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1.
Nucleic Acids Res ; 47(13): 6737-6752, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31147716

RESUMO

Retinoic acid (RA) induces rapid differentiation of embryonic stem cells (ESCs), partly by activating expression of the transcription factor Hoxa1, which regulates downstream target genes that promote ESCs differentiation. However, mechanisms of RA-induced Hoxa1 expression and ESCs early differentiation remain largely unknown. Here, we identify a distal enhancer interacting with the Hoxa1 locus through a long-range chromatin loop. Enhancer deletion significantly inhibited expression of RA-induced Hoxa1 and endoderm master control genes such as Gata4 and Gata6. Transcriptome analysis revealed that RA-induced early ESCs differentiation was blocked in Hoxa1 enhancer knockout cells, suggesting a requirement for the enhancer. Restoration of Hoxa1 expression partly rescued expression levels of ∼40% of genes whose expression changed following enhancer deletion, and ∼18% of promoters of those rescued genes were directly bound by Hoxa1. Our data show that a distal enhancer maintains Hoxa1 expression through long-range chromatin loop and that Hoxa1 directly regulates downstream target genes expression and then orchestrates RA-induced early differentiation of ESCs. This discovery reveals mechanisms of a novel enhancer regulating RA-induced Hoxa genes expression and early ESCs differentiation.


Assuntos
Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos , Proteínas de Homeodomínio/biossíntese , Fatores de Transcrição/biossíntese , Tretinoína/farmacologia , Animais , Sistemas CRISPR-Cas , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Células-Tronco Embrionárias/efeitos dos fármacos , Endoderma/metabolismo , Elementos Facilitadores Genéticos/genética , Edição de Genes , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Ontologia Genética , Proteínas de Homeodomínio/genética , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/genética
2.
Int J Dev Biol ; 63(6-7): 271-280, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31250910

RESUMO

Human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are very attractive cell sources for the treatment of diabetes mellitus, because numerous cells can be obtained using their infinite proliferation potential to overcome the paucity of donor islets. Advances in differentiation protocols make it possible to generate glucose responsive hPSC-beta cells, which can ameliorate hyperglycemia in diabetic mice. These protocols have mainly been based on an adherent culture system. However, in clinical applications, suspension culture methods are more suitable for large-scale culture. There are reports that suspension culture and spheroid formation promote differentiation in various cell types, including hPSCs, but, to our knowledge, there are no reports comparing gene expression patterns between suspension and adherent cultured human iPSCs (hiPSCs) during definitive endoderm (DE) differentiation. In this study, we chose several stage marker genes, not only for DE but also for posterior epiblast and primitive streak, and we examined their time course expression in suspension and adherent cultures by quantitative PT-PCR (qPCR), western blot, flow cytometry and immunocytochemistry. Our results demonstrate that expressions of these marker genes are faster and more strongly induced in suspension culture than in adherent culture during the DE differentiation process, indicating that suspension culture favors DE differentiation.


Assuntos
Adesão Celular , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Endoderma/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Esferoides Celulares/citologia , Técnicas de Cultura de Células , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Endoderma/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Esferoides Celulares/metabolismo
3.
Int J Dev Biol ; 63(3-4-5): 171-186, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31058295

RESUMO

The integration of extracellular signals and lineage-specific transcription factors allows cells to react flexibly to their environment, thus endowing the mammalian embryo with the capacity of regulative development. The combination of genetic and pharmacological tools allowing disruption of the fibroblast growth factor / extracellular signal-regulated kinase (FGF/ERK) pathway, together with animal models expressing lineage-specific reporters provided new insights into the role of this signaling cascade during mammalian development, as well as in embryo-derived stem cells. Here, we combine current knowledge acquired from different mammalian models to consider the universality of this cascade in specifying cellular fate across mammalian species.


