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1.
Nature ; 571(7763): 112-116, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31189957

RESUMO

Size control is fundamental in tissue development and homeostasis1,2. Although the role of cell proliferation in these processes has been widely studied, the mechanisms that control embryo size-and how these mechanisms affect cell fate-remain unknown. Here we use the mouse blastocyst as a model to unravel a key role of fluid-filled lumen in the control of embryo size and specification of cell fate. We find that there is a twofold increase in lumenal pressure during blastocyst development, which translates into a concomitant increase in cell cortical tension and tissue stiffness of the trophectoderm that lines the lumen. Increased cortical tension leads to vinculin mechanosensing and maturation of functional tight junctions, which establishes a positive feedback loop to accommodate lumen growth. When the cortical tension reaches a critical threshold, cell-cell adhesion cannot be sustained during mitotic entry, which leads to trophectoderm rupture and blastocyst collapse. A simple theory of hydraulically gated oscillations recapitulates the observed dynamics of size oscillations, and predicts the scaling of embryo size with tissue volume. This theory further predicts that disrupted tight junctions or increased tissue stiffness lead to a smaller embryo size, which we verified by biophysical, embryological, pharmacological and genetic perturbations. Changes in lumenal pressure and size can influence the cell division pattern of the trophectoderm, and thereby affect cell allocation and fate. Our study reveals how lumenal pressure and tissue mechanics control embryo size at the tissue scale, which is coupled to cell position and fate at the cellular scale.


Assuntos
Diferenciação Celular , Linhagem da Célula , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário , Mecanotransdução Celular/fisiologia , Animais , Blastocisto/citologia , Adesão Celular , Divisão Celular , Forma Celular , Embrião de Mamíferos/anatomia & histologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Junções Íntimas , Vinculina/metabolismo
2.
Nat Commun ; 10(1): 2852, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253764

RESUMO

Cytosine base editors (CBEs) enable programmable C-to-T conversion without DNA double-stranded breaks and homology-directed repair in a variety of organisms, which exhibit great potential for agricultural and biomedical applications. However, all reported cases only involved C-to-T substitution at a single targeted genomic site. Whether C-to-T substitution is effective in multiple sites/loci has not been verified in large animals. Here, by using pigs, an important animal for agriculture and biomedicine, as the subjective animal, we showed that CBEs could efficiently induce C-to-T conversions at multiple sites/loci with the combination of three genes, including DMD, TYR, and LMNA, or RAG1, RAG2, and IL2RG, simultaneously, at the embryonic and cellular levels. CBEs also could disrupt genes (pol gene of porcine endogenous retrovirus) with dozens of copies by introducing multiple premature stop codons. With the CBEs, pigs carrying single gene or multiple gene point mutations were generated through embryo injection or nuclear transfer approach.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Mutação Puntual , Suínos/genética , Desaminase APOBEC-1 , Animais , Sequência de Bases , Proteína 9 Associada à CRISPR , DNA/genética , Técnicas de Cultura Embrionária , Embrião de Mamíferos , Genoma , Técnicas de Transferência Nuclear/veterinária , RNA Guia/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
3.
Nat Commun ; 10(1): 2588, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197172

RESUMO

The brain is a genomic mosaic shaped by cellular responses to genome damage. Here, we manipulate somatic genome stability by conditional Knl1 deletion from embryonic mouse brain. KNL1 mutations cause microcephaly and KNL1 mediates the spindle assembly checkpoint, a safeguard against chromosome missegregation and aneuploidy. We find that following Knl1 deletion, segregation errors in mitotic neural progenitor cells give rise to DNA damage on the missegregated chromosomes. This triggers rapid p53 activation and robust apoptotic and microglial phagocytic responses that extensively eliminate cells with somatic genome damage, thus causing microcephaly. By leaving only karyotypically normal progenitors to continue dividing, these mechanisms provide a second safeguard against brain somatic aneuploidy. Without Knl1 or p53-dependent safeguards, genome-damaged cells are not cleared, alleviating microcephaly, but paradoxically leading to total pre-weaning lethality. Thus, mitotic genome damage activates robust responses to eliminate somatic mutant cells, which if left unpurged, can impact brain and organismal fitness.


