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1.
Chemosphere ; 254: 126900, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32957295

RESUMO

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants in marine environments and have arouse great concern since they pose adverse effects to marine ecosystem. To determine the potential impacts of environmentally relevant PAHs on early life stages of marine fish, this study exposed embryos of marine medaka (Oryzias melastigma) to 0, 2, 10, 50, and 250 µg/L of phenanthrene (Phe), one of the most abundant PAHs. The results demonstrated that Phe exposure decreased hatching rates, delayed hatching time of embryos, and increased deformity rate of newly-hatched larvae. Exposure to 10 and 50 µg/L Phe decreased the survival rate of marine medaka larvae at 28 days post-fertilization (dpf), and no embryo successfully hatched in 250 µg/L Phe exposure group. Morphology results showed that 10, 50, and 250 µg/L Phe exposure significantly retarded the development of embryos, and 2, 10, and 50 µg/L caused yolk sac edema and pericardial edema in newly-hatched larvae, indicating that low concentrations of Phe could induce developmental cardiac toxicity. Furthermore, the changes in the expression of heart development-related genes were determined, and the results showed that Phe-induced cardiac malformation might be related with fgf8, bmp4, smyd1, ATPase and gata4 genes. Overall, environmentally relevant PAHs could disrupt heart morphogenesis and hatching process of marine medaka, which might have profound consequences for sustainability of fish population.


Assuntos
Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Oryzias/crescimento & desenvolvimento , Fenantrenos/toxicidade , Teratogênios/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Ecossistema , Embrião não Mamífero/anormalidades , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Coração/efeitos dos fármacos , Coração/embriologia , Larva/efeitos dos fármacos , Larva/genética , Oryzias/genética , Fenantrenos/análise , Teratogênios/análise , Poluentes Químicos da Água/análise
2.
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
3.
Nature ; 585(7824): 239-244, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32879485

RESUMO

Obligate endosymbiosis, in which distantly related species integrate to form a single replicating individual, represents a major evolutionary transition in individuality1-3. Although such transitions are thought to increase biological complexity1,2,4-6, the evolutionary and developmental steps that lead to integration remain poorly understood. Here we show that obligate endosymbiosis between the bacteria Blochmannia and the hyperdiverse ant tribe Camponotini7-11 originated and also elaborated through radical alterations in embryonic development, as compared to other insects. The Hox genes Abdominal A (abdA) and Ultrabithorax (Ubx)-which, in arthropods, normally function to differentiate abdominal and thoracic segments after they form-were rewired to also regulate germline genes early in development. Consequently, the mRNAs and proteins of these Hox genes are expressed maternally and colocalize at a subcellular level with those of germline genes in the germplasm and three novel locations in the freshly laid egg. Blochmannia bacteria then selectively regulate these mRNAs and proteins to make each of these four locations functionally distinct, creating a system of coordinates in the embryo in which each location performs a different function to integrate Blochmannia into the Camponotini. Finally, we show that the capacity to localize mRNAs and proteins to new locations in the embryo evolved before obligate endosymbiosis and was subsequently co-opted by Blochmannia and Camponotini. This pre-existing molecular capacity converged with a pre-existing ecological mutualism12,13 to facilitate both the horizontal transfer10 and developmental integration of Blochmannia into Camponotini. Therefore, the convergence of pre-existing molecular capacities and ecological interactions-as well as the rewiring of highly conserved gene networks-may be a general feature that facilitates the origin and elaboration of major transitions in individuality.


Assuntos
Formigas/embriologia , Formigas/microbiologia , Bactérias , Evolução Biológica , Regulação da Expressão Gênica no Desenvolvimento/genética , Individualidade , Simbiose/genética , Animais , Formigas/citologia , Formigas/genética , Desenvolvimento Embrionário/genética , Feminino , Genes Homeobox/genética , Herança Materna/genética , Oócitos/citologia , Oócitos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
4.
Nat Commun ; 11(1): 4267, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848148