Assuntos
Blastocisto/metabolismo , Células-Tronco Embrionárias/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Sistema de Sinalização das MAP Quinases , Animais , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Endoderma/citologia , Endoderma/embriologia , Endoderma/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Camadas Germinativas/citologia , Camadas Germinativas/embriologia , Camadas Germinativas/metabolismo , Mamíferos
4.
Methods Mol Biol ; 1975: 53-77, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31062305

RESUMO

Human pluripotent stem cells are defined by their potential to give rise to all of the lineages of an embryo proper. Guiding the differentiation of embryonic stem cells or induced pluripotent stem cells can be achieved by exposing them to a succession of signaling conditions meant to mimic developmental milieus. However, achieving a quantitative understanding of the relationship between proliferation, cell death, and commitment has been difficult due to the inherent heterogeneity of pluripotent stem cells and their differentiation. Here, we describe a computational modeling approach to track the dynamics of germ layer commitment of human embryonic stem cells. We demonstrate that simulations using this model yield specific hypotheses regarding proliferation, cell death, and commitment and that these predictions are consistent with experimental measurements.


Assuntos
Diferenciação Celular , Linhagem da Célula , Endoderma/citologia , Células-Tronco Embrionárias Humanas/citologia , Modelos Teóricos , Células-Tronco Pluripotentes/citologia , Ativinas/metabolismo , Endoderma/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Células-Tronco Pluripotentes/metabolismo
5.
Methods Mol Biol ; 1975: 107-129, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31062307

RESUMO

Studying cell fate dynamics is complicated by the fact that direct in vivo observation of individual cell fate outcomes is usually not possible and only multicellular data of cell clones can be obtained. In this situation, experimental data alone is not sufficient to validate biological models because the hypotheses and the data cannot be directly compared and thus standard statistical tests cannot be leveraged. On the other hand, mathematical modelling can bridge the scales between a hypothesis and measured data via quantitative predictions from a mathematical model. Here, we describe how to implement the rules behind a hypothesis (cell fate outcomes) one-to-one as a stochastic model, how to evaluate such a rule-based model mathematically via analytical calculation or stochastic simulations of the model's Master equation, and to predict the outcomes of clonal statistics for respective hypotheses. We also illustrate two approaches to compare these predictions directly with the clonal data to assess the models.


Assuntos
Diferenciação Celular , Linhagem da Célula , Endoderma/citologia , Modelos Teóricos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Pluripotentes/citologia , Animais , Células Clonais , Endoderma/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes/metabolismo
6.
Nature ; 570(7759): 77-82, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31086336

RESUMO

Ontogeny describes the emergence of complex multicellular organisms from single totipotent cells. This field is particularly challenging in mammals, owing to the indeterminate relationship between self-renewal and differentiation, variation in progenitor field sizes, and internal gestation in these animals. Here we present a flexible, high-information, multi-channel molecular recorder with a single-cell readout and apply it as an evolving lineage tracer to assemble mouse cell-fate maps from fertilization through gastrulation. By combining lineage information with single-cell RNA sequencing profiles, we recapitulate canonical developmental relationships between different tissue types and reveal the nearly complete transcriptional convergence of endodermal cells of extra-embryonic and embryonic origins. Finally, we apply our cell-fate maps to estimate the number of embryonic progenitor cells and their degree of asymmetric partitioning during specification. Our approach enables massively parallel, high-resolution recording of lineage and other information in mammalian systems, which will facilitate the construction of a quantitative framework for understanding developmental processes.


Assuntos
Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endoderma/embriologia , Endoderma/metabolismo , Feminino , Fertilização , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Camundongos , Especificidade de Órgãos/genética , Fenótipo , Análise de Sequência de RNA , Análise de Célula Única
7.
Nat Genet ; 51(6): 999-1010, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110351

RESUMO

Human embryonic stem cells (ESCs) and human induced pluripotent stem cells hold great promise for cell-based therapies and drug discovery. However, homogeneous differentiation remains a major challenge, highlighting the need for understanding developmental mechanisms. We performed genome-scale CRISPR screens to uncover regulators of definitive endoderm (DE) differentiation, which unexpectedly uncovered five Jun N-terminal kinase (JNK)-JUN family genes as key barriers of DE differentiation. The JNK-JUN pathway does not act through directly inhibiting the DE enhancers. Instead, JUN co-occupies ESC enhancers with OCT4, NANOG, SMAD2 and SMAD3, and specifically inhibits the exit from the pluripotent state by impeding the decommissioning of ESC enhancers and inhibiting the reconfiguration of SMAD2 and SMAD3 chromatin binding from ESC to DE enhancers. Therefore, the JNK-JUN pathway safeguards pluripotency from precocious DE differentiation. Direct pharmacological inhibition of JNK significantly improves the efficiencies of generating DE and DE-derived pancreatic and lung progenitor cells, highlighting the potential of harnessing the knowledge from developmental studies for regenerative medicine.


