Expression of apo-aequorin during embryonic development; how much is needed for calcium imaging?

Aequorin is a bioluminescent calcium indicator consisting of a 21 kDa protein (apo-aequorin) that is covalently linked to a lipophilic cofactor (coelenterazine). The aequorin gene can be expressed in a variety of cell lines and tissues, allowing non-invasive calcium imaging of specific cell types. In the present paper, we describe the possibilities and limitations of calcium imaging with genetically introduced apo-aequorin during embryonic development. By injecting aequorin into sea urchin, Drosophila and zebrafish eggs, we found that higher aequorin concentrations are needed in smaller eggs. Our results suggest that for measuring resting levels of free cytosolic calcium, one needs aequorin concentrations of at least 40 μM in sea urchin eggs, 2 μM in Drosophila eggs, and only 0.11 μM in zebrafish eggs. A simple assay was used to determine the absolute concentrations of expressed apo-aequorin and the percentage of aequorin formation in vivo. The use of this assay is illustrated by expression of the aequorin gene in Drosophila oocytes. These oocytes form up to 1 μM apo-aequorin. In our hands, only 0.3% of this apo-aequorin combined with coelenterazine entering from the medium to form aequorin, which was not enough for calcium imaging of the oocytes, but did allow in vivo imaging of the ovaries. From these studies, we conclude that coelenterazine entry into the cell is the rate limiting step in aequorin formation. Based on the rate of coelenterazine uptake in Drosophila, we estimate that complete conversion of 1 μM apo-aequorin would take 50 days in zebrafish eggs, 19 days in Drosophila eggs, 7 days in sea urchin eggs or 18 h in a 10 gm tissue culture cell. Our results suggest that work based on genetically introduced apo-aequorin will be most successful when large amounts of small cells can be incubated in coelenterazine. During embryonic development this would involve introducing coelenterazine into the circulatory system of late stage embryos. Calcium imaging in early stage embryos may be best done by injecting aequorin, which circumvents the slow process of coelenterazine entry.     

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.     

Mathematical model for early development of the sea urchin embryo

In Xenopus and Drosophila, the nucleocytoplasmic ratio controls many aspects of cell-cycle remodeling during the transitory period that leads from fast and synchronous cell divisions of early development to the slow, carefully regulated growth and divisions of somatic cells. After the fifth cleavage in sea urchin embryos, there are four populations of differently sized blastomeres, whose interdivision times are inversely related to size. The inverse relation suggests nucleocytoplasmic control of cell division during sea urchin development as well. To investigate this possibility, we developed a mathematical model based on molecular interactions underlying early embryonic cell-cycle control. Introducing the nucleocytoplasmic ratio explicitly into the molecular mechanism, we are able to reproduce many physiological features of sea urchin development.     

Ars insulator protects transgenes from long-term silencing in sea urchin larva

Reporter genes have been used as a powerful tool to analyze cis-regulatory elements responsible for temporal and spatial expression in the early development of sea urchin. However, here we show that the transgenes introduced into the sea urchin embryos undergo suppression in larval stage. The transgene silencing could be one of major obstacle in the analysis of regulatory regions in the late stages of development. We previously demonstrated that a DNA fragment (ArsI) located in the upstream region of sea urchin (Hemicentrotus pulcherrimus) arylsulfatase gene has the property of an insulator. We show that tandem ArsI prevents silencing of a transgene in sea urchin larvae when the ArsI is fused to the 5′ end of the reporter gene. Furthermore, we demonstrate that DNA of the reporter gene introduced into the sea urchin eggs is methylated during development and that the ArsI protects the transgene from the DNA methylation.     

Cryopreservation of sea urchin embryos (Paracentrotus lividus) applied to marine ecotoxicological studies

Current strategies for marine pollution monitoring are based on the integration of chemical and biological techniques. The sea urchin embryo-larval bioassays are among the biological methods most widely used worldwide. Cryopreservation of early embryos of sea urchins could provide a useful tool to overcome one of the main limitations of such bioassays, the availability of high quality biological material all year round. The present study aimed to determine the suitability of several permeant (dimethyl sulfoxide, Me₂SO; propylene glycol, PG; and ethylene glycol, EG) and non-permeant (trehalose, TRE; polyvinylpyrrolidone, PVP) cryoprotectant agents (CPAs) and their combination, for the cryopreservation of eggs and embryos of the sea urchin Paracentrotus lividus. On the basis of the CPAs toxicity, PG and EG, in combination with PVP, seem to be most suitable for the cryopreservation of P. lividus eggs and embryos. Several freezing procedures were also assayed. The most successful freezing regime consisted on cooling from 4 to −12 °C at 1 °C/min, holding for 2 min for seeding, cooling to −20 °C at 0.5 °C/min, and then cooling to −35 °C at 1 °C/min. Maximum normal larvae percentages of 41.5% and 68.5%, and maximum larval growth values of 42.9% and 60.5%, were obtained for frozen fertilized eggs and frozen blastulae, respectively.     

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.     

