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1.
Cell ; 165(1): 61-74, 2016 Mar 24.
Artículo en Inglés | MEDLINE | ID: mdl-27015307

RESUMEN

The major and essential objective of pre-implantation development is to establish embryonic and extra-embryonic cell fates. To address when and how this fundamental process is initiated in mammals, we characterize transcriptomes of all individual cells throughout mouse pre-implantation development. This identifies targets of master pluripotency regulators Oct4 and Sox2 as being highly heterogeneously expressed between blastomeres of the 4-cell embryo, with Sox21 showing one of the most heterogeneous expression profiles. Live-cell tracking demonstrates that cells with decreased Sox21 yield more extra-embryonic than pluripotent progeny. Consistently, decreasing Sox21 results in premature upregulation of the differentiation regulator Cdx2, suggesting that Sox21 helps safeguard pluripotency. Furthermore, Sox21 is elevated following increased expression of the histone H3R26-methylase CARM1 and is lowered following CARM1 inhibition, indicating the importance of epigenetic regulation. Therefore, our results indicate that heterogeneous gene expression, as early as the 4-cell stage, initiates cell-fate decisions by modulating the balance of pluripotency and differentiation.


Asunto(s)
Proteínas Adaptadoras de Señalización CARD/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción SOXB2/metabolismo , Animales , Blastocisto/metabolismo , Factor de Transcripción CDX2 , Epigénesis Genética , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Proteínas de Homeodominio/genética , Ratones , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/metabolismo , Factores de Transcripción SOXB1/metabolismo , Análisis de la Célula Individual , Factores de Transcripción/genética
2.
Nat Immunol ; 17(12): 1424-1435, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-27695000

RESUMEN

The final stages of restriction to the T cell lineage occur in the thymus after the entry of thymus-seeding progenitors (TSPs). The identity and lineage potential of TSPs remains unclear. Because the first embryonic TSPs enter a non-vascularized thymic rudiment, we were able to directly image and establish the functional and molecular properties of embryonic thymopoiesis-initiating progenitors (T-IPs) before their entry into the thymus and activation of Notch signaling. T-IPs did not include multipotent stem cells or molecular evidence of T cell-restricted progenitors. Instead, single-cell molecular and functional analysis demonstrated that most fetal T-IPs expressed genes of and had the potential to develop into lymphoid as well as myeloid components of the immune system. Moreover, studies of embryos deficient in the transcriptional regulator RBPJ demonstrated that canonical Notch signaling was not involved in pre-thymic restriction to the T cell lineage or the migration of T-IPs.


Asunto(s)
Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Células Progenitoras Linfoides/fisiología , Células Progenitoras Mieloides/fisiología , Receptores Notch/metabolismo , Linfocitos T/fisiología , Timo/inmunología , Animales , Diferenciación Celular , Linaje de la Célula , Movimiento Celular , Células Cultivadas , Feto , Regulación del Desarrollo de la Expresión Génica , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Transducción de Señal
3.
Proc Natl Acad Sci U S A ; 121(23): e2314213121, 2024 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-38805282

RESUMEN

The anterolateral system (ALS) is a major ascending pathway from the spinal cord that projects to multiple brain areas and underlies the perception of pain, itch, and skin temperature. Despite its importance, our understanding of this system has been hampered by the considerable functional and molecular diversity of its constituent cells. Here, we use fluorescence-activated cell sorting to isolate ALS neurons belonging to the Phox2a-lineage for single-nucleus RNA sequencing. We reveal five distinct clusters of ALS neurons (ALS1-5) and document their laminar distribution in the spinal cord using in situ hybridization. We identify three clusters of neurons located predominantly in laminae I-III of the dorsal horn (ALS1-3) and two clusters with cell bodies located in deeper laminae (ALS4 and ALS5). Our findings reveal the transcriptional logic that underlies ALS neuronal diversity in the adult mouse and uncover the molecular identity of two previously identified classes of projection neurons. We also show that these molecular signatures can be used to target groups of ALS neurons using retrograde viral tracing. Overall, our findings provide a valuable resource for studying somatosensory biology and targeting subclasses of ALS neurons.


