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1.
Development ; 151(14)2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-39069943

RESUMO

Naïve epiblast cells in the embryo and pluripotent stem cells in vitro undergo developmental progression to a formative state competent for lineage specification. During this transition, transcription factors and chromatin are rewired to encode new functional features. Here, we examine the role of mitogen-activated protein kinase (ERK1/2) signalling in pluripotent state transition. We show that a primary consequence of ERK activation in mouse embryonic stem cells is elimination of Nanog, which precipitates breakdown of the naïve state gene regulatory network. Variability in pERK dynamics results in heterogeneous loss of Nanog and metachronous state transition. Knockdown of Nanog allows exit without ERK activation. However, transition to formative pluripotency does not proceed and cells collapse to an indeterminate identity. This outcome is due to failure to maintain expression of the central pluripotency factor Oct4. Thus, during formative transition ERK signalling both dismantles the naïve state and preserves pluripotency. These results illustrate how a single signalling pathway can both initiate and secure transition between cell states.


Assuntos
Sistema de Sinalização das MAP Quinases , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero , Células-Tronco Pluripotentes , Animais , Proteína Homeobox Nanog/metabolismo , Proteína Homeobox Nanog/genética , Camundongos , Fator 3 de Transcrição de Octâmero/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Diferenciação Celular/genética , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/metabolismo , Camadas Germinativas/citologia , Redes Reguladoras de Genes , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética
2.
Nature ; 566(7745): 490-495, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30787436

RESUMO

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/genética
3.
Development ; 148(20)2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34932803

RESUMO

A fundamental challenge when studying biological systems is the description of cell state dynamics. During transitions between cell states, a multitude of parameters may change - from the promoters that are active, to the RNAs and proteins that are expressed and modified. Cells can also adopt different shapes, alter their motility and change their reliance on cell-cell junctions or adhesion. These parameters are integral to how a cell behaves and collectively define the state a cell is in. Yet, technical challenges prevent us from measuring all of these parameters simultaneously and dynamically. How, then, can we comprehend cell state transitions using finite descriptions? The recent virtual workshop organised by The Company of Biologists entitled 'Cell State Transitions: Approaches, Experimental Systems and Models' attempted to address this question. Here, we summarise some of the main points that emerged during the workshop's themed discussions. We also present examples of cell state transitions and describe models and systems that are pushing forward our understanding of how cells rewire their state.


Assuntos
Linhagem da Célula/genética , Regiões Promotoras Genéticas/genética , Proteínas/genética , RNA/genética , Adesão Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Junções Intercelulares/genética , Biologia de Sistemas
4.
Development ; 146(6)2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30914406

RESUMO

The power of mouse embryonic stem (ES) cells to colonise the developing embryo has revolutionised mammalian developmental genetics and stem cell research. This power is vulnerable, however, to the cell culture environment, deficiencies in which can lead to cellular heterogeneity, adaptive phenotypes, epigenetic aberrations and genetic abnormalities. Here, we provide detailed methodologies for derivation, propagation, genetic modification and primary differentiation of ES cells in 2i or 2i+LIF media without serum or undefined serum substitutes. Implemented diligently, these procedures minimise variability and deviation, thereby improving the efficiency, reproducibility and biological validity of ES cell experimentation.


Assuntos
Diferenciação Celular/genética , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias Murinas/citologia , Animais , Sistemas CRISPR-Cas , Técnicas de Cultura de Células , Ciclo Celular , Técnicas de Cocultura , Meios de Cultura/química , Humanos , Cariotipagem , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , RNA Interferente Pequeno/genética , Transdução de Sinais
5.
Development ; 145(12)2018 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-29915126

