The translational repressor 4E-BP mediates hypoxia-induced defects in myotome cells.

International audienceCell growth, proliferation, differentiation and survival are influenced by the availability of oxygen. The effect of hypoxia on embryonic cells and the underlying molecular mechanisms to maintain cellular viability are still poorly understood. In this study, we show that hypoxia during Xenopus embryogenesis rapidly leads to a significant developmental delay and to cell apoptosis after prolonged exposure. We provide strong evidence that hypoxia does not affect somitogenesis but affects the number of mitotic cells and muscle-specific protein accumulation in somites, without interfering with the expression of MyoD and MRF4 transcription factors. We also demonstrate that hypoxia reversibly decreases Akt phosphorylation and increases the total amount of the translational repressor 4E-BP, in combination with an increase of the 4E-BP associated with eIF4E. Interestingly, the inhibition of PI3-kinase or mTOR, with LY29002 or rapamycin, respectively, triggers the 4E-BP accumulation in Xenopus embryos. Finally, the overexpression of the non-phosphorylatable 4E-BP protein induces, similar to hypoxia, a decrease in mitotic cells and a decrease in muscle-specific protein accumulation in somites. Taken together, our studies suggest that 4E-BP plays a central role under hypoxia in promoting the cap-independent translation at the expense of cap-dependent translation and triggers specific defects in muscle development

Pax8 and Pax2 are specifically required at different steps of Xenopus pronephros development

AbstractThe respective role of Pax2 and Pax8 in early kidney development in vertebrates is poorly understood. In this report, we have studied the roles of Pax8 and Pax2 in Xenopus pronephros development using a loss-of-function approach. Our results highlight a differential requirement of these two transcription factors for proper pronephros formation. Pax8 is necessary for the earliest steps of pronephric development and its depletion leads to a complete absence of pronephric tubule. Pax2 is required after the establishment of the tubule pronephric anlage, for the expression of several terminal differentiation markers of the pronephric tubule. Neither Pax2 nor Pax8 is essential to glomus development. We further show that Pax8 controls hnf1b, but not lhx1 and Osr2, expression in the kidney field as soon as the mid-neurula stage. Pax8 is also required for cell proliferation of pronephric precursors in the kidney field. It may exert its action through the wnt/beta-catenin pathway since activation of this pathway can rescue MoPax8 induced proliferation defect and Pax8 regulates expression of the wnt pathway components, dvl1 and sfrp3. Finally, we observed that loss of pronephros in Pax8 morphants correlates with an expanded vascular/blood gene expression domain indicating that Pax8 function is important to delimit the blood/endothelial genes expression domain in the anterior part of the dorso-lateral plate

TOPAZ1, a Novel Germ Cell-Specific Expressed Gene Conserved during Evolution across Vertebrates

BACKGROUND: We had previously reported that the Suppression Subtractive Hybridization (SSH) approach was relevant for the isolation of new mammalian genes involved in oogenesis and early follicle development. Some of these transcripts might be potential new oocyte and granulosa cell markers. We have now characterized one of them, named TOPAZ1 for the Testis and Ovary-specific PAZ domain gene. PRINCIPAL FINDINGS: Sheep and mouse TOPAZ1 mRNA have 4,803 bp and 4,962 bp open reading frames (20 exons), respectively, and encode putative TOPAZ1 proteins containing 1,600 and 1653 amino acids. They possess PAZ and CCCH domains. In sheep, TOPAZ1 mRNA is preferentially expressed in females during fetal life with a peak during prophase I of meiosis, and in males during adulthood. In the mouse, Topaz1 is a germ cell-specific gene. TOPAZ1 protein is highly conserved in vertebrates and specifically expressed in mouse and sheep gonads. It is localized in the cytoplasm of germ cells from the sheep fetal ovary and mouse adult testis. CONCLUSIONS: We have identified a novel PAZ-domain protein that is abundantly expressed in the gonads during germ cell meiosis. The expression pattern of TOPAZ1, and its high degree of conservation, suggests that it may play an important role in germ cell development. Further characterization of TOPAZ1 may elucidate the mechanisms involved in gametogenesis, and particularly in the RNA silencing process in the germ lin

