Expression of Na+, K+-ATPase α-Subunit in Animalized and Vegetalized Embryos of the Sea Urchin, Hemicentrotus pulcherrimus.

A marked increase in the Na⁺, K⁺-ATPase activity of sea urchin embryos occurred following an elevation of its mRNA level, revealed by Northern blotting analysis, in developmental period between the swimming blastula and the late gastrula stage. cDNA clone of Na⁺, K⁺-ATPase α-subunit, obtained from γgt10 cDNA library of sea urchin gastrulae, was digested with EcoRl ad Hindlll. The obtained 268 bp cDNA fragment, hybridized to a 4.6 Kb RNA, was used as probe for Northern blotting analysis. The level of Na⁺, K⁺-ATPase mRNA was higher in embryo-wall cell fraction isolated from late gastrulae (ectoderm cells) than the level in the bag fraction, containing mesenchyme cells (mesoderm cells) and archenteron (endoderm cells). The activity of Na⁺, K⁺-ATPase and the level of its mRNA were higher in animalized embryos obtained by pulse treatment with A23187 for 3 hr, starting at the 8–16 cell stage and were considerably lower in vegetalized embryos induced by 3 hr treatment with Li⁺ than that in normal embryos at the post gastrula corresponidng stage. Augmentation of Na⁺, K⁺-ATPase gene expression can be regarded as a marker for ectoderm cell differentiation at the post gastrula stage, which results from determination of cell fate in prehatching period.     

Change in the Activity of Na1, K+-ATPase in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus, during Early Development

The activity of ouabain-sensitive Na⁺, K⁺-ATPase in sea urchin embryos at the morula and the swimming blastula stage was practically the same to that in unfertilized eggs. The activity increased during the period between the mesenchyme blastula and the late gastrula stages. In embryo-wall cell fraction, which contained presumptive ectodermal cells as well as those of other cell lineages at the pre-gastrula stage and ectodermal cells at the late gastrula stage, the Na⁺, K⁺-ATPase activity increased in this developmental period more largely than in another cell fraction, containing mesenchyme cells and archenteron cells. Cycloheximide did not only block the activity increase in this period but also caused evident decrease in the activity in embryos at all examined stages. The activity increase in this period was strongly blocked by the treatment with actinomycin D, starting before the mesenchyme blastula stage, and was not seriously inhibited by the treatment starting at the mesenchyme blastula stage. The treatment starting at the initiation of gastrulation only slightly blocked further increase in the activity. Probably, an accumulation of mRNA encoding Na⁺, K⁺-ATPase occurs mainly in ectodermal cells and is completed up to the early gastrula stage.     

Timers in Early Development of Sea Urchin Embryos

To elucidate the timing mechanisms in the early development of sea urchin embryos, we measured the times of initiation of the first four cleavages, of ciliary movement, of primary mesenchyme cell ingression, and of gastrulation at four temperatures ranging from 11 to 20°C. The times of cleavage and of initiation of ciliary movement showed similar temperature dependency, indicating that these events may be controlled by a common timer (the first timer). Although batches of eggs often showed variation in the period between fertilization and the first cleavage, their subsequent cleavages were more regular. This indicates that the first timer may not start at fertilization. The ingression of mesenchyme cells and the onset of gastrulation showed similar temperature dependency that was higher than that of other events, suggesting the existence of a second timer. Temperature shift experiments indicate that the second timer starts at the mid-blastula (the 8–9th cleavage) stage when divisions of blastomeres become asynchronous.     

Development of Sea Urchin Embryos in Artificial Sea Water Containing Br−in Place of Cl−

In artificial sea water in which the Cl⁻concentration was reduced to less than 10% of that in normal sea water by its replacement with Br⁻, sea urchin eggs were fertilized and developed into abnormal plutei following almost the same time schedule as in natural sea water. These embryos had poorly developed spicules, short pluteus arms, somewhat jagged embryo-walls and quasi-normal archenterons. Similar embryos were obtained in another artificial sea water in which 90% of the Cl⁻concentration in normal sea water was reduced by Br⁻and 10% by acetate. In artificial sea water, in which either 90% of the Cl⁻was replaced by Br⁻or 10% was replaced by acetate, embryos developed into plutei with quasi-normal spicules, pluteus arms and archenterons. These findings indicate that deficiency of Cl⁻results in somewhat abnormal sea urchin embryos. When cells derived from isolated micromeres, were cultured in these Cl⁻-deficient artificial sea waters, containing Br⁻in place of more than 70% of the normal Cl⁻concentration in sea water, spicule formation was strongly inhibited, but pseudopodial cables were well developed. Thus, external Cl⁻seems to be necessary for at least normal formation of spicule rods.     

