Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 8.743
Filtrar
1.
Nat Commun ; 11(1): 4654, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32943640

RESUMO

The shift from maternal to embryonic control is a critical developmental milestone in preimplantation development. Widespread transcriptomic and epigenetic remodeling facilitate this transition from terminally differentiated gametes to totipotent blastomeres, but the identity of transcription factors (TF) and genomic elements regulating embryonic genome activation (EGA) are poorly defined. The timing of EGA is species-specific, e.g., the timing of murine and human EGA differ significantly. To deepen our understanding of mammalian EGA, here we profile changes in open chromatin during bovine preimplantation development. Before EGA, open chromatin is enriched for maternal TF binding, similar to that observed in humans and mice. During EGA, homeobox factor binding becomes more prevalent and requires embryonic transcription. A cross-species comparison of open chromatin during preimplantation development reveals strong similarity in the regulatory circuitry underlying bovine and human EGA compared to mouse. Moreover, TFs associated with murine EGA are not enriched in cattle or humans, indicating that cattle may be a more informative model for human preimplantation development than mice.


Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genoma , Animais , Blastômeros , Bovinos/embriologia , Cromatina/metabolismo , Fertilização , Humanos , Camundongos , Oócitos , Especificidade da Espécie , Fatores de Transcrição/metabolismo
2.
Nat Protoc ; 15(9): 3009-3029, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32796939

RESUMO

Genome editing holds great potential for correcting pathogenic mutations. We developed a method called GOTI (genome-wide off-target analysis by two-cell embryo injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. GOTI directly compares edited and non-edited cells without the interference of genetic background and thus could detect potential off-target variants with high sensitivity. Notably, the GOTI method was designed to detect potential off-target variants of any genome editing tools by the combination of experimental and computational approaches, which is critical for accurate evaluation of the safety of genome editing tools. Here we provide a detailed protocol for GOTI, including mice mating, two-cell embryo injection, embryonic day 14.5 embryo digestion, fluorescence-activated cell sorting, whole-genome sequencing and data analysis. To enhance the utility of GOTI, we also include a computational workflow called GOTI-seq (https://github.com/sydaileen/GOTI-seq) for the sequencing data analysis, which can generate the final genome-wide off-target variants from raw sequencing data directly. The protocol typically takes 20 d from the mice mating to sequencing and 7 d for sequencing data analysis.


Assuntos
Embrião de Mamíferos/metabolismo , Edição de Genes/métodos , Animais , Feminino , Injeções , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação
3.
Open Biol ; 10(8): 200162, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32750256

RESUMO

While initially recognized as causing respiratory disease, the SARS-CoV-2 virus also affects many other organs leading to other complications. It has emerged that advanced age and obesity are risk factors for complications but questions concerning the potential effects on fetal health and successful pregnancy for those infected with SARS-CoV-2 remain largely unanswered. Here, we examine human pre-gastrulation embryos to determine the expression patterns of the genes ACE2, encoding the SARS-CoV-2 receptor, and TMPRSS2, encoding a protease that cleaves both the viral spike protein and the ACE2 receptor to facilitate infection. We show expression and co-expression of these genes in the trophoblast of the blastocyst and syncytiotrophoblast and hypoblast of the implantation stages, which develop into tissues that interact with the maternal blood supply for nutrient exchange. Expression of ACE2 and TMPRSS2 in these tissues raises the possibility for vertical transmission and indicates that further work is required to understand potential risks to implantation, placental health and fetal health that require further study.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/patologia , Embrião de Mamíferos/metabolismo , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Serina Endopeptidases/metabolismo , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Feminino , Humanos , Pandemias , Peptidil Dipeptidase A/genética , Pneumonia Viral/transmissão , Pneumonia Viral/virologia , Gravidez , Primeiro Trimestre da Gravidez , Serina Endopeptidases/genética , Análise de Célula Única , Trofoblastos/metabolismo
4.
PLoS One ; 15(7): e0235799, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32658897