RESUMO

Lineage segregation in the mouse embryo is a finely controlled process dependent upon coordination of signalling pathways and transcriptional responses. Here we employ a conditional deletion system to investigate embryonic patterning and lineage specification in response to loss of Oct4. We first observe ectopic expression of Nanog in Oct4-negative postimplantation epiblast cells. The expression domains of lineage markers are subsequently disrupted. Definitive endoderm expands at the expense of mesoderm; the anterior-posterior axis is positioned more distally and an ectopic posterior-like domain appears anteriorly, suggesting a role for Oct4 in maintaining the embryonic axis. Although primitive streak forms in the presumptive proximal-posterior region, epithelial-to-mesenchymal transition is impeded by an increase of E-cadherin, leading to complete tissue disorganisation and failure to generate germ layers. In explant and in vitro differentiation assays, Oct4 mutants also show upregulation of E-cadherin and Foxa2, suggesting a cell-autonomous phenotype. We confirm requirement for Oct4 in self-renewal of postimplantation epiblast ex vivo Our results indicate a role for Oct4 in orchestrating multiple fates and enabling expansion, correct patterning and lineage choice in the postimplantation epiblast.


Assuntos
Padronização Corporal , Embrião de Mamíferos/metabolismo , Camadas Germinativas/citologia , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem da Célula , Implantação do Embrião , Embrião de Mamíferos/citologia , Endoderma/citologia , Endoderma/metabolismo , Feminino , Gastrulação , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Camadas Germinativas/metabolismo , Imageamento Tridimensional , Masculino , Camundongos , Mutação/genética , Proteína Homeobox Nanog/metabolismo , Fenótipo , Células-Tronco Pluripotentes/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais
6.
Development ; 144(7): 1221-1234, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28174249

RESUMO

Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naïve pluripotency. Here, we examine the initial transition process. The ES cell population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naïve status. Extinction of ES cell identity in single cells is acute. It occurs only after near-complete elimination of naïve pluripotency factors, but precedes appearance of lineage specification markers. Cells newly departed from the ES cell state display features of early post-implantation epiblast and are distinct from primed epiblast. They also exhibit a genome-wide increase in DNA methylation, intermediate between early and late epiblast. These findings are consistent with the proposition that naïve cells transition to a distinct formative phase of pluripotency preparatory to lineage priming.


Assuntos
Rastreamento de Células , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes/citologia , Animais , Linhagem da Célula , Autorrenovação Celular , Metilação de DNA/genética , Regulação para Baixo , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/metabolismo , Genes Reporter , Camadas Germinativas/citologia , Cinética , Camundongos , Células-Tronco Pluripotentes/metabolismo , Transplante de Células-Tronco , Fatores de Transcrição/metabolismo , Transcrição Gênica
7.
EMBO Rep ; 19(8)2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29895711

RESUMO

Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signalling is implicated in initiation of embryonic stem (ES) cell differentiation. The pathway is subject to complex feedback regulation. Here, we examined the ERK-responsive phosphoproteome in ES cells and identified the negative regulator RSK1 as a prominent target. We used CRISPR/Cas9 to create combinatorial mutations in RSK family genes. Genotypes that included homozygous null mutations in Rps6ka1, encoding RSK1, resulted in elevated ERK phosphorylation. These RSK-depleted ES cells exhibit altered kinetics of transition into differentiation, with accelerated downregulation of naïve pluripotency factors, precocious expression of transitional epiblast markers and early onset of lineage specification. We further show that chemical inhibition of RSK increases ERK phosphorylation and expedites ES cell transition without compromising multilineage potential. These findings demonstrate that the ERK activation profile influences the dynamics of pluripotency progression and highlight the role of signalling feedback in temporal control of cell state transitions.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Retroalimentação Fisiológica , Células-Tronco Pluripotentes/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Retroalimentação Fisiológica/efeitos dos fármacos , Humanos , Mutação/genética , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Proteoma/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Bibliotecas de Moléculas Pequenas/farmacologia
8.
Small ; 15(5): e1804576, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30570812