RESUMO

While footprinting analysis of ATAC-seq data can theoretically enable investigation of transcription factor (TF) binding, the lack of a computational tool able to conduct different levels of footprinting analysis has so-far hindered the widespread application of this method. Here we present TOBIAS, a comprehensive, accurate, and fast footprinting framework enabling genome-wide investigation of TF binding dynamics for hundreds of TFs simultaneously. We validate TOBIAS using paired ATAC-seq and ChIP-seq data, and find that TOBIAS outperforms existing methods for bias correction and footprinting. As a proof-of-concept, we illustrate how TOBIAS can unveil complex TF dynamics during zygotic genome activation in both humans and mice, and propose how zygotic Dux activates cascades of TFs, binds to repeat elements and induces expression of novel genetic elements.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação/métodos , Fatores de Transcrição/metabolismo , Ativação Transcricional , Zigoto/metabolismo , Animais , Sítios de Ligação/genética , Desenvolvimento Embrionário/genética , Epigênese Genética , Feminino , Genoma Humano , Proteínas de Homeodomínio/metabolismo , Humanos , Cinética , Camundongos , Regiões Promotoras Genéticas , Estudo de Prova de Conceito , Ligação Proteica/genética , Especificidade da Espécie
5.
PLoS One ; 15(8): e0237167, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764780

RESUMO

The zebrafish Danio rerio is a valuable and common model for scientists in the fields of genetics and developmental biology. Since zebrafish are also amenable to genetic manipulation, modelling of human diseases or behavioral experiments have moved into the focus of zebrafish research. Consequently, gene expression data beyond embryonic and larval stages become more important, yet there is a dramatic knowledge gap of gene expression beyond day four of development. Like in other model organisms, the visualization of spatial and temporal gene expression by whole mount in situ hybridization (ISH) becomes increasingly difficult when zebrafish embryos develop further and hence the growing tissues become dense and less permeable. Here we introduce a modified method for whole mount ISH, which overcomes these penetration and detection problem. The method is an all in one solution that enables the detection and visualization of gene expression patterns up to the late larval stage in a 3D manner without the need for tissue sectioning and offers a valuable extension for whole mount ISH by immunohistochemistry in the zebrafish field.


Assuntos
Biologia do Desenvolvimento/métodos , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Peixe-Zebra/crescimento & desenvolvimento , Animais , Embrião não Mamífero , Imuno-Histoquímica , Hibridização In Situ , Larva/genética , Larva/crescimento & desenvolvimento , Modelos Animais , Peixe-Zebra/genética
6.
Gene ; 761: 145037, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32777526

RESUMO

Primordial germ cells (PGCs) are singled out from somatic cells very early during embryogenesis, then they migrate towards the genital ridge and differentiate into gametes through oogenesis or spermatogenesis. Labeling PGCs with Localized RNAexpression (LRE) technique by fluorescent proteins has been widely applied among teleost species to study the germ cell development and gonad differentiation. In this study, we first cloned and characterized the 3' untranslated regions (3'UTRs) of nanos homolog 1-like (nos1l), dead end (dnd), and vasa in yellow catfish (Pelteobagrus fulvidraco), and then synthesized the GFP-nos1l/dnd/vasa 3'UTR mRNAs. Each of these three 3'UTRs could label PGCs in yellow catfish embryos, of which, vasa 3'UTR exhibited the highest labeling efficiency. To identify the differences in PGCs at embryonic stage, XX all-female and XY all-male yellow catfish embryos were produced and injected with GFP-vasa 3'UTR mRNA. We observed the PGC migration route in these two monosex embryos from 24 hpf to 7 dpf, and found there was no difference between them. Besides, the PGC number was counted at 48 hpf, and the result showed that the average PGC number in XX females (11.3) was significantly larger than that in XY males (8.1).These findings provide an insight into the development of PGCs in yellow catfish embryos and the relationship between embryonicPGCnumberand thelatergonaddifferentiation.


Assuntos
Peixes-Gato/genética , Gametogênese/genética , Células Germinativas/metabolismo , Regiões 3' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Movimento Celular/genética , RNA Helicases DEAD-box/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Proteínas de Peixes/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Gônadas/metabolismo , Masculino , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo
7.
PLoS Genet ; 16(8): e1008967, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32813698

RESUMO

Dysregulation of ribosome production can lead to a number of developmental disorders called ribosomopathies. Despite the ubiquitous requirement for these cellular machines used in protein synthesis, ribosomopathies manifest in a tissue-specific manner, with many affecting the development of the face. Here we reveal yet another connection between craniofacial development and making ribosomes through the protein Paired Box 9 (PAX9). PAX9 functions as an RNA Polymerase II transcription factor to regulate the expression of proteins required for craniofacial and tooth development in humans. We now expand this function of PAX9 by demonstrating that PAX9 acts outside of the cell nucleolus to regulate the levels of proteins critical for building the small subunit of the ribosome. This function of PAX9 is conserved to the organism Xenopus tropicalis, an established model for human ribosomopathies. Depletion of pax9 leads to craniofacial defects due to abnormalities in neural crest development, a result consistent with that found for depletion of other ribosome biogenesis factors. This work highlights an unexpected layer of how the making of ribosomes is regulated in human cells and during embryonic development.


