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2.
Dev Cell ; 39(1): 104-115, 2016 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-27728778

RESUMO

Primordial germ cell (PGC) development is characterized by global epigenetic remodeling, which resets genomic potential and establishes an epigenetic ground state. Here we recapitulate PGC specification in vitro from naive embryonic stem cells and characterize the early events of epigenetic reprogramming during the formation of the human and mouse germline. Following rapid de novo DNA methylation during priming to epiblast-like cells, methylation is globally erased in PGC-like cells. Repressive chromatin marks (H3K9me2/3) and transposable elements are enriched at demethylation-resistant regions, while active chromatin marks (H3K4me3 or H3K27ac) are more prominent at regions that demethylate faster. The dynamics of specification and epigenetic reprogramming show species-specific differences, in particular markedly slower reprogramming kinetics in the human germline. Differences in developmental kinetics may be explained by differential regulation of epigenetic modifiers. Our work establishes a robust and faithful experimental system of the early events of epigenetic reprogramming and regulation in the germline.


Assuntos
Padronização Corporal/genética , Metilação de DNA/genética , Células Germinativas/metabolismo , Animais , Elementos de DNA Transponíveis/genética , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Transcrição Gênica
3.
Cell Rep ; 15(4): 787-800, 2016 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-27149845

RESUMO

It has previously been reported that mouse epiblast stem cell (EpiSC) lines comprise heterogeneous cell populations that are functionally equivalent to cells of either early- or late-stage postimplantation development. So far, the establishment of the embryonic stem cell (ESC) pluripotency gene regulatory network through the widely known chemical inhibition of MEK and GSK3beta has been impractical in late-stage EpiSCs. Here, we show that chemical inhibition of casein kinase 1alpha (CK1alpha) induces the conversion of recalcitrant late-stage EpiSCs into ESC pluripotency. CK1alpha inhibition directly results in the simultaneous activation of the WNT signaling pathway, together with inhibition of the TGFbeta/SMAD2 signaling pathway, mediating the rewiring of the gene regulatory network in favor of an ESC-like state. Our findings uncover a molecular mechanism that links CK1alpha to ESC pluripotency through the direct modulation of WNT and TGFbeta signaling.

5.
Development ; 141(6): 1209-21, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24595287

RESUMO

During gastrulation, epiblast cells are pluripotent and their fate is thought to be constrained principally by their position. Cell fate is progressively restricted by localised signalling cues from areas including the primitive streak. However, it is unknown whether this restriction accompanies, at the individual cell level, a reduction in potency. Investigation of these early transition events in vitro is possible via the use of epiblast stem cells (EpiSCs), self-renewing pluripotent cell lines equivalent to the postimplantation epiblast. Strikingly, mouse EpiSCs express gastrulation stage regional markers in self-renewing conditions. Here, we examined the differentiation potential of cells expressing such lineage markers. We show that undifferentiated EpiSC cultures contain a major subfraction of cells with reversible early primitive streak characteristics, which is mutually exclusive to a neural-like fraction. Using in vitro differentiation assays and embryo grafting we demonstrate that primitive streak-like EpiSCs are biased towards mesoderm and endoderm fates while retaining pluripotency. The acquisition of primitive streak characteristics by self-renewing EpiSCs is mediated by endogenous Wnt signalling. Elevation of Wnt activity promotes restriction towards primitive streak-associated lineages with mesendodermal and neuromesodermal characteristics. Collectively, our data suggest that EpiSC pluripotency encompasses a range of reversible lineage-biased states reflecting the birth of pioneer lineage precursors from a pool of uncommitted EpiSCs similar to the earliest cell fate restriction events taking place in the gastrula stage epiblast.


Assuntos
Camadas Germinativas/citologia , Linha Primitiva/citologia , Via de Sinalização Wnt , Animais , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Gástrula/citologia , Gástrula/embriologia , Gástrula/metabolismo , Gastrulação/fisiologia , Camadas Germinativas/embriologia , Camadas Germinativas/metabolismo , Camundongos , Camundongos Transgênicos , Placa Neural/citologia , Placa Neural/embriologia , Placa Neural/metabolismo , Células-Tronco Pluripotentes/classificação , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Linha Primitiva/embriologia
6.
Curr Opin Genet Dev ; 23(5): 504-11, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23932125

RESUMO

Pluripotency is a property that early embryonic cells possess over a considerable developmental time span. Accordingly, pluripotent cell lines can be established from the pre-implantation or post-implantation mouse embryo as embryonic stem (ES) or epiblast stem (EpiSC) cell lines, respectively. Maintenance of the pluripotent phenotype depends on the function of specific transcription factors (TFs) operating within a pluripotency gene regulatory network (PGRN). As cells move from an ES cell to an EpiSC state, the PGRN changes with expression of some TFs reduced (e.g. Nanog) or eliminated (e.g. Esrrb). Re-expressing such TFs can move cells back to an earlier developmental identity and is being applied to attempt establishment of human cell lines with the properties of mouse ES cells.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Redes Reguladoras de Genes , Células-Tronco Pluripotentes/citologia , Animais , Linhagem da Célula , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Humanos , Camundongos , Células-Tronco Pluripotentes/metabolismo
7.
Cell Stem Cell ; 12(5): 531-45, 2013 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-23642364