RESUMO

Developmental cell biology requires technologies in which the fate of single cells is followed over extended time periods, to monitor and understand the processes of self-renewal, differentiation, and reprogramming. A workflow is presented, in which single cells are encapsulated into droplets (Ø: 80 µm, volume: ≈270 pL) and the droplet compartment is later converted to a hydrogel bead. After on-chip de-emulsification by electrocoalescence, these 3D scaffolds are subsequently arrayed on a chip for long-term perfusion culture to facilitate continuous cell imaging over 68 h. Here, the response of murine embryonic stem cells to different growth media, 2i and N2B27, is studied, showing that the exit from pluripotency can be monitored by fluorescence time-lapse microscopy, by immunostaining and by reverse-transcription and quantitative PCR (RT-qPCR). The defined 3D environment emulates the natural context of cell growth (e.g., in tissue) and enables the study of cell development in various matrices. The large scale of cell cultivation (in 2000 beads in parallel) may reveal infrequent events that remain undetected in lower throughput or ensemble studies. This platform will help to gain qualitative and quantitative mechanistic insight into the role of external factors on cell behavior.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Hidrogéis/farmacologia , Microesferas , Células-Tronco Embrionárias Murinas/citologia , Óptica e Fotônica/métodos , Perfusão , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Fenótipo , Reologia , Fatores de Tempo
10.
Development ; 141(5): 1001-10, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24504341

RESUMO

The transcription factor Oct4 is required in vitro for establishment and maintenance of embryonic stem cells and for reprogramming somatic cells to pluripotency. In vivo, it prevents the ectopic differentiation of early embryos into trophoblast. Here, we further explore the role of Oct4 in blastocyst formation and specification of epiblast versus primitive endoderm lineages using conditional genetic deletion. Experiments involving mouse embryos deficient for both maternal and zygotic Oct4 suggest that it is dispensable for zygote formation, early cleavage and activation of Nanog expression. Nanog protein is significantly elevated in the presumptive inner cell mass of Oct4 null embryos, suggesting an unexpected role for Oct4 in attenuating the level of Nanog, which might be significant for priming differentiation during epiblast maturation. Induced deletion of Oct4 during the morula to blastocyst transition disrupts the ability of inner cell mass cells to adopt lineage-specific identity and acquire the molecular profile characteristic of either epiblast or primitive endoderm. Sox17, a marker of primitive endoderm, is not detected following prolonged culture of such embryos, but can be rescued by provision of exogenous FGF4. Interestingly, functional primitive endoderm can be rescued in Oct4-deficient embryos in embryonic stem cell complementation assays, but only if the host embryos are at the pre-blastocyst stage. We conclude that cell fate decisions within the inner cell mass are dependent upon Oct4 and that Oct4 is not cell-autonomously required for the differentiation of primitive endoderm derivatives, as long as an appropriate developmental environment is established.


Assuntos
Blastocisto/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Animais , Blastocisto/citologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Endoderma/citologia , Endoderma/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Mórula/citologia , Mórula/metabolismo , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Oócitos/citologia , Oócitos/metabolismo , Gravidez , Fatores de Transcrição SOXF/genética , Fatores de Transcrição SOXF/metabolismo , Zigoto/citologia , Zigoto/metabolismo
11.
Philos Trans R Soc Lond B Biol Sci ; 379(1900): 20230050, 2024 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-38432322

RESUMO

Cell state transitions are prevalent in biology, playing a fundamental role in development, homeostasis and repair. Dysregulation of cell state transitions can lead to or occur in a wide range of diseases. In this letter, I explore and highlight the role of post-transcriptional regulatory mechanisms in determining the dynamics of cell state transitions. I propose that regulation of protein levels after transcription provides an under-appreciated regulatory route to obtain fast and sharp transitions between distinct cell states. This article is part of a discussion meeting issue 'Causes and consequences of stochastic processes in development and disease'.