Assuntos
Diferenciação Celular/genética , Endoderma/embriologia , Endoderma/metabolismo , Genoma , Genômica , Sistema de Sinalização das MAP Quinases , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Expressão Gênica , Técnicas de Inativação de Genes , Genes Reporter , Genômica/métodos , Humanos , Células-Tronco Pluripotentes Induzidas , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Modelos Biológicos , Reprodutibilidade dos Testes , Proteínas Smad
8.
In Vitro Cell Dev Biol Anim ; 55(5): 355-367, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30993557

RESUMO

N-terminal acetylation (Nt-acetylation) refers to the acetylation of the free α-amino group at the N-terminus of a polypeptide. While the effects of Nt-acetylation are multifaceted, its most known function is in the acetylation-dependent N-end rule protein degradation pathway (Ac/N-end rule pathway), where Nt-acetylation is recognized as a degron by designated E3 ligases, eventually leading to target degradation by the ubiquitin-proteasome system. Naa10 is the catalytic subunit of the major Nt-acetylation enzyme NatA, which Nt-acetylates proteins whose second amino acid has a small side chain. In humans, NAA10 is the responsible mutated gene in Ogden syndrome and is thought to play important roles in development. However, it is unclear how the Ac/N-end rule pathway affects the differentiation ability of mouse embryonic stem cells (mESCs). We hypothesized that the balance of pluripotency factors may be maintained by the Ac/N-end rule pathway. Thus, we established Naa10 knockout mESCs to test this hypothesis. We found that Naa10 deficiency attenuated differentiation towards the epiblast lineage, deviating towards primitive endoderm. However, this was not caused by disturbing the balance of pluripotency factors, rather by augmenting FGF/MAPK signaling.


Assuntos
Linhagem da Célula/genética , Camadas Germinativas/crescimento & desenvolvimento , Células-Tronco Embrionárias Murinas/metabolismo , Acetiltransferase N-Terminal A/genética , Acetiltransferase N-Terminal E/genética , Acetilação , Animais , Diferenciação Celular/genética , Endoderma/crescimento & desenvolvimento , Endoderma/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Técnicas de Inativação de Genes , Camadas Germinativas/metabolismo , Humanos , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Acetiltransferase N-Terminal A/metabolismo , Acetiltransferase N-Terminal E/metabolismo , Processamento de Proteína Pós-Traducional/genética , Proteólise , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética
9.
Development ; 146(6)2019 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-30814117

RESUMO

Long-range signaling by morphogens and their inhibitors define embryonic patterning yet quantitative data and models are rare, especially in humans. Here, we use a human embryonic stem cell micropattern system to model formation of the primitive streak (PS) by WNT. In the pluripotent state, E-cadherin (E-CAD) transduces boundary forces to focus WNT signaling to the colony border. Following application of WNT ligand, E-CAD mediates a front or wave of epithelial-to-mesenchymal (EMT) conversion analogous to PS extension in an embryo. By knocking out the secreted WNT inhibitors active in our system, we show that DKK1 alone controls the extent and duration of patterning. The NODAL inhibitor cerberus 1 acts downstream of WNT to refine the endoderm versus mesoderm fate choice. Our EMT wave is a generic property of a bistable system with diffusion and we present a single quantitative model that describes both the wave and our knockout data.