Assuntos
Aneuploidia , Microcefalia/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Células-Tronco Neurais/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/genética , Segregação de Cromossomos/genética , Dano ao DNA/genética , Modelos Animais de Doenças , Embrião de Mamíferos , Instabilidade Genômica , Humanos , Cinetocoros/metabolismo , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Cultura Primária de Células , Deleção de Sequência , Fuso Acromático/metabolismo
4.
BMC Genomics ; 20(1): 439, 2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31151386

RESUMO

BACKGROUND: The last decade witnessed a number of genome-wide studies on human pre-implantation, which mostly focused on genes and provided only limited information on repeats, excluding the satellites. Considering the fact that repeats constitute a large portion of our genome with reported links to human physiology and disease, a thorough understanding of their spatiotemporal regulation during human embryogenesis will give invaluable clues on chromatin dynamics across time and space. Therefore, we performed a detailed expression analysis of all repetitive DNA elements including the satellites across stages of human pre-implantation and embryonic stem cells. RESULTS: We uncovered stage-specific expressions of more than a thousand repeat elements whose expressions fluctuated with a mild global decrease at the blastocyst stage. Most satellites were highly expressed at the 4-cell level and expressions of ACRO1 and D20S16 specifically peaked at this point. Whereas all members of the SVA elements were highly upregulated at 8-cell and morula stages, other transposons and small RNA repeats exhibited a high level of variation among their specific subtypes. Our repeat enrichment analysis in gene promoters coupled with expression correlations highlighted potential links between repeat expressions and nearby genes, emphasising mostly 8-cell and morula specific genes together with SVA_D, LTR5_Hs and LTR70 transposons. The DNA methylation analysis further complemented the understanding on the mechanistic aspects of the repeatome's regulation per se and revealed critical stages where DNA methylation levels are negatively correlating with repeat expression. CONCLUSIONS: Taken together, our study shows that specific expression patterns are not exclusive to genes and long non-coding RNAs but the repeatome also exhibits an intriguingly dynamic pattern at the global scale. Repeats identified in this study; particularly satellites, which were historically associated with heterochromatin, and those with potential links to nearby gene expression provide valuable insights into the understanding of key events in genomic regulation and warrant further research in epigenetics, genomics and developmental biology.


Assuntos
DNA/química , Desenvolvimento Embrionário/genética , Expressão Gênica , Sequências Repetitivas de Ácido Nucleico , Metilação de DNA , DNA Satélite/química , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Humanos , Elementos Nucleotídeos Longos e Dispersos , Elementos Nucleotídeos Curtos e Dispersos
5.
Nat Commun ; 10(1): 2883, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253768

RESUMO

A substantial number of mouse genes, about 25%, are embryonically lethal when knocked out. Using current genetic tools, such as the CRISPR-Cas9 system, it is difficult-or even impossible-to produce viable mice with heritable embryonically lethal mutations. Here, we establish a one-step method for microinjection of CRISPR reagents into one blastomere of two-cell embryos to generate viable chimeric founder mice with a heritable embryonically lethal mutation, of either Virma or Dpm1. By examining founder mice, we identify a phenotype and role of Virma in regulating kidney metabolism in adult mice. Additionally, we generate knockout mice with a heritable postnatally lethal mutation, of either Slc17a5 or Ctla-4, and study its function in vivo. This one-step method provides a convenient system that rapidly generates knockout mice possessing lethal phenotypes. This allows relatively easy in vivo study of the associated genes' functions.


Assuntos
Sistemas CRISPR-Cas , Embrião de Mamíferos/fisiologia , Animais , Desenvolvimento Embrionário , Feminino , Edição de Genes/métodos , Engenharia Genética/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Mutação , RNA Guia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
6.
Biochemistry (Mosc) ; 84(3): 190-204, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31221058

