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1.
Nat Commun ; 12(1): 4071, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34210974

RESUMO

Molecular left-right (L-R) asymmetry is established at the node of the mouse embryo as a result of the sensing of a leftward fluid flow by immotile cilia of perinodal crown cells and the consequent degradation of Dand5 mRNA on the left side. We here examined how the fluid flow induces Dand5 mRNA decay. We found that the first 200 nucleotides in the 3' untranslated region (3'-UTR) of Dand5 mRNA are necessary and sufficient for the left-sided decay and to mediate the response of a 3'-UTR reporter transgene to Ca2+, the cation channel Pkd2, the RNA-binding protein Bicc1 and their regulation by the flow direction. We show that Bicc1 preferentially recognizes GACR and YGAC sequences, which can explain the specific binding to a conserved GACGUGAC motif located in the proximal Dand5 3'-UTR. The Cnot3 component of the Ccr4-Not deadenylase complex interacts with Bicc1 and is also required for Dand5 mRNA decay at the node. These results suggest that Ca2+ currents induced by leftward fluid flow stimulate Bicc1 and Ccr4-Not to mediate Dand5 mRNA degradation specifically on the left side of the node.


Assuntos
Embrião de Mamíferos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Estabilidade de RNA/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores CCR4/metabolismo , Regiões 3' não Traduzidas , Animais , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Knockout , Proteínas de Ligação a RNA/genética , Receptores CCR4/genética , Canais de Cátion TRPP/metabolismo , Fatores de Transcrição
2.
Nucleic Acids Res ; 49(11): 6165-6180, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34107020

RESUMO

The current understanding of how overall principles of translational control govern the embryo-to-adult transition in mammals is still far from comprehensive. Herein we profiled the translatomes and transcriptomes of six tissues from the mice at embryonic and adult stages and presented the first report of tissue- and stage-specific translational landscape in mice. We quantified the extent of gene expression divergence among different expression layers, tissues and stages, detected significant changes in gene composition and function underlying these divergences and revealed the changing architecture of translational regulation. We further showed that dynamic translational regulation can be largely achieved via modulation of translational efficiency. Translational efficiency could be altered by alternative splicing (AS), upstream and downstream open reading frames (uORFs and dORFs). We revealed AS-mediated translational repression that was exerted in an event type-dependent manner. uORFs and dORFs exhibited mutually exclusive usage and the opposing effects of translational regulation. Furthermore, we discovered many novel microproteins encoded by long noncoding RNAs and demonstrated their regulatory potential and functional relevance. Our data and analyses will facilitate a better understanding of the complexity of translation and translational regulation across tissue and stage spectra and provide an important resource to the translatome research community.


Assuntos
Regulação da Expressão Gênica , Biossíntese de Proteínas , Processamento Alternativo , Animais , Embrião de Mamíferos/metabolismo , Camundongos Endogâmicos C57BL , Fases de Leitura Aberta , Especificidade de Órgãos , RNA Longo não Codificante/metabolismo , RNA-Seq , Transcriptoma
3.
Nat Commun ; 12(1): 3714, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140513

RESUMO

The mechanism behind transgenerational epigenetic inheritance is unclear, particularly through the maternal grandparental line. We previously showed that disruption of folate metabolism in mice by the Mtrr hypomorphic mutation results in transgenerational epigenetic inheritance of congenital malformations. Either maternal grandparent can initiate this phenomenon, which persists for at least four wildtype generations. Here, we use genome-wide approaches to reveal genetic stability in the Mtrr model and genome-wide differential DNA methylation in the germline of Mtrr mutant maternal grandfathers. We observe that, while epigenetic reprogramming occurs, wildtype grandprogeny and great grandprogeny exhibit transcriptional changes that correlate with germline methylation defects. One region encompasses the Hira gene, which is misexpressed in embryos for at least three wildtype generations in a manner that distinguishes Hira transcript expression as a biomarker of maternal phenotypic inheritance.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Metilação de DNA , Ferredoxina-NADP Redutase/genética , Ácido Fólico/metabolismo , Células Germinativas/metabolismo , Chaperonas de Histonas/metabolismo , Padrões de Herança/genética , Herança Materna/genética , Fatores de Transcrição/metabolismo , Animais , Biomarcadores/metabolismo , Proteínas de Ciclo Celular/genética , Embrião de Mamíferos/metabolismo , Epigênese Genética , Epigenômica , Feminino , Ferredoxina-NADP Redutase/metabolismo , Hereditariedade , Chaperonas de Histonas/genética , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linhagem , Fenótipo , Polimorfismo de Nucleotídeo Único , Espermatozoides/metabolismo , Fatores de Transcrição/genética , Trofoblastos/metabolismo , Sequenciamento Completo do Genoma
4.
Int J Mol Sci ; 22(11)2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073234