Assuntos
Padronização Corporal , Células-Tronco Embrionárias Humanas/citologia , Linha Primitiva/embriologia , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Citoesqueleto de Actina/metabolismo , Animais , Linhagem Celular , Linhagem da Célula , Citocinas/metabolismo , Endoderma/metabolismo , Transição Epitelial-Mesenquimal , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ligantes , Mesoderma/metabolismo , Camundongos , Fenótipo , Células-Tronco Pluripotentes/citologia , Domínios Proteicos , Fator de Crescimento Transformador beta/metabolismo , Proteína Wnt3A/metabolismo
10.
Cell Prolif ; 52(3): e12591, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896067

RESUMO

OBJECTIVES: To date, many efforts have been made to establish porcine embryonic stem (pES) cells without success. Extraembryonic endoderm (XEN) cells can self-renew and differentiate into the visceral endoderm and parietal endoderm. XEN cells are derived from the primitive endoderm of the inner cell mass of blastocysts and may be an intermediate state in cell reprogramming. MATERIALS AND METHODS: Porcine XEN cells (pXENCs) were generated from porcine pluripotent stem cells (pPSCs) and were characterized by RNA sequencing and immunofluorescence analyses. The developmental potential of pXENCs was investigated in chimeric mouse embryos. RESULTS: Porcine XEN cells derived from porcine pPSCs were successfully expanded in N2B27 medium supplemented with bFGF for least 30 passages. RNA sequencing and immunofluorescence analyses showed that pXENCs expressed the murine and canine XEN markers Gata6, Gata4, Sox17 and Pdgfra but not the pluripotent markers Oct4, Sox2 and TE marker Cdx2. Moreover, these cells contributed to the XEN when injected into four-cell stage mouse embryos. Supplementation with Chir99021 and SB431542 promoted the pluripotency of the pXENCs. CONCLUSIONS: We successfully derived pXENCs and showed that supplementation with Chir99021 and SB431542 confer them with pluripotency. Our results provide a new resource for investigating the reprogramming mechanism of porcine-induced pluripotent stem cells.


Assuntos
Endoderma/citologia , Endoderma/embriologia , Suínos/embriologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Técnicas de Cocultura , Cães , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endoderma/metabolismo , Expressão Gênica , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Suínos/genética , Suínos/metabolismo , Quimeras de Transplante
11.
Dev Cell ; 48(3): 361-370.e3, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30753836

RESUMO

The Erk mitogen-activated protein kinase plays diverse roles in animal development. Its widespread reuse raises a conundrum: when a single kinase like Erk is activated, how does a developing cell know which fate to adopt? We combine optogenetic control with genetic perturbations to dissect Erk-dependent fates in the early Drosophila embryo. We find that Erk activity is sufficient to "posteriorize" 88% of the embryo, inducing gut endoderm-like gene expression and morphogenetic movements in all cells within this region. Gut endoderm fate adoption requires at least 1 h of signaling, whereas a 30-min Erk pulse specifies a distinct ectodermal cell type, intermediate neuroblasts. We find that the endoderm-ectoderm cell fate switch is controlled by the cumulative load of Erk activity, not the duration of a single pulse. The fly embryo thus harbors a classic example of dynamic control, where the temporal profile of Erk signaling selects between distinct physiological outcomes.


Assuntos
Ectoderma/citologia , Embrião de Mamíferos/metabolismo , Endoderma/citologia , Regulação da Expressão Gênica no Desenvolvimento , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Drosophila/embriologia , Ectoderma/embriologia , Embrião não Mamífero/metabolismo , Endoderma/embriologia , Endoderma/metabolismo , Morfogênese/genética
12.
Dev Comp Immunol ; 95: 89-95, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30753854

RESUMO

The chicken yolk sac (YS) plays an important role in nutrient absorption and immune function for the developing embryo. The avian ß-defensins (AvBD) are cationic peptides that are important members of the innate immune system. The objective of this study was to profile AvBD mRNA expression patterns and distribution of cells expressing AvBD mRNA in the chicken YS. Expression of AvBD1, 2, 7, and 10 mRNA was low at embryonic day 7 (e7), increased to e9 through e13 and then declined to e19. Using in situ hybridization, AvBD10 mRNA was found to be expressed in endodermal epithelial cells, while AvBD1, 2, and 7 mRNA were expressed in heterophils. The developmental expression pattern and distribution of AvBD mRNA in the YS reveals the importance of these genes to protection of the developing chick embryo.