Stage Specific Effects on Sea Urchin Embryogenesis of Zn2+, Li+, several Inhibitors of cAMP-Phosphodiesterase and Inhibitors of Protein Synthesis

Treatment of sea urchin embryos for 3 hr starting at the 16-64 cell stage with Li⁺ or 3-isobutyl-1-methylxanthine as well as with other inhibitors of cAMP-phosphodiesterase (PDE) and several inhibitors of protein synthesis, resulted in production of vegetalized embryos with a large exogut. However, the same treatment starting at other stages produced hardly any vegetalized embryos. The specific stage for these substances to cause vegetalization is probably the 16-64 cell stage. Treatment with Zn²⁺ between the times of fertilization and hatching, followed by culture in normal sea water produced animalized embryos with little if any archenteron, but the same treatment followed by culture with ethylenediamine-N, N'-diacetic acid (EDDA), a chelator of Zn²⁺, produced quasi-normal plutei. This chelator did not counteract the animalizing effect of Zn²⁺ when culture with EDDA was started at the post-gastrula stage. Treatment of embryos for a long period (1-3 days) starting at the blastula stage with Li⁺ and the inhibitors of PDE and protein synthesis, as well as with Zn²⁺, produced spherical embryos with little or no archenteron. The stages at which these substances produced abnormal embryos with a poor archenteron are post-hatching stages.     

Ribosomal RNA Synthesis in Sea Urchin Embryos: Differential Rates of Accumulation in Chemically-induced Animalized and Vegetalized Larvae

Animalization was induced with evans blue and with Zn⁺⁺ in embryos of Arbacia punctulata and of Lytechinus variegatus , respectively. Li⁺ induced vegetalization in A. punctulata embryos. While animalization did not affect the rate of cleavage, vegetalized embryos exhibited a reduction in cell number at post-morula stages. Mid-gastrulae and corresponding experimental embryos each were labeled for 4 hr with uridine-[5-³H] and with L-[³H-methyl]-methionine. The rate of uptake of each exogenous RNA precursor was similar in control and in experimental embryos. Purified RNA preparations were fractionated by electrophoresis on 2.4% acrylamide+0.5 % agarose gels. Comparison of rates of incorporation of each RNA precursor into 26s and 18s RNAs indicated that on a per cell basis evans blue- and Zn⁺⁺-animalized embryos showed a reduction (0.53–0.56) and Li⁺-vegetalized embryos an enhancement (1.41—1.53) in the rate of accumulation of newly made 26s and 18s RNAs compared to controls (1.00). These results suggest that chemically-induced animalized and vegetalized embryos provide useful tools for studying possible differential gene expression in different embryonic germ layers of the developing sea urchin embryo.     

Changes in Ribosome-associated Proteins during Sea Urchin Development

Ribosomes isolated from unfertilised eggs of the sea urchin, Strongylocentrotus purpuratus , have a higher protein: RNA ratio than ribosomes extracted from blastula stage ribosomes. Approximately 64 additional protein equivalents are found per ribosome. Most of the proteins are of high molecular weight and are tightly bound, being resistant to high-salt and EDTA treatment. The majority of the proteins appear to be basic in nature and remain associated with the 40S subunit on dissociation of the ribosomes. The possible physiological significance of the additional proteins is discussed in terms of the activation of protein synthesis following fertilisation. Sea urchin ribosomes, isolated from various stages of development, showed differential protein-labelling patterns. The high molecular-weight proteins had preferentially higher specific activities and one ribosomal protein was particularly highly labelled, reaching a maximum at the gastrula stage of development. The functional role of this highly labelled protein during development is discussed.     