RESUMO

Embryonic implantation is a key step in the establishment of pregnancy. In the present work, we have carried out an in-depth proteomic analysis of the secretome (extracellular vesicles and soluble proteins) of two bovine blastocysts embryonic trophectoderm primary cultures (BBT), confirming different epithelial-mesenchymal transition stages in these cells. BBT-secretomes contain early pregnancy-related proteins and angiogenic proteins both as cargo in EVs and the soluble fraction. We have demonstrated the functional transfer of protein-containing secretome between embryonic trophectoderm and maternal MSC in vitro using two BBT primary cultures eight endometrial MSC (eMSC) and five peripheral blood MSC (pbMSC) lines. We observed that eMSC and pbMSC chemotax to both the soluble fraction and EVs of the BBT secretome. In addition, in a complementary direction, we found that the pattern of expression of implantation proteins in BBT-EVs changes depending on: (i) their epithelial-mesenchymal phenotype; (ii) as a result of the uptake of eMSC- or pbMSC-EV previously stimulated or not with embryonic signals (IFN-); (iii) because of the stimulation with the endometrial cytokines present in the uterine fluid in the peri-implantation period.


Assuntos
Quimiotaxia , Ectoderma/metabolismo , Implantação do Embrião , Embrião de Mamíferos/metabolismo , Endométrio/metabolismo , Células-Tronco Mesenquimais/metabolismo , Animais , Bovinos , Linhagem Celular , Feminino , Proteômica
5.
Cell Prolif ; 54(7): e13059, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34021643

RESUMO

OBJECTIVES: The genetic instability and DNA damage arise during transcription factor-mediated reprogramming of somatic cells, and its efficiency may be reduced due to abnormal chromatin remodelling. The efficiency in somatic cell nuclear transfer (SCNT)-mediated reprogramming is also very low, and it is caused by development arrest of most reconstituted embryos. MATERIALS AND METHODS: Whether the repair of genetic instability or double-strand breaks (DSBs) during SCNT reprogramming may play an important role in embryonic development, we observed and analysed the effect of Rad 51, a key modulator of DNA damage response (DDR) in SCNT-derived embryos. RESULTS: Here, we observed that the activity of Rad 51 is lower in SCNT eggs than in conventional IVF and found a significantly lower level of DSBs in SCNT embryos during reprogramming. To address this difference, supplementation with RS-1, an activator of Rad51, during the activation of SCNT embryos can increase RAD51 expression and DSB foci and thereby increased the efficiency of SCNT reprogramming. Through subsequent single-cell RNA-seq analysis, we observed the reactivation of a large number of genes that were not expressed in SCNT-2-cell embryos by the upregulation of DDR, which may be related to overcoming the developmental block. Additionally, there may be an independent pathway involving histone demethylase that can reduce reprograming-resistance regions. CONCLUSIONS: This technology can contribute to the production of comparable cell sources for regenerative medicine.


Assuntos
Benzamidas/farmacologia , Reprogramação Celular , Desenvolvimento Embrionário/efeitos dos fármacos , Sulfonamidas/farmacologia , Animais , Reparo do DNA/efeitos dos fármacos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Feminino , Instabilidade Genômica , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Técnicas de Transferência Nuclear , Rad51 Recombinase/metabolismo , Regulação para Cima/efeitos dos fármacos
6.
DNA Res ; 28(2)2021 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-34009337