Assuntos
Deficiências do Desenvolvimento/genética , Desenvolvimento Embrionário/genética , Fator de Transcrição PAX9/genética , Ribossomos/genética , Animais , Nucléolo Celular/genética , Deficiências do Desenvolvimento/patologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Crista Neural/crescimento & desenvolvimento , Crista Neural/metabolismo , Crista Neural/patologia , Biossíntese de Proteínas/genética , RNA Polimerase II/genética , Ribossomos/patologia , Xenopus/genética , Xenopus/crescimento & desenvolvimento
8.
Ecotoxicol Environ Saf ; 205: 111165, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32836160

RESUMO

BACKGROUND: Bisphenol A (BPA) is a well-known xenobiotic endocrine disrupting chemical, with estrogenic activity and many other potential biological effects. Although multiple toxicities have been reported for BPA, molecular mechanisms underlying the transgenerational toxic effects of BPA are still underestimated. METHODS: Parental F0 fish were exposed to 1.0 µM BPA or control (0.1% DMSO, v/v) for 7 days. Eggs (F1) were collected and kept in control medium until 4.5 or 120 h post fertilization (hpf). RNA sequencing (RNA-seq) was conducted on embryos and larvae, to discover differentially expressed genes (DEGs), and then KEGG pathway, GO enrichment and GSEA were performed to interpret functional ontology. Histopathology was performed to explore the morphological and structural alterations in liver tissues of zebrafish larvae (120 hpf) after parental BPA exposure. RESULTS: Parental BPA exposure induced global transcriptomic changes in zebrafish embryos and larvae. For embryos, epigenetic regulation genes were decidedly affected, highlighted epigenotoxicity might involve in the transgenerational effects during embryogenesis and early development. By further investigation on its delayed effects, our RNA-Seq data of larvae suggested ROS metabolic process, apoptosis, p53 and MAPK signaling pathway were concentrated, indicating defensive cellular processes still involved in protecting against BPA toxicity. Furthermore, parental BPA-treated larvae manifested hepatic injury by histopathological analysis. CONCLUSIONS: Parental BPA exposure led to global transcriptomic changes involved in epigenetic regulation, oxidative stress, apoptosis and DNA damage of offspring. These findings advanced the field of the parental-mediated subsequent generational toxic effects of BPA.


Assuntos
Compostos Benzidrílicos/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Larva/efeitos dos fármacos , Fenóis/toxicidade , Transcriptoma/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/genética , Animais , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/genética , Epigênese Genética/efeitos dos fármacos , Perfilação da Expressão Gênica , Larva/genética , Análise de Sequência de RNA , Peixe-Zebra/metabolismo
9.
Science ; 369(6503): 530-537, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32732419

RESUMO

Microglia, immune cells of the central nervous system (CNS), are important for tissue development and maintenance and are implicated in CNS disease, but we lack understanding of human fetal microglia development. Single-cell gene expression and bulk chromatin profiles of microglia at 9 to 18 gestational weeks (GWs) of human fetal development were generated. Microglia were heterogeneous at all studied GWs. Microglia start to mature during this developmental period and increasingly resemble adult microglia with CNS-surveilling properties. Chromatin accessibility increases during development with associated transcriptional networks reflective of adult microglia. Thus, during early fetal development, microglia progress toward a more mature, immune-sensing competent phenotype, and this might render the developing human CNS vulnerable to environmental perturbations during early pregnancy.


