Translation of maternal histone mRNAs in sea urchin embryos: a test of control by 5' cap methylation

Recent results have demonstrated the occurrence of mRNA cap methylation in the sea urchin embryo following fertilization. It has been suggested that this methylation event is responsible for the translational activation of maternal histone mRNAs in these embryos. We have used aphidicolin, an effective inhibitor of both DNA synthesis and cap methylation in cleavage stage sea urchin embryos, to examine the relationship between cap methylation and translation. At 5 micrograms/ml, a dose which rapidly abolishes DNA replication and blocks cleavage, we note no effect on recruitment or translation of maternal alpha-subtype histone mRNAs. This suggests that a postfertilization cap methylation event is not critical to the process of regulation of the translation of stored alpha-subtype histone mRNAs.     

A factor in sea urchin eggs inhibits transcription in isolated nuclei by sea urchin RNA polymerase III

Isolated nuclei from sea urchin embryos synthesize RNA at a rate comparable to other animal cell nuclei. All three RNA polymerases are active as judged by alpha-amanitin sensitivity and hybridization to specific cloned DNAs. Extracts were prepared from sea urchin eggs and embryos by extraction with 0.35 M KCl. None of the crude extracts had a large effect on total RNA synthesis. However, extracts from sea urchin eggs inhibited RNA polymerase III activity in nuclei from blastula and gastrula embryos. There was no effect on the synthesis of ribosomal RNA by RNA polymerase I or on the synthesis of two RNA polymerase II products, histone mRNA and the sea urchin analogue of U1 RNA. The inhibitor is present in two different species of sea urchin and has been 50-fold purified by diethylaminoethylcellulose and hydroxylapatite chromatography. The inhibitor is not present in extracts prepared from sea urchin blastula embryos.     

Maternal messenger RNA and histone synthesis in embryos of the surf clam Spisula solidissima.

Messenger RNA has been isolated from the postribosomal supernatant of Spisula solidissima eggs. This mRNA directs the synthesis of several proteins when added to the ascites or wheat germ cell free system. No histone except F1 is coded for by Spisula egg mRNA, in contrast to what has been reported previously for sea urchin egg mRNA. In sea urchin eggs histone mRNA is among the abundant species of maternal mRNA.Histones have been prepared from Spisula embryos at different development stages and histone synthesis followed by incubation with (¹⁴C)lysine. The analysis by electrophoresis on acrylamide gels indicates that the pattern of synthesis of histones changes during development and that a new histone F1 fraction is actively synthesized from the 32–64 cells stage. In earlier embryos a different F1 histone is synthesized and the mRNA for this protein may be the only histone mRNA present in eggs.     

Dynamics of Ubiquitin Pools in Developing Sea Urchin Embryos

The sea urchin embryo is a closed metabolic system in which embryogenesis is accompanied by significant protein degradation. We report results which are consistent with a function for the ubiquitinmediated proteolytic pathway in selective protein degradation during embryogenesis in this system. Quantitative solid- and solution-phase immunochemical assays, employing anti-ubiquitin antibodies, showed that unfertilized eggs of Strongylocentrotus purpuratus have a high content of unconjugated ubiquitin ( ca . 8 × 10⁸ molecules), and also contain abundant conjugates involving ubiquitin and maternal proteins. The absolute content of ubiquitin in the conjugated form increases about 13-fold between fertilization and the pluteus larva stage; 90% or more of embryonic ubiquitin molecules are conjugated to embryonic proteins in hatched blastulae and later-stage embryos. Qualitatively similar results were obtained with embryos of Lytechinus variegatus . The results of pulse-labeling and immunoprecipitation experiments indicate that synthesis of ubiquitin in S. purpuratus is developmentally regulated, with an overall increase in synthetic rate of 12-fold between fertilization and hatching. Regulation is likely to occur at the level of translation, since others have shown that levels of ubiquitin-encoding mRNA remain virtually constant in echinoid embryos during this developmental interval. The sea urchin embryo should be a useful system for characterizing the role of ubiquitination in embryogenesis.     

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.     

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.     

Nodal and BMP2/4 signaling organizes the oral-aboral axis of the sea urchin embryo

In the sea urchin embryo, the oral-aboral axis is specified after fertilization by mechanisms that are largely unknown. We report that early sea urchin embryos express Nodal and Antivin in the presumptive oral ectoderm and demonstrate that these genes control formation of the oral-aboral axis. Overexpression of nodal converted the whole ectoderm into oral ectoderm and induced ectopic expression of the orally expressed genes goosecoid, brachyury, BMP2/4, and antivin. Conversely, when the function of Nodal was blocked, by injection of an antisense Morpholino oligonucleotide or by injection of antivin mRNA, neither the oral nor the aboral ectoderm were specified. Injection of nodal mRNA into Nodal-deficient embryos induced an oral-aboral axis in a largely non-cell-autonomous manner. These observations suggest that the mechanisms responsible for patterning the oral-aboral axis of the sea urchin embryo may share similarities with mechanisms that pattern the dorsoventral axis of other deuterostomes.     