RESUMO

Myofibres (primary and secondary myofibre) are the basic structure of muscle and the determinant of muscle mass. To explore the skeletal muscle developmental processes from primary myofibres to secondary myofibres in pigs, we conducted an integrative three-dimensional structure of genome and transcriptomic characterization of longissimus dorsi muscle of pig from primary myofibre formation stage [embryonic Day 35 (E35)] to secondary myofibre formation stage (E80). In the hierarchical genomic structure, we found that 11.43% of genome switched compartment A/B status, 14.53% of topologically associating domains are changed intradomain interactions (D-scores) and 2,730 genes with differential promoter-enhancer interactions and (or) enhancer activity from E35 to E80. The alterations of genome architecture were found to correlate with expression of genes that play significant roles in neuromuscular junction, embryonic morphogenesis, skeletal muscle development or metabolism, typically, NEFL, MuSK, SLN, Mef2D and GCK. Significantly, Sox6 and MATN2 play important roles in the process of primary to secondary myofibres formation and increase the regulatory potential score and genes expression in it. In brief, we reveal the genomic reorganization from E35 to E80 and construct genome-wide high-resolution interaction maps that provide a resource for studying long-range control of gene expression from E35 to E80.


Assuntos
Cromatina/metabolismo , Embrião de Mamíferos/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/crescimento & desenvolvimento , Sus scrofa/genética , Transcriptoma , Animais , Montagem e Desmontagem da Cromatina , Sequenciamento de Cromatina por Imunoprecipitação , Desenvolvimento Embrionário , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Músculo Esquelético/metabolismo , Análise de Sequência de RNA , Sus scrofa/crescimento & desenvolvimento , Sus scrofa/metabolismo
7.
Methods Mol Biol ; 2262: 91-103, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977472

RESUMO

Validation of antibody specificity is essential for the accurate evaluation of protein expression. For antibodies that recognize the gene products of the RAS family of oncogenes (HRAS, KRAS, and NRAS), an important challenge is the determination of selectivity for the four nearly identical HRAS, KRAS4A, KRAS4B, and NRAS proteins. With increasing appreciation for the distinct roles of the different RAS proteins in normal and neoplastic cells, there is a need for well-validated antibodies to evaluate the function and expression of the different RAS isoforms. Here we describe our experimental approaches to characterize RAS antibodies for their isoform- and mutant-specificity for use in immunoblot analyses.


Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Mutação , Proteínas ras/genética , Proteínas ras/metabolismo , Animais , Western Blotting , Células Cultivadas , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Camundongos , Isoformas de Proteínas , Proteínas ras/imunologia
8.
Methods Mol Biol ; 2274: 207-215, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050474

RESUMO

Various fluorescent probes for the detection of intracellular reactive oxidative species (ROS) have been developed because ROS levels are closely associated with cellular states. Here, we describe a method for detection of intracellular ROS in living cells using the fluorescent probe, hydroxyphenyl fluorescein (HPF), which detects hydroxyl radicals and peroxynitrite. NIH3T3 cells and p53 knockout (p53-/-) mouse embryonic fibroblasts (MEFs) were transformed by expressing oncogenic RAS using a retrovirus system. The cells were treated with HPF at 37 °C for 30 min, and subsequently, images were acquired using a confocal fluorescence microscope at an excitation wavelength of 488 nm after washing with PBS.


Assuntos
Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Fluoresceínas/química , Fluorescência , Corantes Fluorescentes/química , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Animais , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Radical Hidroxila/análise , Camundongos , Camundongos Knockout , Células NIH 3T3 , Oxirredução , Estresse Oxidativo , Ácido Peroxinitroso/análise , Espectrometria de Fluorescência
9.
Methods Mol Biol ; 2262: 335-346, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977488

RESUMO

RAS proteins are key players in multiple cellular processes. To study the role of RAS proteins individually or in combination, we have developed MEFs that can be rendered RASless, i.e., devoid of all endogenous RAS isoforms. These cells have significantly contributed to our understanding of the requirements for RAS functions in cell proliferation as well as their implications in diverse cellular processes. Here, we describe methods using RASless MEFs to study RAS-dependent cellular activities with special emphasis on proliferation. We provide the details to identify inducers of RAS-independent proliferation in colony assays. We recommend following these stringent guidelines to avoid false-positive results. Moreover, this protocol can be adapted to generate RASless MEFs ectopically expressing RAS variants to interrogate their function in the absence of endogenous RAS isoforms or to perform experiments in the absence of RAS. Finally, we also describe protocols to generate and use RASless MEFs for cell cycle analyses using the FUCCI cell cycle indicator.