RESUMO

Embryonic stem cell (ESC) pluripotency is governed by a gene regulatory network centered on the transcription factors Oct4 and Nanog. To date, robust self-renewing ESC states have only been obtained through the chemical inhibition of signaling pathways or enforced transgene expression. Here, we show that ESCs with reduced Oct4 expression resulting from heterozygosity also exhibit a stabilized pluripotent state. Despite having reduced Oct4 expression, Oct4(+/-) ESCs show increased genome-wide binding of Oct4, particularly at pluripotency-associated enhancers, homogeneous expression of pluripotency transcription factors, enhanced self-renewal efficiency, and delayed differentiation kinetics. Cells also exhibit increased Wnt expression, enhanced leukemia inhibitory factor (LIF) sensitivity, and reduced responsiveness to fibroblast growth factor. Although they are able to maintain pluripotency in the absence of bone morphogenetic protein, removal of LIF destabilizes pluripotency. Our findings suggest that cells with a reduced Oct4 concentration range are maintained in a robust pluripotent state and that the wild-type Oct4 concentration range enables effective differentiation.


Assuntos
Elementos Facilitadores Genéticos/genética , Proteínas de Homeodomínio/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais , Sequência de Bases , Proteínas Morfogenéticas Ósseas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Clonais , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Humanos , Dados de Sequência Molecular , Células-Tronco Pluripotentes/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Soro , Transdução de Sinais/efeitos dos fármacos , Proteínas Wnt/metabolismo
8.
Stem Cells ; 31(8): 1511-22, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23649667

RESUMO

Robust development of the early embryo may benefit from mechanisms that ensure that not all pluripotent cells differentiate at exactly the same time: such mechanisms would build flexibility into the process of lineage allocation. This idea is supported by the observation that pluripotent stem cells differentiate at different rates in vitro. We use a clonal commitment assay to confirm that pluripotent cells commit to differentiate asynchronously even under uniform differentiation conditions. Stochastic variability in expression of the Notch target gene Hes1 has previously been reported to influence neural versus mesodermal differentiation through modulation of Notch activity. Here we report that Hes1 also has an earlier role to delay exit from the pluripotent state into all lineages. The early function of Hes1 to delay differentiation can be explained by an ability of Hes1 to amplify STAT3 responsiveness in a cell-autonomous manner. Variability in Hes1 expression therefore helps to explain why STAT3 responsiveness varies between individual ES cells, and this in turn helps to explain why pluripotent cells commit to differentiate asynchronously.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Receptores Notch/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Diferenciação Celular/fisiologia , Regulação para Baixo , Humanos , Camundongos , Proteína Homeobox Nanog , Transdução de Sinais , Fatores de Transcrição HES-1 , Transfecção
9.
Cell Rep ; 2(6): 1571-8, 2012 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-23200857

RESUMO

Chimera formation after blastocyst injection or morula aggregation is the principal functional assay of the developmental potential of mouse embryonic stem cells (ESCs). This property, which demonstrates functional equivalence between ESCs and the preimplantation epiblast, is not shared by epiblast stem cell (EpiSC) lines. Here, we show that EpiSCs derived either from postimplantation embryos or from ESCs in vitro readily generate chimeras when grafted to postimplantation embryos in whole embryo culture. EpiSC derivatives integrate and differentiate to derivatives of all three embryonic germ layers and primordial germ cells. In contrast, grafted ESCs seldom proliferate in postimplantation embryos, and fail to acquire the identity of their host-derived neighbors. EpiSCs do not incorporate efficiently into embryonic day 8.5 embryos, a stage by which pluripotency has been lost. Thus, chimera formation by EpiSCs requires a permissive environment, the postimplantation epiblast, and demonstrates functional equivalence between this cell type and EpiSCs.


Assuntos
Quimera/embriologia , Embrião de Mamíferos/embriologia , Camadas Germinativas/metabolismo , Células-Tronco/metabolismo , Animais , Linhagem Celular , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/fisiologia , Camadas Germinativas/citologia , Camundongos , Camundongos Transgênicos , Células-Tronco/citologia
10.
EMBO J ; 31(24): 4547-62, 2012 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-23178592