Cyclin D and cdk4 are required for normal development beyond the blastula stage in sea urchin embryos

cdk4 mRNA and protein are constitutively expressed in sea urchin eggs and throughout embryonic development. In contrast, cyclin D mRNA is barely detectable in eggs and early embryos, when the cell cycles consist of alternating S and M phases. Cyclin D mRNA increases dramatically in embryos at the early blastula stage and remains at a constant level throughout embryogenesis. An increase in cdk4 kinase activity occurs concomitantly with the increase in cyclin D mRNA. Ectopic expression of cyclin D mRNA in eggs arrests development before the 16-cell stage and causes eventual embryonic death, suggesting that activation of cyclin D/cdk4 in cleavage cell cycles is lethal to the embryo. In contrast, blocking cyclin D or cdk4 expression with morpholino antisense oligonucleotides results in normal development of early gastrula-stage embryos but abnormal, asymmetric larvae. These results suggest that in sea urchins, cyclin D and cdk4 are required for normal development and perhaps the patterning of the developing embryo, but may not be directly involved in regulating entry into the cell cycle.     

'Unmasking' of stored maternal mRNAs and the activation of protein synthesis at fertilization in sea urchins

The isolation and in vitro assay of maternal mRNPs has led to differing conclusions as to whether maternal mRNAs in sea urchin eggs are in a repressed or 'masked' form. To circumvent the problems involved with in vitro approaches, we have used an in vivo assay to determine if the availability of mRNA and/or components of the translational machinery are limiting protein synthesis in the unfertilized egg. This assay involves the use of a protein synthesis elongation inhibitor to create a situation in the egg in which there is excess translational machinery available to bind mRNA. Eggs were fertilized and the rate of entry into polysomes of individual mRNAs was measured in inhibitor-treated and control embryos using 32P-labeled cDNA probes. The fraction of ribosomes in polysomes and the polysome size were also determined. The results from this in vivo approach provide strong evidence for the coactivation of both mRNAs and components of the translational machinery following fertilization. The average polysome size increases from 7.5 ribosomes per message in 15 min embryos to approximately 10.8 ribosomes in 2 h embryos. This result gives additional support to the idea that translational machinery, as well as mRNA, is activated following fertilization. We also found that individual mRNAs are recruited into polysomes with different kinetics, and that the fraction of an mRNA in polysomes in the unfertilized egg correlates with the rate at which that mRNA is recruited into polysomes following fertilization.     

Characterization of sea urchin unconventional myosins and analysis of their patterns of expression during early embryogenesis

Early sea urchin development requires a dynamic reorganization of both the actin cytoskeleton and cytoskeletal interactions with cellular membranes. These events may involve the activities of multiple members of the superfamily of myosin motor proteins. Using RT-PCR with degenerate myosin primers, we identified 11 myosin mRNAs expressed in unfertilized eggs and coelomocytes of the sea urchin Strongylocentrotus purpuratus. Seven of these sea urchin myosins belonged to myosin classes Igamma, II, V, VI, VII, IX, and amoeboid-type I, and the remaining four may be from novel classes. Sea urchin myosins-V, -VI, -VII, and amoeboid-type-I were either completely or partially cloned and their molecular structures characterized. Sea urchin myosins-V, -VI, -VII, and amoeboid-type-I shared a high degree of sequence identity with their respective family members from vertebrates and they retained their class-specific structure and domain organization. Analysis of expression of myosin-V, -VI, -VII, and amoeboid-type-I mRNAs during development revealed that each myosin mRNA displayed a distinct temporal pattern of expression, suggesting that myosins might be involved in specific events of early embryogenesis. Interestingly, the onset of gastrulation appeared to be a pivotal point in modulation of myosin mRNA expression. The presence of multiple myosin mRNAs in eggs and embryos provides insight into the potential involvement of multiple specific motor proteins in the actin-dependent events of embryo development.     

Inhibitor of translational initiation in sea urchin eggs prevents mRNA utilization

Actively reinitiating cell-free translation systems from sea urchin eggs and embryos have been developed. The extracts retain the overall differences in protein synthetic activity observed in intact eggs and embryos. The effect of combining extracts from eggs and embryos suggests the presence of a dominant inhibitor of translation in the egg. This inhibitor also prevents the initiation of translation in a cell-free system from rabbit reticulocytes. In the reticulocyte system, the egg inhibitor causes the accumulation of 48 S preinitiation complexes, as measured by the accumulation of initiator tRNA and globin mRNA on the small ribosomal subunit. Accumulation of this uncommon intermediate suggests either that the inhibitor prevents the binding of the 60 S ribosomal subunit, or that it prevents migration of the 40 S subunit from the 5' end of mRNA to the first AUG.     