Assuntos
Encéfalo/embriologia , Desenvolvimento Embrionário/imunologia , Feto/imunologia , Microglia/imunologia , Fagocitose/imunologia , Encéfalo/citologia , Separação Celular , Células Cultivadas , Desenvolvimento Embrionário/genética , Redes Reguladoras de Genes , Humanos , Fagocitose/genética , Transcriptoma
10.
PLoS Genet ; 16(7): e1008920, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32697780

RESUMO

Neurofibromatosis type 1 is a monogenetic disorder that predisposes individuals to tumor formation and cognitive and behavioral symptoms. The neuronal circuitry and developmental events underlying these neurological symptoms are unknown. To better understand how mutations of the underlying gene (NF1) drive behavioral alterations, we have examined grooming in the Drosophila neurofibromatosis 1 model. Mutations of the fly NF1 ortholog drive excessive grooming, and increased grooming was observed in adults when Nf1 was knocked down during development. Furthermore, intact Nf1 Ras GAP-related domain signaling was required to maintain normal grooming. The requirement for Nf1 was distributed across neuronal circuits, which were additive when targeted in parallel, rather than mapping to discrete microcircuits. Overall, these data suggest that broadly-distributed alterations in neuronal function during development, requiring intact Ras signaling, drive key Nf1-mediated behavioral alterations. Thus, global developmental alterations in brain circuits/systems function may contribute to behavioral phenotypes in neurofibromatosis type 1.


Assuntos
Proteínas de Drosophila/genética , Desenvolvimento Embrionário/genética , Proteínas do Tecido Nervoso/genética , Neurofibromatose 1/genética , Neurônios/metabolismo , Proteínas Ativadoras de ras GTPase/genética , Animais , Cognição/fisiologia , Modelos Animais de Doenças , Drosophila melanogaster/genética , Embrião não Mamífero , Técnicas de Silenciamento de Genes , Asseio Animal/fisiologia , Humanos , Mutação/genética , Neurofibromatose 1/patologia , Neurônios/patologia
11.
Nat Commun ; 11(1): 3491, 2020 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-32661239

RESUMO

Sperm contributes genetic and epigenetic information to the embryo to efficiently support development. However, the mechanism underlying such developmental competence remains elusive. Here, we investigated whether all sperm cells have a common epigenetic configuration that primes transcriptional program for embryonic development. Using calibrated ChIP-seq, we show that remodelling of histones during spermiogenesis results in the retention of methylated histone H3 at the same genomic location in most sperm cell. This homogeneously methylated fraction of histone H3 in the sperm genome is maintained during early embryonic replication. Such methylated histone fraction resisting post-fertilisation reprogramming marks developmental genes whose expression is perturbed upon experimental reduction of histone methylation. A similar homogeneously methylated histone H3 fraction is detected in human sperm. Altogether, we uncover a conserved mechanism of paternal epigenetic information transmission to the embryo through the homogeneous retention of methylated histone in a sperm cells population.


Assuntos
Metilação de DNA/genética , Epigênese Genética/genética , Animais , Cromatina/genética , Cromatina/metabolismo , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Histonas/genética , Histonas/metabolismo , Masculino , Espermatogênese/genética , Espermatogênese/fisiologia , Xenopus
12.
PLoS One ; 15(7): e0235617, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32634160

RESUMO

Low egg quality and embryonic survival are critical challenges in aquaculture, where assisted reproduction procedures and other factors may impact egg quality. This includes European eel (Anguilla anguilla), where pituitary extract from carp (CPE) or salmon (SPE) is applied to override a dopaminergic inhibition of the neuroendocrine system, preventing gonadotropin secretion and gonadal development. The present study used either CPE or SPE to induce vitellogenesis in female European eel and compared impacts on egg quality and offspring developmental competence with emphasis on the maternal-to-zygotic transition (MZT). Females treated with SPE produced significantly higher proportions of floating eggs with fewer cleavage abnormalities and higher embryonic survival. These findings related successful embryogenesis to higher abundance of mRNA transcripts of genes involved in cell adhesion, activation of MZT, and immune response (dcbld1, epcam, oct4, igm) throughout embryonic development. The abundance of mRNA transcripts of cldnd, foxr1, cea, ccna1, ccnb1, ccnb2, zar1, oct4, and npm2 was relatively stable during the first eight hours, followed by a drop during MZT and low levels thereafter, indicating transfer and subsequent clearance of maternal mRNA. mRNA abundance of zar1, epcam, and dicer1 was associated with cleavage abnormalities, while mRNA abundance of zar1, sox2, foxr1, cldnd, phb2, neurod4, and neurog1 (before MZT) was associated with subsequent embryonic survival. In a second pattern, low initial mRNA abundance with an increase during MZT and higher levels persisting thereafter indicating the activation of zygotic transcription. mRNA abundance of ccna1, npm2, oct4, neurod4, and neurog1 during later embryonic development was associated with hatch success. A deviating pattern was observed for dcbld1, which mRNA levels followed the maternal-effect gene pattern but only for embryos from SPE treated females. Together, the differences in offspring production and performance reported in this study show that PE composition impacts egg quality and embryogenesis and in particular, the transition from initial maternal transcripts to zygotic transcription.


