Translational control genes in the sea urchin genome

Sea urchin eggs and early cleavage stage embryos provide an example of regulated gene expression at the level of translation. The availability of the sea urchin genome offers the opportunity to investigate the "translational control" toolkit of this model system. The annotation of the genome reveals that most of the factors implicated in translational control are encoded by nonredundant genes in echinoderm, an advantage for future functional studies. In this paper, we focus on translation factors that have been shown or suggested to play crucial role in cell cycle and development of sea urchin embryos. Addressing the cap-binding translational control, three closely related eIF4E genes (class I, II, III) are present, whereas its repressor 4E-BP and its activator eIF4G are both encoded by one gene. Analysis of the class III eIF4E proteins in various phyla shows an echinoderm-specific amino acid substitution. Furthermore, an interaction site between eIF4G and poly(A)-binding protein is uncovered in the sea urchin eIF4G proteins and is conserved in metazoan evolution. In silico screening of the sea urchin genome has uncovered potential new regulators of eIF4E sharing the common eIF4E recognition motif. Taking together, these data provide new insights regarding the strong requirement of cap-dependent translation following fertilization. The genome analysis gives insights on the complexity of eEF1B structure and motifs of functional relevance, involved in the translational control of gene expression at the level of elongation. Finally, because deregulation of translation process can lead to diseases and tumor formation in humans, the sea urchin orthologs of human genes implicated in human diseases and signaling pathways regulating translation were also discussed.     

eIF4E/4E-BP dissociation and 4E-BP degradation in the first mitotic division of the sea urchin embryo

The mRNA's cap-binding protein eukaryotic translation initiation factor (eIF)4E is a major target for the regulation of translation initiation. eIF4E activity is controlled by a family of translation inhibitors, the eIF4E-binding proteins (4E-BPs). We have previously shown that a rapid dissociation of 4E-BP from eIF4E is related with the dramatic rise in protein synthesis that occurs following sea urchin fertilization. Here, we demonstrate that 4E-BP is destroyed shortly following fertilization and that 4E-BP degradation is sensitive to rapamycin, suggesting that proteolysis could be a novel means of regulating 4E-BP function. We also show that eIF4E/4E-BP dissociation following fertilization is sensitive to rapamycin. Furthermore, while rapamycin modestly affects global translation rates, the drug strongly inhibits cyclin B de novo synthesis and, consequently, precludes the completion of the first mitotic cleavage. These results demonstrate that, following sea urchin fertilization, cyclin B translation, and thus the onset of mitosis, are regulated by a rapamycin-sensitive pathway. These processes are effected at least in part through eIF4E/4E-BP complex dissociation and 4E-BP degradation.     

Inhibition of translation and modification of translation factors during apoptosis induced by the DNA-damaging agent MMS in sea urchin embryos

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.     

eIF4E association with 4E-BP decreases rapidly following fertilization in sea urchin

The eukaryotic translation initiation factor (eIF) 4F facilitates the recruitment of ribosomes to the mRNA 5' end. The 4E-BPs are small proteins with hypophosphorylated forms that interact with the cap binding protein eIF4E, preventing its interaction with eIF4G, thereby preventing ribosome interaction with mRNA. In sea urchin, fertilization triggers a rapid rise in protein synthesis. Here, we demonstrate that a 4E-BP homologue exists and is associated with eIF4E in unfertilized eggs. We also show that 4E-BP/eIF4E association diminishes a few minutes following fertilization. This decrease is correlated with a decrease in the total amount of 4E-BP in combination with an increase in the phosphorylation of the protein. We propose that 4E-BP acts as a repressor of protein synthesis in unfertilized sea urchin eggs and that 4E-BP/eIF4E dissociation plays an important role in the rise in protein synthesis that occurs shortly following fertilization.     