RESUMO

Hematopoietic stem cells (HSCs) were the first stem cells discovered in humans. A. A. Maximov proposed an idea of blood stem cells that was confirmed later by McCulloch and Till experimentally. HSCs were the first type of stem cells to be used in clinics and ever since are being continually used. Indeed, a single HSC transplanted intravenously is capable of giving rise to all types of blood cells. In recent decades, human and animal HSC origin, development, hierarchy, and gene signature have been extensively investigated. Due to the constant need for donor blood and HSCs suitable for therapeutic transplants, the experimental possibility of obtaining HSCs in vitro by directed differentiation of pluripotent stem cells (PSCs) has been considered in recent years. However, despite all efforts, it is not yet possible to reproduce in vitro the ontogenesis of HSCs and obtain cells capable of long-term maintenance of hematopoiesis. The study of hematopoiesis in embryonic development facilitates the establishment and improvement of protocols for deriving blood cells from PCSs and allows a better understanding of the pathogenesis of various types of proliferative blood diseases, anemia, and immunodeficiency. This review focuses on the development of hematopoiesis in mammalian ontogenesis.


Assuntos
Embrião de Mamíferos/citologia , Células-Tronco Hematopoéticas/citologia , Animais , Diferenciação Celular , Humanos
7.
Nat Commun ; 10(1): 2487, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31171776

RESUMO

Lack or excess expression of the surface ectoderm-expressed transcription factor Grainyhead-like2 (Grhl2), each prevent spinal neural tube closure. Here we investigate the causative mechanisms and find reciprocal dysregulation of epithelial genes, cell junction components and actomyosin properties in Grhl2 null and over-expressing embryos. Grhl2 null surface ectoderm shows a shift from epithelial to neuroepithelial identity (with ectopic expression of N-cadherin and Sox2), actomyosin disorganisation, cell shape changes and diminished resistance to neural fold recoil upon ablation of the closure point. In contrast, excessive abundance of Grhl2 generates a super-epithelial surface ectoderm, in which up-regulation of cell-cell junction proteins is associated with an actomyosin-dependent increase in local mechanical stress. This is compatible with apposition of the neural folds but not with progression of closure, unless myosin activity is inhibited. Overall, our findings suggest that Grhl2 plays a crucial role in regulating biomechanical properties of the surface ectoderm that are essential for spinal neurulation.


Assuntos
Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Tubo Neural/embriologia , Células Neuroepiteliais/metabolismo , Neurulação/genética , Fatores de Transcrição/genética , Actomiosina/genética , Actomiosina/metabolismo , Animais , Fenômenos Biomecânicos , Caderinas/metabolismo , Ectoderma/citologia , Ectoderma/embriologia , Ectoderma/metabolismo , Células Epiteliais/metabolismo , Junções Intercelulares/genética , Junções Intercelulares/metabolismo , Camundongos , Tubo Neural/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Estresse Mecânico , Fatores de Transcrição/metabolismo
8.
Nat Commun ; 10(1): 2792, 2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31243271

RESUMO

The Deciphering the Mechanisms of Developmental Disorders programme has analysed the morphological and molecular phenotypes of embryonic and perinatal lethal mouse mutant lines in order to investigate the causes of embryonic lethality. Here we show that individual whole-embryo RNA-seq of 73 mouse mutant lines (>1000 transcriptomes) identifies transcriptional events underlying embryonic lethality and associates previously uncharacterised genes with specific pathways and tissues. For example, our data suggest that Hmgxb3 is involved in DNA-damage repair and cell-cycle regulation. Further, we separate embryonic delay signatures from mutant line-specific transcriptional changes by developing a baseline mRNA expression catalogue of wild-type mice during early embryogenesis (4-36 somites). Analysis of transcription outside coding sequence identifies deregulation of repetitive elements in Morc2a mutants and a gene involved in gene-specific splicing. Collectively, this work provides a large scale resource to further our understanding of early embryonic developmental disorders.


Assuntos
Embrião de Mamíferos/metabolismo , Análise de Sequência de RNA , Transcrição Genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Mutação , Transcriptoma
9.
BMC Bioinformatics ; 20(1): 294, 2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31142274

