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.     

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.     

VEGETALIZATION OF SEA URCHIN LARVAE INDUCED WITH cAMP PHOSPHODIESTERASE INHIBITORS

Treatment of sea urchin embryos with cAMP phosphodiesterase (PDE)-inhibitors such as caffeine (4×10⁻³ M), theophylline (8×10⁻³M), or nicotinamide (10⁻²M), at the morula stage for only a couple of hours, yields vegetalized larvae. Most of the embryos treated with these reagents before the morula stage develop to blastulae filled with mcsenchyme-like cells. Almost all embryos at the blastula stage develop normally even if they are treated with a PDE-inhibitor for a considerable period. The rate of ³H-valine incorporation into protein in the morulae is reduced by caffeine and theophylline, but does not decrease in the presence of nicotinamide. Actinomycin D cancels the vegetalizing effect of PDE-inhibitors on the morulae.     

PREVENTION BY HYDROXYUREA OF VEGETALIZATION OF SEA URCHIN LARVAE INDUCED BY cAMP PHOSPHODIESTERASE INHIBITORS

During 3-hr treatment of the morulae of sea urchin with cAMP phosphodiesterase (PDE) inhibitors, which produce vegetalized larvae, incorporation of ³H-thymidine into DNA occurs at almost the same rate as in control embryos. DNase I digests the newly synthesized DNA in chromatin isolated from morulae treated with PDE-inhibitor (caffeine) faster than that isoloated from normal morulae whereas it dogests DNA isolated from chromatin in caffeine treated embryos at almost the same rate as that in normal embryos. Hydroxyurea, an inhibitor of nucleside diphosphate reductase, prevents the vegetalizing effect of PDE-inhibitor on the development of sea urchin embrys.     

Synthesis of Proteins Enriched in Li+- Induced Vegetalized Embryos of Sea Urchin during Early Development

Sea urchin embryos were vegetalized by a pulse treatment with 60 mM Li⁺ between 2.5 hr and 6 hr after fertilization at 20°C. Normal and Li⁺ -treated embryos were exposed to [³⁵S]-methionine for 2 hr at various stages and [³⁵S]-labeled proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). On fluorograph of 2D-PAGE at the pre-hatching blastula stage, significant difference of labeled proteins between normal and vegetalized embryos was not observed in the range from neutral to acidic pH, but pls of several proteins were found to be shifted toward alkaline pH. At the mesechyme blastula stage, five major proteins [M.W. 36 K, 43 K (two species), 71 K and 150 K] were enriched in Li⁺-treated embryos among a few hundreds of synthesized proteins. At the late gastrula stage, the labeling intensities of these proteins except for one of 43 K proteins increased remarkably in Li⁺-treated embryos. Furthermore, two proteins (M.W. 105 K and 135 K) were also enriched in Li⁺-treated embryos at this stage. At the prism stage, these proteins enriched in Li⁺-treated embryos became hardly detectable, and the synthesis of at least four proteins (M.W. about 20 K, 41 K, 43 K and 200 K<) appeared to increase in normal embryos, but not in Li⁺-treated embryos. Synthesis of proteins eniched in Li⁺-treated embryos probably support endodermal cell differentiation.     

CHANGE IN THE GLYCOGEN CONTENT OF SEA URCHIN EGGS DURING EARLY DEVELOPMENT

During development of eggs of the sea urchins, Pseudocenrotus depressus and Anthocidaris crassispina , the glycogen level is maintained from the time of fertilization to the swimming blastula stage and then decreases rapidly in the early gastrula stage. During development of eggs of Clypeaster japonicus. Hemicentrotus pulcherrimus and Mespilia globulus the glycogen content decreases slowly from the time of fertilization to the mesenchyme blastula stage, and then more rapidly during gastrulation. The amounts of glycogen mobilized in the embryos from the time of fertilization to the morula stage correspond to 67% of the amount of O₂ consumed in Mespilia eggs, 62% in Clypeaster eggs, 30% in Hemicentrotus eggs and 0–4% in Anthocidaris and Pseudocentrotus eggs. The main energy source in early development seems to differ in different species. When eggs and embryos were incubated with [¹⁴C]glucose for 10min, considarable ¹⁴C-radioactivity accumulated in the glycogen fraction. The rate of [¹⁴C]glucose incorporation into glycogen increased gradually during the first 6 hr after fertilization (up to the morula stage), decreases during the next 4 hr (up to the early blastula stage), and then increased again.     

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.     