Caulerpenyne blocks MBP kinase activation controlling mitosis in sea urchin eggs

In a previous study, we demonstrated that caulerpenyne (Cyn), a natural sesquiterpene having an antiproliferative potency, blocked the mitotic cycle of sea urchin embryos at metaphase and inhibited the phosphorylation of several proteins, but did not affect histone H1 kinase activation (Pesando et al, 1998, Eur. J. Cell Biol. 77, 19-26). Here, we show that concentrations of Cyn that blocked the first division of the sea urchin Paracentrotus lividus embryos in a metaphase-like stage (45 microM) also inhibited the stimulation of mitogen-activated protein kinase (MAPK) activity in vivo as measured in treated egg extracts using myelin basic protein (MBP) as a substrate (MBPK). However, Cyn had no effect on MBP phosphorylation when added in vitro to an untreated egg extract taken at the time of metaphase, suggesting that Cyn acts on an upstream activation process. PD 98059 (40 microM), a previously characterized specific synthetic inhibitor of MAPK/extracellular signal-regulated kinase-1 (MEK1), also blocked sea urchin eggs at metaphase in a way very similar to Cyn. Both molecules induced similar inhibitory effects on MBP kinase activation in vivo, but had no direct effect on MBP kinase activity in vitro, whereas they did not affect H1 kinase activation neither in vivo nor in vitro. As a comparison, butyrolactone 1 (100 microM), a known inhibitor of H1 kinase activity, did inhibit H1 kinase of sea urchin eggs in vivo and in vitro, and blocked the sea urchin embryo mitotic cycle much before metaphase. Immunoblots of mitotic extracts, treated with anti-active MAP-kinase antibody, showed that both Cyn and PD 98059 reduced the phosphorylation of p42 MAP kinase (Erk2) in vivo. Our overall results suggest that Cyn blocks the sea urchin embryo mitotic cycle at metaphase by inhibiting an upstream phosphorylation event in the MBPK activation pathway. They also show that H1 kinase and MBPK activation can be dissociated from each other in this model system.     

The Relative Contributions of Newly Synthesized and Stored Messages to Hl Histone Synthesis in Interspecies Hybrid Echinoid Embryos

We have measured the ratio of incoropation of ³H-lysine into the maternal and paternal forms of Hl histones synthesized by the interordinal hybrid embryo which results from the fertilization of sand dollar eggs with sea urchin sperm. This ratio has been used to calculate the relative contributions of newly transcribed and stored Hl histone mRNA to the synthesis of Hl histone at five different stages of development. These calculations are based on the assumption that histone mRNA of both parental types is transcribed with equal efficiency from the genome and that these RNAs are translated with equal efficiency in the cytoplasm of the hybrid embryos. On this basis, we have estimated that the contribution of new mRNA respresents 80% of total Hl histone synthesis at the 16–32 cell stage, 54% at the hatching blastula stage, 40% at the mesenchyme blastula stage, and 100% after gastrulation.
These data are discussed in the light of presently known parameters of histone and histone mRNA synthesis in echinoderm embryos.     

A Century of Sea Urchin Development

SYNOPSIS. In the last quarter of the nineteenth century severalinvestigators including Richard and Oskar Hertwig, Theodor Boveri,Hans Driesch, Curt Herbst, T. H. Morgan and others turned theirattention to sea urchin eggs and early embryos. This favorablecombination of outstanding investigators and the sea urchinembryo as an experimental organism contributed to a fundamentalunderstanding of the cell, fertilization and heredity. The advantagesof the sea urchin continued to be recognized as experimentalembryologists used these embryos to develop the concepts ofgradients, regulative development and inductive interactions.Then, as developmental biology arose from chemical embryology,the sea urchin embryo once again emerged as an ideal experimentalanimal, pivotal in the understanding of the molecular and developmentalbiology of eukaryotic organisms.     

Wnt6 activates endoderm in the sea urchin gene regulatory network

In the sea urchin, entry of β-catenin into the nuclei of the vegetal cells at 4th and 5th cleavages is necessary for activation of the endomesoderm gene regulatory network. Beyond that, little is known about how the embryo uses maternal information to initiate specification. Here, experiments establish that of the three maternal Wnts in the egg, Wnt6 is necessary for activation of endodermal genes in the endomesoderm GRN. A small region of the vegetal cortex is shown to be necessary for activation of the endomesoderm GRN. If that cortical region of the egg is removed, addition of Wnt6 rescues endoderm. At a molecular level, the vegetal cortex region contains a localized concentration of Dishevelled (Dsh) protein, a transducer of the canonical Wnt pathway; however, Wnt6 mRNA is not similarly localized. Ectopic activation of the Wnt pathway, through the expression of an activated form of β-catenin, of a dominant-negative variant of GSK-3β or of Dsh itself, rescues endomesoderm specification in eggs depleted of the vegetal cortex. Knockdown experiments in whole embryos show that absence of Wnt6 produces embryos that lack endoderm, but those embryos continue to express a number of mesoderm markers. Thus, maternal Wnt6 plus a localized vegetal cortical molecule, possibly Dsh, is necessary for endoderm specification; this has been verified in two species of sea urchin. The data also show that Wnt6 is only one of what are likely to be multiple components that are necessary for activation of the entire endomesoderm gene regulatory network.     

