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1.
Development ; 146(14)2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31320324

RESUMO

Activation of the ERK signalling pathway is essential for the differentiation of the inner cell mass (ICM) during mouse preimplantation development. We show here that ERK phosphorylation occurs in ICM precursor cells, in differentiated primitive endoderm (PrE) cells as well as in the mature, formative state epiblast (Epi). We further show that DUSP4 and ETV5, factors often involved in negative-feedback loops of the FGF pathway, are differently regulated. Whereas DUSP4 presence clearly depends on ERK phosphorylation in PrE cells, ETV5 localises mainly to Epi cells. Unexpectedly, ETV5 accumulation does not depend on direct activation by ERK but requires NANOG activity. Indeed ETV5, like Fgf4 expression, is not present in Nanog mutant embryos. Our results lead us to propose that in pluripotent early Epi cells, NANOG induces the expression of both Fgf4 and Etv5 to enable the differentiation of neighbouring cells into the PrE while protecting the Epi identity from autocrine signalling.


Assuntos
Blastocisto/metabolismo , Desenvolvimento Embrionário/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , Sistema de Sinalização das MAP Quinases , Animais , Massa Celular Interna do Blastocisto/citologia , Massa Celular Interna do Blastocisto/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator 4 de Crescimento de Fibroblastos/genética , Fator 4 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Proteínas Tirosina Fosfatases/fisiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
2.
Cell Mol Life Sci ; 78(2): 757-768, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32405722

RESUMO

The acquisition of cell identity is associated with developmentally regulated changes in the cellular histone methylation signatures. For instance, commitment to neural differentiation relies on the tightly controlled gain or loss of H3K27me3, a hallmark of polycomb-mediated transcriptional gene silencing, at specific gene sets. The KDM6B demethylase, which removes H3K27me3 marks at defined promoters and enhancers, is a key factor in neurogenesis. Therefore, to better understand the epigenetic regulation of neural fate acquisition, it is important to determine how Kdm6b expression is regulated. Here, we investigated the molecular mechanisms involved in the induction of Kdm6b expression upon neural commitment of mouse embryonic stem cells. We found that the increase in Kdm6b expression is linked to a rearrangement between two 3D configurations defined by the promoter contact with two different regions in the Kdm6b locus. This is associated with changes in 5-hydroxymethylcytosine (5hmC) levels at these two regions, and requires a functional ten-eleven-translocation (TET) 3 protein. Altogether, our data support a model whereby Kdm6b induction upon neural commitment relies on an intronic enhancer the activity of which is defined by its TET3-mediated 5-hmC level. This original observation reveals an unexpected interplay between the 5-hmC and H3K27me3 pathways during neural lineage commitment in mammals. It also questions to which extent KDM6B-mediated changes in H3K27me3 level account for the TET-mediated effects on gene expression.


Assuntos
Dioxigenases/metabolismo , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Histona Desmetilases com o Domínio Jumonji/genética , Neurogênese , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Células Cultivadas , Dioxigenases/genética , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Técnicas de Silenciamento de Genes , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Regulação para Cima
3.
Nat Commun ; 13(1): 3550, 2022 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-35729116

RESUMO

The epiblast is the source of all mammalian embryonic tissues and of pluripotent embryonic stem cells. It differentiates alongside the primitive endoderm in a "salt and pepper" pattern from inner cell mass (ICM) progenitors during the preimplantation stages through the activity of NANOG, GATA6 and the FGF pathway. When and how epiblast lineage specification is initiated is still unclear. Here, we show that the coordinated expression of pluripotency markers defines epiblast identity. Conversely, ICM progenitor cells display random cell-to-cell variability in expression of various pluripotency markers, remarkably dissimilar from the epiblast signature and independently from NANOG, GATA6 and FGF activities. Coordination of pluripotency markers expression fails in Nanog and Gata6 double KO (DKO) embryos. Collectively, our data suggest that NANOG triggers epiblast specification by ensuring the coordinated expression of pluripotency markers in a subset of cells, implying a stochastic mechanism. These features are likely conserved, as suggested by analysis of human embryos.