RESUMO

BACKGROUND: Biochemical networks are often described through static or time-averaged measurements of the component macromolecules. Temporal variation in these components plays an important role in both describing the dynamical nature of the network as well as providing insights into causal mechanisms. Few methods exist, specifically for systems with many variables, for analyzing time series data to identify distinct temporal regimes and the corresponding time-varying causal networks and mechanisms. RESULTS: In this study, we use well-constructed temporal transcriptional measurements in a mammalian cell during a cell cycle, to identify dynamical networks and mechanisms describing the cell cycle. The methods we have used and developed in part deal with Granger causality, Vector Autoregression, Estimation Stability with Cross Validation and a nonparametric change point detection algorithm that enable estimating temporally evolving directed networks that provide a comprehensive picture of the crosstalk among different molecular components. We applied our approach to RNA-seq time-course data spanning nearly two cell cycles from Mouse Embryonic Fibroblast (MEF) primary cells. The change-point detection algorithm is able to extract precise information on the duration and timing of cell cycle phases. Using Least Absolute Shrinkage and Selection Operator (LASSO) and Estimation Stability with Cross Validation (ES-CV), we were able to, without any prior biological knowledge, extract information on the phase-specific causal interaction of cell cycle genes, as well as temporal interdependencies of biological mechanisms through a complete cell cycle. CONCLUSIONS: The temporal dependence of cellular components we provide in our model goes beyond what is known in the literature. Furthermore, our inference of dynamic interplay of multiple intracellular mechanisms and their temporal dependence on one another can be used to predict time-varying cellular responses, and provide insight on the design of precise experiments for modulating the regulation of the cell cycle.


Assuntos
Ciclo Celular/genética , Redes Reguladoras de Genes , Algoritmos , Animais , Pontos de Checagem do Ciclo Celular/genética , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fase G1/genética , Genes cdc , Camundongos , Fatores de Tempo
10.
Nat Rev Genet ; 20(7): 373, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31114033
11.
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
12.
Acta Neurobiol Exp (Wars) ; 79(1): 92-100, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31038488

RESUMO

In a previous study, methylenedioxypyrovalerone (MDPV), a designer drug of the cathinone family, caused selective enhancement of Caspase3 immunoreactive (Casp3+) apoptotic cells in the nucleus accumbens (NAc) of 7­day­old mice. To further elaborate on the mechanism underlying MDPV­elicited apoptosis, here, we investigated the appearance of Casp3+ cells in developing neural tube explants of E12.5 mice, following MDPV treatment in vitro. Apoptotic cells appeared in large number in the pallium as radial progenitor cells and multipolar neurons, and in the subpallium including the future NAc, both in control and MDPV treated specimens. MDPV did not cause gross morphological changes in the neural tube or in the abundance of Casp3+ cells, based on a visual impression, though quantification was not attempted. We also studied the changes in NMDA receptor (NMDAR) protein subunits NR1 and NR2B in the NAc of 7­day­old MDPV treated and control mice, using western blotting of tissue obtained by selective dissection. In MDPV treated animals, expression of NR2B was lower than in the control animals, whereas expression of NR1 did not differ significantly from controls. The findings indicate that, during early postembryonic development, downregulation of the NR2B receptor subunit (at this time predominant in the NMDAR) is accompanied by a decreased viability of neurons. Decreased viability is expressed, in this case, as enhanced susceptibility to stimulation by MDPV - essentially a robust dopaminergic agent, potently affecting the neurons of the NAc. The findings are likely relevant to dopaminergic/NMDAR interactions and a potential pro­survival role of the NR2B subunit in critical phases of neural development.


Assuntos
Apoptose/efeitos dos fármacos , Benzodioxóis/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Núcleo Accumbens/citologia , Pirrolidinas/farmacologia , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Caspase 3/metabolismo , Embrião de Mamíferos , Camundongos , Camundongos Endogâmicos BALB C , Tubo Neural/citologia , Tubo Neural/efeitos dos fármacos , Núcleo Accumbens/efeitos dos fármacos
13.
Anim Reprod Sci ; 205: 150-155, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31076217