Asunto(s)
Proteínas de Homeodominio , Animales , Ratones , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Médula Espinal/citología , Médula Espinal/metabolismo , Neuronas/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Masculino , Núcleo Celular/metabolismo , Núcleo Celular/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
4.
PLoS Genet ; 19(10): e1010913, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37796765

RESUMEN

The genetic code is one of the most highly conserved features across life. Only a few lineages have deviated from the "universal" genetic code. Amongst the few variants of the genetic code reported to date, the codons UAA and UAG virtually always have the same translation, suggesting that their evolution is coupled. Here, we report the genome and transcriptome sequencing of a novel uncultured ciliate, belonging to the Oligohymenophorea class, where the translation of the UAA and UAG stop codons have changed to specify different amino acids. Genomic and transcriptomic analyses revealed that UAA has been reassigned to encode lysine, while UAG has been reassigned to encode glutamic acid. We identified multiple suppressor tRNA genes with anticodons complementary to the reassigned codons. We show that the retained UGA stop codon is enriched in the 3'UTR immediately downstream of the coding region of genes, suggesting that there is functional drive to maintain tandem stop codons. Using a phylogenomics approach, we reconstructed the ciliate phylogeny and mapped genetic code changes, highlighting the remarkable number of independent genetic code changes within the Ciliophora group of protists. According to our knowledge, this is the first report of a genetic code variant where UAA and UAG encode different amino acids.


Asunto(s)
Aminoácidos , Cilióforos , Aminoácidos/genética , Secuencia de Aminoácidos , Código Genético , Cilióforos/genética , Codón de Terminación
5.
Trends Genet ; 38(8): 831-843, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35537880

RESUMEN

Single-cell transcriptomic approaches have revolutionised the study of complex biological systems, with the routine measurement of gene expression in thousands of cells enabling construction of whole-organism cell atlases. However, the transcriptome is just one layer amongst many that coordinate to define cell type and state and, ultimately, function. In parallel with the widespread uptake of single-cell RNA-seq (scRNA-seq), there has been a rapid emergence of methods that enable multiomic profiling of individual cells, enabling parallel measurement of intercellular heterogeneity in the genome, epigenome, transcriptome, and proteomes. Linking measurements from each of these layers has the potential to reveal regulatory and functional mechanisms underlying cell behaviour in healthy development and disease.


Asunto(s)
Análisis de la Célula Individual , Transcriptoma , Perfilación de la Expresión Génica , Análisis de Secuencia de ARN , Análisis de la Célula Individual/métodos , Transcriptoma/genética
6.
Nat Immunol ; 13(4): 412-9, 2012 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-22344248

RESUMEN

The stepwise commitment from hematopoietic stem cells in the bone marrow to T lymphocyte-restricted progenitors in the thymus represents a paradigm for understanding the requirement for distinct extrinsic cues during different stages of lineage restriction from multipotent to lineage-restricted progenitors. However, the commitment stage at which progenitors migrate from the bone marrow to the thymus remains unclear. Here we provide functional and molecular evidence at the single-cell level that the earliest progenitors in the neonatal thymus had combined granulocyte-monocyte, T lymphocyte and B lymphocyte lineage potential but not megakaryocyte-erythroid lineage potential. These potentials were identical to those of candidate thymus-seeding progenitors in the bone marrow, which were closely related at the molecular level. Our findings establish the distinct lineage-restriction stage at which the T cell lineage-commitment process transits from the bone marrow to the remote thymus.


Asunto(s)
Linfocitos B/citología , Linaje de la Célula/inmunología , Células Progenitoras Linfoides/citología , Células Mieloides/citología , Células Precursoras de Linfocitos B/citología , Linfocitos T/citología , Animales , Separación Celular , Citometría de Flujo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Células Progenitoras Linfoides/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Timo/citología
8.
Nature ; 552(7684): 239-243, 2017 12 14.
Artículo en Inglés | MEDLINE | ID: mdl-29186120

RESUMEN

The foundations of mammalian development lie in a cluster of embryonic epiblast stem cells. In response to extracellular matrix signalling, these cells undergo epithelialization and create an apical surface in contact with a cavity, a fundamental event for all subsequent development. Concomitantly, epiblast cells transit through distinct pluripotent states, before lineage commitment at gastrulation. These pluripotent states have been characterized at the molecular level, but their biological importance remains unclear. Here we show that exit from an unrestricted naive pluripotent state is required for epiblast epithelialization and generation of the pro-amniotic cavity in mouse embryos. Embryonic stem cells locked in the naive state are able to initiate polarization but fail to undergo lumenogenesis. Mechanistically, exit from naive pluripotency activates an Oct4-governed transcriptional program that results in expression of glycosylated sialomucin proteins and the vesicle tethering and fusion events of lumenogenesis. Similarly, exit of epiblasts from naive pluripotency in cultured human post-implantation embryos triggers amniotic cavity formation and developmental progression. Our results add tissue-level architecture as a new criterion for the characterization of different pluripotent states, and show the relevance of transitions between these states during development of the mammalian embryo.