Assuntos
Ciclo Celular , Proliferação de Células , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Mutação , Proteínas ras/administração & dosagem , Proteínas ras/metabolismo , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Camundongos , Camundongos Knockout , Proteínas ras/genética
10.
Methods Mol Biol ; 2262: 397-409, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977491

RESUMO

Costello syndrome (CS), characterized by a developmental delay and a failure to thrive, is also associated with an impaired lipid and energy metabolism. White adipose tissue is a central sensor of whole-body energy homeostasis, and HRAS hyperactivation may affect adipocyte differentiation and mature adipocyte homeostasis. An extremely useful tool for delineating in vitro intrinsic cellular signaling leading to metabolic alterations during adipogenesis is mouse embryonic fibroblasts, known to differentiate into adipocytes in response to adipogenesis-stimulating factors. Here, we describe in detail the isolation and maintenance of CS HRAS G12V mouse embryonic fibroblasts, their differentiation into adipocytes, and an assessment of adipocyte differentiation.


Assuntos
Adipócitos/patologia , Diferenciação Celular , Síndrome de Costello/patologia , Modelos Animais de Doenças , Fibroblastos/patologia , Mutação , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Adipócitos/metabolismo , Adipogenia , Animais , Síndrome de Costello/genética , Síndrome de Costello/metabolismo , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Feminino , Fibroblastos/metabolismo , Homeostase , Técnicas In Vitro , Masculino , Camundongos , Camundongos Knockout
11.
Methods Mol Biol ; 2262: 361-395, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977490

RESUMO

Animal models have become in recent years a crucial tool to understand the physiological and pathological roles of many cellular proteins. They allow analysis of the functional consequences of [1] complete or partial (time- or organ-limited) removal of specific proteins (knockout animals), [2] the exchange of a wild-type allele for a mutant or truncated version found in human illnesses (knock-in), or [3] the effect of overexpression of a given protein in the whole body or in specific organs (transgenic mice). In this regard, the study of phenotypes in Ras GEF animal models has allowed researchers to find specific functions for otherwise very similar proteins, uncovering their role in physiological contexts such as memory formation, lymphopoiesis, photoreception, or body homeostasis. In addition, mouse models have been used to unveil the functional role of Ras GEFs under pathological conditions, including Noonan syndrome, skin tumorigenesis, inflammatory diseases, diabetes, or ischemia among others. In the following sections, we will describe the methodological approaches employed for Ras GEF animal model analyses, as well as the main discoveries made.


Assuntos
Modelos Animais de Doenças , Marcação de Genes/métodos , Homeostase , Isquemia/patologia , Neoplasias/patologia , Fatores ras de Troca de Nucleotídeo Guanina/metabolismo , Animais , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Isquemia/genética , Isquemia/metabolismo , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Neurogênese , Fatores ras de Troca de Nucleotídeo Guanina/antagonistas & inibidores , Fatores ras de Troca de Nucleotídeo Guanina/genética
12.
Nat Genet ; 53(4): 539-550, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33821003

RESUMO

Parental epigenomes are established during gametogenesis. While they are largely reset after fertilization, broad domains of Polycomb repressive complex 2 (PRC2)-mediated formation of lysine 27-trimethylated histone H3 (H3K27me3) are inherited from oocytes in mice. How maternal H3K27me3 is established and inherited by embryos remains elusive. Here, we show that PRC1-mediated formation of lysine 119-monoubiquititinated histone H2A (H2AK119ub1) confers maternally heritable H3K27me3. Temporal profiling of H2AK119ub1 dynamics revealed that atypically broad H2AK119ub1 domains are established, along with H3K27me3, during oocyte growth. From the two-cell stage, H2AK119ub1 is progressively deposited at typical Polycomb targets and precedes H3K27me3. Reduction of H2AK119ub1 by depletion of Polycomb group ring finger 1 (PCGF1) and PCGF6-essential components of variant PRC1 (vPRC1)-leads to H3K27me3 loss at a subset of genes in oocytes. The gene-selective H3K27me3 deficiency is irreversibly inherited by embryos, causing loss of maternal H3K27me3-dependent imprinting, embryonic sublethality and placental enlargement at term. Collectively, our study unveils preceding dynamics of H2AK119ub1 over H3K27me3 at the maternal-to-zygotic transition, and identifies PCGF1/6-vPRC1 as an essential player in maternal epigenetic inheritance.