RESUMO

ATP-dependent chromatin-remodeling complexes epigenetically modulate transcription of target genes to impact a variety of developmental processes. Our lab previously demonstrated that CHD4-a central ATPase and catalytic enzyme of the NuRD chromatin-remodeling complex-plays an important role in murine embryonic endothelial cells by transcriptionally regulating vascular integrity at midgestation. Since NuRD complexes can incorporate the ATPase CHD3 as an alternative to CHD4, we questioned whether the CHD3 enzyme likewise modulates vascular development or integrity. We generated a floxed allele of Chd3 but saw no evidence of lethality or vascular anomalies when we deleted it in embryonic endothelial cells in vivo (Chd3ECKO). Furthermore, double-deletion of Chd3 and Chd4 in embryonic endothelial cells (Chd3/4ECKO) did not dramatically alter the timing and severity of embryonic phenotypes seen in Chd4ECKO mutants, indicating that CHD3 does not play a cooperative role with CHD4 in early vascular development. However, excision of Chd3 at the epiblast stage of development with a Sox2-Cre line allowed us to generate global heterozygous Chd3 mice (Chd3Δ/+), which were subsequently intercrossed and revealed partial lethality of Chd3Δ/Δ mutants prior to weaning. Tissues from surviving Chd3Δ/Δ mutants helped us confirm that CHD3 was efficiently deleted in these animals and that CHD3 is highly expressed in the gonads and brains of adult wildtype mice. Therefore, Chd3-flox mice will be beneficial for future studies about roles for this chromatin-remodeling enzyme in viable embryonic development and in gonadal and brain physiology.


Assuntos
Vasos Sanguíneos/embriologia , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/embriologia , Animais , Vasos Sanguíneos/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/metabolismo , Perda do Embrião/genética , Perda do Embrião/metabolismo , Embrião de Mamíferos/metabolismo , Feminino , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos
5.
Nature ; 582(7811): 253-258, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32523119

RESUMO

Tissue sculpting during development has been attributed mainly to cellular events through processes such as convergent extension or apical constriction1,2. However, recent work has revealed roles for basement membrane remodelling in global tissue morphogenesis3-5. Upon implantation, the epiblast and extraembryonic ectoderm of the mouse embryo become enveloped by a basement membrane. Signalling between the basement membrane and these tissues is critical for cell polarization and the ensuing morphogenesis6,7. However, the mechanical role of the basement membrane in post-implantation embryogenesis remains unknown. Here we demonstrate the importance of spatiotemporally regulated basement membrane remodelling during early embryonic development. Specifically, we show that Nodal signalling directs the generation and dynamic distribution of perforations in the basement membrane by regulating the expression of matrix metalloproteinases. This basement membrane remodelling facilitates embryo growth before gastrulation. The establishment of the anterior-posterior axis8,9 further regulates basement membrane remodelling by localizing Nodal signalling-and therefore the activity of matrix metalloproteinases and basement membrane perforations-to the posterior side of the embryo. Perforations on the posterior side are essential for primitive-streak extension during gastrulation by rendering the basement membrane of the prospective primitive streak more prone to breaching. Thus spatiotemporally regulated basement membrane remodelling contributes to the coordination of embryo growth, morphogenesis and gastrulation.


Assuntos
Membrana Basal/embriologia , Membrana Basal/metabolismo , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Animais , Membrana Basal/citologia , Blastocisto/citologia , Blastocisto/metabolismo , Embrião de Mamíferos/citologia , Matriz Extracelular/metabolismo , Feminino , Gástrula/embriologia , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos , Ligantes da Sinalização Nodal/metabolismo , Linha Primitiva/citologia , Linha Primitiva/embriologia , Linha Primitiva/metabolismo
6.
Nat Commun ; 11(1): 3153, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561758