Asunto(s)
Embrión de Mamíferos/citología , Morfogénesis , Células Madre Pluripotentes/citología , Amnios/citología , Animales , Tipificación del Cuerpo , Colágeno , Combinación de Medicamentos , Femenino , Regulación del Desarrollo de la Expresión Génica , Estratos Germinativos/citología , Glicosilación , Células Madre Embrionarias Humanas/citología , Humanos , Laminina , Masculino , Ratones , Células Madre Embrionarias de Ratones/citología , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteoglicanos , Sialomucinas/metabolismo , Esferoides Celulares/citología
9.
Nature ; 541(7636): 233-236, 2017 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-28052056

RESUMEN

Metastasis is the leading cause of death for cancer patients. This multi-stage process requires tumour cells to survive in the circulation, extravasate at distant sites, then proliferate; it involves contributions from both the tumour cell and tumour microenvironment ('host', which includes stromal cells and the immune system). Studies suggest the early steps of the metastatic process are relatively efficient, with the post-extravasation regulation of tumour growth ('colonization') being critical in determining metastatic outcome. Here we show the results of screening 810 mutant mouse lines using an in vivo assay to identify microenvironmental regulators of metastatic colonization. We identify 23 genes that, when disrupted in mouse, modify the ability of tumour cells to establish metastatic foci, with 19 of these genes not previously demonstrated to play a role in host control of metastasis. The largest reduction in pulmonary metastasis was observed in sphingosine-1-phosphate (S1P) transporter spinster homologue 2 (Spns2)-deficient mice. We demonstrate a novel outcome of S1P-mediated regulation of lymphocyte trafficking, whereby deletion of Spns2, either globally or in a lymphatic endothelial-specific manner, creates a circulating lymphopenia and a higher percentage of effector T cells and natural killer (NK) cells present in the lung. This allows for potent tumour cell killing, and an overall decreased metastatic burden.


Asunto(s)
Proteínas de Transporte de Anión/genética , Proteínas de Transporte de Anión/metabolismo , Genoma/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Animales , Proteínas de Transporte de Anión/deficiencia , Línea Celular Tumoral , Movimiento Celular , Modelos Animales de Enfermedad , Femenino , Genómica , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Linfopenia/genética , Linfopenia/patología , Lisofosfolípidos/metabolismo , Masculino , Ratones , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Linfocitos T/citología , Linfocitos T/inmunología , Microambiente Tumoral
10.
Nature ; 535(7611): 289-293, 2016 07 14.
Artículo en Inglés | MEDLINE | ID: mdl-27383781

RESUMEN

In mammals, specification of the three major germ layers occurs during gastrulation, when cells ingressing through the primitive streak differentiate into the precursor cells of major organ systems. However, the molecular mechanisms underlying this process remain unclear, as numbers of gastrulating cells are very limited. In the mouse embryo at embryonic day 6.5, cells located at the junction between the extra-embryonic region and the epiblast on the posterior side of the embryo undergo an epithelial-to-mesenchymal transition and ingress through the primitive streak. Subsequently, cells migrate, either surrounding the prospective ectoderm contributing to the embryo proper, or into the extra-embryonic region to form the yolk sac, umbilical cord and placenta. Fate mapping has shown that mature tissues such as blood and heart originate from specific regions of the pre-gastrula epiblast, but the plasticity of cells within the embryo and the function of key cell-type-specific transcription factors remain unclear. Here we analyse 1,205 cells from the epiblast and nascent Flk1(+) mesoderm of gastrulating mouse embryos using single-cell RNA sequencing, representing the first transcriptome-wide in vivo view of early mesoderm formation during mammalian gastrulation. Additionally, using knockout mice, we study the function of Tal1, a key haematopoietic transcription factor, and demonstrate, contrary to previous studies performed using retrospective assays, that Tal1 knockout does not immediately bias precursor cells towards a cardiac fate.