Probable Role of Allylisothiocyanate-Sensitive H+, K+-ATPase in Spicule Calcification in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus

In embryos of the sea urchin, Hemicentrotus pulcherrimus , as well as in cultured cells derived from isolated micromeres, spicule formation was inhibited by allylisothiocyanate, an inhibitor of H⁺, K⁺-ATPase, at above 0.5 μM and was almost completely blocked at above 10 μM. Amiloride, an inhibitor of Na⁺, H⁺ antiporter, at above 100 μM exerted only slight inhibitory effect, if any, on spicule formation. Intravesicular acidification, determined using [ dimethylamine -¹⁴C]-aminopyrine as a pH probe, was observed in the presence of ATP and 200 mM KCl in microsome fraction obtained from embryos at the post gastrula stage, at which embryos underwent spicule calcification. Intravesicular acidification and K⁺-dependent ATPase activity were almost completely inhibited by allylisothiocyanate at 10 μM. Allylisothiocyanate-sensitive ATPase activity was found mainly in the mesenchyme cells with spicules isolated from prisms. H⁺, K⁺-ATPase, an H⁺ pump, probably mediates H⁺ release to accelerate CaCO₃ deposition from Ca²⁺, CO₂ and H₂O in the primary mesenchyme cells. Intravesicular acidification was stimulated by valinomycin at the late gastrula and the prism stages but not at the pluteus stage. K⁺ permeability probably increases after the prism stage to activate H⁺ release.     

Proteins ADP-ribosylated in Nuclei and Plasma Membrane Vesicles Isolated from Sea Urchin Embryos at Various Stages of Early Development

The ADP-ribosylations of proteins in nuclei, plasma membrane vesicles, mitochondria, microsome vesicles and the soluble fraction of sea urchin embryos isolated at various stages of development were examined by measuring the radioactivities of proteins after exposure of these subcellular fractions to [adenosine-¹⁴C]NAD or [adenylate-³²P]NAD. ADP-ribosylation of proteins was detected only in the nuclear and plasma membrane fractions. In the nuclear fraction, the rate of ADP-ribosylation of the histone fraction did not change appreciably during early development. In the TCA-insoluble protein fraction of the nuclei, the rate of ADP-ribosylation increased from fertilization to the morula stage, then decreased and again increased from the mesenchyme blastula to the late gastrula stage. After exposure of the nuclear fraction to [adenylate-³²P]NAD, a protein band with a molecular weight of 90 kDa was detected by SDS-polyacrylamide gel electrophoresis and radioautography at all stages examined. Its labeling intensity indicated that its ADP-ribosylation is higher at the morula and late gastrula stages than at other stages. In the plasma membrane fraction, proteins with molecular weights of 22 and 68 kDa were ADP-ribosylated and their rates of ADP-ribosylation hardly changed during early development.     

Vegetalization Induced by Procaine and Tetracaine in Sea Urchin Embryos

Vegetalization of sea urchin embryos was induced by the treatment with procaine and tetracaine, inhibitors of Ca²⁺mobilization, for 3 hr starting 3–5 hr after insemination at 20°C. The treatment starting 7 hr after insemination sometimes produced similar type of vegetalized embryos. The pulse treatment starting at the other stages hardly yielded vegetalized embryos. The stages at which these compounds were effective to produce vegetalized embryos were almost the same to those for Li⁺to make embryos vegetalized. On the basis of known inhibitory effects of tetracaine, procaine and Li⁺on Ca²⁺mobilization, we postulate that Ca²⁺dependent reactions participate in the process of cell determination at these stages. Inhibitory effects of procaine, tetracaine and Li⁺on Ca²⁺dependent induction of fertilization membrane formation, found in the present study, indicate that these compounds block Ca²⁺mobilization in sea urchin eggs.     

Stage Specific Effects of Zn2+ on Sea Urchin Embryogenesis

The treatment of sea urchin embryos by Zn²⁺ followed by culture with Zn²⁺-specific chelators such as ethylenediamine-N, N'-diacetic acid and N-hydroxyethylethylenediamine-N, N', N'-triacetic acid, was performed at various developmental stages to find out specific stages for Zn²⁺ to induce abnormal differentiation. The treatment with 1 mM ZnSO₄ at 20°C during a period including two spans of development between 0 and 8 hr and between 14 and 16 hr post fertilization yielded permanent blastulae. Zn²⁺-treatment during the former span produced abnormal prisms and plutei with small archenteron. The treatment for a period including only the latter span failed to produce abnormal ones. Zn²⁺-treatment during a period including the gastrula stage also produced abnormal spherical embryos. Without the culture with these chelators, abnormal embryos were produced by Zn²⁺-treatment performed at any stages before gastrulation. A high zinc amount in the embryos just after the treatment became as low as in normal embryos soon after the culture with these chelators and was maintained during the culture without them. These results indicate that zinc retention occurs in the Zn²⁺-treated embryos and causes abnormal differentiation when the treated embryos develop in normal sea water through the Zn²⁺-specific periods of development.     