RESUMO

Mouse embryos acquire global DNA methylation of their genome during implantation. However the exact roles of DNA methyltransferases (DNMTs) in embryos have not been studied comprehensively. Here we systematically analyze the consequences of genetic inactivation of Dnmt1, Dnmt3a and Dnmt3b on the methylome and transcriptome of mouse embryos. We find a strict division of function between DNMT1, responsible for maintenance methylation, and DNMT3A/B, solely responsible for methylation acquisition in development. By analyzing severely hypomethylated embryos, we uncover multiple functions of DNA methylation that is used as a mechanism of repression for a panel of genes including not only imprinted and germline genes, but also lineage-committed genes and 2-cell genes. DNA methylation also suppresses multiple retrotransposons and illegitimate transcripts from cryptic promoters in transposons and gene bodies. Our work provides a thorough analysis of the roles of DNA methyltransferases and the importance of DNA methylation for transcriptome integrity in mammalian embryos.


Assuntos
DNA (Citosina-5-)-Metiltransferases , Metilação de DNA , Desenvolvimento Embrionário/genética , Animais , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , Metilação de DNA/fisiologia , Embrião de Mamíferos/metabolismo , Epigenômica , Regulação da Expressão Gênica , Genoma , Camundongos , Transcriptoma
7.
PLoS One ; 15(6): e0234246, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32502225

RESUMO

INTRODUCTION: Esophageal atresia with or without tracheoesophageal fistula (EA/TEF) occurs approximately 1 in 3.500 live births representing the most common malformation of the upper digestive tract. Only half a century ago, EA/TEF was fatal among affected newborns suggesting that the steady birth prevalence might in parts be due to mutational de novo events in genes involved in foregut development. METHODS: To identify mutational de novo events in EA/TEF patients, we surveyed the exome of 30 case-parent trios. Identified and confirmed de novo variants were prioritized using in silico prediction tools. To investigate the embryonic role of genes harboring prioritized de novo variants we performed targeted analysis of mouse transcriptome data of esophageal tissue obtained at the embryonic day (E) E8.5, E12.5, and postnatal. RESULTS: In total we prioritized 14 novel de novo variants in 14 different genes (APOL2, EEF1D, CHD7, FANCB, GGT6, KIAA0556, NFX1, NPR2, PIGC, SLC5A2, TANC2, TRPS1, UBA3, and ZFHX3) and eight rare de novo variants in eight additional genes (CELSR1, CLP1, GPR133, HPS3, MTA3, PLEC, STAB1, and PPIP5K2). Through personal communication during the project, we identified an additional EA/TEF case-parent trio with a rare de novo variant in ZFHX3. In silico prediction analysis of the identified variants and comparative analysis of mouse transcriptome data of esophageal tissue obtained at E8.5, E12.5, and postnatal prioritized CHD7, TRPS1, and ZFHX3 as EA/TEF candidate genes. Re-sequencing of ZFHX3 in additional 192 EA/TEF patients did not identify further putative EA/TEF-associated variants. CONCLUSION: Our study suggests that rare mutational de novo events in genes involved in foregut development contribute to the development of EA/TEF.


Assuntos
DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/metabolismo , Atresia Esofágica/genética , Exoma/genética , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas Repressoras/genética , Fístula Traqueoesofágica/genética , Animais , Humanos , Camundongos , Sequenciamento Completo do Exoma
8.
Nat Commun ; 11(1): 2958, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32528010

RESUMO

The high incidence of aneuploidy in the embryo is considered the principal cause for low human fecundity. However, the prevalence of aneuploidy dramatically declines as pregnancy progresses, with the steepest drop occurring as the embryo completes implantation. Despite the fact that the plasticity of the embryo in dealing with aneuploidy is fundamental to normal development, the mechanisms responsible for eliminating aneuploid cells are unclear. Here, using a mouse model of chromosome mosaicism, we show that aneuploid cells are preferentially eliminated from the embryonic lineage in a p53-dependent process involving both autophagy and apoptosis before, during and after implantation. Moreover, we show that diploid cells in mosaic embryos undertake compensatory proliferation during the implantation stages to confer embryonic viability. Together, our results indicate a close link between aneuploidy, autophagy, and apoptosis to refine the embryonic cell population and ensure only chromosomally fit cells proceed through development of the fetus.


