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1.
Development ; 148(19)2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34608934

RESUMO

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expansion of the CAG repeats in the huntingtin gene (HTT). Although HD has been shown to have a developmental component, how early during human embryogenesis the HTT-CAG expansion can cause embryonic defects remains unknown. Here, we demonstrate a specific and highly reproducible CAG length-dependent phenotypic signature in a synthetic model for human gastrulation derived from human embryonic stem cells (hESCs). Specifically, we observed a reduction in the extension of the ectodermal compartment that is associated with enhanced activin signaling. Surprisingly, rather than a cell-autonomous effect, tracking the dynamics of TGFß signaling demonstrated that HTT-CAG expansion perturbs the spatial restriction of activin response. This is due to defects in the apicobasal polarization in the context of the polarized epithelium of the 2D gastruloid, leading to ectopic subcellular localization of TGFß receptors. This work refines the earliest developmental window for the prodromal phase of HD to the first 2 weeks of human development, as modeled by our 2D gastruloids.


Assuntos
Linhagem da Célula , Polaridade Celular , Camadas Germinativas/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Proteína Huntingtina/metabolismo , Ativinas/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Camadas Germinativas/citologia , Camadas Germinativas/embriologia , Células-Tronco Embrionárias Humanas/citologia , Humanos , Proteína Huntingtina/genética , Camundongos , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Expansão das Repetições de Trinucleotídeos
2.
Nat Commun ; 12(1): 5771, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34599190

RESUMO

Germline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that endow epiblast cells with the competence to respond to germline inductive signals remain unknown. Single-cell RNA sequencing across multiple stages of an in vitro PGC-like cells (PGCLC) differentiation system shows that PGCLC genes initially expressed in the naïve pluripotent stage become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). In contrast, the decommissioning of enhancers associated with these germline genes is incomplete. Namely, a subset of these enhancers partly retain H3K4me1, accumulate less heterochromatic marks and remain accessible and responsive to transcriptional activators. Subsequently, as in vitro germline competence is lost, these enhancers get further decommissioned and lose their responsiveness to transcriptional activators. Importantly, using H3K4me1-deficient cells, we show that the loss of this histone modification reduces the germline competence of EpiLC and decreases PGCLC differentiation efficiency. Our work suggests that, although H3K4me1 might not be essential for enhancer function, it can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions.


Assuntos
Elementos Facilitadores Genéticos , Células Germinativas/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Animais , Diferenciação Celular , Cromatina/metabolismo , Embrião de Mamíferos/citologia , Regulação da Expressão Gênica , Células Germinativas/citologia , Camadas Germinativas/citologia , Masculino , Metilação , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/citologia , Mutação/genética , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , RNA-Seq , Análise de Célula Única , Sítio de Iniciação de Transcrição , Transcrição Genética
3.
Cells ; 10(9)2021 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-34571882

RESUMO

The role of the pluripotency factor NANOG during the second embryonic lineage differentiation has been studied extensively in mouse, although species-specific differences exist. To elucidate the role of NANOG in an alternative model organism, we knocked out NANOG in fibroblast cells and produced bovine NANOG-knockout (KO) embryos via somatic cell nuclear transfer (SCNT). At day 8, NANOG-KO blastocysts showed a decreased total cell number when compared to controls from SCNT (NT Ctrl). The pluripotency factors OCT4 and SOX2 as well as the hypoblast (HB) marker GATA6 were co-expressed in all cells of the inner cell mass (ICM) and, in contrast to mouse Nanog-KO, expression of the late HB marker SOX17 was still present. We blocked the MEK-pathway with a MEK 1/2 inhibitor, and control embryos showed an increase in NANOG positive cells, but SOX17 expressing HB precursor cells were still present. NANOG-KO together with MEK-inhibition was lethal before blastocyst stage, similarly to findings in mouse. Supplementation of exogenous FGF4 to NANOG-KO embryos did not change SOX17 expression in the ICM, unlike mouse Nanog-KO embryos, where missing SOX17 expression was completely rescued by FGF4. We conclude that NANOG mediated FGF/MEK signaling is not required for HB formation in the bovine embryo and that another-so far unknown-pathway regulates HB differentiation.