Change in the Triglyceride Level in Sea Urchin Eggs and Embryos During Early Development

Triglycerides in the embryos of the sea urchin, Anthocidaris crassispina , analyzed by gas-liquid chromatography, distributed in a range of carbon numbers between 42 and 58 in the sum of three fatty acid residues. During the development until gastrulation, the levels of triglycerides with 48, 56 and 58 carbon numbers decreased at constant rates and the levels of the others decreased at specific stages different with one another, respectively. Thereafter, the amounts of all triglycerides decreased simultaneously. The amount of oxygen consumed in the embryos is enough for the oxidation of mobilized triglycerides during post-hatching period but is not during pre-hatching period. The levels of neutral glycerides increased gradually during pre-hatching period and thereafter decreased. The fatty acid level also increased during pre-hatching and post-hatching period. These suggest that the cleavage of triglycerides and the oxidation of their cleavage-products occur during whole span of early development. During pre-hatching period, the break down of triglycerides is probably higher in its rate than the rate of their oxidation, resulting in the increase in the levels of neutral glycerides, as well as fatty acids.     

Effect of α,α'-dipyridyl on Exogut Formation in Vegetalized Embryos of the Sea Urchin

In presumptive vegetalized embryos, obtained by 3-hr treatment with chloramphenicol at the 16–32 cell stage, the rates of [¹⁴C]proline incorporation into the collagen fraction and production of the [¹⁴C]hydroxyproline residues increased during development between 16 hr (equivalent to mesenchyme blastula stage) and 40 hr (the early pluteus stage) after fertilization at 20°C. In presumptive vegetalized embryos, the radioactivity of [¹⁴C]hydroxyproline residues was higher at the mesenchyme blastula stage (16 hr after fertilization), but lower at the post-gastrula stage than in normal embryos. In normal embryos at the post-gastrula stage, [¹⁴C]hydroxyproline residues were mainly found in isolated spicules, and the amounts of [¹⁴C]hydroxyproline residues in other parts were much lower than in vegetalized embryos, which had few, if any, spicules. α, α'-Dipyridyl, an inhibitor of prolyl hydroxylase, inhibited the hydroxylation of [¹⁴C]proline residues in presumptive vegetalized and normal embryos, and blocked the formation of the archenteron and exogut.     

Changes in the Activities of H+, K+-ATPase and Na+, K+-ATPase in Cultured Cells Derived from Micromeres of Sea Urchin Embryos with Special Reference to Their Roles in Spicule Rod Formation

In cultured cells derived from micromeres isolated at the 16-cell stage of sea urchin embryos, the activity of H⁺, K⁺-ATPase became detectable after 15 hr of culture, when the cells started to form spicules, and then increased reaching a plateau from 25 hr of culture. The Na⁺, K⁺-ATPase activity of isolated micromeres increased to a maximum at 20 hr of culture and thereafter decreased gradually. Allylisothiocyanate, an inhibitor of H⁺, K⁺-ATPase, caused a decrease in intracellular pH (pHi) accompanied by blockage of ⁴⁵Ca deposition in spicule rods in spicule-forming cells at 30 hr of culture. Ouabain and amiloride had scarcely any effect on the pHi or ⁴⁵, deposition. In cultured cells exposed to nifedipine, which blocked ⁴⁵Ca deposition in spicule rods, allylisothiocyanate did not cause any decrease in pHi. These results show that H⁺, which is generated in the overall reaction to produce CaCO₃ from Ca²⁺ and HCO₃⁻, is probably released from the cells mainly in the reaction catalyzed by H⁺, K⁺-ATPase to maintain successive production of CaCO₃.     

