experimental approaches and analysis tools applicable to dissecting a broad range of both normal and perturbed cellular differentiation landscapes.
Assuntos
Linhagem da Célula/fisiologia , Sistema Hematopoético/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula/genética , Epigenômica , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Camundongos , Proteína Meis1/metabolismo , Fator de Transcrição PAX5/genética , Fator de Transcrição PAX5/metabolismo , Células Precursoras de Linfócitos B , Fatores de Transcrição/genéticaRESUMO
Thymopoiesis depends on the recruitment and expansion of bone marrow-derived progenitor populations; tight regulation of these processes is required for maintenance of the homeostasis of the T lineage. Lyl-1, a transcription factor that regulates hematopoietic progenitors, is expressed in thymocyte progenitors until T cell commitment. Here we demonstrate a requirement for Lyl-1 in lymphoid specification and the maintenance of early T lineage progenitors (ETPs). Lyl-1 deficiency resulted in profound defects in the generation of lymphoid-primed multipotent progenitors (LMPPs), common lymphoid progenitors (CLPs) and ETPs. Lyl-1-deficient ETPs and thymocyte progenitors at the CD4(-)CD8(-) double-negative 2 (DN2) stage showed more apoptosis, blocked differentiation and impaired population expansion. We identified Gfi1 as a critical transcriptional target of Lyl-1-mediated lymphopoiesis of T cells. Thus, Lyl-1 is a pivotal component of a transcriptional program that controls the lymphoid specification and maintenance of ETPs.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Progenitoras Linfoides/fisiologia , Linfopoese , Proteínas de Neoplasias/metabolismo , Linfócitos T/imunologia , Animais , Apoptose/imunologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células da Medula Óssea/fisiologia , Antígenos CD4/biossíntese , Antígenos CD8/biossíntese , Linhagem da Célula , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Progenitoras Linfoides/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Linfócitos T/fisiologia , Timócitos/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Hematopoietic stem cells (HSCs) are of major clinical importance, and finding methods for their in vitro generation is a prime research focus. We show here that the cell cycle inhibitor p57Kip2/Cdkn1c limits the number of emerging HSCs by restricting the size of the sympathetic nervous system (SNS) and the amount of HSC-supportive catecholamines secreted by these cells. This regulation occurs at the SNS progenitor level and is in contrast to the cell-intrinsic function of p57Kip2 in maintaining adult HSCs, highlighting profound differences in cell cycle requirements of adult HSCs compared with their embryonic counterparts. Furthermore, this effect is specific to the aorta-gonad-mesonephros (AGM) region and shows that the AGM is the main contributor to early fetal liver colonization, as early fetal liver HSC numbers are equally affected. Using a range of antagonists in vivo, we show a requirement for intact ß2-adrenergic signaling for SNS-dependent HSC expansion. To gain further molecular insights, we have generated a single-cell RNA-sequencing data set of all Ngfr+ sympathoadrenal cells around the dorsal aorta to dissect their differentiation pathway. Importantly, this not only defined the relevant p57Kip2-expressing SNS progenitor stage but also revealed that some neural crest cells, upon arrival at the aorta, are able to take an alternative differentiation pathway, giving rise to a subset of ventrally restricted mesenchymal cells that express important HSC-supportive factors. Neural crest cells thus appear to contribute to the AGM HSC niche via 2 different mechanisms: SNS-mediated catecholamine secretion and HSC-supportive mesenchymal cell production.