Assuntos
Anguilla/fisiologia , Carpas/metabolismo , Desenvolvimento Embrionário , Oogênese , Hipófise/metabolismo , Salmão/metabolismo , Anguilla/crescimento & desenvolvimento , Animais , Ciclina A1/genética , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Feminino , Proteínas de Peixes/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Fator 3 de Transcrição de Octâmero/genética , Oogênese/efeitos dos fármacos , Hipófise/química , Hormônios Hipofisários/farmacologia , RNA Mensageiro/metabolismo , Zigoto/efeitos dos fármacos , Zigoto/crescimento & desenvolvimento , Zigoto/metabolismo
13.
Nucleic Acids Res ; 48(15): 8374-8392, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32619237

RESUMO

The core-promoter, a stretch of DNA surrounding the transcription start site (TSS), is a major integration-point for regulatory-signals controlling gene-transcription. Cellular differentiation is marked by divergence in transcriptional repertoire and cell-cycling behaviour between cells of different fates. The role promoter-associated gene-regulatory-networks play in development-associated transitions in cell-cycle-dynamics is poorly understood. This study demonstrates in a vertebrate embryo, how core-promoter variations define transcriptional output in cells transitioning from a proliferative to cell-lineage specifying phenotype. Assessment of cell proliferation across zebrafish embryo segmentation, using the FUCCI transgenic cell-cycle-phase marker, revealed a spatial and lineage-specific separation in cell-cycling behaviour. To investigate the role differential promoter usage plays in this process, cap-analysis-of-gene-expression (CAGE) was performed on cells segregated by cycling dynamics. This analysis revealed a dramatic increase in tissue-specific gene expression, concurrent with slowed cycling behaviour. We revealed a distinct sharpening in TSS utilization in genes upregulated in slowly cycling, differentiating tissues, associated with enhanced utilization of the TATA-box, in addition to Sp1 binding-sites. In contrast, genes upregulated in rapidly cycling cells carry broad distribution of TSS utilization, coupled with enrichment for the CCAAT-box. These promoter features appear to correspond to cell-cycle-dynamic rather than tissue/cell-lineage origin. Moreover, we observed genes with cell-cycle-dynamic-associated transitioning in TSS distribution and differential utilization of alternative promoters. These results demonstrate the regulatory role of core-promoters in cell-cycle-dependent transcription regulation, during embryo-development.


Assuntos
Redes Reguladoras de Genes/genética , Regiões Promotoras Genéticas/genética , Sítio de Iniciação de Transcrição , Transcrição Genética , Animais , Sítios de Ligação/genética , Ciclo Celular/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Desenvolvimento Embrionário/genética , Humanos , Morfogênese/genética , Fator de Transcrição Sp1/genética , TATA Box/genética , Peixe-Zebra/genética
14.
Nucleic Acids Res ; 48(15): 8431-8444, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32667642