Assuntos
Aneuploidia , Apoptose/fisiologia , Autofagia/fisiologia , Animais , Apoptose/genética , Autofagia/genética , Proliferação de Células/genética , Proliferação de Células/fisiologia , Diploide , Implantação do Embrião , Embrião de Mamíferos/metabolismo , Embriologia , Desenvolvimento Embrionário/fisiologia , Feminino , Imunofluorescência , Camadas Germinativas/metabolismo , Camundongos , Mosaicismo
9.
Nat Cell Biol ; 22(6): 663-673, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32393887

RESUMO

The linear ubiquitin chain assembly complex (LUBAC), which consists of HOIP, SHARPIN and HOIL-1L, promotes NF-κB activation and protects against cell death by generating linear ubiquitin chains. LUBAC contains two RING-IBR-RING (RBR) ubiquitin ligases (E3), and the HOIP RBR is responsible for catalysing linear ubiquitination. We found that HOIL-1L RBR plays a crucial role in regulating LUBAC. HOIL-1L RBR conjugates monoubiquitin onto all LUBAC subunits, followed by HOIP-mediated conjugation of linear chains onto monoubiquitin, and these linear chains attenuate the functions of LUBAC. The introduction of E3-defective HOIL-1L mutants into cells augmented linear ubiquitination, which protected the cells against Salmonella infection and cured dermatitis caused by reduced LUBAC levels due to SHARPIN loss. Our results reveal a regulatory mode of E3 ligases in which the accessory E3 in LUBAC downregulates the main E3 by providing preferred substrates for autolinear ubiquitination. Thus, inhibition of HOIL-1L E3 represents a promising strategy for treating severe infections or immunodeficiency.


Assuntos
Proteínas de Transporte/fisiologia , Morte Celular , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Dermatite de Contato/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Salmonelose Animal/imunologia , Ubiquitina-Proteína Ligases/fisiologia , Ubiquitina/metabolismo , Animais , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Dermatite de Contato/metabolismo , Dermatite de Contato/patologia , Embrião de Mamíferos/imunologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Fibroblastos/imunologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , Salmonella/patogenicidade , Salmonelose Animal/metabolismo , Salmonelose Animal/patologia , Índice de Gravidade de Doença , Transdução de Sinais , Ubiquitinação
10.
Nat Cell Biol ; 22(6): 740-750, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32393889

RESUMO

Cytidine base editors are powerful genetic tools that catalyse cytidine to thymidine conversion at specific genomic loci, and further improvement of the editing range and efficiency is critical for their broader applications. Through insertion of a non-sequence-specific single-stranded DNA-binding domain from Rad51 protein between Cas9 nickase and the deaminases, serial hyper cytidine base editors were generated with substantially increased activity and an expanded editing window towards the protospacer adjacent motif in both cell lines and mouse embryos. Additionally, hyeA3A-BE4max selectively catalysed cytidine conversion in TC motifs with a broader editing range and much higher activity (up to 257-fold) compared with eA3A-BE4max. Moreover, hyeA3A-BE4max specifically generated a C-to-T conversion without inducing bystander mutations in the haemoglobin gamma gene promoter to mimic a naturally occurring genetic variant for amelioration of ß-haemoglobinopathy, suggesting the therapeutic potential of the improved base editors.


