Playing pools

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Glial regulation of GnRH neurones

International audienc

Mise en évidence d’une transition endothéliale/mésenchymateuse dans les lésions vasculaires pulmonaires d’hypertension artérielle pulmonaire par microscopie corrélative

National audienceL'hypertension artérielle pulmonaire résulte de l'obturation des artères pulmonaires de diamètre inférieur à 500 μm. Par microscopie corrélative, nous avons pu démontrer que cette obturation était la conséquence d'une trans-différenciation des cellules endothéliales en cellules mésenchymateuses

Depletion of double homeobox proteins in bovine zygotes abolishes blastocyst formation

International audienceIt remains unclear which factors—maternal or embryonic—are responsible for reprogramming the mammalian epigenome following fertilisation. Recently, overexpression of double homeobox (DUX) transcription factors—a family of proteins conserved among placental mammals—was found to induce a totipotent-like state in human and murine stem cells. In humans and mice, DUX transcription is activated in zygotes, suggesting it could be an early regulator of embryonic genome activation (EGA). Moreover, DUX4 has been reported to interact with histone acetyltransferases (HATs) in human cells, suggesting it could also be involved in epigenetic reprogramming. However, it is unclear whether DUX proteins are essential for development, as knockout studies in mice have found variable effects on EGA and blastocyst formation. Moreover, the function of DUX in embryos has yet to be established in any other mammal, despite recent reports that mice may not be the most appropriate model for human development. Here, we evaluate the function of DUX during the first divisions of the developing bovine embryo. Public annotations of DUX4 in cattle were largely based on predictions from the analogous human locus, so bovine DUX4 transcripts were manually reconstructed from published short-read RNA-seq data. The predicted bovine DUX4 protein resembled human DUX4 (E-value 2e-42), including homology at the HAT-binding domain. Using qPCR and immunofluorescence, we find that DUX4 is absent in the oocyte but present in zygotes by 22 h post-fertilisation (hpf), similar to what has been described in humans. Notably, this is well before minor EGA in both species, suggesting that DUX4 may be the first activated gene in the developing embryo. Conversely, the only other DUX family member present in the bovine genome, DUXA, is not appreciably expressed until the 8-cell stage (major EGA). To determine the function of DUX proteins in bovine development, we depleted zygotes for either DUX4 or DUXA through electroporation with siRNA at 10–14 hpf. The knock-down was verified by RT-qPCR and immunofluorescence. We find that DUX4 and DUXA depletion significantly reduced blastocyst formation from 44% (56/128) for controls to 2.5% (3/122) and 5.5% (7/126), respectively. Moreover, DUX4 depletion causes downregulation of EGA-associated genes, suggesting that DUX4 is a major regulator of EGA, as in the mouse. Considering the interaction of DUX4 with HATs in human cells, it is possible that DUX4 activates target gene expression through histone acetylation. Using CUT&Tag, we are currently investigating the effect of DUX4 depletion on genome-wide histone acetylation. Overall, these data indicate that zygotic expression of DUX is conserved among placental mammals, and that these factors serve as key regulators of EGA

ETUDE DES REMANIEMENTS ÉPIGÉNÉTIQUES AU MOMENT DE L'EGA CHEZ LE BOVIN À L'AIDE D'APPROCHES CORRÉLATIVES EN MICROSCOPIE OPTIQUE

International audienceLors du développement embryonnaire précoce, les génomes parentaux provenant respectivement du spermatozoïde et de l'ovocyte, sont fortement remaniés. Ces remaniements permettent une reprogrammation épigénétique de la chromatine et sont concomitants avec la mise en route de la transcription de l'embryon. Le projet vise à étudier l'impact de l'utilisation de semences de taureau ayant des profils de méthylation différents et un profil de sncRNA contrastés, sur cette reprogrammation épigénétique autour de l'activation majeure du génome embryonnaire (EGA) i.e. au stade 8-cellules. Pour cela nous avons privilégié la détection de modification post traductionnelle d'histone (H3K4me3 et H3K9me3) par immunofluorescence sur embryon in toto des stades 2-cellules à Morula. Nous avons à la fois utilisé un microscope confocal pour déterminer la localisation en 3D dans le noyau du signal de fluorescence et sur les mêmes embryons, un microscope photonique équipé d'un module Apotome pour estimer la quantité de fluorescence dans les noyaux. Ces deux approches complémentaires nous ont permis d'acquérir des données quantitatives et qualitatives pour la comparaison de la distribution de ces marques d'épigénétiques entre des taureaux ayant des profils de méthylation et de sncRNA spermatique contrastés

Transient Changes of Metabolism at the Pronuclear Stage in Mice Influences Skeletal Muscle Phenotype in Adulthood

International audienceSkeletal muscle has a remarkable plasticity, and its phenotype is strongly influenced by hormones, transcription factors, and physical activity. However, whether skeletal phenotype can be oriented or not during early embryonic stages has never been investigated. Here, we report that pyruvate as the only source of carbohydrate in the culture medium of mouse one cell stage embryo influenced the establishment of the muscular phenotype in adulthood. We found that pyruvate alone induced changes in the contractile phenotype of the skeletal muscle in a sexually dependent manner. For male mice, a switch to a more glycolytic phenotype was recorded, whereas, in females, the pyruvate induced a switch to a more oxidative phenotype. In addition, the influence of pyruvate on the contractile phenotypes was confirmed in two mouse models of muscle hypertrophy: the well-known myostatin deficient mouse (Mstn−/−) and a mouse carrying a specific deletion of p43, a mitochondrial triiodothyronine receptor. Finally, to understand the link between these adult phenotypes and the early embryonic period, we assessed the levels of two histone H3 post-translational modifications in presence of pyruvate alone just after the wave of chromatin reprogramming specific of the first cell cycle. We showed that H3K4 acetylation level was decreased in Mstn−/− 2-cell embryos, whereas no difference was found for H3K27 trimethylation level, whatever the genotype. These findings demonstrate for the first time that changes in the access of energy substrate during the very first embryonic stage can induce a precocious orientation of skeletal muscle phenotype in adulthood

Transcription of rRNA in early mouse embryos promotes chromatin reorganization and expression of major satellite repeats

International audienceDuring the first cell cycles of early development, the chromatin of the embryo is highly reprogrammed while the embryonic genome starts its own transcription. The spatial organization of the genome is an important process that contributes to regulating gene transcription in time and space. It has, however, been poorly studied in the context of early embryos. To study the cause-and-effect link between transcription and spatial organization in embryos, we focused on ribosomal genes, which are silent initially but start to be transcribed in 2-cell mouse embryos. We demonstrated that ribosomal sequences and early unprocessed rRNAs are spatially organized in a very particular manner between 2-cell and 16-cell stage. By using drugs that interfere with ribosomal DNA transcription, we showed that this organization – which is totally different in somatic cells – depends on an active transcription of ribosomal genes and induces a unique chromatin environment that favors transcription of major satellite sequences once the 4-cell stage has been reached

A new culture model of rabbit trophoblastic cells to explore cell function and transplacental transfers

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