Asunto(s)
Embrión de Mamíferos/citología , Gastrulación/genética , Perfilación de la Expresión Génica , Mesodermo/citología , Mesodermo/embriología , Análisis de la Célula Individual , Transcriptoma/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Linaje de la Célula/genética , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Eritropoyesis/genética , Gástrula/citología , Gástrula/metabolismo , Mesodermo/metabolismo , Ratones , Proteínas Proto-Oncogénicas/deficiencia , Análisis de Secuencia de ADN , Proteína 1 de la Leucemia Linfocítica T Aguda
11.
Trends Genet ; 33(2): 155-168, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-28089370

RESUMEN

Single-cell sequencing provides information that is not confounded by genotypic or phenotypic heterogeneity of bulk samples. Sequencing of one molecular type (RNA, methylated DNA or open chromatin) in a single cell, furthermore, provides insights into the cell's phenotype and links to its genotype. Nevertheless, only by taking measurements of these phenotypes and genotypes from the same single cells can such inferences be made unambiguously. In this review, we survey the first experimental approaches that assay, in parallel, multiple molecular types from the same single cell, before considering the challenges and opportunities afforded by these and future technologies.


Asunto(s)
Metilación de ADN/genética , Metaboloma/genética , Proteoma/genética , Transcriptoma/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Análisis de la Célula Individual/métodos
12.
Genome Res ; 27(3): 451-461, 2017 03.
Artículo en Inglés | MEDLINE | ID: mdl-28087841

RESUMEN

The immune system of vertebrate species consists of many different cell types that have distinct functional roles and are subject to different evolutionary pressures. Here, we first analyzed conservation of genes specific for all major immune cell types in human and mouse. Our results revealed higher gene turnover and faster evolution of trans-membrane proteins in NK cells compared with other immune cell types, and especially T cells, but similar conservation of nuclear and cytoplasmic protein coding genes. To validate these findings in a distant vertebrate species, we used single-cell RNA sequencing of lck:GFP cells in zebrafish and obtained the first transcriptome of specific immune cell types in a nonmammalian species. Unsupervised clustering and single-cell TCR locus reconstruction identified three cell populations, T cells, a novel type of NK-like cells, and a smaller population of myeloid-like cells. Differential expression analysis uncovered new immune-cell-specific genes, including novel immunoglobulin-like receptors, and neofunctionalization of recently duplicated paralogs. Evolutionary analyses confirmed the higher gene turnover of trans-membrane proteins in NK cells compared with T cells in fish species, suggesting that this is a general property of immune cell types across all vertebrates.


Asunto(s)
Evolución Molecular , Células Asesinas Naturales/inmunología , Receptores de IgG/genética , Transcriptoma , Proteínas de Pez Cebra/genética , Animales , Células Cultivadas , Secuencia Conservada , Humanos , Células Asesinas Naturales/citología , Ratones , Análisis de la Célula Individual , Pez Cebra/genética , Pez Cebra/inmunología
13.
Nat Methods ; 14(4): 381-387, 2017 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-28263961

RESUMEN

Single-cell RNA sequencing (scRNA-seq) has become an established and powerful method to investigate transcriptomic cell-to-cell variation, thereby revealing new cell types and providing insights into developmental processes and transcriptional stochasticity. A key question is how the variety of available protocols compare in terms of their ability to detect and accurately quantify gene expression. Here, we assessed the protocol sensitivity and accuracy of many published data sets, on the basis of spike-in standards and uniform data processing. For our workflow, we developed a flexible tool for counting the number of unique molecular identifiers (https://github.com/vals/umis/). We compared 15 protocols computationally and 4 protocols experimentally for batch-matched cell populations, in addition to investigating the effects of spike-in molecular degradation. Our analysis provides an integrated framework for comparing scRNA-seq protocols.


Asunto(s)
Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Animales , Células Madre Embrionarias/fisiología , Congelación , Ratones , Poli A , ARN Mensajero , Sensibilidad y Especificidad , Análisis de Secuencia de ARN/normas , Análisis de Secuencia de ARN/estadística & datos numéricos , Análisis de la Célula Individual/normas , Análisis de la Célula Individual/estadística & datos numéricos , Flujo de Trabajo
14.
Nat Methods ; 13(3): 229-232, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26752769

RESUMEN

We report scM&T-seq, a method for parallel single-cell genome-wide methylome and transcriptome sequencing that allows for the discovery of associations between transcriptional and epigenetic variation. Profiling of 61 mouse embryonic stem cells confirmed known links between DNA methylation and transcription. Notably, the method revealed previously unrecognized associations between heterogeneously methylated distal regulatory elements and transcription of key pluripotency genes.