Translational activation of maternal mRNA encoding the heat-shock protein hsp90 during sea urchin embryogenesis

Changes in protein synthesis induced by heat shock of Strongylocentrotus purpuratus gastrulae were analyzed bt two-dimensional electrophoresis. Hyperthermia induces the synthesis of polypeptides having molecular masses of 90, 70, 50, 40, and 38 kDa. One of these, hsp90, appears as a pair of polypeptides which comigrates with proteins synthesized at normal temperature in eggs and embryos; these comigrating spots produce indistinguishable patterns upon electrophoretic analysis of partial V8 protease digests, indicating that hsp90 is synthesized throughout embryogenesis. The relative rate of incorporation of methionine into hsp90 is low in eggs and zygotes, but increases abruptly in morulae, constituting a rare and striking change in protein synthesis during early development. Cell-free translation analyses indicate that most of the mRNA encoding hsp90 resides in the pool of free ribonucleoprotein particles in eggs and early embryos, but shifts to polysomes by the 64-cell stage while remaining constant in mass. Thus the increase in synthesis of hsp90 appears to be via the selective activation of translation of a stored maternal mRNA. The shift of hsp90 mRNA to polysomes is accompanied by polyadenylation. Heat shock of eggs or zygotes did not result in translational activation of hsp90 mRNA. The sea urchin hsp90 doublet of spots comigrates with hsp90 induced by heat shock of chicken embryo fibroblasts, a conserved protein abundant in many cells of a variety of species.     

The relative contributions of newly synthesized and stored messages to Hl histone synthesis in interspecies hybrid echinoid embryos.

We have measured the ratio of incorporation of 3H-lysine into the maternal and paternal forms of Hl histones synthesized by the interordinal hybrid embryo which results from the fertilization of sand dollar eggs with sea urchin sperm. This ratio has been used to calculate the relative contributions of newly transcribed and stored Hl histones mRNA to the synthesis of Hl histone at five different stages of development. These calculations are based on the assumption that histone mRNA of both parental types is transcribed with equal efficiency from the genome and that these RNAs are translated with equal efficiency in the cytoplasm of the hybrid embryos. On this basis, we have estimated that the contribution of new mRNA represents 80% of total Hl histone synthesis at the 16--32 cell stage, 54% at the hatching blastula stage, 40% at the mesenchyme blastula stage, and 100% after gastrulation. These data are discussed in the light of presently known parameters of histone and histone mRNA synthesis in echinoderm embryos.     

Changes in rates of protein synthesis and eukaryotic initiation factor-4 inhibitory activity in cell-free translation systems of sea urchin eggs and early cleavage stage embryos.

The characteristics of cell-free translation systems prepared from unfertilized eggs and early cleavage stage embryos of the sea urchin, Strongylocentrotus purpuratus, closely reflect the developmentally regulated changes in protein synthesis initiation observed in vivo. Cell-free translation systems prepared over the first 0-6 h following fertilization show gradually increasing activities, mimicking the changes observed in vivo. The mechanisms underlying these increases are complex and occur at several levels. One factor contributing to the rise in protein synthetic rate is the gradual increase in eukaryotic initiation factor (eIF)-4 activity. This is correlated with the progressive inactivation of an inhibitor of eIF-4 function, which can be reactivated by in vitro manipulations. The relatively slow activation of eIF-4 follows similar kinetics to the increased utilization of maternal mRNA and ribosomes, in contrast to the rapid rise in maternal mRNA activation, and the increase in eIF-2B activity. This slow release from eIF-4 inhibition following a rapid release from eIF-2B inhibition and increased mRNA availability is reflected in the pattern of initiator tRNA binding to the small ribosomal subunit observed in cell-free translation systems. In translation systems from unfertilized eggs, initiator tRNA is unable to interact with the small ribosomal subunit, consistent with an initial block in both eIF-2B and eIF-4 activity. In translation systems from 30-min embryos, 48 S preinitiation complexes accumulate, reflecting the release from inhibition of mRNA availability and eIF-2B activity, but continued low activity of eIF-4. The accumulation of initiator tRNA in 48 S preinitiation complexes disappears gradually in translation systems from later embryos, as eIF-4 is slowly released from inhibition.