MORPHOGENETIC SUBSTANCES FOUND IN THE EMBRYOS OF SEA URCHIN, WITH SPECIAL REFERENCE TO THE ANTI-VEGETALIZING SUBSTANCE

When sea urchin embryos up to the 2-cell stage are treated with 5 × 10⁻³ M chloramphenicol for a short period, small blastulae filled with mesenchyme-like cells (type A) are formed. If a homogenate of the embryos up to the 2-cell stage is introduced just after the chloramphenicol treatment, almost all embryos developed to normal plutei. Chloramphenicol treatment, started at the 8 ∽ 32-cell stages, induces vegetalized larvae (type B), and presumptive vegetalized ones develop normally after treatment with a homogenate of the embryos at the 16 ∽ 64-cell stages. If embryos are treated in the same manner at 7 hr after insemination, abnormal embryos which seem to be bipolar ones (type C) are observed at 40 hr after the treatment. These also develop normally, if a homogenate of embryos at the stages from unfertilized egg to gastrula is added to them at the end of the chloramphenicol treatment. The substances having these activities are named morphogenetic substances α, β and γ, respectively. The morphogenetic substance β (anti-vegetalizing substance) is heat stable and its molecular weight is less than 10,000.     

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.     

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.     

Exogut Formation by the Treatment of Sea Urchin Embryos with Ascorbate and α-Ketoglutarate

In sea urchin embryos at the stages from hatch out to the pluteus stage, [¹⁴C]proline incorporation into hot trichloroacetic acid TCA-extractable proteins occurred during an exposure to [¹⁴C]proline for 3 hrs at 20°C. The rate of [¹⁴C]proline incorporation into hot TCA-extractable proteins was higher in gastrulae and plutei than in blastulae. Percentage of [¹⁴C]hydroxyproline residue to whole radioactivity of the hot TCA-extractable proteins was quite low at the blastula stage and increased exponentially during futher development. Production of [¹⁴C]hydroxyproline residue at the blastula stage, as well as at the later stages, was stimulated by ascorbate and α-ketoglutarate, activators of protocollagen proline hydroxylase, and inhibited by α, α'-dipyridyl, an inhibitor of this enzyme. It is also probable that the enzyme in the embryos is not fully activated because of low amounts of activating substances. These suggest that blastulae,…, also have a potency of protocollagen hydroxylation. Blastula kept in sea water containing ascorbateand α-ketoglutarate became undeveloped embryo with large exogut. Gastrula developed normally to pluteus even in the presence of these compounds. The embryos, kept in sea water containing these compounds from fertilization to hatch out, also developed normally. Exogut formation in the embryos treated by these compounds, as well as normal archenteron formation, was inhibited by α, α'-dipyridyl.     

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 Insulin-Binding Capacity of the Plasma Membrane Fraction during Culture in vitro of Cells Derived from Micromeres of 16-Cell-Stage Sea Urchin Embryos

In cultured cells derived from isolated micromeres of 16-cell stage sea urchin embryos, which undergo insulin-induced pseudopodial cable growth, specific and reversible insulin binding by a 52-kDa protein, probably an insulin receptor in the plasma membrane, is augmented during 5 h of culture without any change in the dissociation constant (Kuno et al : 1994). The increase in insulin-binding capacity in micromere-derived cells was only minimally blocked by actinomycin D and cycloheximide, which inhibited [U-³H]uridine incorporation into RNA and [³⁵S]methionine incorporation into protein, respectively. Insulin binding capacity was found in the plasma membrane fraction and the microsome fraction of isolated micromeres. The capacity in the plasma membrane fraction increased, accompanied by its decrease in the microsome fraction, during 5 h of culture of micromere-derived cells. The insulin receptor is probably accumulated in microsomes of presumptive micromeres prior to the 16-cell stage and transferred to the plasma membrane, resulting in an increase in the insulin binding capacity of micromere-derived cells during 5 h of culture.     

SOME OBSERVATIONS OF ABNORMAL EMBRYOS INDUCED BY SHORT-PERIOD TREATMENT WITH CHLORAMPHENICOL DURING EARLY DEVELOPMENT OF SEA URCHIN

Sea urchin embryos, which were treated with 5 × 10⁻³ M chloramphenicol for 1 to 4 hr at certain stages before hatching, developed to several types of abnormal embryos. No significant effect on the shape of the embryo was observed when the concentrations of chloramphenicol used in the short-period treatment were lower than 2 × 10⁻³ M. Embryos up to the 2-cell stage, treated with 5 × 10⁻³ M chloramphenicol for a short period, became small blastulae filled with mesenchyme-like cells (type A). A similar effect of puromycin (2 μg/ml) was also observed at this stage. When the chloramphenicol treatment (for 1 to 4 hr) was applied at 8 ∽ 32-cell stages, vegetalized larvae were produced (type B). Embryos treated with chloramphenicol at 7 hr after insemination at 20°C, developed to another type of abnormal larva different from the previous types (type C). A concentration of puromycin (2 μg/ml) which inhibited protein synthesis to the same degree as 5 × 10⁻³ M chloramphenicol, induced only type A. Between these chloramphenicol-sensitive stages, there were chloramphenicol-insensitive stages for forming abnormal embryos.     

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.     