Assuntos
Embrião de Mamíferos/metabolismo , Epigênese Genética , Histonas/genética , Herança Materna , Complexo Repressor Polycomb 1/genética , Animais , Embrião de Mamíferos/citologia , Epigenoma , Feminino , Fertilização/genética , Histonas/metabolismo , Lisina/metabolismo , Masculino , Camundongos , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Gravidez , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Espermatozoides/citologia , Espermatozoides/metabolismo , Ubiquitinação , Zigoto/citologia , Zigoto/crescimento & desenvolvimento , Zigoto/metabolismo
13.
Int J Mol Sci ; 22(8)2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33917060

RESUMO

Basic and translational research in reproductive medicine can provide new insights with the application of scanning probe microscopies, such as atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM). These microscopies, which provide images with spatial resolution well beyond the optical resolution limit, enable users to achieve detailed descriptions of cell topography, inner cellular structure organization, and arrangements of single or cluster membrane proteins. A peculiar characteristic of AFM operating in force spectroscopy mode is its inherent ability to measure the interaction forces between single proteins or cells, and to quantify the mechanical properties (i.e., elasticity, viscoelasticity, and viscosity) of cells and tissues. The knowledge of the cell ultrastructure, the macromolecule organization, the protein dynamics, the investigation of biological interaction forces, and the quantification of biomechanical features can be essential clues for identifying the molecular mechanisms that govern responses in living cells. This review highlights the main findings achieved by the use of AFM and SNOM in assisted reproductive research, such as the description of gamete morphology; the quantification of mechanical properties of gametes; the role of forces in embryo development; the significance of investigating single-molecule interaction forces; the characterization of disorders of the reproductive system; and the visualization of molecular organization. New perspectives of analysis opened up by applying these techniques and the translational impacts on reproductive medicine are discussed.


Assuntos
Microscopia de Varredura por Sonda/métodos , Medicina Reprodutiva/métodos , Animais , Fenômenos Biomecânicos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/ultraestrutura , Células Germinativas/citologia , Células Germinativas/metabolismo , Células Germinativas/ultraestrutura , Humanos , Microscopia de Força Atômica/métodos , Microscopia de Varredura por Sonda/normas , Imagem Molecular/métodos , Imagem Molecular/normas , Medicina Reprodutiva/normas , Imagem Individual de Molécula/métodos
14.
Nat Genet ; 53(5): 694-706, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33833454

RESUMO

Characterization of the progression of cellular states during human embryogenesis can provide insights into the origin of pediatric diseases. We examined the transcriptional states of neural crest- and mesoderm-derived lineages differentiating into adrenal glands, kidneys, endothelium and hematopoietic tissue between post-conception weeks 6 and 14 of human development. Our results reveal transitions connecting the intermediate mesoderm and progenitors of organ primordia, the hematopoietic system and endothelial subtypes. Unexpectedly, by using a combination of single-cell transcriptomics and lineage tracing, we found that intra-adrenal sympathoblasts at that stage are directly derived from nerve-associated Schwann cell precursors, similarly to local chromaffin cells, whereas the majority of extra-adrenal sympathoblasts arise from the migratory neural crest. In humans, this process persists during several weeks of development within the large intra-adrenal ganglia-like structures, which may also serve as reservoirs of originating cells in neuroblastoma.


Assuntos
Linhagem da Célula , Embrião de Mamíferos/metabolismo , Neuroblastoma/embriologia , Neuroblastoma/genética , Análise de Célula Única , Sistema Simpático-Suprarrenal/embriologia , Transcriptoma/genética , Animais , Células Cromafins/metabolismo , Células Cromafins/patologia , Análise por Conglomerados , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Humanos , Lactente , Camundongos , Células-Tronco Neurais/metabolismo , Neuroblastoma/patologia , Células de Schwann/metabolismo , Células de Schwann/patologia , Microambiente Tumoral
15.
Biochem Biophys Res Commun ; 553: 37-43, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33765557