RESUMO

Genome-wide passive DNA demethylation in cleavage-stage mouse embryos is related to the cytoplasmic localization of the maintenance methyltransferase DNMT1. However, recent studies provided evidences of the nuclear localization of DNMT1 and its contribution to the maintenance of methylation levels of imprinted regions and other genomic loci in early embryos. Using the DNA adenine methylase identification method, we identified Dnmt1-binding regions in four- and eight-cell embryos. The unbiased distribution of Dnmt1 peaks in the genic regions (promoters and CpG islands) as well as the absence of a correlation between the Dnmt1 peaks and the expression levels of the peak-associated genes refutes the active participation of Dnmt1 in the transcriptional regulation of genes in the early developmental period. Instead, Dnmt1 was found to associate with genomic retroelements in a greatly biased fashion, particularly with the LINE1 (long interspersed nuclear elements) and ERVK (endogenous retrovirus type K) sequences. Transcriptomic analysis revealed that the transcripts of the Dnmt1-enriched retroelements were overrepresented in Dnmt1 knockdown embryos. Finally, methyl-CpG-binding domain sequencing proved that the Dnmt1-enriched retroelements, which were densely methylated in wild-type embryos, became demethylated in the Dnmt1-depleted embryos. Our results indicate that Dnmt1 is involved in the repression of retroelements through DNA methylation in early mouse development.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/genética , Metilação de DNA/genética , Desenvolvimento Embrionário/genética , Genômica , Retroelementos/genética , Animais , Ilhas de CpG/genética , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos , Perfilação da Expressão Gênica , Genoma/genética , Impressão Genômica/genética , Camundongos , Fatores de Transcrição/genética
15.
Aquat Toxicol ; 226: 105562, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32668346

RESUMO

Fish are exposed to steroids of different classes in contaminated waters, but their effects are not sufficiently understood. Here we employed an anti-sense technique using morpholino oligonucleotides to knockdown the glucocorticoid receptors (GRs, GRα and GRß) and androgen receptor (AR) to investigate their role in physiological and transcriptional responses. To this end, zebrafish embryos were exposed to clobetasol propionate (CLO), androstenedione (A4) and mixtures containing different classes of steroids. CLO caused a decrease of spontaneous muscle contraction and increase of heart rate, as well as transcriptional induction of pepck1, fkbp5, sult2st3 and vitellogenin (vtg1) at 24 and/or 48 h post fertilization (hpf). Knockdown of GRs eliminated these effects, while knockdown of AR decreased the ar transcript but caused no expressional changes, except induction of sult2st3 after exposure to A4 at 24 hpf. Exposure to a mixture of 6 steroids comprising progesterone (P4) and three progestins, cyproterone acetate, dienogest, drospirenone, 17ß-estradiol (E2) and CLO caused a significant induction of pepck1, sult2st3, vtg1 and per1a. Knockdown of GRs eliminated the physiological effects and the up-regulation of vtg1, sult2st3, pepck1, fkbp5 and per1a. Thus, as with CLO, responses in mixtures were regulated by GRs independently from the presence of other steroids. Exposure to a mixture comprising A4, CLO, E2 and P4 caused induction of vtg1, cyp19b, sult2st3 and fkbp5. Knockdown of AR had no effect, indicating that regulation of these genes occurred by the GRs and estrogen receptor (ER). Our findings show that in early embryos GRs cause vtg1 and sult2st3 induction in addition to known glucocorticoid target genes. Each steroid receptor regulated its own target genes in steroid mixtures independently from other steroids. However, enhanced expressional induction occurred for vtg1 and fkbp5 in steroid mixtures, indicating an interaction/cross-talk between GRs and ER. These findings have importance for the understanding of molecular effects of steroid mixtures.


Assuntos
Embrião não Mamífero/efeitos dos fármacos , Receptores Androgênicos/metabolismo , Receptores de Glucocorticoides/metabolismo , Esteroides/toxicidade , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/metabolismo , Animais , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Receptores Androgênicos/genética , Receptores de Glucocorticoides/genética , Transdução de Sinais , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
16.
Proc Natl Acad Sci U S A ; 117(30): 17864-17875, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32669432

RESUMO

Early pregnancy loss affects ∼15% of all implantation-confirmed human conceptions. However, evolutionarily conserved molecular mechanisms that regulate self-renewal of trophoblast progenitors and their association with early pregnancy loss are poorly understood. Here, we provide evidence that transcription factor TEAD4 ensures survival of postimplantation mouse and human embryos by controlling self-renewal and stemness of trophoblast progenitors within the placenta primordium. In an early postimplantation mouse embryo, TEAD4 is selectively expressed in trophoblast stem cell-like progenitor cells (TSPCs), and loss of Tead4 in postimplantation mouse TSPCs impairs their self-renewal, leading to embryonic lethality before embryonic day 9.0, a developmental stage equivalent to the first trimester of human gestation. Both TEAD4 and its cofactor, yes-associated protein 1 (YAP1), are specifically expressed in cytotrophoblast (CTB) progenitors of a first-trimester human placenta. We also show that a subset of unexplained recurrent pregnancy losses (idiopathic RPLs) is associated with impaired TEAD4 expression in CTB progenitors. Furthermore, by establishing idiopathic RPL patient-specific human trophoblast stem cells (RPL-TSCs), we show that loss of TEAD4 is associated with defective self-renewal in RPL-TSCs and rescue of TEAD4 expression restores their self-renewal ability. Unbiased genomics studies revealed that TEAD4 directly regulates expression of key cell cycle genes in both mouse and human TSCs and establishes a conserved transcriptional program. Our findings show that TEAD4, an effector of the Hippo signaling pathway, is essential for the establishment of pregnancy in a postimplantation mammalian embryo and indicate that impairment of the Hippo signaling pathway could be a molecular cause for early human pregnancy loss.