Assuntos
Regulação da Expressão Gênica , Homeostase
12.
Nat Commun ; 14(1): 4022, 2023 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-37419903

RESUMO

Biomechanical cues are instrumental in guiding embryonic development and cell differentiation. Understanding how these physical stimuli translate into transcriptional programs will provide insight into mechanisms underlying mammalian pre-implantation development. Here, we explore this type of regulation by exerting microenvironmental control over mouse embryonic stem cells. Microfluidic encapsulation of mouse embryonic stem cells in agarose microgels stabilizes the naive pluripotency network and specifically induces expression of Plakoglobin (Jup), a vertebrate homolog of ß-catenin. Overexpression of Plakoglobin is sufficient to fully re-establish the naive pluripotency gene regulatory network under metastable pluripotency conditions, as confirmed by single-cell transcriptome profiling. Finally, we find that, in the epiblast, Plakoglobin was exclusively expressed at the blastocyst stage in human and mouse embryos - further strengthening the link between Plakoglobin and naive pluripotency in vivo. Our work reveals Plakoglobin as a mechanosensitive regulator of naive pluripotency and provides a paradigm to interrogate the effects of volumetric confinement on cell-fate transitions.


Assuntos
Desenvolvimento Embrionário , Camadas Germinativas , Animais , Camundongos , Humanos , gama Catenina/genética , gama Catenina/metabolismo , Diferenciação Celular/genética , Camadas Germinativas/metabolismo , Desenvolvimento Embrionário/genética , Perfilação da Expressão Gênica , Blastocisto/metabolismo , Mamíferos/genética
13.
Stem Cell Reports ; 16(5): 1117-1141, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33979598

RESUMO

Detailed studies of the embryo allow an increasingly mechanistic understanding of development, which has proved of profound relevance to human disease. The last decade has seen in vitro cultured stem cell-based models of embryo development flourish, which provide an alternative to the embryo for accessible experimentation. However, the usefulness of any stem cell-based embryo model will be determined by how accurately it reflects in vivo embryonic development, and/or the extent to which it facilitates new discoveries. Stringent benchmarking of embryo models is thus an important consideration for this growing field. Here we provide an overview of means to evaluate both the properties of stem cells, the building blocks of most embryo models, as well as the usefulness of current and future in vitro embryo models.


Assuntos
Embrião de Mamíferos/fisiologia , Modelos Biológicos , Animais , Desenvolvimento Embrionário , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Humanos , Padrões de Referência
14.
Nat Commun ; 12(1): 6132, 2021 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-34675200

RESUMO

Studies of mechanical signalling are typically performed by comparing cells cultured on soft and stiff hydrogel-based substrates. However, it is challenging to independently and robustly control both substrate stiffness and extracellular matrix tethering to substrates, making matrix tethering a potentially confounding variable in mechanical signalling investigations. Moreover, unstable matrix tethering can lead to poor cell attachment and weak engagement of cell adhesions. To address this, we developed StemBond hydrogels, a hydrogel in which matrix tethering is robust and can be varied independently of stiffness. We validate StemBond hydrogels by showing that they provide an optimal system for culturing mouse and human pluripotent stem cells. We further show how soft StemBond hydrogels modulate stem cell function, partly through stiffness-sensitive ERK signalling. Our findings underline how substrate mechanics impact mechanosensitive signalling pathways regulating self-renewal and differentiation, indicating that optimising the complete mechanical microenvironment will offer greater control over stem cell fate specification.


Assuntos
Técnicas de Cultura de Células/instrumentação , Matriz Extracelular/química , Hidrogéis/química , Células-Tronco Pluripotentes/citologia , Animais , Fenômenos Biomecânicos , Adesão Celular , Diferenciação Celular , Células Cultivadas , Matriz Extracelular/metabolismo , Humanos , Mecanotransdução Celular , Camundongos , Células-Tronco Pluripotentes/química , Células-Tronco Pluripotentes/metabolismo
15.
Lab Chip ; 20(14): 2580-2591, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32573646