Assuntos
Proteínas Aviárias/genética , Desenvolvimento Embrionário/imunologia , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Saco Vitelino/imunologia , beta-Defensinas/genética , Animais , Proteínas Aviárias/imunologia , Embrião de Galinha , Galinhas , Endoderma/citologia , Endoderma/imunologia , Endoderma/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , RNA Mensageiro/metabolismo , Saco Vitelino/crescimento & desenvolvimento , Saco Vitelino/metabolismo , beta-Defensinas/imunologia
13.
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
14.
PLoS Genet ; 15(2): e1007996, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30763319

RESUMO

Pharyngeal pouches, a series of outpocketings that bud from the foregut endoderm, are essential to the formation of craniofacial skeleton as well as several important structures like parathyroid and thymus. However, whether pharyngeal pouch progenitors exist in the developing gut tube remains unknown. Here, taking advantage of cell lineage tracing and transgenic ablation technologies, we identified a population of nkx2.3+ pouch progenitors in zebrafish embryos and demonstrated an essential requirement of ectodermal BMP2b for their specification. At early somite stages, nkx2.3+ cells located at lateral region of pharyngeal endoderm give rise to the pouch epithelium except a subpopulation expressing pdgfαa rather than nkx2.3. A small-scale screen of chemical inhibitors reveals that BMP signaling is necessary to specify these progenitors. Loss-of-function analyses show that BMP2b, expressed in the pharyngeal ectoderm, actives Smad effectors in endodermal cells to induce nkx2.3+ progenitors. Collectively, our study provides in vivo evidence for the existence of pouch progenitors and highlights the importance of BMP2b signaling in progenitor specification.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Padronização Corporal/fisiologia , Proteína Morfogenética Óssea 2/genética , Linhagem da Célula/genética , Linhagem da Célula/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endoderma/embriologia , Endoderma/metabolismo , Proteínas de Homeodomínio/genética , Mutação , Faringe/embriologia , Faringe/metabolismo , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismo , Somitos/embriologia , Somitos/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
15.
Dev Biol ; 449(1): 1-13, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30797757

RESUMO

Wnt proteins can activate different intracellular signaling pathways. These pathways need to be tightly regulated for proper cardiogenesis. The canonical Wnt/ß-catenin inhibitor Dkk1 has been shown to be sufficient to trigger cardiogenesis in gain-of-function experiments performed in multiple model systems. Loss-of-function studies however did not reveal any fundamental function for Dkk1 during cardiogenesis. Using Xenopus laevis as a model we here show for the first time that Dkk1 is required for proper differentiation of cardiomyocytes, whereas specification of cardiomyocytes remains unaffected in absence of Dkk1. This effect is at least in part mediated through regulation of non-canonical Wnt signaling via Wnt11. In line with these observations we also found that Isl1, a critical regulator for specification of the common cardiac progenitor cell (CPC) population, acts upstream of Dkk1.


Assuntos
Diferenciação Celular , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Miocárdio/citologia , Via de Sinalização Wnt , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Animais , Biomarcadores/metabolismo , Padronização Corporal , Sistema Digestório/embriologia , Sistema Digestório/metabolismo , Regulação para Baixo/genética , Embrião não Mamífero/metabolismo , Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Homeodomínio LIM/metabolismo , Mesoderma/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Organogênese/genética , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo
16.
Curr Stem Cell Res Ther ; 14(3): 278-289, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30674265

RESUMO

The discovery of small non-coding RNAs began an interesting era in cellular and molecular biology. To date, miRNAs are the best recognized non-coding RNAs for maintenance and differentiation of pluripotent stem cells including embryonic stem cells (ES), induced pluripotent stem cells (iPSC), and cancer stem cells. ES cells are defined by their ability to self-renew, teratoma formation, and to produce numerous types of differentiated cells. Dual capacity of ES cells for self-renewal and differentiation is controlled by specific interaction with the neighboring cells and intrinsic signaling pathways from the level of transcription to translation. The ES cells have been the suitable model for evaluating the function of non-coding RNAs and in specific miRNAs. So far, the general function of the miRNAs in ES cells has been assessed in mammalian and non-mammalian stem cells. Nowadays, the evolution of sequencing technology led to the discovery of numerous miRNAs in human and mouse ES cells that their expression levels significantly changes during proliferation and differentiation. Several miRNAs have been identified in ectoderm, mesoderm, and endoderm cells, as well. This review would focus on recent knowledge about the expression and functional roles of miRNAs in embryonic and lineage-specific stem cells. It also describes that miRNAs might have essential roles in orchestrating the Waddington's landscape structure during development.