Assuntos
Autorrenovação Celular/genética , Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário/genética , Proteínas Musculares/genética , Fatores de Transcrição/genética , Trofoblastos/citologia , Trofoblastos/metabolismo , Aborto Habitual/etiologia , Aborto Habitual/metabolismo , Aborto Espontâneo/etiologia , Aborto Espontâneo/metabolismo , Animais , Biomarcadores , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Implantação do Embrião , Feminino , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Imuno-Histoquímica , Camundongos , Proteínas Musculares/metabolismo , Placenta/metabolismo , Gravidez , Fatores de Transcrição/metabolismo
17.
PLoS Biol ; 18(7): e3000561, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32702011

RESUMO

Maternal ß-catenin activity is essential and critical for dorsal induction and its dorsal activation has been thoroughly studied. However, how the maternal ß-catenin activity is suppressed in the nondorsal cells remains poorly understood. Nanog is known to play a central role for maintenance of the pluripotency and maternal -zygotic transition (MZT). Here, we reveal a novel role of Nanog as a strong repressor of maternal ß-catenin signaling to safeguard the embryo against hyperactivation of maternal ß-catenin activity and hyperdorsalization. In zebrafish, knockdown of nanog at different levels led to either posteriorization or dorsalization, mimicking zygotic or maternal activation of Wnt/ß-catenin activities, and the maternal zygotic mutant of nanog (MZnanog) showed strong activation of maternal ß-catenin activity and hyperdorsalization. Although a constitutive activator-type Nanog (Vp16-Nanog, lacking the N terminal) perfectly rescued the MZT defects of MZnanog, it did not rescue the phenotypes resulting from ß-catenin signaling activation. Mechanistically, the N terminal of Nanog directly interacts with T-cell factor (TCF) and interferes with the binding of ß-catenin to TCF, thereby attenuating the transcriptional activity of ß-catenin. Therefore, our study establishes a novel role for Nanog in repressing maternal ß-catenin activity and demonstrates a transcriptional switch between ß-catenin/TCF and Nanog/TCF complexes, which safeguards the embryo from global activation of maternal ß-catenin activity.


Assuntos
Desenvolvimento Embrionário/genética , Proteína Homeobox Nanog/metabolismo , Transativadores/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , beta Catenina/metabolismo , Animais , Padronização Corporal/genética , Núcleo Celular/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Masculino , Mutação/genética , Proteína Homeobox Nanog/química , Proteína Homeobox Nanog/genética , Ligação Proteica , Transporte Proteico , Proteínas Repressoras/metabolismo , Transcrição Genética , Via de Sinalização Wnt/genética , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética , Zigoto/metabolismo
18.
Proc Natl Acad Sci U S A ; 117(27): 15694-15701, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571922

RESUMO

The p53 family member p73 has a complex gene structure, including alternative promoters and alternative splicing of the 3' UTR. This results in a complex range of isoforms whose biological relevance largely remains to be determined. By deleting exon 13 (which encodes a sterile α motif) from the Trp73 gene, we selectively engineered mice to replace the most abundantly expressed C-terminal isoform, p73α, with a shorter product of alternative splicing, p73ß. These mice (Trp73 Δ13/Δ13 ) display severe neurodevelopmental defects with significant functional and morphological abnormalities. Replacement of p73α with p73ß results in the depletion of Cajal-Retzius (CR) cells in embryonic stages, thus depriving the developing hippocampus of the pool of neurons necessary for correct hippocampal architecture. Consequently, Trp73 Δ13/Δ13 mice display severe hippocampal dysgenesis, reduced synaptic functionality and impaired learning and memory capabilities. Our data shed light on the relevance of p73 alternative splicing and show that the full-length C terminus of p73 is essential for hippocampal development.