Probable Contribution of Protein Phosphorylation by Protein Kinase C to Spicule Formation in Sea Urchin Embryos

The formation of spicules and development of pluteus arms in sea urchin embryos were strongly blocked by H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) but were not affected by HA1004 ( N -(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride). Archenteron formation occurred normally in the presence of these compounds. Late gastrulae (28 hr after fertilization) were exposed to ³²Pi for 30 min at 20°C, and then dissociated and their primary mesenchyme cells with spicules, embryo-wall cells and archenteron cells were separated. Then, the radioactivities in the protein fractions of these separated cells were measured. Results showed that culture of embryos with H-7 strongly inhibited ³²p incorporation into proteins in primary mesenchyme cells but caused little inhibition of its incorporations in embryo-wall cells and archenteron cells. The effective concentrations of H-7 for inhibition of ³²p incorporation were within the range that blocked spicule formation and growth of pluteus arms in embryos. HA1004 only slightly inhibited ³²p incorporation into protein in mesenchyme cells, embryo-wall cells and archenteron cells of embryos exposed to ³²Pi. Protein kinase C activity was detectable only in isolated primary mesenchyme cells associated with spicule structures. These suggest that phosphorylation of proteins by protein kinase C contributes to the formation of spicule structures.     

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.     

Animalizing Effect of A23187 on Sea Urchin Embryos

Pulse treatment of sea urchin embryos with 3 μM A23187 for 2 hr starting at a stage in initial 10 hr period of development at 20°C, followed by a culture in normal sea water up to the pluteus corresponding stage (45 hr after fertilization), yielded many large exogastrulae with thin embryo walls. The pulse treatment starting at a time between 10 and 13 hr after fertilization yielded considerable number of large prisms and gastrulae having thin embryo walls. Probably, the pulse treatment exerts stimulating effects on ectodermal cell determination in whole span of pre-hatching period to produce animalized embryos. On the other hand, pulse treatment with A23187 in pre-hatching period exerts stage-specific effects on gut formation. Embryos, thus treated for 2 hr starting at stages between 3 and 5 hr after fertilization, produced quite small exoguts but those treated at stages between 7 and 8 hr formed well developed and long exoguts. In embryos treated at the other stages than above, guts or exoguts were almost the same in their size to those in normal ones. These effects of A23187 on morphogenesis were canceled by procaine, tetracaine and ruthenium red. Probably, artificial Ca²⁺signal induced by A23187 alters the determination of cell fates, programmed in pre-hatching period.     

Synthesis of Collagen-Like Proteins in Embryonic Organs of the Sea Urchin, Hemicentrotus pulcherrimus

In sea urchin embryos exposed to ¹⁴C-proline at 20°C for 3 hr at the gastrula, prism or pluteus stage, ¹⁴C-radioactivity was found in hot acid-extractable proteins, in which more than 4% of the radioactivity was detectable in hydroxyproline residues. In these embryos, ¹⁴C-radioactivity in collagen-like proteins was found in the archenteron, spicule and embryo-wall cells. The rate of synthesis of collagen-like proteins was highest in the archenteron in the mid-gastrula stage, in the embryo-wall cells in the prism stage and in the spicule in the pluteus stage. The rate of synthesis decreased in the archenteron and increased in embryo-wall cells in the period between the mid- and late-gastrula stages, when the rate of synthesis in the spicule was quite low. Thereafter, the rate decreased slightly in the embryo-wall cells, was maintained in archenteron and increased markedly in the spicule. The rates of synthesis of collagen-like proteins are high in these embryonic organs at stages at which development and growth respectively, occur in embryos. Therefore, synthesis of collagen-like proteins probably supports morphogenesis in these embryonic organs.     

STUDIES ON THE POLYSACCHARIDE-SULFATING SYSTEM IN SEA URCHIN EMBRYOS

The sulfating system in sea urchin embryos was examined, using the labeled precursor inorganic [³⁵S]sulfate in vivo and [³⁵S]3'-phosphoadenosine 5'-phosphosulfate ([³⁵S]PAPS) in a cell-free system. In vivo incorporation of [³⁵pS]sulfate into the trichloroacetic acid (TCA)-insolubte fraction increased gradually during sea urchin development, whereas radioactivity of [³⁵S]sulfate contained in the TCA-soluble fraction showed a conspicuous peak at the late gastrula stage.
In a cell-free system, the particulate fraction showed marked incorporation of [³⁵pS]JPAPS. This sulfating activity was highest at pH 6.4 to 7.2 and at 27°C, and it was strongly inhibited by Hg ²⁺and p-chloromercuribenzoic acid.
The sulfating activity was quite low in fertilized eggs, but then increased rapidly up to the swimming blastula stage. The activity in the particulate fraction precipitated at 10,000 xg increased gradually and that in the particulate fraction precipitated at 100,000 xg was almost constant from the swimming blastula stage to the pluteus stage.