Assuntos
Embrião de Mamíferos/metabolismo , Camadas Germinativas/metabolismo , Proteína Homeobox Nanog/genética , Animais , Apoptose/efeitos dos fármacos , Blastocisto/citologia , Blastocisto/metabolismo , Sistemas CRISPR-Cas , Bovinos , Diferenciação Celular , Linhagem da Célula , Embrião de Mamíferos/citologia , Edição de Genes , Camadas Germinativas/citologia , Camadas Germinativas/efeitos dos fármacos , Camundongos , Camundongos Knockout , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteína Homeobox Nanog/deficiência , Proteína Homeobox Nanog/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Fatores de Transcrição SOXF/metabolismo , Transdução de Sinais
4.
Cells ; 10(9)2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34571922

RESUMO

The ribosome, which is present in all three domains of life, plays a well-established, critical role in the translation process by decoding messenger RNA into protein. Ribosomal proteins, in contrast, appear to play non-translational roles in growth, differentiation, and disease. We recently discovered that ribosomes are involved in reverting cellular potency to a multipotent state. Ribosomal incorporation (the uptake of free ribosome by living cells) can direct the fate of both somatic and cancer cells into multipotency, allowing them to switch cell lineage. During this process, both types of cells experienced cell-cycle arrest and cellular stress while remaining multipotent. This review provides a molecular perspective on current insights into ribosome-induced multipotency and sheds light on how a common stress-associated mechanism may be involved. We also discuss the impact of this phenomenon on cancer cell reprogramming and its potential in cancer therapy.


Assuntos
Linhagem da Célula , Camadas Germinativas/citologia , Neoplasias/patologia , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/fisiologia , Animais , Diferenciação Celular , Camadas Germinativas/metabolismo , Humanos , Neoplasias/genética , Neoplasias/metabolismo , RNA Mensageiro/genética , Proteínas Ribossômicas/genética
5.
Nat Commun ; 12(1): 5140, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34446708

RESUMO

The difficulty of studying post-implantation development in mammals has sparked a flurry of activity to develop in vitro models, termed embryoids, based on self-organizing pluripotent stem cells. Previous approaches to derive embryoids either lack the physiological morphology and signaling interactions, or are unconducive to model post-gastrulation development. Here, we report a bioengineering-inspired approach aimed at addressing this gap. We employ a high-throughput cell aggregation approach to simultaneously coax mouse embryonic stem cells into hundreds of uniform epiblast-like aggregates in a solid matrix-free manner. When co-cultured with mouse trophoblast stem cell aggregates, the resulting hybrid structures initiate gastrulation-like events and undergo axial morphogenesis to yield structures, termed EpiTS embryoids, with a pronounced anterior development, including brain-like regions. We identify the presence of an epithelium in EPI aggregates as the major determinant for the axial morphogenesis and anterior development seen in EpiTS embryoids. Our results demonstrate the potential of EpiTS embryoids to study peri-gastrulation development in vitro.


Assuntos
Embrião de Mamíferos/embriologia , Camundongos/embriologia , Células-Tronco Embrionárias Murinas/citologia , Animais , Bioengenharia , Biomimética , Diferenciação Celular , Proliferação de Células , Implantação do Embrião , Embrião de Mamíferos/citologia , Corpos Embrioides/citologia , Desenvolvimento Embrionário , Feminino , Camadas Germinativas/citologia , Humanos , Morfogênese , Trofoblastos/citologia
6.
PLoS One ; 16(7): e0254763, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34320001

RESUMO

Understanding the processes by which the mammalian embryo implants in the maternal uterus is a long-standing challenge in embryology. New insights into this morphogenetic event could be of great importance in helping, for example, to reduce human infertility. During implantation the blastocyst, composed of epiblast, trophectoderm and primitive endoderm, undergoes significant remodelling from an oval ball to an egg cylinder. A main feature of this transformation is symmetry breaking and reshaping of the epiblast into a "cup". Based on previous studies, we hypothesise that this event is the result of mechanical constraints originating from the trophectoderm, which is also significantly transformed during this process. In order to investigate this hypothesis we propose MG# (MechanoGenetic Sharp), an original computational model of biomechanics able to reproduce key cell shape changes and tissue level behaviours in silico. With this model, we simulate epiblast and trophectoderm morphogenesis during implantation. First, our results uphold experimental findings that repulsion at the apical surface of the epiblast is essential to drive lumenogenesis. Then, we provide new theoretical evidence that trophectoderm morphogenesis indeed can dictate the cup shape of the epiblast and fosters its movement towards the uterine tissue. Our results offer novel mechanical insights into mouse peri-implantation and highlight the usefulness of agent-based modelling methods in the study of embryogenesis.