RESUMO

NANOG, OCT4 and SOX2 form the core network of transcription factors supporting embryonic stem (ES) cell self-renewal. While OCT4 and SOX2 expression is relatively uniform, ES cells fluctuate between states of high NANOG expression possessing high self-renewal efficiency, and low NANOG expression exhibiting increased differentiation propensity. NANOG, OCT4 and SOX2 are currently considered to activate transcription of each of the three genes, an architecture that cannot readily account for NANOG heterogeneity. Here, we examine the architecture of the Nanog-centred network using inducible NANOG gain- and loss-of-function approaches. Rather than activating itself, Nanog activity is autorepressive and OCT4/SOX2-independent. Moreover, the influence of Nanog on Oct4 and Sox2 expression is minimal. Using Nanog:GFP reporters, we show that Nanog autorepression is a major regulator of Nanog transcription switching. We conclude that the architecture of the pluripotency gene regulatory network encodes the capacity to generate reversible states of Nanog transcription via a Nanog-centred autorepressive loop. Therefore, cellular variability in self-renewal efficiency is an emergent property of the pluripotency gene regulatory network.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica/fisiologia , Redes Reguladoras de Genes/genética , Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/genética , Imunoprecipitação da Cromatina , Retroalimentação Fisiológica , Citometria de Fluxo , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde , Hibridização in Situ Fluorescente , Camundongos , Proteína Homeobox Nanog , Reação em Cadeia da Polimerase Via Transcriptase Reversa
11.
Cell Stem Cell ; 11(4): 477-90, 2012 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-23040477

RESUMO

Embryonic stem cell (ESC) self-renewal efficiency is determined by the level of Nanog expression. However, the mechanisms by which Nanog functions remain unclear, and in particular, direct Nanog target genes are uncharacterized. Here we investigate ESCs expressing different Nanog levels and Nanog(-/-) cells with distinct functionally inducible Nanog proteins to identify Nanog-responsive genes. Surprisingly, these constitute a minor fraction of genes that Nanog binds. Prominent among Nanog-reponsive genes is Estrogen-related receptor b (Esrrb). Nanog binds directly to Esrrb, enhances binding of RNAPolII, and stimulates Esrrb transcription. Overexpression of Esrrb in ESCs maintains cytokine-independent self-renewal and pluripotency. Remarkably, this activity is retained in Nanog(-/-) ESCs. Moreover, Esrrb can reprogram Nanog(-/-) EpiSCs and can rescue stalled reprogramming in Nanog(-/-) pre-iPSCs. Finally, Esrrb deletion abolishes the defining ability of Nanog to confer LIF-independent ESC self-renewal. These findings are consistent with the functional placement of Esrrb downstream of Nanog.


Assuntos
Proteínas de Homeodomínio/metabolismo , Células-Tronco Neurais/fisiologia , Células-Tronco Pluripotentes/fisiologia , Receptores de Estrogênio/metabolismo , Animais , Fusão Celular , Linhagem Celular , Proliferação de Células , Sobrevivência Celular/genética , Reprogramação Celular/genética , Quimera , Técnicas de Cultura Embrionária , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Interleucina-6/metabolismo , Camundongos , Análise em Microsséries , Proteínas Mutantes/genética , Proteína Homeobox Nanog , Receptores de Estrogênio/genética , Receptores de OSM-LIF/genética , Transgenes/genética
12.
Development ; 139(13): 2288-98, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22669820

RESUMO

The transcription factors Nanog and Oct4 regulate pluripotency in the pre-implantation epiblast and in derivative embryonic stem cells. During post-implantation development, the precise timing and mechanism of the loss of pluripotency is unknown. Here, we show that in the mouse, pluripotency is extinguished at the onset of somitogenesis, coincident with reduced expression and chromatin accessibility of Oct4 and Nanog regulatory regions. Prior to somitogenesis expression of both Nanog and Oct4 is regionalized. We show that pluripotency tracks the in vivo level of Oct4 and not Nanog by assessing the ability to reactivate or maintain Nanog expression in cell culture. Enforced Oct4 expression in somitogenesis-stage tissue provokes rapid reopening of Oct4 and Nanog chromatin, Nanog re-expression and resuscitates moribund pluripotency. Our data suggest that decreasing Oct4 expression is converted to a sudden drop in competence to maintain pluripotency gene regulatory network activity that is subsequently stabilized by epigenetic locks.


Assuntos
Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Células Cultivadas , Cromatina/metabolismo , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Proteína Homeobox Nanog
13.
Philos Trans R Soc Lond B Biol Sci ; 366(1575): 2230-7, 2011 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-21727128

RESUMO

Stem cells are defined by the simultaneous possession of the seemingly incongruent properties of self-renewal and multi-lineage differentiation potential. To maintain a stem cell population, these opposing forces must be balanced. Transcription factors that function to direct pluripotent cell identity are not all equally distributed throughout the pluripotent cell population. While Oct4 levels are relatively homogeneous, other transcription factors, such as Nanog, are more heterogeneously expressed. Moreover, Oct4 positive cells fluctuate between states of high Nanog expression associated with a high probability of self-renewal and low Nanog expression associated with an increased propensity to differentiate. As embryonic stem (ES) cells transit to the more developmentally advanced epiblast stem cell (EpiSC) state, the levels of pluripotency transcription factors are modulated. Such modulations are blunted in cells that overexpress Nanog and this may underlie the resistance of Nanog-overexpressing cells to transit to an EpiSC state. Interestingly, increasing the levels of Nanog in EpiSC can facilitate reversion to the ES cell state. Together these observations suggest that Nanog lies close to the top of the hierarchy of pluripotent transcription factor regulation.


Assuntos
Regulação da Expressão Gênica/fisiologia , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Animais , Fator 3 de Transcrição de Octâmero/genética
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