Asunto(s)
Células Madre Embrionarias/fisiología , Epigénesis Genética/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Elementos Reguladores de la Transcripción/genética , Factores de Transcripción/genética , Animales , Secuencia de Bases , Células Cultivadas , Ratones , Datos de Secuencia Molecular
15.
Nature ; 502(7470): 232-6, 2013 Oct 10.
Artículo en Inglés | MEDLINE | ID: mdl-23934107

RESUMEN

The blood system is maintained by a small pool of haematopoietic stem cells (HSCs), which are required and sufficient for replenishing all human blood cell lineages at millions of cells per second throughout life. Megakaryocytes in the bone marrow are responsible for the continuous production of platelets in the blood, crucial for preventing bleeding--a common and life-threatening side effect of many cancer therapies--and major efforts are focused at identifying the most suitable cellular and molecular targets to enhance platelet production after bone marrow transplantation or chemotherapy. Although it has become clear that distinct HSC subsets exist that are stably biased towards the generation of lymphoid or myeloid blood cells, we are yet to learn whether other types of lineage-biased HSC exist or understand their inter-relationships and how differently lineage-biased HSCs are generated and maintained. The functional relevance of notable phenotypic and molecular similarities between megakaryocytes and bone marrow cells with an HSC cell-surface phenotype remains unclear. Here we identify and prospectively isolate a molecularly and functionally distinct mouse HSC subset primed for platelet-specific gene expression, with enhanced propensity for short- and long-term reconstitution of platelets. Maintenance of platelet-biased HSCs crucially depends on thrombopoietin, the primary extrinsic regulator of platelet development. Platelet-primed HSCs also frequently have a long-term myeloid lineage bias, can self-renew and give rise to lymphoid-biased HSCs. These findings show that HSC subtypes can be organized into a cellular hierarchy, with platelet-primed HSCs at the apex. They also demonstrate that molecular and functional priming for platelet development initiates already in a distinct HSC population. The identification of a platelet-primed HSC population should enable the rational design of therapies enhancing platelet output.


Asunto(s)
Plaquetas/citología , Diferenciación Celular , Células Madre Hematopoyéticas/citología , Animales , Linaje de la Célula/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Linfocitos/citología , Masculino , Ratones , Ratones Endogámicos C57BL
16.
Proteomics ; 18(18): e1700312, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29644800

RESUMEN

Cells are a fundamental unit of life, and the ability to study the phenotypes and behaviors of individual cells is crucial to understanding the workings of complex biological systems. Cell phenotypes (epigenomic, transcriptomic, proteomic, and metabolomic) exhibit dramatic heterogeneity between and within the different cell types and states underlying cellular functional diversity. Cell genotypes can also display heterogeneity throughout an organism, in the form of somatic genetic variation-most notably in the emergence and evolution of tumors. Recent technical advances in single-cell isolation and the development of omics approaches sensitive enough to reveal these aspects of cell identity have enabled a revolution in the study of multicellular systems. In this review, we discuss the technologies available to resolve the genomes, epigenomes, transcriptomes, proteomes, and metabolomes of single cells from a wide variety of living systems.


Asunto(s)
Biomarcadores/análisis , Linaje de la Célula , Epigenómica/métodos , Genómica/métodos , Metabolómica/métodos , Proteómica/métodos , Análisis de la Célula Individual/métodos , Animales , Humanos , Fenotipo
17.
Nat Methods ; 12(6): 519-22, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25915121

RESUMEN

The simultaneous sequencing of a single cell's genome and transcriptome offers a powerful means to dissect genetic variation and its effect on gene expression. Here we describe G&T-seq, a method for separating and sequencing genomic DNA and full-length mRNA from single cells. By applying G&T-seq to over 220 single cells from mice and humans, we discovered cellular properties that could not be inferred from DNA or RNA sequencing alone.