Assuntos
Sistemas CRISPR-Cas , Citidina/genética , Proteínas de Ligação a DNA/metabolismo , Edição de Genes , Mutação , Rad51 Recombinase/metabolismo , Animais , Diferenciação Celular , Citidina/química , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Domínios Proteicos , Rad51 Recombinase/genética
11.
Proc Natl Acad Sci U S A ; 117(22): 12182-12191, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32414917

RESUMO

In multicellular organisms, paralogs from gene duplication survive purifying selection by evolving tissue-specific expression and function. Whether this genetic redundancy is also selected for within a single cell type is unclear for multimember paralogs, as exemplified by the four obligatory Lef/Tcf transcription factors of canonical Wnt signaling, mainly due to the complex genetics involved. Using the developing mouse lung as a model system, we generate two quadruple conditional knockouts, four triple mutants, and various combinations of double mutants, showing that the four Lef/Tcf genes function redundantly in the presence of at least two Lef/Tcf paralogs, but additively upon losing additional paralogs to specify and maintain lung epithelial progenitors. Prelung-specification, pan-epithelial double knockouts have no lung phenotype; triple knockouts have varying phenotypes, including defective branching and tracheoesophageal fistulas; and the quadruple knockout barely forms a lung, resembling the Ctnnb1 mutant. Postlung-specification deletion of all four Lef/Tcf genes leads to branching defects, down-regulation of progenitor genes, premature alveolar differentiation, and derepression of gastrointestinal genes, again phenocopying the corresponding Ctnnb1 mutant. Our study supports a monotonic, positive signaling relationship between CTNNB1 and Lef/Tcf in lung epithelial progenitors as opposed to reported repressor functions of Lef/Tcf, and represents a thorough in vivo analysis of cell-type-specific genetic redundancy among the four Lef/Tcf paralogs.


Assuntos
Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Pulmão/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/fisiologia , Células-Tronco/metabolismo , beta Catenina/metabolismo , Animais , Diferenciação Celular , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Feminino , Fator 1-alfa Nuclear de Hepatócito/fisiologia , Pulmão/citologia , Camundongos , Camundongos Knockout , Análise de Célula Única , Células-Tronco/citologia , Proteína 1 Semelhante ao Fator 7 de Transcrição/fisiologia , Proteína 2 Semelhante ao Fator 7 de Transcrição/fisiologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética
12.
Nature ; 581(7806): 77-82, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32376949

RESUMO

Grafts of spinal-cord-derived neural progenitor cells (NPCs) enable the robust regeneration of corticospinal axons and restore forelimb function after spinal cord injury1; however, the molecular mechanisms that underlie this regeneration are unknown. Here we perform translational profiling specifically of corticospinal tract (CST) motor neurons in mice, to identify their 'regenerative transcriptome' after spinal cord injury and NPC grafting. Notably, both injury alone and injury combined with NPC grafts elicit virtually identical early transcriptomic responses in host CST neurons. However, in mice with injury alone this regenerative transcriptome is downregulated after two weeks, whereas in NPC-grafted mice this transcriptome is sustained. The regenerative transcriptome represents a reversion to an embryonic transcriptional state of the CST neuron. The huntingtin gene (Htt) is a central hub in the regeneration transcriptome; deletion of Htt significantly attenuates regeneration, which shows that Htt has a key role in neural plasticity after injury.


Assuntos
Proliferação de Células/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Regeneração Nervosa/genética , Células-Tronco Neurais/citologia , Neurônios/metabolismo , Neurônios/patologia , Transcrição Genética , Animais , Axônios/patologia , Axônios/fisiologia , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Proteína Huntingtina/genética , Camundongos , Células-Tronco Neurais/transplante , Plasticidade Neuronal , Neurônios/citologia , Neurônios/transplante , Biossíntese de Proteínas , Tratos Piramidais/citologia , Tratos Piramidais/metabolismo , Tratos Piramidais/patologia , RNA-Seq , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/patologia , Transcriptoma
13.
Nat Commun ; 11(1): 2325, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32393762

RESUMO

Common polygenic diseases result from compounded risk contributed by multiple genetic variants, meaning that simultaneous correction or introduction of single nucleotide variants is required for disease modeling and gene therapy. Here, we show precise, efficient, and simultaneous multiplex base editing of up to three target sites across 11 genes/loci in cynomolgus monkey embryos using CRISPR-based cytidine- and adenine-base editors. Unbiased whole genome sequencing demonstrates high specificity of base editing in monkey embryos. Our data demonstrate feasibility of multiplex base editing for polygenic disease modeling in primate zygotes.