EFFECTS OF TWO PROTEASE INHIBITORS ON SEA URCHIN AND AMPHIBIAN EGG DEVELOPMENT*

After a review of our present knowledge about the effects of proteases and protease inhibitors on cell growth and egg fertilization, the results of experiments where sea urchin and amphibian eggs were treated with two protease inhibitors (TPCK, TLCK) are described. Cleavage was hardly affected, but gastrulation quickly stopped or was incomplete. The morphogenetic abnormalities which follow can be explained by abnormal gastrulation and other factors: persistence of remnants of the fertilization membrane in sea urchin larvae and dissociation of the ectoderm cells in Xenopus embryos.     

Conservation of the WD-repeat, microtubule-binding protein, EMAP, in sea urchins, humans, and the nematode C. elegans

The echinoderm microtubule-associated protein (EMAP) is the most abundant microtubule-binding protein in the first cleavage mitotic apparatus in sea urchin embryos. The first goal of this study was to determine whether there is sufficient EMAP in the egg and embryo to modify microtubule dynamics during the early cleavages divisions and whether EMAP functions at a specific time or place in the embryo. To accomplish this goal, we examined the relative abundance, tissue distribution, and temporal pattern of EMAP expression during embryonic development. The second goal of this study was to identify important functional domains within the EMAP coding sequence. A conserved sequence might reveal a potential microtubule-binding domain. We cloned, sequenced and compared overlapping EMAP cDNAs from two different sea urchin species that diverged approximately 80 million years ago, and compared these with cDNA sequences from a vertebrate and nematode species. From quantitative immunoblots, we determined the EMAP concentration in eggs to be 4 μM. The steady-state levels of EMAP mRNA and protein accumulated during development, and all three germ layers expressed EMAP. During the early stages of development, EMAP and tubulin were both abundant in the ectoderm, mesoderm and endoderm. However, during late gastrulation and the formation of the early pluteus larvae, EMAP was enriched in the mesoderm, while tubulin staining was most abundant in the archenteron. These results indicate that EMAP may have tissue-specific functions in the late stage embryo. To identify conserved functional domains, we compared the predicted amino acid sequence encoded by Strongylocentrotus purpuratus and Lytechinus variegatus EMAP cDNAs, and determined that these two sea urchin EMAPs were 95% conserved and shared an identical domain organization. A parsimonious analysis of these sea urchin protein sequences, as well as human and C. elegans EMAP sequences was used to construct a gene tree. Together these results suggest that EMAP is an important microtubule protein required at all developmental stages of sea urchins, and whose cellular function may be conserved amongst metazoans. Received: 2 March 1999 / Accepted: 28 June 1999     

Alteration of Ca2+ homeostasis of sea urchin embryos by retinoid CD 367, dual effect on egg cleavage and embryonic development

The effect of a light stable retinoid (CD 367) was studied on sea urchin embryos. CD 367 did not affect sperm-egg interaction. In a range of concentrations between 10 and 100 μM, CD 367 delayed the first and the second cleavages. When added after fertilization, micromolar amounts of CD 367 delayed hatching and produced embryonic abnormalities in a dose-dependent manner. Mesodermal cells, primary (PMC) and secondary (SMC) mesenchyme cells migration was particularly disturbed, leading to exogastrulations and calcified spicules malformations. Concentrations of CD 367 higher than 8 μM were embryolethal. Micromolar amount of CD 367 increased plasmalemma Ca²⁺ permeability of fertilized eggs but not of unfertilized eggs. CD 367 inhibited ATP-dependent intra-cellular sequestration of Ca²⁺ in a range of concentrations similar to those affecting egg cleavage and embryonic structures. Since we were unable to detect nuclear receptors for CD 367 in sea urchin eggs and ovocytes, these effects probably are not related to interaction of the retinoid with members of the RAR family, to which CD 367 has a high affinity, but rather to its toxicity by the means of some unknown mechanisms.     

Variation among females in egg lipid content and developmental success of echinoderms from McMurdo Sound,Antarctica

Egg lipid and protein content of different females of Antarctic echinoderms in McMurdo Sound, Antarctica, were measured to assess variation among females and developmental success. Egg triacylglycerol content of the Antarctic sea urchin Sterechinus neumayeri, when less than 70 ng, correlated with embryos that failed to develop past the 4-day-old blastula stage. In contrast asteroids (Odontaster meridionalis, O. validus, Acodontaster hodgsoni) all produced eggs that developed normally, even with variable egg lipid content. This difference might be related to dietary sources for more herbivorous sea urchins compared to more omnivorous and predatory asteroids. Low egg lipid content, with resulting poor embryonic survivorship, suggests that herbivorous sea urchins may be under unusual levels of nutritional stress in McMurdo Sound during the time frame studied (2004–2005). This nutritional stress might be related to the presence of large icebergs, known to have reduced primary production in the Ross Sea area.     