Assuntos
Endoderma/citologia , Camadas Germinativas/citologia , Modelos Biológicos , Animais , Proliferação de Células , Implantação do Embrião , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Endoderma/metabolismo , Camadas Germinativas/metabolismo , Camundongos
7.
Commun Biol ; 4(1): 802, 2021 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-34183774

RESUMO

Regeneration of the testis from pluripotent stem cells is a real challenge, reflecting the complexity of the interaction of germ cells and somatic cells. Here we report the generation of testicular somatic cell-like cells (TesLCs) including Sertoli cell-like cells (SCLCs) from mouse embryonic stem cells (ESCs) in xeno-free culture. We find that Nr5a1/SF1 is critical for interaction between SCLCs and PGCLCs. Intriguingly, co-culture of TesLCs with epiblast-like cells (EpiLCs), rather than PGCLCs, results in self-organised aggregates, or testicular organoids. In the organoid, EpiLCs differentiate into PGCLCs or gonocyte-like cells that are enclosed within a seminiferous tubule-like structure composed of SCLCs. Furthermore, conditioned medium prepared from TesLCs has a robust inducible activity to differentiate EpiLCs into PGCLCs. Our results demonstrate conditions for in vitro reconstitution of a testicular environment from ESCs and provide further insights into the generation of sperm entirely in xeno-free culture.


Assuntos
Células-Tronco Embrionárias/citologia , Camadas Germinativas/citologia , Espermatozoides/citologia , Testículo/citologia , Animais , Diferenciação Celular , Técnicas de Cocultura , Masculino , Camundongos , Organoides/citologia , Transcriptoma
8.
Nat Commun ; 12(1): 3679, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140473

RESUMO

Following implantation, the human embryo undergoes major morphogenetic transformations that establish the future body plan. While the molecular events underpinning this process are established in mice, they remain unknown in humans. Here we characterise key events of human embryo morphogenesis, in the period between implantation and gastrulation, using single-cell analyses and functional studies. First, the embryonic epiblast cells transition through different pluripotent states and act as a source of FGF signals that ensure proliferation of both embryonic and extra-embryonic tissues. In a subset of embryos, we identify a group of asymmetrically positioned extra-embryonic hypoblast cells expressing inhibitors of BMP, NODAL and WNT signalling pathways. We suggest that this group of cells can act as the anterior singalling centre to pattern the epiblast. These results provide insights into pluripotency state transitions, the role of FGF signalling and the specification of anterior-posterior axis during human embryo development.


Assuntos
Implantação do Embrião/genética , Desenvolvimento Embrionário , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Camadas Germinativas/metabolismo , Análise de Célula Única/métodos , Via de Sinalização Wnt , Proteína Morfogenética Óssea 1/antagonistas & inibidores , Linhagem da Célula , Células Cultivadas , Implantação do Embrião/fisiologia , Embrião de Mamíferos , Fatores de Crescimento de Fibroblastos/metabolismo , Gastrulação/fisiologia , Camadas Germinativas/citologia , Humanos , Processamento de Imagem Assistida por Computador , Família Multigênica , Proteína Nodal/antagonistas & inibidores , RNA-Seq , Análise Espaço-Temporal
9.
Biochem J ; 478(9): 1809-1825, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33988704

RESUMO

Epithelial plasticity involved the terminal and transitional stages that occur during epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET), both are essential at different stages of early embryonic development that have been co-opted by cancer cells to undergo tumor metastasis. These processes are regulated at multiple instances, whereas the post-transcriptional regulation of key genes mediated by microRNAs is gaining major attention as a common and conserved pathway. In this review, we focus on discussing the latest findings of the cellular and molecular basis of the less characterized process of MET during embryonic development, with special attention to the role of microRNAs. Although we take in consideration the necessity of being cautious when extrapolating the obtained evidence, we propose some commonalities between early embryonic development and cancer progression that can shed light into our current understanding of this complex event and might aid in the design of specific therapeutic approaches.