RESUMO

As oocytes and embryos of pigs have greater lipid content in the cytoplasm than those of other species, supplementation of the medium for in vitro maturation (IVM) of oocytes with omega-3 polyunsaturated fatty acids (PUFA) may help to improve embryo development. This study was conducted to evaluate effects of the inclusion of the docosaexaenoic (DHA) and of the eicosapentaenoic acids (EPA) in the IVM medium on the development of pig oocytes and on the lipid content of oocytes and embryos. In all experiments, control media consisted of porcine follicular fluid and oocytes were activated through parthenogenesis. In Experiment 1, there were four treatments for each PUFA: one control; and three treatments including EPA or DHA in the IVM medium at 12.5 µM, 25.0 µM and 50.0 µM). In Experiment 2, inclusion of 50 µM DHA was compared against the control. Cleavage rates in the IVM medium including 12.5 µM EPA and blastocyst development rates in media at any EPA concentration were less than for the control in Experiment 1 (P < 0.05). Compared to the control, inclusion of 50 µM DHA in the IVM medium was related to greater cleavage rates and greater number of embryo cells, in Experiment 1, and lesser lipid content in oocytes after 22 and 44 h and in embryos after 7 days, in Experiment 2 (both P < 0.05). Addition of DHA in the IVM medium may benefit the development of pig oocytes, but EPA appears to be cytotoxic.


Assuntos
Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Embrião de Mamíferos/química , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/efeitos dos fármacos , Suínos/embriologia , Animais , Ácidos Docosa-Hexaenoicos/administração & dosagem , Relação Dose-Resposta a Droga , Ácido Eicosapentaenoico/administração & dosagem , Feminino , Metabolismo dos Lipídeos , Partenogênese , Suínos/fisiologia
14.
Nat Commun ; 10(1): 2146, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086175

RESUMO

Posterior fossa type A (PFA) ependymomas exhibit very low H3K27 methylation and express high levels of EZHIP (Enhancer of Zeste Homologs Inhibitory Protein, also termed CXORF67). Here we find that a conserved sequence in EZHIP is necessary and sufficient to inhibit PRC2 catalytic activity in vitro and in vivo. EZHIP directly contacts the active site of the EZH2 subunit in a mechanism similar to the H3 K27M oncohistone. Furthermore, expression of H3 K27M or EZHIP in cells promotes similar chromatin profiles: loss of broad H3K27me3 domains, but retention of H3K27me3 at CpG islands. We find that H3K27me3-mediated allosteric activation of PRC2 substantially increases the inhibition potential of EZHIP and H3 K27M, providing a mechanism to explain the observed loss of H3K27me3 spreading in tumors. Our data indicate that PFA ependymoma and DIPG are driven in part by the action of peptidyl PRC2 inhibitors, the K27M oncohistone and the EZHIP 'oncohistone-mimic', that dysregulate gene silencing to promote tumorigenesis.


Assuntos
Neoplasias Encefálicas/genética , Ependimoma/genética , Glioma/genética , Proteínas Oncogênicas/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Animais , Neoplasias Encefálicas/patologia , Carcinogênese/genética , Linhagem Celular Tumoral , Cromatina/metabolismo , Ilhas de CpG , Fossa Craniana Posterior , Conjuntos de Dados como Assunto , Embrião de Mamíferos , Ependimoma/patologia , Fibroblastos , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Glioma/patologia , Células HEK293 , Histonas , Humanos , Camundongos , Proteínas Oncogênicas/genética , Cultura Primária de Células , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
15.
Horm Metab Res ; 51(5): 315-325, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31071736

RESUMO

BACKGROUND: The objective of the work was to investigate the cycle characteristics and outcomes of infertile women with nonclassic 21-hydroxylase deficiency (21-OHD) undergoing in vitro fertilization (IVF). METHODS: Twenty-five infertile nonclassic 21-OHD patients were retrospectively observed. From cycle day 3, patients were given human menopausal gonadotropin (HMG) 150 IU/d or 225 IU/d daily. Dexamethasone was administered orally at 0.75 mg/d. Ovulation was co-triggered by human chorionic gonadotropin (hCG) and triptorelin. Binary logistic regression was performed to quantify the effect of IVF parameters on embryo transfer cycle outcomes. The receiver operating characteristic (ROC) curve was used to determine cutoff points of the selected confounders for predicting pregnant probabilities. RESULTS: In controlled ovarian hyperstimulation (COH) cycles, there was a trend that the viable embryos group consisted of more polycystic ovary (PCO) patients. In embryo transfer (ET) cycles, differences were detected in the usage rate of dexamethasone and the minimum progesterone (P4) and total testosterone (TT) values between the non-pregnant group and the biochemical pregnant group. Binary logistic regression analysis confirmed that decreasing minimum P4 and body mass index (BMI) value was respectively correlated with increasing pregnancy probability. ROC analysis proved that the cutoff values for minimum P4 and BMI were 0.45 ng/ml and 23.36 kg/m2. CONCLUSION: In COH cycles, the ultrasonographic appearance of ovary helps to predict the number of viable embryos. In ET cycles, dexamethasone obviously improves the pregnancy rate. If the minimum P4 value before endometrial transformation cannot be kept below 0.45 ng/ml, we may consider cycle cancellation. Moreover, it is suggested that BMI of nonclassic 21-OHD women is regulated below 23.36 kg/m2.