Signal transduction pathways that contribute to CDK1/cyclin B activation during the first mitotic division in sea urchin embryos

In sea urchins, fertilization triggers a rapid rise in protein synthesis necessary for activation of CDK1/cyclin B, the universal cell cycle regulator. It has been shown that FRAP/mTOR is required for eIF4E release from the translational repressor 4E-BP, a process that occurs upstream of de novo cyclin B synthesis. Here, we investigate whether PI 3-kinase acts independently or upstream from FRAP/mTOR in the signal transduction pathway that links fertilization to the activation of the CDK1/cyclin B complex in sea urchin egg. We found that wortmannin, a potent inhibitor of PI 3-kinase, partially inhibited the global increase in protein synthesis triggered by fertilization. Furthermore, wortmannin treatment induced partial inhibition of cyclin B translation triggered by fertilization, in correlation with an intermediate effect of the drug on 4E-BP degradation and on the dissociation of the 4E-BP/eIF4E complex induced by fertilization. Our results presented here suggest that PI 3-kinase activity is required for completion of mitotic divisions of the sea urchin embryo. Incubation of eggs with wortmannin or microinjection of wortmannin or LY 294002 affects drastically mitotic divisions induced by fertilization. In addition, we found that wortmannin treatment inhibits dephosphorylation of the tyrosine inhibitory site of CDK1. Taken together, these data suggest that PI 3-kinase acts upstream of at least two independent targets that function in the CDK1/cyclin B activation triggered by fertilization of sea urchin oocytes. We discuss the significance of these results concerning the cascade of reactions that impinge upon the activation of the CDK1/cyclin B complex that follows sea urchin oocyte fertilization.     

Hbox1 and Hbox7 are involved in pattern formation in sea urchin embryos

In spite of their potential importance in evolution, there is little information about Hox genes in animal groups that are related to ancestors of deuterostome. It has been reported that only two Hox genes (Hbox1 and Hbox7) are expressed significantly in sea urchin embryos. Expression of Hbox1 protein is restricted to the aboral ectoderm, and Hbox7 expression is restricted to oral ectoderm, endoderm and secondary mesenchyme cells in sea urchin embryos after the gastrula stage. With the aim of gaining insight into the role of Hbox1 and Hbox7 in sea urchin development, Hbox1 and Hbox7 overexpression experiments were performed. Overexpression of Hbox1 repressed the development of oral ectoderm, endoderm and mesenchyme cells. On the contrary, overexpression of Hbox7 repressed the development of aboral ectoderm and primary mesenchyme cells. The data suggest that Hbox1 and Hbox7 are expressed in distinct non-overlapping territories, and overexpression of either one inhibits territory-specific gene expression in the domain of the other. It is proposed that an important function of both Hbox1 and Hbox7 genes is to maintain specific territorial gene expression by each one, in its domain of expression, while repressing the expression of the other in this same domain.     

Rho-kinase in sea urchin eggs and embryos

The activation of sea urchin eggs at fertilization provides an ideal system for studying the molecular events involved in cellular activation. Rho GTPases, which are key signaling enzymes in eukaryotes, are involved in sustaining the activation of sea urchin eggs; however, their downstream effectors have not yet been characterized. In somatic cells, RhoA regulates a serine/threonine kinase known as Rho-kinase (ROCK). The activity of ROCK in early sea urchin development has been inferred, but not tested directly. A ROCK gene was identified in the sea urchin (Strongylocentrotus purpuratus) genome and the sequence of its cDNA determined. The sea urchin ROCK (SpROCK) sequence predicts a protein of 158 kDa with >72% and 45% identities with different protein orthologues of the kinase catalytic domain and the complete protein sequence, respectively. SpROCK mRNA levels are high in unfertilized eggs and decrease to 35% after 15 min postfertilization and remain low up to the 4 cell stage. Antibodies to the human ROCK-I kinase domain revealed SpROCK to be concentrated in the cortex of eggs and early embryos. Co-immunoprecipitation assays indicate that RhoA and SpROCK are physically associated. This association is destroyed by treatment with the C3 exoenzyme and with the ROCK antagonist H-1152. H-1152 also inhibited DNA synthesis in embryos. We conclude that the Rho-dependent signaling pathway, via SpROCK, is essential for early embryonic development.     