Assuntos
Desenvolvimento Embrionário/genética , Transição Epitelial-Mesenquimal/genética , MicroRNAs/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Progressão da Doença , Embrião de Mamíferos , Regulação Neoplásica da Expressão Gênica , Camadas Germinativas/citologia , Camadas Germinativas/crescimento & desenvolvimento , Camadas Germinativas/metabolismo , Humanos , MicroRNAs/classificação , MicroRNAs/metabolismo , Metástase Neoplásica , Proteínas de Neoplasias/classificação , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais , Somitos/citologia , Somitos/crescimento & desenvolvimento , Somitos/metabolismo
10.
Nature ; 595(7865): 85-90, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33981037

RESUMO

The ontogeny of the human haematopoietic system during fetal development has previously been characterized mainly through careful microscopic observations1. Here we reconstruct a phylogenetic tree of blood development using whole-genome sequencing of 511 single-cell-derived haematopoietic colonies from healthy human fetuses at 8 and 18 weeks after conception, coupled with deep targeted sequencing of tissues of known embryonic origin. We found that, in healthy fetuses, individual haematopoietic progenitors acquire tens of somatic mutations by 18 weeks after conception. We used these mutations as barcodes and timed the divergence of embryonic and extra-embryonic tissues during development, and estimated the number of blood antecedents at different stages of embryonic development. Our data support a hypoblast origin of the extra-embryonic mesoderm and primitive blood in humans.


Assuntos
Linhagem da Célula/genética , Desenvolvimento Embrionário/genética , Sistema Hematopoético/embriologia , Sistema Hematopoético/metabolismo , Mutação , Células Sanguíneas/citologia , Células Sanguíneas/metabolismo , Células Clonais/citologia , Células Clonais/metabolismo , Análise Mutacional de DNA , Feto/citologia , Feto/embriologia , Feto/metabolismo , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Saúde , Sistema Hematopoético/citologia , Humanos , Cariotipagem , Masculino , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/metabolismo , Taxa de Mutação , Especificidade de Órgãos/genética , Fatores de Tempo , Sequenciamento Completo do Genoma , Fluxo de Trabalho
11.
Development ; 148(10)2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33999993

RESUMO

In mammals, the pre-gastrula proximal epiblast gives rise to primordial germ cells (PGCs) or somatic precursors in response to BMP4 and WNT signaling. Entry into the germline requires activation of a naïve-like pluripotency gene regulatory network (GRN). Recent work has shown that suppression of OTX2 expression in the epiblast by BMP4 allows cells to develop a PGC fate in a precise temporal window. However, the mechanisms by which OTX2 suppresses PGC fate are unknown. Here, we show that, in mice, OTX2 prevents epiblast cells from activating the pluripotency GRN by direct repression of Oct4 and Nanog. Loss of this control during PGC differentiation in vitro causes widespread activation of the pluripotency GRN and a deregulated response to LIF, BMP4 and WNT signaling. These abnormalities, in specific cell culture conditions, result in massive germline entry at the expense of somatic mesoderm differentiation. Increased generation of PGCs also occurs in mutant embryos. We propose that the OTX2-mediated repressive control of Oct4 and Nanog is the basis of the mechanism that determines epiblast contribution to germline and somatic lineage.


Assuntos
Células Germinativas/citologia , Camadas Germinativas/citologia , Proteína Homeobox Nanog/antagonistas & inibidores , Fator 3 de Transcrição de Octâmero/antagonistas & inibidores , Fatores de Transcrição Otx/metabolismo , Animais , Proteína Morfogenética Óssea 4/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento/genética , Fator Inibidor de Leucemia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Pluripotentes/citologia , Via de Sinalização Wnt/fisiologia
12.
PLoS Comput Biol ; 17(3): e1008571, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33684098

RESUMO

During early mammalian embryo development, a small number of cells make robust fate decisions at particular spatial locations in a tight time window to form inner cell mass (ICM), and later epiblast (Epi) and primitive endoderm (PE). While recent single-cell transcriptomics data allows scrutinization of heterogeneity of individual cells, consistent spatial and temporal mechanisms the early embryo utilize to robustly form the Epi/PE layers from ICM remain elusive. Here we build a multiscale three-dimensional model for mammalian embryo to recapitulate the observed patterning process from zygote to late blastocyst. By integrating the spatiotemporal information reconstructed from multiple single-cell transcriptomic datasets, the data-informed modeling analysis suggests two major processes critical to the formation of Epi/PE layers: a selective cell-cell adhesion mechanism (via EphA4/EphrinB2) for fate-location coordination and a temporal attenuation mechanism of cell signaling (via Fgf). Spatial imaging data and distinct subsets of single-cell gene expression data are then used to validate the predictions. Together, our study provides a multiscale framework that incorporates single-cell gene expression datasets to analyze gene regulations, cell-cell communications, and physical interactions among cells in complex geometries at single-cell resolution, with direct application to late-stage development of embryogenesis.