RESUMO

Combining live imaging with the ability to retrieve individual cells of interest remains a technical challenge. Combining imaging with precise cell retrieval is of particular interest when studying highly dynamic or transient, asynchronous, or heterogeneous cell biological and developmental processes. Here, we present a method to encapsulate live cells in a 3D hydrogel matrix, via hydrogel bead compartmentalisation. Using a small-scale screen, we optimised matrix conditions for the culture and multilineage differentiation of mouse embryonic stem cells. Moreover, we designed a custom microfluidic platform that is compatible with live imaging. With this platform we can long-term culture and subsequently extract individual cells-in-beads by media flow only, obviating the need for enzymatic cell removal from the platform. Specific beads may be extracted from the platform in isolation, without disrupting the adjacent beads. We show that we can differentiate mouse embryonic stem cells, monitor reporter expression by live imaging, and retrieve individual beads for functional assays, correlating reporter expression with functional response. Overall, we present a highly flexible 3D cell encapsulation and microfluidic platform that enables both monitoring of cellular dynamics and retrieval for molecular and functional assays.


Assuntos
Técnicas de Cultura de Células , Microfluídica , Animais , Diferenciação Celular , Células Cultivadas , Células Clonais , Camundongos
16.
Cell Stem Cell ; 24(5): 785-801.e7, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31031137

RESUMO

The gene regulatory network (GRN) of naive mouse embryonic stem cells (ESCs) must be reconfigured to enable lineage commitment. TCF3 sanctions rewiring by suppressing components of the ESC transcription factor circuitry. However, TCF3 depletion only delays and does not prevent transition to formative pluripotency. Here, we delineate additional contributions of the ETS-family transcription factor ETV5 and the repressor RBPJ. In response to ERK signaling, ETV5 switches activity from supporting self-renewal and undergoes genome relocation linked to commissioning of enhancers activated in formative epiblast. Independent upregulation of RBPJ prevents re-expression of potent naive factors, TBX3 and NANOG, to secure exit from the naive state. Triple deletion of Etv5, Rbpj, and Tcf3 disables ESCs, such that they remain largely undifferentiated and locked in self-renewal, even in the presence of differentiation stimuli. Thus, genetic elimination of three complementary drivers of network transition stalls developmental progression, emulating environmental insulation by small-molecule inhibitors.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Neurônios/fisiologia , Células-Tronco Pluripotentes/fisiologia , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Autorrenovação Celular , Proteínas de Ligação a DNA/genética , Técnicas de Inativação de Genes , Redes Reguladoras de Genes , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , RNA Interferente Pequeno/genética , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/genética
17.
Nat Cell Biol ; 20(2): 127-134, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29311656

RESUMO

During gastrulation, cell types from all three germ layers are specified and the basic body plan is established 1 . However, molecular analysis of this key developmental stage has been hampered by limited cell numbers and a paucity of markers. Single-cell RNA sequencing circumvents these problems, but has so far been limited to specific organ systems 2 . Here, we report single-cell transcriptomic characterization of >20,000 cells immediately following gastrulation at E8.25 of mouse development. We identify 20 major cell types, which frequently contain substructure, including three distinct signatures in early foregut cells. Pseudo-space ordering of somitic progenitor cells identifies dynamic waves of transcription and candidate regulators, which are validated by molecular characterization of spatially resolved regions of the embryo. Within the endothelial population, cells that transition from haemogenic endothelial to erythro-myeloid progenitors specifically express Alox5 and its co-factor Alox5ap, which control leukotriene production. Functional assays using mouse embryonic stem cells demonstrate that leukotrienes promote haematopoietic progenitor cell generation. Thus, this comprehensive single-cell map can be exploited to reveal previously unrecognized pathways that contribute to tissue development.