Assuntos
Endoderma , Camadas Germinativas , Animais , Blastocisto/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/metabolismo , Humanos , Mamíferos/genética , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo
4.
Methods Mol Biol ; 2214: 1-10, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32944899

RESUMO

A couple of days after fertilization of a mouse oocyte by a sperm, two sequential cell differentiation events segregate pluripotent cells that can be identified by the presence of specific markers. Early mammalian embryos are relatively easy to recover as they are not yet implanted in the uterus matrix. Several decades of experimentation have enabled to find appropriate media to culture them, and therefore provide an excellent way to test different experimental setups such as the use of signaling inhibitors. We provide here a commonly used protocol to culture preimplantation embryos as well as a method to detect pluripotent cells in blastocysts.


Assuntos
Blastocisto/citologia , Técnicas de Cultura Embrionária/métodos , Camundongos/embriologia , Células-Tronco Pluripotentes/citologia , Animais , Embrião de Mamíferos/citologia , Imunofluorescência/métodos , Microscopia de Fluorescência/métodos
5.
Cell Stem Cell ; 28(9): 1625-1640.e6, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34004179

RESUMO

Understanding lineage specification during human pre-implantation development is a gateway to improving assisted reproductive technologies and stem cell research. Here we employ pseudotime analysis of single-cell RNA sequencing (scRNA-seq) data to reconstruct early mouse and human embryo development. Using time-lapse imaging of annotated embryos, we provide an integrated, ordered, and continuous analysis of transcriptomics changes throughout human development. We reveal that human trophectoderm/inner cell mass transcriptomes diverge at the transition from the B2 to the B3 blastocyst stage, just before blastocyst expansion. We explore the dynamics of the fate markers IFI16 and GATA4 and show that they gradually become mutually exclusive upon establishment of epiblast and primitive endoderm fates, respectively. We also provide evidence that NR2F2 marks trophectoderm maturation, initiating from the polar side, and subsequently spreads to all cells after implantation. Our study pinpoints the precise timing of lineage specification events in the human embryo and identifies transcriptomics hallmarks and cell fate markers.


Assuntos
Desenvolvimento Embrionário , Transcriptoma , Animais , Blastocisto , Linhagem da Célula/genética , Desenvolvimento Embrionário/genética , Camadas Germinativas , Humanos , Camundongos , Transcriptoma/genética
6.
Nat Cell Biol ; 22(4): 389-400, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32231305

RESUMO

In mouse embryonic stem cells (mESCs), chemical blockade of Gsk3α/ß and Mek1/2 (2i) instructs a self-renewing ground state whose endogenous inducers are unknown. Here we show that the axon guidance cue Netrin-1 promotes naive pluripotency by triggering profound signalling, transcriptomic and epigenetic changes in mESCs. Furthermore, we demonstrate that Netrin-1 can substitute for blockade of Gsk3α/ß and Mek1/2 to sustain self-renewal of mESCs in combination with leukaemia inhibitory factor and regulates the formation of the mouse pluripotent blastocyst. Mechanistically, we reveal how Netrin-1 and the balance of its receptors Neo1 and Unc5B co-regulate Wnt and MAPK pathways in both mouse and human ESCs. Netrin-1 induces Fak kinase to inactivate Gsk3α/ß and stabilize ß-catenin while increasing the phosphatase activity of a Ppp2r2c-containing Pp2a complex to reduce Erk1/2 activity. Collectively, this work identifies Netrin-1 as a regulator of pluripotency and reveals that it mediates different effects in mESCs depending on its receptor dosage, opening perspectives for balancing self-renewal and lineage commitment.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/genética , Receptores de Netrina/genética , Netrina-1/genética , Receptores de Superfície Celular/genética , Via de Sinalização Wnt/genética , Animais , Linhagem Celular , Embrião de Mamíferos , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismo , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 2/genética , MAP Quinase Quinase 2/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos SCID , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de Netrina/metabolismo , Netrina-1/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Receptores de Superfície Celular/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
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