Assuntos
Hiperplasia Suprarrenal Congênita/patologia , Fertilização In Vitro , Indução da Ovulação , Resultado da Gravidez , Adulto , Índice de Massa Corporal , Transferência Embrionária , Embrião de Mamíferos/metabolismo , Feminino , Humanos , Gravidez , Curva ROC
16.
Int J Mol Sci ; 20(9)2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31072004

RESUMO

Deciphering how signaling pathways interact during development is necessary for understanding the etiopathogenesis of congenital malformations and disease. In several embryonic structures, components of the Hedgehog and retinoic acid pathways, two potent players in development and disease are expressed and operate in the same or adjacent tissues and cells. Yet whether and, if so, how these pathways interact during organogenesis is, to a large extent, unclear. Using genetic and experimental approaches in the mouse, we show that during development of ontogenetically different organs, including the tail, genital tubercle, and secondary palate, Sonic hedgehog (SHH) loss-of-function causes anomalies phenocopying those induced by enhanced retinoic acid signaling and that SHH is required to prevent supraphysiological activation of retinoic signaling through maintenance and reinforcement of expression of the Cyp26 genes. Furthermore, in other tissues and organs, disruptions of the Hedgehog or the retinoic acid pathways during development generate similar phenotypes. These findings reveal that rigidly calibrated Hedgehog and retinoic acid activities are required for normal organogenesis and tissue patterning.


Assuntos
Família 26 do Citocromo P450/genética , Desenvolvimento Embrionário/genética , Proteínas Hedgehog/genética , Ácido Retinoico 4 Hidroxilase/genética , Animais , Apoptose/genética , Diferenciação Celular/genética , Embrião de Mamíferos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Organogênese/genética , Transdução de Sinais/genética , Dente/crescimento & desenvolvimento , Dente/metabolismo , Tretinoína/metabolismo
17.
BMC Genomics ; 20(1): 386, 2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101013

RESUMO

BACKGROUND: Adenovirus protein, Gam1, triggers the proteolytic destruction of the E1 SUMO-activating enzyme. Microinjection of an empirically determined amount of Gam1 mRNA into one-cell Xenopus embryos can reduce SUMOylation activity to undetectable, but nonlethal, levels, enabling an examination of the role of this post-translational modification during early vertebrate development. RESULTS: We find that SUMOylation-deficient embryos consistently exhibit defects in neural tube and heart development. We have measured differences in gene expression between control and embryos injected with Gam1 mRNA at three developmental stages: early gastrula (immediately following the initiation of zygotic transcription), late gastrula (completion of the formation of the three primary germ layers), and early neurula (appearance of the neural plate). Although changes in gene expression are widespread and can be linked to many biological processes, three pathways, non-canonical Wnt/PCP, snail/twist, and Ets-1, are especially sensitive to the loss of SUMOylation activity and can largely account for the predominant phenotypes of Gam1 embryos. SUMOylation appears to generate different pools of a given transcription factor having different specificities with this post-translational modification involved in the regulation of more complex, as opposed to housekeeping, processes. CONCLUSIONS: We have identified changes in gene expression that underlie the neural tube and heart phenotypes resulting from depressed SUMOylation activity. Notably, these developmental defects correspond to the two most frequently occurring congenital birth defects in humans, strongly suggesting that perturbation of SUMOylation, either globally or of a specific protein, may frequently be the origin of these pathologies.