RESUMO

Previously, we reported that the presence of multiple day 7 (D7) bovine embryos in the uterus induces systemic immune responses in circulating polymorphonuclear neutrophils (PMNs), but with unknown mechanism. Thus, this study aimed to investigate the direct impact of D7 bovine embryo on PMNs' immune responses in vitro and whether these PMNs can amplify and transfer embryo signals further to another PMN population. PMNs were directly stimulated by embryo culture media (ECM) or interferon tau (IFNT) (10 ng/ml) followed by evaluating mRNA expression by real-time PCR and phenotypic analysis by flow cytometry. To test whether PMNs can transfer embryo signals to a new PMN population, PMNs triggered by ECM or IFNT, were thoroughly washed and diluted to remove any media components, and again were incubated in fresh culture media for 3 h, from which culture supernatants were collected and used as PMN conditioned media (CM) to stimulate a new PMN population. Similar to ECM, IFNT directly stimulated expressions of IFNs (IFNA, IFNG), interferon-stimulated genes (ISGs; OAS1, ISG15, MX1), STAT1, TGFB and IL8, and downregulated TNFA in PMNs. Flow cytometrical analyses demonstrated that IFNT stimulated expressions of pregnancy-related phenotypic markers, CD16 and arginase-1 (ARG1), in PMNs. Most importantly, PMN CM induced ISGs and STAT1 mRNA in fresh PMNs. Since IFNT directly upregulated IFNA expression in PMNs, the impact of IFNA on PMNs' immune responses was further tested. Stimulation of PMNs with IFNA, especially at a low level (1 pg/ml), induced IFNT-like immune responses comparable to those induced by PMN CM. Together, these findings indicated that D7 bovine embryos induce direct anti-inflammatory responses with upregulation of ISGs expressions in PMNs mainly via IFNT. Additionally, PMNs can amplify and transfer embryo signals to a new PMN population in a cell-to-cell communication mechanism possibly mediated in part by IFNA. Such a novel immunological crosstalk might contribute to embryo tolerance and pregnancy establishment in cattle.


Assuntos
Embrião de Mamíferos/imunologia , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica , Interferon Tipo I/imunologia , Neutrófilos/imunologia , Proteínas da Gravidez/imunologia , Gravidez/genética , Gravidez/imunologia , Animais , Arginase/genética , Bovinos , Meios de Cultivo Condicionados/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Imunidade Inata , Técnicas In Vitro , Interferon Tipo I/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Fenótipo , Proteínas da Gravidez/farmacologia , Receptores de IgG/genética
16.
PLoS Comput Biol ; 17(3): e1008571, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33684098

RESUMO

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


Assuntos
Desenvolvimento Embrionário/genética , Camadas Germinativas , Modelos Biológicos , Transcriptoma/genética , Animais , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/fisiologia , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Camadas Germinativas/fisiologia , Camundongos , Análise de Célula Única
17.
Biochem Biophys Res Commun ; 552: 98-105, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33743353

RESUMO

SET domain-containing 2 (SETD2), the primary methyltransferase for histone 3 lysine-36 trimethylation (H3K36me3) in mammals, is associated with many hematopoietic diseases when mutated. Previous works have emphasized its role in maintaining adult hematopoietic stem cells or tumorigenesis, however, whether and how SETD2 regulates erythropoiesis during embryonic development is relatively unexplored. In this study, using a conditional SETD2 knockout (KO) mouse model, we reveal that SETD2 plays an essential role in fetal erythropoiesis. Loss of Setd2 in hematopoietic cells ablates H3K36me3, and leads to anemia with a significant decrease in erythroid cells in the peripheral blood at E18.5. This is due to impaired erythroblast differentiation in both spleen and liver. We also find increased proportions of nucleated erythrocytes in the blood of Setd2 KO embryos. Lastly, we ascribe embryonic erythropoiesis-related genes Vegfc, Vegfr3, and Prox1, as likely downstream targets of SETD2 regulation. Our study reveals a critical role of SETD2 in fetal erythropoiesis that precedes adult hematopoiesis, and provide unique insights into the defects in erythroid lineages, such as anemia.