Inhibition of cysteine protease activity disturbs DNA replication and prevents mitosis in the early mitotic cell cycles of sea urchin embryos

Recent findings suggested that the role of cysteine proteases would not be limited to protein degradation in lysosomes but would also play regulatory functions in more specific cell mechanisms. We analyzed here the role of these enzymes in the control of cell cycle during embryogenesis. The addition of the potent cysteine protease inhibitor E64d to newly fertilized sea urchin eggs disrupted cell cycle progression, affecting nuclear as well as cytoplasmic characteristic events. Monitoring BrdU incorporation in E64d treated eggs demonstrated that DNA replication is severely disturbed. Moreover, this drug treatment inhibited male histones degradation, a step that is necessary for sperm chromatin remodeling and precedes the initiation of DNA replication in control eggs. This inhibition likely explains the DNA replication disturbance and suggests that S phase initiation requires cysteine protease activity. In turn, activation of the DNA replication checkpoint could be responsible for the consecutive block of nuclear envelope breakdown (NEB). However, in sea urchin early embryos this checkpoint doesn't control the mitotic cytoplasmic events that are not tightly coupled with NEB. Thus the fact that microtubule spindle is not assembled and cyclin B-cdk1 not activated under E64d treatment more likely rely on a distinct mechanism. Immunofluorescence experiments indicated that centrosome organization was deficient in absence of cysteine protease activity. This potentially accounts for mitotic spindle disruption and for cyclin B mis-localization in E64d treated eggs. We conclude that cysteine proteases are essential to trigger S phase and to promote M phase entry in newly fertilized sea urchin eggs.     

Effects of retinoic acid and dimethylsulfoxide on the morphogenesis of the sea urchin embryo

Sea urchin embryos of the species Paracentrotus lividus were treated continuously with different concentrations of all-trans retinoic acid (RA) or dimethylsulfoxide (DMSO) at different developmental stages. A delay in embryonic development was observed when embryos were cultured in the presence of 2x10^?5 M RA, between 1 and 12 hours of development. Hence, at 48 hours of development, while control embryos had reached the pluteus stage, RA-treated embryos were at the prism stage. At 72 hours of development RA-treated embryos recovered and continued normal development reaching the pluteus stage. No effect was observed when treatment was performed before 1 hour or after 12 hours of developmet. DMSO treatment had no effect on normal sea urchin embryo development, although we observed that pigment cells, clearly visible at the pluteus stage, become visible earlier with respect to control embryos. This report confirms the advantages that the sea urchin embryo offers for the study of problems in cellular and developmental biology.     

HpSulf,a heparan sulfate 6-O-endosulfatase,is involved in the regulation of VEGF signaling during sea urchin development

Cell surface heparan sulfate proteoglycans (HSPGs) play significant roles in the regulation of developmental signaling, including vascular endothelial growth factor (VEGF), fibroblast growth factor, Wnt and bone morphogenetic protein signaling, through modification of their sulfation patterns. Recent studies have revealed that one of the functions of heparan sulfate 6-O-endosulfatase (Sulf) is to remove the sulfate from the 6-O position of HSPGs at the cell surface, thereby regulating the binding activities of heparan sulfate (HS) chains to numerous ligands and receptors in animal species. In this study, we focused on the sea urchin Hemicentrotus pulcherrimus homolog of Sulf (HpSulf), and analyzed its expression pattern and functions during development. HpSulf protein was present throughout development and localized at cell surface of all blastomeres. In addition, the HS-specific epitope 10E4 was detected at the cell surface and partially colocalized with HpSulf. Knockdown of HpSulf using morpholino antisense oligonucleotides (MO) caused abnormal morphogenesis, and the development of MO-injected embryos was arrested before the hatched blastula stage, indicating that HpSulf is necessary for the early developmental process of sea urchin embryos. Furthermore, we found that injection of HpSulf mRNA suppressed the abnormal skeleton induced by overexpression of HpVEGF mRNA, whereas injection of an inactive form of HpSulf mRNA, containing mutated cysteines in the sulfatase domain, did not have this effect. Taken together, these results suggest that HpSulf is involved in the regulation of various signal transductions, including VEGF signaling, during sea urchin development.     