Assuntos
Embrião de Mamíferos/patologia , Regulação da Expressão Gênica no Desenvolvimento , Cardiopatias Congênitas/genética , Defeitos do Tubo Neural/genética , Sumoilação , Proteínas de Xenopus/metabolismo , Animais , Embrião de Mamíferos/metabolismo , Feminino , Perfilação da Expressão Gênica , Cardiopatias Congênitas/patologia , Masculino , Defeitos do Tubo Neural/patologia , Proteínas Virais/administração & dosagem , Xenopus laevis
18.
Genome Biol ; 20(1): 101, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118069

RESUMO

Base editing installs a precise nucleotide change in specific gene loci without causing a double-strand break. Its efficiency in human embryos is generally low, limiting its utility in functional genetic studies. Here, we report that injecting base editors into human cleaving two-cell and four-cell embryos results in much higher (up to 13-fold) homozygotic nucleotide substitution efficiency as opposed to MII oocytes or zygotes. Furthermore, as a proof-of-principle study, a point mutation can be efficiently corrected by our method. Our study indicates that human cleaving embryos provide an efficient base editing window for robust gene disruption and correction.


Assuntos
Pesquisas com Embriões , Embrião de Mamíferos , Edição de Genes/métodos , Humanos
19.
Int J Mol Sci ; 20(9)2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-31035421

RESUMO

Embryo implantation in the mink follows the pattern of many carnivores, in that preimplantation embryo diapause occurs in every gestation. Details of the gene expression and regulatory networks that terminate embryo diapause remain poorly understood. Illumina RNA-Seq was used to analyze global gene expression changes in the mink uterus during embryo diapause and activation leading to implantation. More than 50 million high quality reads were generated, and assembled into 170,984 unigenes. A total of 1684 differential expressed genes (DEGs) in uteri with blastocysts in diapause were compared to the activated embryo group (p < 0.05). Among these transcripts, 1527 were annotated as known genes, including 963 up-regulated and 564 down-regulated genes. The gene ontology terms for the observed DEGs, included cellular communication, phosphatase activity, extracellular matrix and G-protein couple receptor activity. The KEGG pathways, including PI3K-Akt signaling pathway, focal adhesion and extracellular matrix (ECM)-receptor interactions were the most enriched. A protein-protein interaction (PPI) network was constructed, and hub nodes such as VEGFA, EGF, AKT, IGF1, PIK3C and CCND1 with high degrees of connectivity represent gene clusters expected to play an important role in embryo activation. These results provide novel information for understanding the molecular mechanisms of maternal regulation of embryo activation in mink.


Assuntos
Blastocisto/metabolismo , Útero/metabolismo , Animais , Blastocisto/fisiologia , Implantação do Embrião/genética , Implantação do Embrião/fisiologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Vison , Gravidez , Transcriptoma/genética , Útero/fisiologia
20.
Nat Commun ; 10(1): 2316, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31127120

RESUMO

Chromatin looping allows enhancer-bound regulatory factors to influence transcription. Large domains, referred to as topologically associated domains, participate in genome organization. However, the mechanisms underlining interactions within these domains, which control gene expression, are not fully understood. Here we report that activation of embryonic myogenesis is associated with establishment of long-range chromatin interactions centered on Pax3-bound loci. Using mass spectrometry and genomic studies, we identify the ubiquitously expressed LIM-domain binding protein 1 (Ldb1) as the mediator of looping interactions at a subset of Pax3 binding sites. Ldb1 is recruited to Pax3-bound elements independently of CTCF-Cohesin, and is necessary for efficient deposition of H3K4me1 at these sites and chromatin looping. When Ldb1 is deleted in Pax3-expressing cells in vivo, specification of migratory myogenic progenitors is severely impaired. These results highlight Ldb1 requirement for Pax3 myogenic activity and demonstrate how transcription factors can promote formation of sub-topologically associated domain interactions involved in lineage specification.


Assuntos
Cromossomos de Mamíferos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Domínio LIM/metabolismo , Desenvolvimento Muscular , Fator de Transcrição PAX3/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Cromatina/metabolismo , Cromossomos de Mamíferos/química , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos , Feminino , Fibroblastos , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Histonas/metabolismo , Humanos , Proteínas com Domínio LIM/genética , Masculino , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas
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