Assuntos
Proteínas Ativadoras de 5-Lipoxigenase/genética , Araquidonato 5-Lipoxigenase/genética , Leucotrienos/genética , Organogênese/genética , Animais , Linhagem da Célula , Desenvolvimento Embrionário/genética , Gastrulação/genética , Células-Tronco Hematopoéticas/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Leucotrienos/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Transdução de Sinais , Análise de Célula Única
18.
Stem Cell Reports ; 9(1): 77-91, 2017 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-28669603

RESUMO

Naive mouse embryonic stem cells (ESCs) can develop multiple fates, but the cellular and molecular processes that enable lineage competence are poorly characterized. Here, we investigated progression from the ESC ground state in defined culture. We utilized downregulation of Rex1::GFPd2 to track the loss of ESC identity. We found that cells that have newly downregulated this reporter have acquired capacity for germline induction. They can also be efficiently specified for different somatic lineages, responding more rapidly than naive cells to inductive cues. Inhibition of autocrine NODAL signaling did not alter kinetics of exit from the ESC state but compromised both germline and somatic lineage specification. Transient inhibition prior to loss of ESC identity was sufficient for this effect. Genetic ablation of Nodal reduced viability during early differentiation, consistent with defective lineage specification. These results suggest that NODAL promotes acquisition of multi-lineage competence in cells departing naive pluripotency.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias Murinas/citologia , Proteína Nodal/metabolismo , Animais , Linhagem Celular , Linhagem da Célula , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Proteína Nodal/genética , Transdução de Sinais
19.
Cell Rep ; 20(5): 1215-1228, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28768204

RESUMO

The mouse inner cell mass (ICM) segregates into the epiblast and primitive endoderm (PrE) lineages coincident with implantation of the embryo. The epiblast subsequently undergoes considerable expansion of cell numbers prior to gastrulation. To investigate underlying regulatory principles, we performed systematic single-cell RNA sequencing (seq) of conceptuses from E3.5 to E6.5. The epiblast shows reactivation and subsequent inactivation of the X chromosome, with Zfp57 expression associated with reactivation and inactivation together with other candidate regulators. At E6.5, the transition from epiblast to primitive streak is linked with decreased expression of polycomb subunits, suggesting a key regulatory role. Notably, our analyses suggest elevated transcriptional noise at E3.5 and within the non-committed epiblast at E6.5, coinciding with exit from pluripotency. By contrast, E6.5 primitive streak cells became highly synchronized and exhibit a shortened G1 cell-cycle phase, consistent with accelerated proliferation. Our study systematically charts transcriptional noise and uncovers molecular processes associated with early lineage decisions.


Assuntos
Proliferação de Células/fisiologia , Embrião de Mamíferos/embriologia , Gástrula/embriologia , Gastrulação/fisiologia , Transcrição Gênica/fisiologia , Animais , Gástrula/citologia , Camundongos
20.
Stem Cell Reports ; 1(1): 66-78, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24052943

RESUMO

Mammalian primordial germ cells (PGCs) are unipotent progenitors of the gametes. Nonetheless, they can give rise directly to pluripotent stem cells in vitro or during teratocarcinogenesis. This conversion is inconsistent, however, and has been difficult to study. Here, we delineate requirements for efficient resetting of pluripotency in culture. We demonstrate that in defined conditions, routinely 20% of PGCs become EG cells. Conversion can occur from the earliest specified PGCs. The entire process can be tracked from single cells. It is driven by leukemia inhibitory factor (LIF) and the downstream transcription factor STAT3. In contrast, LIF signaling is not required during germ cell ontogeny. We surmise that ectopic LIF/STAT3 stimulation reconstructs latent pluripotency and self-renewal. Notably, STAT3 targets are significantly upregulated in germ cell tumors, suggesting that dysregulation of this pathway may underlie teratocarcinogenesis. These findings demonstrate that EG cell formation is a robust experimental system for exploring mechanisms involved in reprogramming and cancer.


Assuntos
Diferenciação Celular , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Células Germinativas/citologia , Células-Tronco Pluripotentes/citologia , Animais , Células-Tronco Embrionárias/metabolismo , Células Germinativas/metabolismo , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismo , Camundongos , Neoplasias Embrionárias de Células Germinativas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Regulação para Cima
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