Cyclin E and its associated cdk activity do not cycle during early embryogenesis of the sea Urchin

Female sea urchins store their gametes as haploid eggs. The zygote enters S-phase 1 h after fertilization, initiating a series of cell cycles that lack gap phases. We have cloned cyclin E from the sea urchin Strongylocentrotus purpuratus. Cyclin E is synthesized during oogenesis, is present in the germinal vesicle, and is released into the egg cytoplasm at oocyte maturation. Cyclin E synthesis is activated at fertilization, although there is no increase in cyclin E protein levels due to continuous turnover of the protein. Cyclin E protein levels decline in morula embryos, while cyclin E mRNA levels remain high. After the blastula stage, cyclin E mRNA and protein levels are very low, and cyclin E expression is predominant only in cells that are actively dividing. These include cells in the left coelomic pouch, which forms the adult rudiment in the embryo. The cyclin E present in the egg is complexed with a protein kinase. Activity of the cyclin E/cdk2 changes little during the initial cell cycles. In particular, cyclin E-cdk2 levels remain high during both S-phase and mitosis. Our results suggest that progression through the early embryonic cell cycles in the sea urchin does not require fluctuations in cyclin E kinase activity.     

The role of lysyl oxidase and collagen crosslinking during sea urchin development

Lysyl oxidase, the only enzyme involved in collagen crosslinking, is shown to be present in embryos of the sea urchin Strongylocentrotus purpuratus. The enzyme specific activity increases over six-fold during development, showing the greatest rise during gastrulation and prism larva formation. The enzyme is inhibited by the specific inhibitor, beta-aminoproprionitrile (BAPN). Continuous BAPN treatment of S. purpuratus and Lytechinus pictus embryos from late cleavage stages onward increases the amount of noncrosslinked collagen present in prism larvae. When BAPN is added at the 128- or 256-cell stage it causes developmental arrest at the mesenchyme blastula stage. Embryos can be maintained in the arrested state for at least 96 h and will resume normal development and morphogenesis following BAPN removal. If BAPN is added after the mesenchyme blastula stage, it has little adverse effect on development; consequently nonspecific toxic effects of the drug are unlikely. The results suggest that lysyl oxidase and collagen crosslinking play a vital role in primary mesenchyme migration, gastrulation, and morphogenesis during sea urchin development and indicate that BAPN may be very useful in studying the extracellular matrix-cell interactions at the cellular and molecular level.     

CANCELING EFFECT OF 2, 4-DINITROPHENOL ON THE VEGETALIZATION OF SEA URCHIN LARVAE INDUCED WITH CAFFEINE*

The treatment of the sea urchin morulae with both caffeine and 2, 4-dinitrophenol (DNP) for a couple of hours exerts no harmful effect on the development of sea urchin, whereas the tretment with caffeine alone yields vegetalized larvae. As long as the morulae are kept in the pressence of DNP alone, further development or the embryos is arrested, but the treated embryos develop normally after they are transferred into plain sea water. Hence, DNP is supposed to cancel vegetalizing effect of caffeine on the sea urchin morulae. When the embryos were kept in sea water containing respective radioactive precursors of macromolecules and caffeine, the radioactivity in the DNA fraction is slightly higher and those in the RNA and protein fraction are slightly lower than those of control ones (without the caffeine treatment). In the presence of DNP, the radioactivity in these macromolecules is very low in the caffeine-treated embroyos as well as in the control.     

Effect of millimeter waves on the early development of the mouse and sea urchin embryo]

The action of nonthermal electromagnetic radiation (EMR) of the millimeter range on the early development of murine and sea urchin embryos was investigated. An MRTA-01E-03 generator with a frequency of 54-78 GHz and radiation intensity of 0.06 mWt/cm2 was used. The embryos were irradiated during 30 min at the stage of two blastomeres. The number of murine embryos that reached the blastocyst stage increased (up to 97.3% in comparison with 87.5% in control). The total time of cultivation up to the blastocyst stage was also shorter (72 h) than in control (96 h). The irradiation had effect on the development of sea urchin embryos only if embryos with a weakened viability were tested. The results indicate that millimeter electromagnetic radiation has a stimulating effect on the early development of embryos, increasing the resistance of embryos to unfavorable environmental conditions.     