Assuntos
Edição de Genes/métodos , Animais , Sequência de Bases , Embrião de Mamíferos/metabolismo , Éxons/genética , Feto/metabolismo , Fígado/metabolismo , Macaca fascicularis/embriologia , Mutação/genética
14.
Nat Commun ; 11(1): 1813, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286279

RESUMO

The oocyte cytoplasm can reprogram the somatic cell nucleus into a totipotent state, but with low efficiency. The spatiotemporal chromatin organization of somatic cell nuclear transfer (SCNT) embryos remains elusive. Here, we examine higher order chromatin structures of mouse SCNT embryos using a low-input Hi-C method. We find that donor cell chromatin transforms to the metaphase state rapidly after SCNT along with the dissolution of typical 3D chromatin structure. Intriguingly, the genome undergoes a mitotic metaphase-like to meiosis metaphase II-like transition following activation. Subsequently, weak chromatin compartments and topologically associating domains (TADs) emerge following metaphase exit. TADs are further removed until the 2-cell stage before being progressively reestablished. Obvious defects including stronger TAD boundaries, aberrant super-enhancer and promoter interactions are found in SCNT embryos. These defects are partially caused by inherited H3K9me3, and can be rescued by Kdm4d overexpression. These observations provide insight into chromatin architecture reorganization during SCNT embryo development.


Assuntos
Cromatina/metabolismo , Embrião de Mamíferos/metabolismo , Técnicas de Transferência Nuclear , Animais , Reprogramação Celular , Desenvolvimento Embrionário , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Histonas/metabolismo , Lisina/metabolismo , Metáfase , Metilação , Camundongos , Regiões Promotoras Genéticas/genética , Zigoto/metabolismo
15.
Toxicology ; 439: 152466, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32315717

RESUMO

Glyphosate is the most popular herbicide used in modern agriculture, and its use has been increasing substantially since its introduction. Accordingly, glyphosate exposure from food and water, the environment, and accidental and occupational venues has also increased. Recent studies have demonstrated a relationship between glyphosate exposure and a number of disorders such as cancer, immune and metabolic disorders, endocrine disruption, imbalance of intestinal flora, cardiovascular disease, and infertility; these results have given glyphosate a considerable amount of media and scientific attention. Notably, glyphosate is a powerful metal chelator, which could help explain some of its effects. Recently, our findings on 2,3-dimercapto-1-propanesulfonic acid, another metal chelator, showed deterioration of oocyte quality. Here, to generalize, we investigated the effects of glyphosate (0 - 300 µM) on metaphase II mouse oocyte quality and embryo damage to obtain insight on its mechanisms of cellular action and the tolerance of oocytes and embryos towards this chemical. Our work shows for the first time that glyphosate exposure impairs metaphase II mouse oocyte quality via two mechanisms: 1) disruption of the microtubule organizing center and chromosomes such as anomalous pericentrin formation, spindle fiber destruction and disappearance, and defective chromosomal alignment and 2) substantial depletion of intracellular zinc bioavailability and enhancement of reactive oxygen species accumulation. Similar effects were found in embryos. These results may help clarify the effects of glyphosate exposure on female fertility and provide counseling and preventative steps for excessive glyphosate intake and resulting oxidative stress and reduced zinc bioavailability.