Assuntos
Linhagem da Célula/genética , Células-Tronco Embrionárias/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , MicroRNAs/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neurais/metabolismo , Animais , Comunicação Celular , Diferenciação Celular , Proliferação de Células , Ectoderma/citologia , Ectoderma/metabolismo , Células-Tronco Embrionárias/citologia , Endoderma/citologia , Endoderma/metabolismo , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Mesoderma/citologia , Mesoderma/metabolismo , MicroRNAs/classificação , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neurais/citologia , Transdução de Sinais
17.
Nature ; 565(7740): 480-484, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30651642

RESUMO

The embryonic gut tube is a cylindrical structure from which the respiratory and gastrointestinal tracts develop1. Although the early emergence of the endoderm as an epithelial sheet2,3 and later morphogenesis of the definitive digestive and respiratory organs4-6 have been investigated, the intervening process of gut tube formation remains relatively understudied7,8. Here we investigate the molecular control of macroscopic forces underlying early morphogenesis of the gut tube in the chick embryo. The gut tube has been described as forming from two endodermal invaginations-the anterior intestinal portal (AIP) towards the rostral end of the embryo and the caudal intestinal portal (CIP) at the caudal end-that migrate towards one another, internalizing the endoderm until they meet at the yolk stalk (umbilicus in mammals)1,6. Migration of the AIP to form foregut has been descriptively characterized8,9, but the hindgut is likely to form by a distinct mechanism that has not been fully explained10. We find that the hindgut is formed by collective cell movements through a stationary CIP, rather than by movement of the CIP itself. Further, combining in vivo imaging, biophysics and mathematical modelling with molecular and embryological approaches, we identify a contractile force gradient that drives cell movements in the hindgut-forming endoderm, enabling tissue-scale posterior extension of the forming hindgut tube. The force gradient, in turn, is established in response to a morphogenic gradient of fibroblast growth factor signalling. As a result, we propose that an important positive feedback arises, whereby contracting cells draw passive cells from low to high fibroblast growth factor levels, recruiting them to contract and pull more cells into the elongating hindgut. In addition to providing insight into the early gut development, these findings illustrate how large-scale tissue level forces can be traced to developmental signals during vertebrate morphogenesis.


Assuntos
Trato Gastrointestinal/embriologia , Morfogênese , Animais , Padronização Corporal , Movimento Celular , Embrião de Galinha , Endoderma/citologia , Endoderma/embriologia , Endoderma/metabolismo , Fator 8 de Crescimento de Fibroblasto/metabolismo , Trato Gastrointestinal/citologia , Trato Gastrointestinal/metabolismo , Transdução de Sinais
18.
Development ; 146(2)2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30630826

RESUMO

Defects in the middle ear ossicles - malleus, incus and stapes - can lead to conductive hearing loss. During development, neural crest cells (NCCs) migrate from the dorsal hindbrain to specific locations in pharyngeal arch (PA) 1 and 2, to form the malleus-incus and stapes, respectively. It is unclear how migratory NCCs reach their proper destination in the PA and initiate mesenchymal condensation to form specific ossicles. We show that secreted molecules sonic hedgehog (SHH) and bone morphogenetic protein 4 (BMP4) emanating from the pharyngeal endoderm are important in instructing region-specific NCC condensation to form malleus-incus and stapes, respectively, in mouse. Tissue-specific knockout of Shh in the pharyngeal endoderm or Smo (a transducer of SHH signaling) in NCCs causes the loss of malleus-incus condensation in PA1 but only affects the maintenance of stapes condensation in PA2. By contrast, knockout of Bmp4 in the pharyngeal endoderm or Smad4 (a transducer of TGFß/BMP signaling) in the NCCs disrupts NCC migration into the stapes region in PA2, affecting stapes formation. These results indicate that region-specific endodermal signals direct formation of specific middle ear ossicles.