Hyperoxia alters the expression and phosphorylation of multiple factors regulating translation initiation

Hyperoxia is cytotoxic and depresses many cellular metabolic functions including protein synthesis. Translational control is exerted primarily during initiation by two mechanisms: 1) through inhibition of translation initiation complex formation via sequestration of the cap-binding protein, eukaryotic initiation factor (eIF) 4E, with inhibitory 4E-binding proteins (4E-BP); and 2) by prevention of eIF2-GTP-tRNA(i)(Met) formation and eIF2B activity by phosphorylated eIF2alpha. In this report, exposure of human lung fibroblasts to 95% O2 decreased the incorporation of thymidine into DNA at 6 h and the incorporation of leucine into protein beginning at 12 h. The reductions in DNA and protein synthesis were accompanied by increased phosphorylation of eIF4E protein and reduced phosphorylation of 4E-BP1. At 24 h, hyperoxia shifted 4E-BP1 phosphorylation to lesser-phosphorylated isoforms, increased eIF4E expression, and increased the association of eIF4E with 4E-BP1. Although hyperoxia did not change eIF2alpha expression, it increased its phosphorylation at Ser51, but not until 48 h. In addition, the activation of eIF2alpha was not accompanied by the formation of stress granules. These findings suggest that hyperoxia diminishes protein synthesis by increasing eIF4E phosphorylation and enhancing the affinity of 4E-BP1 for eIF4E.     

4E-BP1, a multifactor regulated multifunctional protein

Eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) is a member of a family of translation repressor proteins, and a well-known substrate of mechanistic target of rapamycin (mTOR) signaling pathway. Phosphorylation of 4E-BP1 causes its release from eIF4E to allow cap-dependent translation to proceed. Recently, 4E-BP1 was shown to be phosphorylated by other kinases besides mTOR, and overexpression of 4E-BP1 was found in different human carcinomas. In this review, we summarize the novel findings on mTOR independent 4E-BP1 phosphorylation in carcinomas. The implications of overexpression and possible multi-function of 4E-BP1 are also discussed.     

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

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.     

Brachyury homolog (HpTa) is involved in the formation of archenteron and secondary mesenchyme cell differentiation in the sea urchin embryo

Sea urchin Brachyury homolog (HpTa) is expressed exclusively in the vegetal plate and secondary mesenchyme cells in the embryos of sea urchin Hemicentrotus pulcherrimus. In order to gain insights into the role of HpTa during sea urchin development, we designed experiments to perturb the embryo by inducing ectopic overexpression of HpTa by injecting fertilized eggs with HpTa mRNA. The overexpression of HpTa resulted in suppression of the formation of vegetal plate and secondary mesenchyme cells. We assume that the interaction of HpTa with unknown factors is required for the activation of the HpTa target genes, and that the excess amount of HpTa proteins produced from injected HpTa mRNA depletes the co-factors. In consequence, the target genes of HpTa would be repressed by the overexpression of HpTa. We suggest that HpTa is involved in the formation of the vegetal plate and the differentiation of secondary mesenchyme cells.     

Apoptosis in early development of the sea urchin, Strongylocentrotus purpuratus

Apoptosis provides metazoans remarkable developmental flexibility by (1) eliminating damaged undifferentiated cells early in development and then (2) sculpting, patterning, and restructuring tissues during successive stages thereafter. We show here that apoptotic programmed cell death is infrequent and not obligatory during early embryogenesis of the purple sea urchin, Strongylocentrotus purpuratus. During the first 30 h of urchin development, fewer than 20% of embryos exhibit any cell death. Cell death during the cleavage stages consists of necrotic or pathological cell death, while cell death during the blastula and gastrula stages is random and predominantly caspase-mediated apoptosis. Apoptosis remains infrequent during the late blastula stage followed by a gradual increase in frequency during gastrulation. Even after prolonged exposure during the cleavage period to chemical stress, apoptosis occurs in less than 50% of embryos and always around the pre-hatching stage. Embryonic suppression of apoptosis through caspase inhibition leads to functionally normal larvae that can survive to metamorphosis, but in the presence of inducers of apoptosis, caspase inhibition leads to deformed larvae and reduced survival. Remarkably, however, pharmacological induction of apoptosis, while reducing overall survival, also significantly accelerates development of the survivors such that metamorphosis occurs up to a week before controls.