Assuntos
Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Glicina/análogos & derivados , Herbicidas/toxicidade , Metáfase/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Zinco/metabolismo , Animais , Cromossomos/efeitos dos fármacos , Feminino , Glicina/toxicidade , Infertilidade Feminina/induzido quimicamente , Infertilidade Feminina/patologia , Camundongos , Microtúbulos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Gravidez , Fuso Acromático/efeitos dos fármacos
16.
Med Sci Monit ; 26: e920520, 2020 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-32188838

RESUMO

BACKGROUND Freshly isolated mouse embryonic fibroblasts (MEFs) have great proliferation capacity but quickly enter senescent state after several rounds of cell cycle, a process called premature senescence. Cellular senescence can be induced by various stresses such as telomere erosion, DNA damage, and oncogenic signaling. But the contribution of other molecules, such as growth factors, to cellular senescence is incompletely understood. This study aimed to compare the gene expression difference between non-senescent and senescent MEFs to identify the key molecule(s) involved in the spontaneous senescence of MEFs. MATERIAL AND METHODS Primary MEFs were isolated from E12.5 pregnant C57/BL6 mice. The cells were continuously cultured in Dulbecco's Modified Eagle Medium for 9 passages. SA-ß-Gal staining was used as an indicator of cell senescence. The supernatant from primary MEFs (P1 medium) or Passage 6 MEFs (P6 medium) were used to culture freshly isolated MEFs to observe the effects on cell senescence state. Gene expression profiles of primary and senescent MEFs were investigated by RNA-Seq to find the key genes involved in cell senescence. Adipocyte differentiation assay was used to evaluate the stemness of MEFs cultured in FGF2-stimulated medium. RESULTS The senescence of MEFs cultured in the P1 medium was alleviated when compared to the P6 medium. Downregulation of FGF2 expression was revealed by RNA-Seq and further confirmed by real-time quantitative polymerase chain reaction and western blot. FGF2-stimulated medium also had anti-senescence function and could maintain the differentiation ability of MEFs. CONCLUSIONS The premature senescence of MEFs was at least partially caused by FGF2 deficiency. Exogenous FGF2 could alleviate the senescent phenotype.


Assuntos
Senescência Celular/fisiologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fibroblastos/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Dano ao DNA , Embrião de Mamíferos/metabolismo , Feminino , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais
17.
Proc Natl Acad Sci U S A ; 117(13): 7236-7244, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32184326

RESUMO

Spatial cellular organization is fundamental for embryogenesis. Remarkably, coculturing embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) recapitulates this process, forming embryo-like structures. However, mechanisms driving ESC-TSC interaction remain elusive. We describe specialized ESC-generated cytonemes that react to TSC-secreted Wnts. Cytoneme formation and length are controlled by actin, intracellular calcium stores, and components of the Wnt pathway. ESC cytonemes select self-renewal-promoting Wnts via crosstalk between Wnt receptors, activation of ionotropic glutamate receptors (iGluRs), and localized calcium transients. This crosstalk orchestrates Wnt signaling, ESC polarization, ESC-TSC pairing, and consequently synthetic embryogenesis. Our results uncover ESC-TSC contact-mediated signaling, reminiscent of the glutamatergic neuronal synapse, inducing spatial self-organization and embryonic cell specification.


Assuntos
Comunicação Celular/fisiologia , Células-Tronco Embrionárias/metabolismo , Pseudópodes/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Drosophila , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/fisiologia , Camundongos , Trofoblastos/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia
18.
Medicine (Baltimore) ; 99(13): e19591, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32221078

RESUMO

To systematically analyze the potential of embryo implantation through comparison between the number of surviving blastomeres, the growth, and implantation rate.Retrospective analysis on implantation rate and the growth of prefreeze-postthaw embryos with different blastomeres in 1487 frozen embryo transfer cycles.In groups of postthaw embryos without damage, implantation rate and the average number of blastomere growth increased significantly with increasing number of blastomeres. The implantation rate and the number of blastomeres of embryos with 8-8c (the number of blastomeres in prefreeze embryo-the number of blastomeres in postthaw embryo) continued to grow at a significantly higher rate than that of 5-5c and 6-6c (P < .05). In groups of embryos with the same number of blastomeres before freezing and with partial damage after resuscitation, the implantation rates were lower and the average numbers of blastomere growth reduced as the number of damaged blastomeres increased. For embryos with good quality before freezing, 1 to 3 damaged blastomeres in postthawed embryos did not affect the development and implantation rate. Both implantation rate and growth rate of embryos with 8-6c were significantly higher than those of embryos with 6-6c (P < .05).The number of surviving blastomeres and growth in frozen-thawed embryos could be important index to predict embryo development potential and clinical outcome of implantation. For embryos with good quality, a small amount of damaged blastomeres would not weaken embryo development potential and implantation rate after being thawed.