Assuntos
Desenvolvimento Embrionário/genética , Camadas Germinativas , Modelos Biológicos , Transcriptoma/genética , Animais , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/fisiologia , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Camadas Germinativas/fisiologia , Camundongos , Análise de Célula Única
13.
Science ; 371(6535): 1249-1253, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33737485

RESUMO

Although cell lineage information is fundamental to understanding organismal development, very little direct information is available for humans. We performed high-depth (250×) whole-genome sequencing of multiple tissues from three individuals to identify hundreds of somatic single-nucleotide variants (sSNVs). Using these variants as "endogenous barcodes" in single cells, we reconstructed early embryonic cell divisions. Targeted sequencing of clonal sSNVs in different organs (about 25,000×) and in more than 1000 cortical single cells, as well as single-nucleus RNA sequencing and single-nucleus assay for transposase-accessible chromatin sequencing of ~100,000 cortical single cells, demonstrated asymmetric contributions of early progenitors to extraembryonic tissues, distinct germ layers, and organs. Our data suggest onset of gastrulation at an effective progenitor pool of about 170 cells and about 50 to 100 founders for the forebrain. Thus, mosaic mutations provide a permanent record of human embryonic development at very high resolution.


Assuntos
Linhagem da Célula , Gastrulação , Mutação , Células-Tronco Neurais/citologia , Prosencéfalo/citologia , Adolescente , Adulto , Divisão Celular , Células Clonais/citologia , Desenvolvimento Embrionário/genética , Feminino , Gástrula/citologia , Variação Genética , Camadas Germinativas/citologia , Humanos , Masculino , Neurônios/citologia , Organogênese , Polimorfismo de Nucleotídeo Único , Prosencéfalo/embriologia , Análise de Célula Única , Sequenciamento Completo do Genoma
14.
Science ; 371(6529)2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33542111

RESUMO

During development, cells progress from a pluripotent state to a more restricted fate within a particular germ layer. However, cranial neural crest cells (CNCCs), a transient cell population that generates most of the craniofacial skeleton, have much broader differentiation potential than their ectodermal lineage of origin. Here, we identify a neuroepithelial precursor population characterized by expression of canonical pluripotency transcription factors that gives rise to CNCCs and is essential for craniofacial development. Pluripotency factor Oct4 is transiently reactivated in CNCCs and is required for the subsequent formation of ectomesenchyme. Furthermore, open chromatin landscapes of Oct4+ CNCC precursors resemble those of epiblast stem cells, with additional features suggestive of priming for mesenchymal programs. We propose that CNCCs expand their developmental potential through a transient reacquisition of molecular signatures of pluripotency.


Assuntos
Crista Neural/embriologia , Células-Tronco Pluripotentes/fisiologia , Animais , Diferenciação Celular/genética , Movimento Celular , Embrião de Mamíferos , Camadas Germinativas/citologia , Camundongos , Crista Neural/citologia , Crista Neural/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , RNA-Seq , Transcrição Genética , Transcriptoma
15.
Int J Mol Sci ; 22(3)2021 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-33513663

RESUMO

The association of members of the enterovirus family with pregnancy complications up to miscarriages is under discussion. Here, infection of two different human induced pluripotent stem cell (iPSC) lines and iPSC-derived primary germ-layer cells with coxsackievirus B3 (CVB3) was characterized as an in vitro cell culture model for very early human development. Transcriptomic analysis of iPSC lines infected with recombinant CVB3 expressing enhanced green fluorescent protein (EGFP) revealed a reduction in the expression of pluripotency genes besides an enhancement of genes involved in RNA metabolism. The initial distribution of CVB3-EGFP-positive cells within iPSC colonies correlated with the distribution of its receptor coxsackie- and adenovirus receptor (CAR). Application of anti-CAR blocking antibodies supported the requirement of CAR, but not of the co-receptor decay-accelerating factor (DAF) for infection of iPSC lines. Among iPSC-derived germ-layer cells, mesodermal cells were especially vulnerable to CVB3-EGFP infection. Our data implicate further consideration of members of the enterovirus family in the screening program of human pregnancies. Furthermore, iPSCs with their differentiation capacity into cell populations of relevant viral target organs could offer a reliable screening approach for therapeutic intervention and for assessment of organ-specific enterovirus virulence.