Facteurs de traduction et mécanismes de surveillance du cycle cellulaire

Cell division is a highly regulated process and when a problem occurs, the cell cycle checkpoints are activated. When cell cycle checkpoints are defective, pathological disease, as cancer, can occur. The implication of translation factors during checkpoints activation was studied in sea urchin embryo model. The work realized during this PhD demonstrated a functional DNA damage checkpoint during the first cell division of sea urchin embryo. The eIF2α phosphorylation was shown to be implicated in translational activation after fertilization and in translation inhibition after MMS treatment, a DNA alkylating molecule. This study shows an expression of functional 4E-BP protein, a translational inhibitor, after induction of DNA damages by radiomimetic drug (bleomycin), hypoxic stress or heavy metal (ChromeIII) treatment in sea urchin embryo. We demonstrated that, after MMS treatment, which doesn’t induced 4E-BP expression, the eIF4G protein was modified, degraded or cleaved as a function of drug dose. Our work support interest and knowledge on translational factors implication when checkpoint was mobilisated after DNA damages or cellular stress in sea urchin embryo. These results are a starting point to study new regulations of translational factors eIF4E, eIF4G and 4E-BP when cell is directed toward survival or cell death pathways.La division cellulaire est un processus physiologique extrêmement régulé et la moindre anomalie entraîne la mobilisation de points de surveillance. Lorsque qu’un problème survient dans le contrôle de la division, cela peut entraîner l’apparition de pathologie grave comme le cancer. L’étude de l’implication des facteurs de traduction lors de la mobilisation des points de surveillance a été réalisée en profitant des atouts du modèle de l’embryon d’oursin. Les travaux réalisés au cours de cette thèse, ont montré que le point de surveillance de l'ADN endommagé (arrêt du cycle-réparation-apoptose) était fonctionnel dès le premier cycle suivant la fécondation dans l'embryon d'oursin. Nous avons également mis en évidence que dans les embryons d'oursin, la phosphorylation de la protéine eIF2α est impliquée dans l'augmentation de synthèse protéique induite par la fécondation et dans l’inhibition de synthèse protéique induite par un traitement avec une molécule alkylant l’ADN, le MMS. Nos études montrent que la protéine 4E-BP, un inhibiteur de la traduction, est surexprimée et fonctionnelle en réponse à la mobilisation du point de surveillance de l'ADN endommagé par une molécule radiomimétique (bléomycine), à un stress hypoxique ou lors de l'exposition à un métal lourd (ChromeIII) dans l’embryon d’oursin. Enfin, nous démontrons que lors du traitement des embryons par le MMS, qui n'induit pas de surexpression de 4E-BP, la protéine eIF4G est modifiée, dégradée ou clivée selon la dose de drogue utilisée. Nos études renforcent l’intérêt et les connaissances sur l’implication des facteurs de traduction lors de la mobilisation des points de surveillance du cycle cellulaire dans l’embryon d’oursin mis en situation de stress et/ou d'endommagement de l'ADN. Ainsi les résultats obtenus permettent de poser les bases de nouvelles régulations des facteurs de traduction eIF4E et eIF4G et de leur inhibiteur 4E-BP permettant de contrôler la synthèse protéique lorsque la cellule va s'engager dans la voie de survie ou de mort cellulaire

Chromium(III) triggers the DNA-damaged checkpoint of the cell cycle and induces a functional increase of 4E-BP.

International audienceUsing sea urchin early embryos as a pertinent model, chromium(III) provoked cell cycle arrest and induced apoptosis. The molecular machinery of translation initiation was investigated. Chromium provoked a time- and dose-dependent increase in the level of 4E-BP protein, the natural regulator of the cap-dependent initiation factor 4E (eIF4E). The 4E-BP increase was the result of 4E-BP stabilization and appeared functional for physiological eIF4E binding, removal of eIF4E from the initiation factor eIF4G, and almost full inhibition of cap-dependent translation in vivo. The protein 4E-BP may be involved in the biological pathway of apoptosis associated with the activation of the DNA-damaged checkpoint of the cell cycle

Characterization of potential TRPP2 regulating proteins in early Xenopus embryos

International audienceTransient receptor potential cation channel‐2 (TRPP2) is a nonspecific Ca2+‐dependent cation channel with versatile functions including control of extracellular calcium entry at the plasma membrane, release of intracellular calcium ([Ca2+]i) from internal stores of endoplasmic reticulum, and calcium‐dependent mechanosensation in the primary cilium. In early Xenopus embryos, TRPP2 is expressed in cilia of the gastrocoel roof plate (GRP) involved in the establishment of left‐right asymmetry, and in nonciliated kidney field (KF) cells, where it plays a central role in early specification of nephron tubule cells dependent on [Ca2+]i signaling. Identification of proteins binding to TRPP2 in embryo cells can provide interesting clues about the mechanisms involved in its regulation during these various processes. Using mass spectrometry, we have therefore characterized proteins from late gastrula/early neurula stage embryos coimmunoprecipitating with TRPP2. Binding of three of these proteins, golgin A2, protein kinase‐D1, and disheveled‐2 has been confirmed by immunoblotting analysis of TRPP2‐coprecipitated proteins. Expression analysis of the genes, respectively, encoding these proteins, golga2, prkd1, and dvl2 indicates that they are likely to play a role in these two regions. Golga2 and prkd1 are expressed at later stage in the developing pronephric tubule where golgin A2 and protein kinase‐D1 might also interact with TRPP2. Colocalization experiments using exogenously expressed fluorescent versions of TRPP2 and dvl2 in GRP and KF reveal that these two proteins are generally not coexpressed, and only colocalized in discrete region of cells. This was observed in KF cells, but does not appear to occur in the apical ciliated region of GRP cells

L'embryon d'oursin, le point de surveillance de l'ADN endommagé de la division cellulaire et les mécanismes à l'origine de la cancérisation

La division cellulaire est essentielle pour l'hérédité, le maintien et l'évolution du monde vivant. Lors d'une lésion de l'ADN au cours de la division cellulaire, les "points de surveillance (= checkpoints) de l'ADN endommagé" exécutent les fonctions d'arrêt du cycle, de la réparation de l'ADN et de l'orientation vers la mort cellulaire par apoptose lorsqu'une réparation est impossible. À propos de l'origine des cancers, deux concepts majeurs se renforcent de jour en jour : les cancers s'initient par un dysfonctionnement des points de surveillance de l'ADN endommagé et les cancers naissent de la transformation de cellules souches "normales" en cellules souches "cancéreuses". Ce dernier concept modifie la définition même des cancers puisqu'il est démontré qu'une cellule souche "cancéreuse" suffit pour générer la tumeur, bien avant les signes cliniques de la maladie. Le développement précoce de l'oursin représente un excellent modèle expérimental pour appréhender l'analyse du fonctionnement des points de surveillance du cycle de division, car il présente l'ensemble des éléments de régulation, comme le montrent l'analyse du génome complet et l'existence d'un point de surveillance de l'ADN endommagé tout à fait opérationnel. Le modèle biologique du développement précoce de l'oursin, dont l'oeuf constitue une cellule souche par excellence, permet d'aborder l'étude de l'origine de la cancérisation. Dans le domaine de la toxicologie et de l'implication de nouvelles molécules en matière de santé, le modèle peut être utilisé pour prédire le risque de cancer dû à des molécules ou des combinaisons de molécules, bien avant le moindre signe clinique de la maladie. C'est ainsi que le risque cancérigène d'un herbicide d'usage intensif dans le monde, le Roundup (Marque déposée par Monsanto Company, Saint-Louis, USA.), dont le glyphosate est l'élément actif, a pu être démontré. Le modèle expérimental de l'embryon d'oursin permet ainsi de progresser considérablement dans la prévention des cancers par la connaissance des produits à risques et d'envisager de nouvelles formes de diagnostic précoce de la maladie par la mise en évidence de marqueurs moléculaires. Prévention et diagnostic précoce sont deux des éléments décisifs de la lutte contre le cancer

[Sea urchin embryo, DNA-damaged cell cycle checkpoint and the mechanisms initiating cancer development]

International audienceCell division is an essential process for heredity, maintenance and evolution of the whole living kingdom. Sea urchin early development represents an excellent experimental model for the analysis of cell cycle checkpoint mechanisms since embryonic cells contain a functional DNA-damage checkpoint and since the whole sea urchin genome is sequenced. The DNA-damaged checkpoint is responsible for an arrest in the cell cycle when DNA is damaged or incorrectly replicated, for activation of the DNA repair mechanism, and for commitment to cell death by apoptosis in the case of failure to repair. New insights in cancer biology lead to two fundamental concepts about the very first origin of cancerogenesis. Cancers result from dysfunction of DNA-damaged checkpoints and cancers appear as a result of normal stem cell (NCS) transformation into a cancer stem cell (CSC). The second aspect suggests a new definition of "cancer", since CSC can be detected well before any clinical evidence. Since early development starts from the zygote, which is a primary stem cell, sea urchin early development allows analysis of the early steps of the cancerization process. Although sea urchins do not develop cancers, the model is alternative and complementary to stem cells which are not easy to isolate, do not divide in a short time and do not divide synchronously. In the field of toxicology and incidence on human health, the sea urchin experimental model allows assessment of cancer risk from single or combined molecules long before any epidemiologic evidence is available. Sea urchin embryos were used to test the worldwide used pesticide Roundup that contains glyphosate as the active herbicide agent; it was shown to activate the DNA-damage checkpoint of the first cell cycle of development. The model therefore allows considerable increase in risk evaluation of new products in the field of cancer and offers a tool for the discovery of molecular markers for early diagnostic in cancer biology. Prevention and early diagnosis are two decisive elements of human cancer therapy