Assuntos
Ossículos da Orelha/embriologia , Endoderma/embriologia , Endoderma/metabolismo , Crista Neural/citologia , Transdução de Sinais , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Movimento Celular , Sobrevivência Celular , Deleção de Genes , Proteínas Hedgehog , Bigorna/embriologia , Bigorna/metabolismo , Martelo/embriologia , Martelo/metabolismo , Camundongos , Modelos Biológicos , Crista Neural/embriologia , Crista Neural/metabolismo , Especificidade de Órgãos , Faringe/embriologia , Fenótipo , Estribo/embriologia , Estribo/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador beta/metabolismo
19.
Nat Protoc ; 14(2): 350-378, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30617351

RESUMO

Molecular and embryology studies have demonstrated that mouse pre-implantation embryo development is a process of progressive cell fate determination. At the time of implantation, three cell lineages are present in the developing blastocyst: the trophectoderm (TE), the epiblast (Epi) and the primitive endoderm (PrE). From these early embryo cells, trophoblast stem (TS) cells, embryonic stem (ES) cells and extra-embryonic endoderm stem (XEN) cells can be derived. Recently, we derived stem cells with blastomere-like features from mouse cleavage-stage embryos, which we named expanded-potential stem cells (EPSCs). Here, we provide detailed protocols that describe how to establish EPSCs from single eight-cell-stage blastomeres or whole eight-cell pre-implantation mouse embryos, or by conversion of mouse ES cells or induced pluripotent stem (iPS) cells reprogrammed from fibroblasts. It takes 2-3 weeks to derive EPSCs from each cell source. The EPSCs derived from these protocols can differentiate into all embryonic and extra-embryonic lineages when implanted into chimeras. Furthermore, bona fide TS and XEN cell lines can be derived from EPSCs in vitro.


Assuntos
Técnicas de Cultura de Células , Linhagem da Célula , Desenvolvimento Embrionário , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Embrionárias Murinas/citologia , Trofoblastos/citologia , Animais , Biomarcadores/metabolismo , Blastômeros/citologia , Blastômeros/metabolismo , Fator de Transcrição CDX2/genética , Fator de Transcrição CDX2/metabolismo , Diferenciação Celular , Proliferação de Células , Reprogramação Celular , Endoderma/citologia , Endoderma/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Cultura Primária de Células , Trofoblastos/metabolismo
20.
Proc Natl Acad Sci U S A ; 116(4): 1384-1393, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30606801

RESUMO

Upon virus infection, pluripotent stem cells neither induce nor respond to canonical type I interferons (IFN-I). To better understand this biology, we characterized induced pluripotent stem cells (iPSCs) as well as their differentiated parental or rederived counterparts. We confirmed that only iPSCs failed to respond to viral RNA, IFN-I, or viral infection. This lack of response could be phenocopied in fibroblasts with the expression of a reprogramming factor which repressed the capacity to induce canonical antiviral pathways. To ascertain the consequences of restoring the antiviral response in the context of pluripotency, we engineered a system to engage these defenses in iPSCs. Inducible expression of a recombinant virus-activated transcription factor resulted in the successful reconstitution of antiviral defenses through the direct up-regulation of IFN-I-stimulated genes. Induction of the antiviral signature in iPSCs, even for a short duration, resulted in the dysregulation of genes associated with all three germ layers despite maintaining pluripotency markers. Trilineage differentiation of these same cells showed that engagement of the antiviral defenses compromised ectoderm and endoderm formation and dysregulated the development of mesodermal sublineages. In all, these data suggest that the temporal induction of the antiviral response primes iPSCs away from pluripotency and induces numerous aberrant gene products upon differentiation. Together these results suggest that the IFN-I system and pluripotency may be incompatible with each other and thus explain why stem cells do not utilize the canonical antiviral system.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Interferon Tipo I/metabolismo , Antivirais/farmacologia , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Reprogramação Celular/fisiologia , Ectoderma/efeitos dos fármacos , Ectoderma/metabolismo , Ectoderma/fisiologia , Ectoderma/virologia , Endoderma/efeitos dos fármacos , Endoderma/metabolismo , Endoderma/fisiologia , Endoderma/virologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Fibroblastos/virologia , Camadas Germinativas/efeitos dos fármacos , Camadas Germinativas/metabolismo , Camadas Germinativas/fisiologia , Camadas Germinativas/virologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/virologia , RNA Viral/genética , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia
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