Assuntos
Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , Infecções por Coxsackievirus/metabolismo , Infecções por Coxsackievirus/virologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Camadas Germinativas/metabolismo , Camadas Germinativas/virologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Antígenos CD55/genética , Antígenos CD55/metabolismo , Linhagem Celular , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/genética , Infecções por Coxsackievirus/genética , Ectoderma/metabolismo , Endoderma/metabolismo , Enterovirus Humano B/metabolismo , Enterovirus Humano B/patogenicidade , Perfilação da Expressão Gênica , Camadas Germinativas/citologia , Interações entre Hospedeiro e Microrganismos/genética , Humanos , Células-Tronco Pluripotentes Induzidas/virologia , Mesoderma/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA/genética , RNA/metabolismo
16.
Development ; 148(1)2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33199445

RESUMO

Anterior mesoderm (AM) and definitive endoderm (DE) progenitors represent the earliest embryonic cell types that are specified during germ layer formation at the primitive streak (PS) of the mouse embryo. Genetic experiments indicate that both lineages segregate from Eomes-expressing progenitors in response to different Nodal signaling levels. However, the precise spatiotemporal pattern of the emergence of these cell types and molecular details of lineage segregation remain unexplored. We combined genetic fate labeling and imaging approaches with single-cell RNA sequencing (scRNA-seq) to follow the transcriptional identities and define lineage trajectories of Eomes-dependent cell types. Accordingly, all cells moving through the PS during the first day of gastrulation express Eomes AM and DE specification occurs before cells leave the PS from Eomes-positive progenitors in a distinct spatiotemporal pattern. ScRNA-seq analysis further suggested the immediate and complete separation of AM and DE lineages from Eomes-expressing cells as last common bipotential progenitor.


Assuntos
Linhagem da Célula , Endoderma/citologia , Endoderma/metabolismo , Gastrulação , Mesoderma/citologia , Mesoderma/metabolismo , Alelos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/citologia , Camundongos , Modelos Biológicos , Linha Primitiva/embriologia , Linha Primitiva/metabolismo , Células-Tronco/metabolismo , Proteínas com Domínio T/metabolismo , Fatores de Tempo , Transcrição Genética
17.
Dev Biol ; 470: 84-94, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33217407

RESUMO

At implantation, the mouse embryo undergoes a critical transformation which requires the precise spatiotemporal control of signalling pathways necessary for morphogenesis and developmental progression. The role played by such signalling pathways during this transition are largely unexplored, due to the inaccessibility of the embryo during the implantation when it becomes engulfed by uterine tissues. Genetic studies demonstrate that mutant embryos for BMPs die around gastrulation. Here we have aimed to dissect the role of BMPs during pre-to post-implantation transition by using a protocol permitting the development of the embryo beyond implantation stages in vitro and using stem cells to mimic post-implantation tissue organisation. By assessing both the canonical and non-canonical mechanisms of BMP, we show that the loss of canonical BMP activity compromises the extra-embryonic ectoderm development. Our analyses demonstrate that BMP signalling maintains stem cell populations within both embryonic/extra-embryonic tissues during pre-to post-implantation development. These results may provide insight into the role played by BMP signalling in controlling early embryogenesis.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Ectoderma/embriologia , Implantação do Embrião , Desenvolvimento Embrionário , Transdução de Sinais , Animais , Morte Celular , Linhagem da Célula , Ectoderma/citologia , Técnicas de Cultura Embrionária , Células-Tronco Embrionárias/citologia , Camadas Germinativas/citologia , Camadas Germinativas/embriologia , Camundongos , Morfogênese , Trofoblastos/citologia
18.
Nature ; 589(7840): 103-109, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33239783