Assuntos
Diferenciação Celular/genética , Eritroblastos/metabolismo , Eritropoese/genética , Feto/metabolismo , Histona-Lisina N-Metiltransferase/genética , Animais , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Eritroblastos/citologia , Eritrócitos/citologia , Eritrócitos/metabolismo , Feto/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo
18.
Dokl Biochem Biophys ; 496(1): 48-51, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33689075

RESUMO

Somatic Cell Nuclear Transfer (SCNT) technique was used to produce the first viable cloned cattle offspring in Russia. Whole-genome SNP genotyping confirmed that the cloned calf was identical to the fibroblast cell line that was used for SCNT. CRISPR/Cas9 approach was subsequently used to knock out genes for beta-lactoglobulin gene (PAEP) and the beta-lactoglobulin-like protein gene (LOC100848610) in the fibroblast cells. Gene editing (GE) efficiency was 4.4% for each of these genes. We successfully obtained single-cell-derived fibroblast colonies containing PAEP and LOC100848610 knockouts, which will be used to produce beta-lactoglobulin-deficient cattle.


Assuntos
Animais Geneticamente Modificados/genética , Sistemas CRISPR-Cas , Bovinos/genética , Clonagem de Organismos/métodos , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Edição de Genes/métodos , Animais , Animais Geneticamente Modificados/embriologia , Bovinos/embriologia , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Técnicas de Inativação de Genes/métodos , Técnicas de Transferência Nuclear
19.
FASEB J ; 35(4): e21545, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33729606

RESUMO

The neural tube is the first critically important structure that develops in the embryo. It serves as the primordium of the central nervous system; therefore, the proper formation of the neural tube is essential to the developing organism. Neural tube defects (NTDs) are severe congenital defects caused by failed neural tube closure during early embryogenesis. The pathogenesis of NTDs is complicated and still not fully understood even after decades of research. While it is an ethically impossible proposition to investigate the in vivo formation process of the neural tube in human embryos, a newly developed technology involving the creation of neural tube organoids serves as an excellent model system with which to study human neural tube formation and the occurrence of NTDs. Herein we reviewed the recent literature on the process of neural tube formation, the progress of NTDs investigations, and particularly the exciting potential to use neural tube organoids to model the cellular and molecular mechanisms underlying the etiology of NTDs.


Assuntos
Sistema Nervoso Central/crescimento & desenvolvimento , Embrião de Mamíferos/metabolismo , Defeitos do Tubo Neural/etiologia , Tubo Neural/metabolismo , Organoides/patologia , Animais , Modelos Animais de Doenças , Embrião de Mamíferos/patologia , Humanos , Tubo Neural/patologia , Defeitos do Tubo Neural/metabolismo , Organoides/crescimento & desenvolvimento
20.
Int J Mol Sci ; 22(4)2021 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33673337

RESUMO

Notch signaling is critical for controlling a variety of cell fate decisions during metazoan development and homeostasis. This unique, highly conserved signaling pathway relies on cell-to-cell contact, which triggers the proteolytic release of the cytoplasmic domain of the membrane-anchored transcription factor Notch from the membrane. A disintegrin and metalloproteinase (ADAM) proteins are crucial for Notch activation by processing its S2 site. While ADAM10 cleaves Notch1 under physiological, ligand-dependent conditions, ADAM17 mainly cleaves Notch1 under ligand-independent conditions. However, the mechanism(s) that regulate the distinct contributions of these ADAMs in Notch processing remain unclear. Using cell-based assays in mouse embryonic fibroblasts (mEFs) lacking ADAM10 and/or ADAM17, we aimed to clarify what determines the relative contributions of ADAM10 and ADAM17 to ligand-dependent or ligand-independent Notch processing. We found that EDTA-stimulated ADAM17-dependent Notch1 processing is rapid and requires the ADAM17-regulators iRhom1 and iRhom2, whereas the Delta-like 4-induced ligand-dependent Notch1 processing is slower and requires ADAM10. The selectivity of ADAM17 for EDTA-induced Notch1 processing can most likely be explained by a preference for ADAM17 over ADAM10 for the Notch1 cleavage site and by the stronger inhibition of ADAM10 by EDTA. The physiological ADAM10-dependent processing of Notch1 cannot be compensated for by ADAM17 in Adam10-/- mEFs, or by other ADAMs shown here to be able to cleave the Notch1 cleavage site, such as ADAMs9, 12, and 19. Collectively, these results provide new insights into the mechanisms underlying the substrate selectivity of ADAM10 and ADAM17 towards Notch1.


Assuntos
Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Proteínas de Membrana/metabolismo , Proteólise , Receptor Notch1/metabolismo , Proteína ADAM10/genética , Proteína ADAM17/genética , Secretases da Proteína Precursora do Amiloide/genética , Animais , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Receptor Notch1/genética
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