Assuntos
Processamento Alternativo/genética , Desenvolvimento Embrionário/genética , Hipocampo/crescimento & desenvolvimento , Proteína Tumoral p73/genética , Animais , Apoptose/genética , Hipocampo/metabolismo , Humanos , Células Intersticiais de Cajal/metabolismo , Aprendizagem/fisiologia , Memória/fisiologia , Camundongos , Neurônios/metabolismo , Regiões Promotoras Genéticas
19.
Proc Natl Acad Sci U S A ; 117(27): 15702-15711, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32576691

RESUMO

Mammalian cells contain two isoforms of RNA polymerase III (Pol III) that differ in only a single subunit, with POLR3G in one form (Pol IIIα) and the related POLR3GL in the other form (Pol IIIß). Previous research indicates that POLR3G and POLR3GL are differentially expressed, with POLR3G expression being highly enriched in embryonic stem cells (ESCs) and tumor cells relative to the ubiquitously expressed POLR3GL. To date, the functional differences between these two subunits remain largely unexplored, especially in vivo. Here, we show that POLR3G and POLR3GL containing Pol III complexes bind the same target genes and assume the same functions both in vitro and in vivo and, to a significant degree, can compensate for each other in vivo. Notably, an observed defect in the differentiation ability of POLR3G knockout ESCs can be rescued by exogenous expression of POLR3GL. Moreover, whereas POLR3G knockout mice die at a very early embryonic stage, POLR3GL knockout mice complete embryonic development without noticeable defects but die at about 3 wk after birth with signs of both general growth defects and potential cerebellum-related neuronal defects. The different phenotypes of the knockout mice likely reflect differential expression levels of POLR3G and POLR3GL across developmental stages and between tissues and insufficient amounts of total Pol III in vivo.


Assuntos
Cerebelo/crescimento & desenvolvimento , Desenvolvimento Embrionário/genética , Neurônios/metabolismo , RNA Polimerase III/genética , Animais , Sítios de Ligação/genética , Diferenciação Celular/genética , Cerebelo/patologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Camundongos , Camundongos Knockout , Neurônios/patologia , Ligação Proteica/genética , Isoformas de Proteínas/genética , Subunidades Proteicas/genética
20.
Life Sci ; 256: 117895, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32502545

RESUMO

AIMS: We aimed to investigate the effect of sperm miR-34c on early human embryonic development kinetics and clinical outcomes of in vitro fertilization (IVF) patients. MATERIALS AND METHODS: After oocyte insemination, residual sperm specimens were collected from 58 patients undergoing IVF. miR-34c expression levels in sperm, oocytes, zygotes, and embryos/blastocysts were detected with qRT-PCR, and embryonic development kinetics were observed using time-lapse technology. To confirm the role of miR-34c in regulation of early embryonic development, miR-34c siRNA was injected into zygotes obtained from in vitro-matured oocytes. A ROC curve was used to determine the cutoff value. Comparisons of embryonic development kinetics and clinical outcomes were performed according to the cutoff value. KEY FINDINGS: The miR-34c expression level was highest in 3PN zygotes, but was not expressed in human oocytes. In the miR-34c siRNA group, embryonic development kinetic parameters t2, t3, t4, and t5 were significantly prolonged, but the cleavage rate and high-quality embryo rate were lower than in the control group. The levels of sperm miR-34c were negatively correlated with t5 and positively correlated with rates of blastocyst formation, high-quality blastocysts, and pregnancy. The miR-34c levels and the blastocyst formation rate were higher in the pregnancy group (p < 0.05). Logistic regression analysis showed that sperm miR-34c level was significantly correlated with pregnancy (OR: 5.056, 95% CI: 1.560-16.384; p = 0.007). SIGNIFICANCE: The sperm miR-34c expression level is associated with embryonic development kinetics and clinical outcomes. Thus, miR-34c expression is beneficial to embryonic development and may be used as an indicator of IVF outcomes.


Assuntos
Desenvolvimento Embrionário , MicroRNAs/metabolismo , Espermatozoides/metabolismo , Adulto , Blastocisto/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Cinética , Masculino , MicroRNAs/genética , Oócitos/metabolismo , Gravidez , Curva ROC
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