Relation of Nodal expression to the specification of the dorsal‐ventral axis and tissue patterning in the starfish Patiria pectinifera

In this study, we attempted to reveal fundamental aspects of starfish embryogenesis, particularly embryonic axis specification or determination, in Patiria pectinifera. We first cloned PpNodal, which is known to play an important role in the specification of the embryonic axis in a wide range of animals, and studied its expression profile. PpNodal expression was first detected at the mid‐blastula stage and showed a single peak around the onset of gastrulation. These features of Nodal expression were shifted to later stages by several hours, compared with those of sea urchin embryos. After the gastrulation started, the expression level became gradually lowered up to the early bipinnaria stage, while the expression level became drastically lowered in sea urchin embryos during gastrulation. The localized Nodal expression in the presumptive oral region was not observed in starfish embryos, unlike in sea urchin embryos. Furthermore, SB431542, an inhibitor of Nodal receptor, did not affect the formation of the DV axis, although it caused the loss of left‐right asymmetry. In contrast to this, SB525334, a specific inhibitor of TGF‐beta receptor, caused the complete loss of the DV axis. Thus, the usage of signaling molecules during early embryogenesis likely varies among echinoderm classes.     

Fluorescence staining of living cells with fluorescamine

Summary The interaction of fluorescamine with living plant and animal cells was investigated to determine which subcellular structures and molecular species might react with the dye and to assess its effects on cell viability and function.Plasma and nuclear membranes ofXenopus erythrocytes, mitochondria of sea urchin sperm, growing apices of Timothy root hairs, and various organelles ofNitella andEuglena were labelled as judged by fluorescence microscopy. Cytoplasmic fluorescence was particulate inNitella and easily displaced by moderate centrifugal fields in sea urchin eggs. Chloroplasts and nuclei isolated from cells labelledin vivo exhibited fluorescamine dependent fluorescence.Reaction seemed to have little or no effect on cell viability (Euglena) photoautotrophic growth (Euglena), cell motility (sperm), fertilizability (sperm or egg), embryonic development (sea urchin), or cytoplasmic streaming (Nitella, Timothy).Quantitative fluorometric analysis of thein vivo reactants in sperm indicated a reaction preference for phospholipid over protein compared to control cells dissociated in SDS prior to labelling. The bulk of labelled lipid was phosphatidylethanolamine.These results suggest that fluorescamine is a true vital dye which can label the cell surface as well as penetrate deeply within cells to label a variety of organelles. The distribution of fluorescence and results of chemical analysis suggest thatin vivo the dye may preferentially react with membrane.     

CDK5 is present in sea urchin and starfish eggs and embryos and can interact with p35, cyclin E and cyclin B3

While most cyclin‐dependent kinases (CDKs) are involved in cell cycle control, CDK5 is mostly known for crucial functions in neurogenesis. However, we cloned sea urchin CDK5 from a two‐cell stage cDNA library and found that the protein is present in eggs and embryos, up to the pluteus stage, but without associated kinase activity. To investigate the potential for nonneuronal roles, we screened a starfish cDNA library with the yeast two‐hybrid system, for possible CDK5 partners. Interactions with clones expressing part of cyclin B3 and cyclin E proteins were found and the full‐length cyclins were cloned. These interactions were verified in vitro but not in extracts of starfish oocytes and embryos, at any stages, despite the presence of detectable amounts of CDK5, cyclin B3, and cyclin E. We then looked for p35, the CDK5‐specific activator, and cloned the sea urchin ortholog. A sea urchin‐specific anomaly in the amino acid sequence is the absence of N‐terminal myristoylation signal, but nucleotide environment analysis suggests a much higher probability of translation initiation on the second methionine(Met44), that is associated with a conserved myristoylation signal. p35 was found to associate with CDK5 and, when bacterially produced, to confer protein kinase activity to CDK5 immunoprecipitated from sea urchin eggs and embryos. However, p35 mRNA expression was found to begin only at the end of the blastula stage, and the protein was undetectable at any embryonic stage, suggesting a neuronal role beginning in late larval stages. Mol. Reprod. Dev. 77: 449–461, 2010. © 2010 Wiley‐Liss, Inc.     

CHANGE IN THE ACTIVITY OF LIPASE IN SEA URCHIN EGGS AND EMBRYOS DURING EARLY DEVELOPMENT*

During the early development of the sea urchin, Anthocidaris crassispina, the activity of lipase was maintained at the same level as in unfertilized eggs until the mesenchymal blastula stage (20 hr culture at 20°C) and then increased gradually after gastrulation. The activity in the embryos kept in SO^2?₄-free artificial sea water changed in a similar manner to that in those kept in normal sea water, during the development until 36 hr of fertilization. At 48 hr, the activity in the embryos, which had developed to the permanent blastulae in SO^2?₄-free sea water, was markedly lower than in normal plutei and was similar to that in unfertilized eggs. The lipase activity in fertilized eggs 30 min after fertilization, which was almost the same as that in unfertilized eggs was found mainly to be localized in the precipitate fraction obtained by the centrifugation at 12,000 x g for 20 min, whereas the activity in unfertilized eggs was found in the precipitate by the centrifugation at 105,000 x g for 60 min. Ca²⁺, adenosine 3′, 5′-cyclic monophosphate (cAMP) and guanosine 3′, 5′-cyclic monophosphate (cGMP) had no effect on the lipase activity.     

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.