RESUMO

Mammalian telomeres protect chromosome ends from aberrant DNA repair1. TRF2, a component of the telomere-specific shelterin protein complex, facilitates end protection through sequestration of the terminal telomere repeat sequence within a lariat T-loop structure2,3. Deleting TRF2 (also known as TERF2) in somatic cells abolishes T-loop formation, which coincides with telomere deprotection, chromosome end-to-end fusions and inviability3-9. Here we establish that, by contrast, TRF2 is largely dispensable for telomere protection in mouse pluripotent embryonic stem (ES) and epiblast stem cells. ES cell telomeres devoid of TRF2 instead activate an attenuated telomeric DNA damage response that lacks accompanying telomere fusions, and propagate for multiple generations. The induction of telomere dysfunction in ES cells, consistent with somatic deletion of Trf2 (also known as Terf2), occurs only following the removal of the entire shelterin complex. Consistent with TRF2 being largely dispensable for telomere protection specifically during early embryonic development, cells exiting pluripotency rapidly switch to TRF2-dependent end protection. In addition, Trf2-null embryos arrest before implantation, with evidence of strong DNA damage response signalling and apoptosis specifically in the non-pluripotent compartment. Finally, we show that ES cells form T-loops independently of TRF2, which reveals why TRF2 is dispensable for end protection during pluripotency. Collectively, these data establish that telomere protection is solved by distinct mechanisms in pluripotent and somatic tissues.


Assuntos
Cromossomos de Mamíferos/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/deficiência , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Sobrevivência Celular , Cromossomos de Mamíferos/genética , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Pluripotentes/citologia , Telômero/genética , Proteína 2 de Ligação a Repetições Teloméricas/genética , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo
19.
Nat Protoc ; 16(1): 309-326, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33311712

RESUMO

Early post-implantation human embryonic development has been challenging to study due to both technical limitations and ethical restrictions. Proper modeling of the process is important for infertility and toxicology research. Here we provide details of the design and implementation of a microfluidic device that can be used to model human embryo development. The microfluidic human embryo model is established from human pluripotent stem cells (hPSCs), and the resulting structures exhibit molecular and cellular features resembling the progressive development of the early post-implantation human embryo. The compartmentalized configuration of the microfluidic device allows the formation of spherical hPSC clusters in prescribed locations in the device, enabling the two opposite regions of each hPSC cluster to be exposed to two different exogenous chemical environments. Under such asymmetrical chemical conditions, several early post-implantation human embryo developmental landmarks, including lumenogenesis of the epiblast and the resultant pro-amniotic cavity, formation of a bipolar embryonic sac, and specification of primordial germ cells and gastrulating cells (or mesendoderm cells), can be robustly recapitulated using the microfluidic device. The microfluidic human embryo model is compatible with high-throughput studies, live imaging, immunofluorescence staining, fluorescent in situ hybridization, and single-cell sequencing. This protocol takes ~5 d to complete, including microfluidic device fabrication (2 d), cell seeding (1 d), and progressive development of the microfluidic model until gastrulation-like events occur (1-2 d).


Assuntos
Células-Tronco Embrionárias Humanas/citologia , Técnicas Analíticas Microfluídicas/instrumentação , Células-Tronco Pluripotentes/citologia , Linhagem Celular , Desenvolvimento Embrionário , Desenho de Equipamento , Camadas Germinativas/citologia , Humanos , Técnicas Analíticas Microfluídicas/métodos
20.
Methods Mol Biol ; 2214: 41-58, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32944902

RESUMO

Pluripotent stem cells (PSCs) are the in vitro counterpart of the pluripotent epiblast of the mammalian embryo with the capacity to generate all cell types of the adult organism. During development, the three definitive germ layers are specified and simultaneously spatially organized. In contrast, differentiating PSCs tend to generate cell fates in a spatially disorganized manner. This has limited the in vitro study of specific cell-cell interactions and patterning mechanisms that occur in vivo. Here we describe a protocol to differentiate mouse PSCs in a spatially organized manner on micropatterned surfaces. Micropatterned chips comprise many colonies of uniform size and geometry facilitating a robust quantitative analysis of patterned fate specification. Furthermore, multiple factors may be simultaneously manipulated with temporal accuracy to probe the dynamic interactions regulating these processes. The micropattern system is scalable, providing a valuable tool to generate material for large-scale analysis and biochemical experiments that require substantial amounts of starting material, difficult to obtain from early embryos.


Assuntos
Materiais Biocompatíveis/química , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células-Tronco Pluripotentes/citologia , Animais , Técnicas de Cultura de Células/instrumentação , Células Cultivadas , Ectoderma/citologia , Endoderma/citologia , Desenho de Equipamento , Gastrulação , Camadas Germinativas/citologia , Camundongos , Propriedades de Superfície
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