Assuntos
Blastômeros/metabolismo , Criopreservação , Implantação do Embrião/fisiologia , Embrião de Mamíferos/metabolismo , Fatores Etários , Endométrio/citologia , Estradiol/sangue , Feminino , Humanos , Mórula/metabolismo , Estudos Retrospectivos
19.
Nat Commun ; 11(1): 1357, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170114

RESUMO

Embryonic Stem Cell (ESC) differentiation requires complex cell signalling network dynamics, although the key molecular events remain poorly understood. Here, we use phosphoproteomics to identify an FGF4-mediated phosphorylation switch centred upon the key Ephrin receptor EPHA2 in differentiating ESCs. We show that EPHA2 maintains pluripotency and restrains commitment by antagonising ERK1/2 signalling. Upon ESC differentiation, FGF4 utilises a bimodal strategy to disable EPHA2, which is accompanied by transcriptional induction of EFN ligands. Mechanistically, FGF4-ERK1/2-RSK signalling inhibits EPHA2 via Ser/Thr phosphorylation, whilst FGF4-ERK1/2 disrupts a core pluripotency transcriptional circuit required for Epha2 gene expression. This system also operates in mouse and human embryos, where EPHA receptors are enriched in pluripotent cells whilst surrounding lineage-specified trophectoderm expresses EFNA ligands. Our data provide insight into function and regulation of EPH-EFN signalling in ESCs, and suggest that segregated EPH-EFN expression coordinates cell fate with compartmentalisation during early embryonic development.


Assuntos
Diferenciação Celular/fisiologia , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Proteômica/métodos , Receptor EphA2/metabolismo , Animais , Diferenciação Celular/genética , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Efrina-A2 , Fator 4 de Crescimento de Fibroblastos/metabolismo , Humanos , Ligantes , Sistema de Sinalização das MAP Quinases , Camundongos , Fosforilação , Receptor EphA2/genética , Transdução de Sinais
20.
Nature ; 582(7812): 405-409, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32076263

RESUMO

Gastruloids are three-dimensional aggregates of embryonic stem cells that display key features of mammalian development after implantation, including germ-layer specification and axial organization1-3. To date, the expression pattern of only a small number of genes in gastruloids has been explored with microscopy, and the extent to which genome-wide expression patterns in gastruloids mimic those in embryos is unclear. Here we compare mouse gastruloids with mouse embryos using single-cell RNA sequencing and spatial transcriptomics. We identify various embryonic cell types that were not previously known to be present in gastruloids, and show that key regulators of somitogenesis are expressed similarly between embryos and gastruloids. Using live imaging, we show that the somitogenesis clock is active in gastruloids and has dynamics that resemble those in vivo. Because gastruloids can be grown in large quantities, we performed a small screen that revealed how reduced FGF signalling induces a short-tail phenotype in embryos. Finally, we demonstrate that embedding in Matrigel induces gastruloids to generate somites with the correct rostral-caudal patterning, which appear sequentially in an anterior-to-posterior direction over time. This study thus shows the power of gastruloids as a model system for exploring development and somitogenesis in vitro in a high-throughput manner.


Assuntos
Gástrula , Células-Tronco Embrionárias Murinas/citologia , Organoides/citologia , Organoides/embriologia , Análise de Célula Única , Somitos/citologia , Somitos/embriologia , Transcriptoma , Animais , Colágeno , Combinação de Medicamentos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Feminino , Gástrula/citologia , Gástrula/embriologia , Gástrula/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Laminina , Masculino , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Organoides/metabolismo , Proteoglicanas , RNA-Seq , Somitos/metabolismo , Fatores de Tempo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA