RESUMO
Development of the Xenopus pronephros relies on renal precursors grouped at neurula stage into a specific region of dorso-lateral mesoderm called the kidney field. Formation of the kidney field at early neurula stage is dependent on retinoic (RA) signaling acting upstream of renal master transcriptional regulators such as pax8 or lhx1. Although lhx1 might be a direct target of RA-mediated transcriptional activation in the kidney field, how RA controls the emergence of the kidney field remains poorly understood. In order to better understand RA control of renal specification of the kidney field, we have performed a transcriptomic profiling of genes affected by RA disruption in lateral mesoderm explants isolated prior to the emergence of the kidney field and cultured at different time points until early neurula stage. Besides genes directly involved in pronephric development (pax8, lhx1, osr2, mecom), hox (hoxa1, a3, b3, b4, c5 and d1) and the hox co-factor meis3 appear as a prominent group of genes encoding transcription factors (TFs) downstream of RA. Supporting the idea of a role of meis3 in the kidney field, we have observed that meis3 depletion results in a severe inhibition of pax8 expression in the kidney field. Meis3 depletion only marginally affects expression of lhx1 and aldh1a2 suggesting that meis3 principally acts upstream of pax8. Further arguing for a role of meis3 and hox in the control of pax8, expression of a combination of meis3, hoxb4 and pbx1 in animal caps induces pax8 expression, but not that of lhx1. The same combination of TFs is also able to transactivate a previously identified pax8 enhancer, Pax8-CNS1. Mutagenesis of potential PBX-Hox binding motifs present in Pax8-CNS1 further allows to identify two of them that are necessary for transactivation. Finally, we have tested deletions of regulatory sequences in reporter assays with a previously characterized transgene encompassing 36.5 âkb of the X. tropicalis pax8 gene that allows expression of a truncated pax8-GFP fusion protein recapitulating endogenous pax8 expression. This transgene includes three conserved pax8 enhancers, Pax8-CNS1, Pax8-CNS2 and Pax8-CNS3. Deletion of Pax8-CNS1 alone does not affect reporter expression, but deletion of a 3.5 âkb region encompassing Pax8-CNS1 and Pax8-CNS2 results in a severe inhibition of reporter expression both in the otic placode and kidney field domains.
Assuntos
Pronefro , Tretinoína , Animais , Xenopus laevis/genética , Xenopus laevis/metabolismo , Tretinoína/farmacologia , Tretinoína/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Pronefro/metabolismo , Rim/metabolismo , Família Aldeído Desidrogenase 1 , Retinal Desidrogenase/metabolismoRESUMO
Transient 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.
Assuntos
Proteínas Desgrenhadas/genética , Canais de Cátion TRPP/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética , Animais , Cálcio/metabolismo , Cílios/genética , Retículo Endoplasmático/genética , Células Epiteliais/metabolismo , Gástrula/embriologia , Gástrula/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Rim/embriologia , Rim/metabolismo , Transdução de Sinais/genética , Xenopus laevis/embriologiaRESUMO
In Xenopus laevis embryos, kidney field specification is dependent on retinoic acid (RA) and coincides with a dramatic increase of Ca(2+) transients, but the role of Ca(2+) signaling in the kidney field is unknown. Here, we identify TRPP2, a member of the transient receptor potential (TRP) superfamily of channel proteins encoded by the pkd2 gene, as a central component of Ca(2+) signaling in the kidney field. TRPP2 is strongly expressed at the plasma membrane where it might regulate extracellular Ca(2+) entry. Knockdown of pkd2 in the kidney field results in the downregulation of pax8, but not of other kidney field genes (lhx1, osr1 and osr2). We further show that inhibition of Ca(2+) signaling with an inducible Ca(2+) chelator also causes downregulation of pax8, and that pkd2 knockdown results in a severe inhibition of Ca(2+) transients in kidney field explants. Finally, we show that disruption of RA results both in an inhibition of intracellular Ca(2+) signaling and of TRPP2 incorporation into the plasma membrane of kidney field cells. We propose that TRPP2-dependent Ca(2+) signaling is a key component of pax8 regulation in the kidney field downstream of RA-mediated non-transcriptional control of TRPP2.
Assuntos
Sinalização do Cálcio/fisiologia , Embrião não Mamífero/embriologia , Rim/embriologia , Fatores de Transcrição Box Pareados/metabolismo , Canais de Cátion TRPP/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Embrião não Mamífero/citologia , Rim/citologia , Fator de Transcrição PAX8 , Fatores de Transcrição Box Pareados/genética , Canais de Cátion TRPP/genética , Proteínas de Xenopus/genética , Xenopus laevisRESUMO
Dystroglycan (Dg) is a transmembrane receptor for laminin that must be expressed at the right time and place in order to be involved in notochord morphogenesis. The function of Dg was examined in Xenopus laevis embryos by knockdown of Dg and overexpression and replacement of the endogenous Dg with a mutated form of the protein. This analysis revealed that Dg is required for correct laminin assembly, for cell polarization during mediolateral intercalation and for proper differentiation of vacuoles. Using mutations in the cytoplasmic domain, we identified two sites that are involved in cell polarization and are required for mediolateral cell intercalation, and a site that is required for vacuolation. Furthermore, using a proteomic analysis, the cytoskeletal non-muscle myosin IIA has been identified for the first time as a molecular link between the Dg-cytoplasmic domain and cortical actin. The data allowed us to identify the adhesome laminin-Dg-myosin IIA as being required to maintain the cortical actin cytoskeleton network during vacuolation, which is crucial to maintain the shape of notochordal cells.
Assuntos
Distroglicanas/metabolismo , Laminina/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Notocorda/embriologia , Organogênese/fisiologia , Vacúolos/fisiologia , Xenopus laevis/embriologia , Animais , Western Blotting , Bromodesoxiuridina , Polaridade Celular/fisiologia , Técnicas de Silenciamento de Genes , Imunoprecipitação , Hibridização In Situ , Morfolinos/genética , Proteômica , Espectrometria de Massas em TandemRESUMO
The 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.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Fator de Transcrição PAX2/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Pronefro/embriologia , Via de Sinalização Wnt/fisiologia , Proteínas de Xenopus/metabolismo , Xenopus/embriologia , Animais , Bromodesoxiuridina , Primers do DNA/genética , Hibridização In Situ , Fator de Transcrição PAX8 , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , Via de Sinalização Wnt/genética , Xenopus/genéticaRESUMO
Meiotic recombination is a mandatory process for sexual reproduction. We identified a protein specifically implicated in meiotic homologous recombination that we named: meiosis specific with OB domain (MEIOB). This protein is conserved among metazoan species and contains single-strand DNA binding sites similar to those of RPA1. Our studies in vitro revealed that both recombinant and endogenous MEIOB can be retained on single-strand DNA. Those in vivo demonstrated the specific expression of Meiob in early meiotic germ cells and the co-localization of MEIOB protein with RPA on chromosome axes. MEIOB localization in Dmc1 (-/-) spermatocytes indicated that it accumulates on resected DNA. Homologous Meiob deletion in mice caused infertility in both sexes, due to a meiotic arrest at a zygotene/pachytene-like stage. DNA double strand break repair and homologous chromosome synapsis were impaired in Meiob (-/-) meiocytes. Interestingly MEIOB appeared to be dispensable for the initial loading of recombinases but was required to maintain a proper number of RAD51 and DMC1 foci beyond the zygotene stage. In light of these findings, we propose that RPA and this new single-strand DNA binding protein MEIOB, are essential to ensure the proper stabilization of recombinases which is required for successful homology search and meiotic recombination.
Assuntos
Pareamento Cromossômico/genética , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Recombinação Homóloga/genética , Meiose/genética , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Células Germinativas , Humanos , Masculino , Camundongos , Rad51 Recombinase/genética , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Espermatócitos/metabolismoRESUMO
Cell 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.
Assuntos
Hipóxia/patologia , Biossíntese de Proteínas , Proteínas Repressoras/metabolismo , Somitos/metabolismo , Somitos/patologia , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Apoptose/efeitos dos fármacos , Contagem de Células , Hipóxia Celular/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/enzimologia , Embrião não Mamífero/patologia , Fator de Iniciação 4E em Eucariotos/metabolismo , Hipóxia/metabolismo , Mitose/efeitos dos fármacos , Modelos Biológicos , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Proteínas Musculares/metabolismo , Oxigênio/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Somitos/efeitos dos fármacos , Xenopus laevis/embriologiaRESUMO
The mechanisms regulating germ line sex determination and meiosis initiation are poorly understood. Here, we provide evidence for the involvement of homeobox Msx transcription factors in foetal meiosis initiation in mammalian germ cells. Upon meiosis initiation, Msx1 and Msx2 genes are strongly expressed in the foetal ovary, possibly stimulated by soluble factors found there: bone morphogenetic proteins Bmp2 and Bmp4, and retinoic acid. Analysis of Msx1/Msx2 double mutant embryos revealed a majority of undifferentiated germ cells remaining in the ovary and, importantly, a decrease in the number of meiotic cells. In vivo, the Msx1/Msx2 double-null mutation prevented full activation of Stra8, a gene required for meiosis. In F9 cells, Msx1 can bind to Stra8 regulatory sequences and Msx1 overexpression stimulates Stra8 transcription. Collectively, our data demonstrate for the first time that some homeobox genes are required for meiosis initiation in the female germ line.
Assuntos
Proteínas de Homeodomínio/fisiologia , Fator de Transcrição MSX1/fisiologia , Meiose/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular Tumoral , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Fator de Transcrição MSX1/genética , Fator de Transcrição MSX1/metabolismo , Camundongos , Mutação , Técnicas de Cultura de Órgãos , Ovário/fisiologia , Proteínas/metabolismo , Tretinoína/metabolismo , Tretinoína/fisiologiaRESUMO
BACKGROUND INFORMATION: In Xenopus, the functional kidney of the tadpole, the pronephros, forms from the kidney field, which is specified at completion of gastrulation. Specification of the kidney field requires retinoic acid (RA) signalling during gastrulation, while fibroblast growth factor (FGF) signals inhibit should be inhibit this process. RESULTS: We have analysed the functional interactions taking place during gastrulation between RA and FGF signals in the lateral marginal zone (LMZ), that is in the environment of unspecified pronephric mesoderm precursors. Inhibition of FGF receptor (FGFR) signalling with SU5402 does not significantly affect expression of genes encoding RA metabolism enzymes and RA receptor-α in LMZ explants. Furthermore, SU5402 has no effect on the expression of hoxa1, a major RA target in the LMZ, showing that FGF is not antagonising RA in the LMZ. Disruption of RA signalling affects FGF ligand production to some extent, especially FGF8b, but the strongest effect is the down-regulation of the mitogen-activated protein kinase phosphatase-3 (MKP3)-encoding gene, mkp3. A strong up-regulation of mkp3 occurs in response to exogenous RA. This effect is reduced in a context of FGFR inhibition, suggesting that RA and FGF signals are co-operating upstream of mkp3. Mkp3 knockdown results in an inhibition of the kidney field markers pax8 and lhx1 and in a defective development of the pronephros. CONCLUSIONS: FGF is not negatively influencing pronephric specification by antagonising RA signalling. Functional interactions between RA and FGF rather take place at the level of the transcriptional regulation of mkp3, indicating that RA may antagonise FGF signalling at the level of the extracellular signal-regulated kinase (Erk) pathway. A fine tuning of Erk signalling by MKP3 is important for the proper establishment of the kidney field.
Assuntos
Rim/embriologia , Rim/enzimologia , Fosfoproteínas Fosfatases/metabolismo , Tretinoína/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Fosfatase 6 de Especificidade Dupla , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Rim/metabolismo , Masculino , Fosfoproteínas Fosfatases/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Xenopus laevis/genéticaRESUMO
Vibrio harveyi is a marine bacterial pathogen responsible for episodic abalone epidemics associated with massive mortalities in France, Japan, and Australia. The aim of this study was the understanding of a possible role of the p38 MAPK in abalone haemocyte responses towards this bacterium. First, the pathogenicity of different V. harveyi strains was compared in both immersion and injection trials, and clear differences were detected. The three strains, ORM4, 04/092, and 05/053, all isolated from moribund abalone, induced up to 80% mortalities in immersion or injection challenges (LD(50) (ORM4) = 2.5 x 10(2) CFU animal(-1)). The two strains, LMG 4044T and LMG 7890 were non-pathogenic towards abalone in immersion trials, and needed very high numbers for killing by intramuscular injections (LD(50) = 8.9 x 10(4) and 1.6 x 10(5) CFU animal(-1), respectively). To start unraveling the mechanism explaining these differences, the p38-MAPK, a keyplayer in antimicrobial immune response, was studied. The non-pathogenic strain, LMG 7890 can be eliminated by abalone haemocytes and induces haemocyte phagocytosis and high ROS production. With different concentrations of a p38-specific inhibitor, SB203580, p38 implication was shown. This inhibitor reduced phagocytosis and ROS induction leading to LMG 7890 proliferation. In the case of the pathogenic ORM4 which can not be eliminated by abalone haemocytes, no phagocytosis and ROS production was induced, and a retarded p38 activation was observed. Taken together, our results suggest that p38 MAPK modulation may be one of the ways of virulent V. harveyi to attack its host and escape abalone immune response.
Assuntos
Hemócitos/imunologia , Sistema de Sinalização das MAP Quinases , Vibrio/patogenicidade , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Gastrópodes/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Vibrio/classificação , Vibrioses/imunologia , VirulênciaRESUMO
The POU (Pit-Oct-Unc) genes encode a large transcription factor family comprising 6 classes (pou1f to pou6f ) involved in many developmental processes, such as cell commitment and differentiation. The pou3f class contains four members (pou3f1, pou3f2, pou3f3, pou3f4) characterized by expression in ectodermal tissue derivatives, such as nervous system and otic vesicle, during mammalian development. In order to obtain insights into the potential conservation of this class of transcription factors in vertebrates, we carried out a phylogenetic analysis and a comprehensive comparative study of pou3f expression in the frog Xenopus laevis. All vertebrates examined possessed members of the four pou3f subfamilies, excepting the zebrafish, which lacked a pou3f4 gene. Whole mount in situ hybridization and real-time quantitative polymerase chain reaction (RT-qPCR) analyses revealed that Xenopus pou3f genes were expressed in the forming neural tube and their expression was maintained in the brain, mostly in the dorsal part, at tailbud stages. The pou3f2, pou3f3, and pou3f4 genes were also expressed in the developing otic vesicle, and pou3f1 in some cells of the epidermis. Besides ectodermal derivatives, pou3f3 and pou3f4 were expressed in the developing kidney. Their expression started at the early tailbud stage in the pronephric anlage and partly overlapped. In the mature pronephric tubule, pou3f3 was restricted to the intermediate tubule, while pou3f4 was also expressed in the distal and connecting tubule. Together, our results highlight a significant conservation of pou3f gene expression in vertebrates and indicate that they may have distinct but also redundant functions during neural and renal development.
Assuntos
Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Rim/metabolismo , Fatores do Domínio POU/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Desenvolvimento Embrionário/fisiologia , Rim/embriologia , Organogênese/fisiologia , Fatores do Domínio POU/genética , Proteínas de Xenopus/genética , Xenopus laevis/embriologiaRESUMO
Translational regulation of gene expression in eukaryotes can rapidly and accurately control cell activity in response to stimuli or when rapidly dividing. There is increasing evidence for a key role of the elongation step in this process. Elongation factor-1 (eEF1), which is responsible for aminoacyl-tRNA transfer on the ribosome, is comprised of two entities: a G-protein named eEF1A and a nucleotide exchange factor, eEF1B. The multifunctional nature of eEF1A, as well as its oncogenic potential, is currently the subject of a number of studies. Until recently, less work has been done on eEF1B. This review describes the macromolecular complexity of eEF1B, its multiple phosphorylation sites and numerous cellular partners, which lead us to suggest an essential role for the factor in the control of gene expression, particularly during the cell cycle.
Assuntos
Fator 1 de Elongação de Peptídeos/química , Fator 1 de Elongação de Peptídeos/fisiologia , Animais , Regulação da Expressão Gênica , Humanos , Complexos Multiproteicos , Fator 1 de Elongação de Peptídeos/genética , Fosforilação , FilogeniaRESUMO
Arid5b belongs to the ARID family of transcription factors characterised by a helix-turn-helix motif- based DNA-binding domain called ARID (A-T Rich Interaction Domain). In human, alternative splicing leads to long and short isoforms (isoform1 and 2, respectively) which differ in their N-terminal part. In this study, we report the cloning and expression pattern of Xenopus laevis arid5b. We have isolated a full length cDNA that shows homology with the human arid5b isoform1. Furthermore, 5'RACE experiments revealed the presence of a shorter isoform equivalent to the human isoform2. Temporal expression analysis by RT-qPCR indicated that X. laevis arid5b isoform1 and isoform2 are differentially expressed during development. Isoform1 is strongly expressed maternally, while isoform2 expression is essentially restricted to tailbud stages. Spatial expression analysis by whole mount in situ showed that arid5b is predominantly expressed in the developing pronephros. Arid5b mRNAs are detected in the antero-dorsal part of the pronephros anlage at the early tailbud stage and later on, in the proximal part of the pronephric tubule. RT-qPCR analyses with primers that allow to discriminate isoform1 from isoform2 showed that the latter is enriched in the pronephros anlage. In agreement with a specific pronephric signature of the isoform2, we also observed that isoform2 but not isoform1 is upregulated in animal caps induced to form pronephric tissue in response to activin A and retinoic acid. These results indicate that the two arid5b isoforms are differentially expressed and likely play different roles during early Xenopus development.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Pronefro/metabolismo , Isoformas de Proteínas/genética , Fatores de Transcrição/genética , Proteínas de Xenopus/genética , Animais , Proteínas de Ligação a DNA/metabolismo , Embrião não Mamífero/metabolismo , Pronefro/embriologia , Isoformas de Proteínas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
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 line.
Assuntos
Evolução Molecular , Células Germinativas/metabolismo , Proteínas de Homeodomínio/metabolismo , Meiose/fisiologia , Ovário/metabolismo , Testículo/metabolismo , Vertebrados/fisiologia , Sequência de Aminoácidos , Animais , Western Blotting , Etiquetas de Sequências Expressas , Feminino , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/citologia , Proteínas de Homeodomínio/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Ovário/citologia , Filogenia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Ovinos , Testículo/citologiaRESUMO
Translational control was investigated in sea urchin eggs and embryos in response to the DNA-damaging agent methyl methanesulfonate (MMS). We have shown in this report that exposure of sea urchin embryos to MMS induces drastic effects on protein synthesis activity, and on translation factors level, integrity and post-translational modifications. In response to the treatment of embryos by the DNA-damaging agent MMS, protein synthesis is inhibited independently of the translation inhibitor 4E-BP and in correlation with phosphorylation of the translation factor eIF2alpha subunit. Furthermore, a low molecular weight form of translation initiation factor eIF4G is detected correlatively with MMS-induced apoptosis. We propose that modifications of translation factors play an important role in protein synthesis modulation that occurs during DNA-damage induced apoptosis.
Assuntos
Apoptose , Dano ao DNA/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Ribossômicas/metabolismo , Animais , Embrião não Mamífero , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação Eucariótico 4G/análise , Metanossulfonato de Metila/farmacologia , Óvulo , Fosforilação , Proteínas Ribossômicas/efeitos dos fármacos , Ouriços-do-MarRESUMO
Using 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.
Assuntos
Ciclo Celular/efeitos dos fármacos , Cloretos/toxicidade , Compostos de Cromo/toxicidade , Dano ao DNA , Embrião não Mamífero/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/genética , Relação Dose-Resposta a Droga , Embrião não Mamífero/química , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/análise , Fator de Iniciação 4E em Eucariotos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/análise , Óvulo/química , Óvulo/efeitos dos fármacos , Óvulo/metabolismo , Ligação Proteica , Ouriços-do-Mar/embriologia , Ouriços-do-Mar/fisiologia , Fatores de TempoRESUMO
Cell 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.
Assuntos
Ciclo Celular/fisiologia , Dano ao DNA , Embrião não Mamífero/fisiologia , Neoplasias/fisiopatologia , Ouriços-do-Mar/embriologia , Animais , Evolução Biológica , Ciclo Celular/genética , Embrião não Mamífero/citologia , Feminino , Masculino , Modelos Biológicos , Neoplasias/genética , Reprodução , Ouriços-do-Mar/genéticaRESUMO
The 4E-binding proteins (4E-BPs) regulate the cap-dependent eukaryotic initiation factor 4E (eIF4E). The level of 4E-BP protein is regulated during early development of sea urchin embryos. Fertilization leads to the rapid disappearance of the protein that reappears later in development. We show that two important cellular stresses, hypoxia and bleomycin prolonged checkpoint mobilization provoked the overexpression of the protein 4E-BP in developing sea urchin embryos. Hypoxia resulted after 1 h in a reversible gradual increase in the protein 4E-BP level. At 20 h, the protein 4E-BP had reached the level existing in the unfertilized eggs. Bleomycin used as a DNA-damaging agent for checkpoint activation, provoked cell cycle inhibition and after prolonged exposure (20 h), induced the expression of the protein 4E-BP. The effect of bleomycin on 4E-BP protein overexpression was dose-dependent between 0.4 and 1.2 mM. The role of the overexpression of the protein 4E-BP is discussed in relation with cellular stress responses.