Regulation of Microtubule Synthesis and Utilization during Early Embryonic Development of the Sea Urchin

Microtubules deployed during early development of the sea urchinembryo are derived both from a preexisting pool of subunitspresent in the egg and from microtubule protein subunits synthesizedin the embryo. Several aspects of microtubule protein synthesisand utilization are reviewed. Microtubule protein synthesisin early development utilizes oogenetic messenger RNA species.Translation of this mRNA is under regulation. Microtubule proteinsynthesis rises concomitantly with overall protein synthesisat fertilization, but rises at a relatively higher rate laterin cleavage stages. Microtubule protein labeled with [³H]-leucinein early development is incorporated into cilia, indicatingthat newly synthesized protein enters the pool of subunits usedin organelle assembly. The microtubule protein pool comprisesabout 1%of the soluble protein of the egg, and remains constantin size at least until the blastula stage. Direct pool sizeestimates are consistent with results of experiments on recruitmentof microtubule protein subunits into the mitotic apparatus andinto regenerating cilia. Soluble and particulate colchicinebinding fractions, which have been reported from several systems,appear to be present in sea urchin embryos. The possible roleof such fractions are discussed, as are aspects of the regulationof ciliary assembly.     

Inhibitory Effect of Omeprazole, a Specific Inhibitor of H+, K+-ATPase, on Spicule Formation in Sea Urchin Embryos and in Cultured Micromere-Derived Cells.

Embryos kept with omeprazole, a specific H⁺, K⁺-ATPase inhibitor, in a period of development between the mesenchyme blastula and the pluteus corresponding stage became abnormal plutei having quite small spicules, somewhat poor pluteus arms and apparently normal archenterons. In micro-mere-derived cells, kept with omeprazole at pH 8.2 in a period between 15 and 40 hr of culture at 20°C, omeprazole strongly inhibited spicule formation but did not block the outgrowth of pseudopodial cables, in which spicule rods were to be formed. These indicate that omeprazole probably exerts no obvious inhibitory effects other than spicule rods formation. Omeprazole-sensitive H⁺, K⁺-ATPase, an H⁺pump, seems to be indispensable for CaCO₃ deposition (formation of spicule rod) in these spicule forming cells. H⁺, produced in overall reaction for CaCO₃ formation: Ca²⁺+ CO₂+H₂O°CaCO₃+2H⁺, is probably released from the cells by this H⁺pump and hence, this reaction tends to go to CaCO₃ production to form spicule rods. Omeprazole, known to become effective following its conversion to a specific inhibitor of H⁺, K⁺-ATPase at acidic pH, is able to inhibit formation of spicule rod at alkaline pH in sea water. This is probably due to an acidification of sea water near the cell surface by H⁺ejection in H⁺, K⁺-ATPase reaction.     

Mg2+- or Ca2+-Activated ATPase in Squid Giant Fiber Axoplasm

A divalent cation-activated ATPase in axoplasm from the squid giant axon is described. The enzyme requires Mg2+ or Ca2+, has a K+ optimum of 60 mM, and has a pH optimum of 7.5. Several nucleotide triphosphates other than ATP can serve as substrates. The enzyme is inhibited by excess ATP or Mg2+. The enzyme is enriched in a rapidly sedimenting fraction of the axoplasm, and is eluted in the exclusion volume of a Sepharose 4B column, suggesting that it is associated with a highly aggregated structure. Comparison of the properties of enzyme with those of myosin and Na+-K+-ATPase suggests that differs from both of these enzymes. The enzyme has many similarities to vertebrate nerve ATPases previously described. The demonstration of the presence of this ATPase in squid axoplasm proves the neuronal localization of the enzyme.     

Excretion of ions (Cd2+, Li+, Na+ and Cl−) by Tamarix aphylla

Excretion of minerals by the NaCl-resistant and comparatively cadmium-resistant tree Tamarix aphylla (L.) Karst, was investigated. Cd²⁺ was excreted by plants exposed for 1–10 days to 9 or 45 μ M Cd²⁺ solutions. Excretion of this toxic ion increased considerably with time but was less than 5% of the quantities that had been accumulated in the shoots. Excretion of Na⁺ and Cl⁻ was positively correlated with NaCl concentration (1.5, 10, 50 m M ) of the medium. The Na⁺/Cl⁻ ratios of the excrete were positively correlated with the concentration of the treatment solution. Ca²⁺ excretion decreased with increasing NaCl concentrations of the solution. Excretion of K⁺ and Mg²⁺ was only little affected by NaCl. Excretion of Li⁺ occurred whenever this element was supplied in the uptake solution; daily excretion rates of Li⁺ increased with time. The ecological significance of excretion is discussed in relation to the low selectivity of the mechanism in T. aphylla .