Asunto(s)
ADN/genética , Genómica/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Mensajero/genética , Animales , Línea Celular Tumoral , Humanos , Ratones
18.
Eur J Immunol ; 46(4): 846-56, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26694097

RESUMEN

Intrathymic T-cell development is critically dependent on cortical and medullary thymic epithelial cells (TECs). Both epithelial subsets originate during early thymus organogenesis from progenitor cells that express the thymoproteasome subunit ß5t, a typical feature of cortical TECs. Using in vivo lineage fate mapping, we demonstrate in mice that ß5t(+) TEC progenitors give rise to the medullary TEC compartment early in life but significantly limit their contribution once the medulla has completely formed. Lineage-tracing studies at single cell resolution demonstrate for young mice that the postnatal medulla is expanded from individual ß5t(+) cortical progenitors located at the cortico-medullary junction. These results therefore not only define a developmental window during which the expansion of medulla is efficiently enabled by progenitors resident in the thymic cortex, but also reveal the spatio-temporal dynamics that control the growth of the thymic medulla.


Asunto(s)
Células Epiteliales/citología , Complejo de la Endopetidasa Proteasomal/metabolismo , Linfocitos T/citología , Timo/citología , Timo/embriología , Animales , Diferenciación Celular , Linaje de la Célula/inmunología , Proliferación Celular , Doxiciclina/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Organogénesis/fisiología , Células Madre/citología , Linfocitos T/inmunología
19.
Genome Res ; 24(12): 1918-31, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25224068

RESUMEN

Promiscuous gene expression (PGE) by thymic epithelial cells (TEC) is essential for generating a diverse T cell antigen receptor repertoire tolerant to self-antigens, and thus for avoiding autoimmunity. Nevertheless, the extent and nature of this unusual expression program within TEC populations and single cells are unknown. Using deep transcriptome sequencing of carefully identified mouse TEC subpopulations, we discovered a program of PGE that is common between medullary (m) and cortical TEC, further elaborated in mTEC, and completed in mature mTEC expressing the autoimmune regulator gene (Aire). TEC populations are capable of expressing up to 19,293 protein-coding genes, the highest number of genes known to be expressed in any cell type. Remarkably, in mouse mTEC, Aire expression alone positively regulates 3980 tissue-restricted genes. Notably, the tissue specificities of these genes include known targets of autoimmunity in human AIRE deficiency. Led by the observation that genes induced by Aire expression are generally characterized by a repressive chromatin state in somatic tissues, we found these genes to be strongly associated with H3K27me3 marks in mTEC. Our findings are consistent with AIRE targeting and inducing the promiscuous expression of genes previously epigenetically silenced by Polycomb group proteins. Comparison of the transcriptomes of 174 single mTEC indicates that genes induced by Aire expression are transcribed stochastically at low cell frequency. Furthermore, when present, Aire expression-dependent transcript levels were 16-fold higher, on average, in individual TEC than in the mTEC population.


Asunto(s)
Autoantígenos/genética , Células Epiteliales/metabolismo , Silenciador del Gen , Proteínas del Grupo Polycomb/genética , Timo/citología , Timo/metabolismo , Factores de Transcripción/genética , Acetilación , Animales , Autoantígenos/inmunología , Cromatina/genética , Cromatina/metabolismo , Análisis por Conglomerados , Biología Computacional , Expresión Génica , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Orden Génico , Marcación de Gen , Sitios Genéticos , Vectores Genéticos/genética , Genómica/métodos , Histonas/metabolismo , Ratones , Ratones Transgénicos , Especificidad de Órganos/genética , Proteínas del Grupo Polycomb/metabolismo , Transducción de Señal , Análisis de la Célula Individual , Timo/inmunología , Factores de Transcripción/metabolismo , Transcriptoma , Proteína AIRE
20.
PLoS Genet ; 10(1): e1004126, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24497842

RESUMEN

Advances in whole-genome and whole-transcriptome amplification have permitted the sequencing of the minute amounts of DNA and RNA present in a single cell, offering a window into the extent and nature of genomic and transcriptomic heterogeneity which occurs in both normal development and disease. Single-cell approaches stand poised to revolutionise our capacity to understand the scale of genomic, epigenomic, and transcriptomic diversity that occurs during the lifetime of an individual organism. Here, we review the major technological and biological breakthroughs achieved, describe the remaining challenges to overcome, and provide a glimpse into the promise of recent and future developments.


Asunto(s)
Genómica/métodos , Análisis de la Célula Individual/métodos , Transcriptoma , Biología Computacional/métodos , Epigenómica , Heterogeneidad Genética , Variación Genética , Humanos , Análisis de Secuencia de ADN
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