Ryanodine Activates Sea Urchin Eggs

We have studied the effect on sea urchin eggs of ryanodine, a plant alkaloid that causes muscle contraction by opening calcium channels in the sarcoplasmic reticulum terminal cisternae. Ryanodine, although it is less effective that IP₃, produces full or partial activation in 62% of injected sea urchin eggs. In addition ryanodine inhibits in a dose dependant manner ⁴⁵Ca pumping in the isolated egg cortex or in eggs permeabilized with digitonin. Efflux experiments show that in fact ryanodine as IP₃ stimulates the release of calcium sequestered intracellularly. We further show that these effects of ryanodine are inhibited by Mg⁺⁺, ruthenium red and heparin. Our results suggest that ryanodine-sensitive intracellular calcium channels exist in the sea urchin egg.     

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.     

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.     

Lithium-induced teratogenesis in frog embryos prevented by a polyphosphoinositide cycle intermediate or a diacylglycerol analog

Microinjection of LiCl into prospective ventral blastomeres of the 32-cell Xenopus embryo gives rise to duplication of dorsoanterior structures such as the notochord, neural tube, eyes, and cement gland. We report here that this teratogenic effect of Li+ is prevented by coinjection of equimolar myo-inositol, an intermediate of the polyphosphoinositide cycle. In contrast, epi-inositol, a nonbiological positional isomer of inositol not employed in this cycle, is ineffective at rescuing Li+-injected embryos. Treatment of embryos at stage 7 with the tumor promoter, phorbol myristate acetate (an analog of the polyphosphoinositide cycle-derived second messenger, diacylglycerol), also prevents dorsoanterior duplication of Li+ embryos, while the nontransforming analog, phorbol myristate acetate-4-O-methyl ether, is without effect. Both of these rescuing agents are without obvious effects on development when administered alone (i.e., without Li+). Li+-selective microelectrode measurements demonstrate that intracellular Li+ levels are identical when Li+ is injected with or without myo-inositol. Clonal analysis shows that blastomeres injected with Li+ plus myo-inositol make a normal contribution of progeny to the later embryo. Because Li+ is a well-established inhibitor of the polyphosphoinositide cycle and can thereby have profound effects on cellular myo-inositol and diacylglycerol levels, these observations concerning inositol-mediated rescue suggest a role for altered polyphosphoinositide cycle activity in lithium-induced teratogenesis.     

The Effects of Chemical Animalizing and Vegetalizing Agents and of Cell Dissociation on the Synthesis of 5S RNA and Transfer RNA in Cleaving Sea Urchin Embryos

The effect of altering normal cell associations and interactions on the synthesis of 5S RNA and transfer RNA (tRNA) was studied in cleaving embryos of the sea urchin, Arbacia punctulata. Cell interactions were altered: (1) by culturing cleaving embryos in the animalizing agent, Evans Blue, and in the vegetalizing agent, Li⁺ as LiCl and (2) by culturing dissociated cells. Control and experimental embryos each were labeled from 3 h to 6 h post fertilization with [8-³H]-guanosine. Sixteen-cell embryos, whose GTP precursor pools had been preloaded, were dissociated, labeled and cultured under conditions which prevent reaggregation. Quantitative measurements of rates of accumulation of newly synthesized 5S RNA and tRNA showed that these rates are similar in cleaving sea urchin embryos and in corresponding embryos cultured in the presence of Evans Blue and of Li⁺. In addition, cells dissociated from cleavage embryos and maintained under conditions which prevent reaggregation retained the ability to synthesize 5S RNA and tRNA. These results suggest that normal cell associations and interactions are not necessary for the synthesis of 5S RNA and tRNA to occur in cleaving sea urchin embryos.     

Electrical coupling of blastomeres in early embryos of ascidians and sea urchins

In the early embryos of ascidians and sea urchins, blastomeres are in electrical communication; however, the type and extent of interaction is related to the basic characteristics of the embryo. In the mosaic-like structure of the ascidian embryo, blastomeres have a coupling ratio of about 1 throughout the division cycle. Coupling is facilitated by the extremely low conductance of the non-junctional membrane and possibly mediated via specialized low resistance junctions. Sea urchin embryos do not have specialized low resistance junctions; however, blastomeres are electrically coupled, probably via cytoplasmic bridges, during the first half of the division cycle. The coupling ratio in sea urchins, initially about 0.3, progressively decreases, together with the conductance of the nonjunctional membrane. During the latter half of the division cycle blastomeres are uncoupled; however, a structural junction appears at the periphery, which may play a role in their destiny.     

SCN— Blocks Hardening of the Fertilization Envelope of Sea Urchin Eggs and Adhesion of Blastomeres

Sea urchin eggs kept in artificial sea water (ASW) containing 0.01–0.3 M NaSCN in place of NaCI from within 2 min after insemination formed thin, enlarged fertilization envelopes, which were broken on mild agitation of egg suspensions more easily than those formed in Ca²⁺-free ASW. The blastomeres of almost all embryos derived from eggs treated with 0.2M SCN^— for 1 hr dissociated spontaneously, and did not reassociate with other blastomeres appreciably. Thus SCN^— probably denaturated some compound(s) participating in blastomere binding and hardening of the fertilization envelope. Abnormal arrangements of blastomeres, probably due to incomplete blastomere dissociation, were observed in embryos derived from eggs treated with 0.1 M SCN^— for 1 hr. Treatment of fertilized or unfertilized eggs with 0.05–0.1 M SCN^— for a short period caused concentration-dependent block of morphogenic processes such as formation of the archenteron and pluteus arms in the post-hatching period. The effects of SCN^— on morphogenesis were not inhibited by furosemide or 4,4'-diisothiocyano 2,2'-disulfonic stilbene. Presumably, the denaturation of several compounds in the egg surface by SCN^— causes abnormal morphogenesis of embryos. The inhibitory effects of SCN^— on hardening of the fertilization envelope, blastomere binding and morphogenesis were greater in the absence of Ca²⁺.     

Micromere Differentiation in the Sea Urchin Embryo: Two-Dimensional Gel Electrophoretic Analysis of Newly Synthesized Proteins

A method for large-scale culture of isolated blastomeres of sea urchin embryos in spinner flasks was developed. Micromeres and meso-, macromeres isolated from sea urchin embryos at the 16-cell stage were cultured by this method and the patterns of protein synthesis by their descendants were examined by two-dimensional gel electrophoresis of [³⁵S] methionine-labeled proteins. Six distinct proteins with molecular weights of 140–kDa, 105–kDa, 43–kDa, 32–kDa, and 28–kDa (two components) were specifically synthesized by differentiating micromeres. Quantitative analysis of the two-dimensional gel patterns demonstrated that all these proteins, except the 32–kDa protein, appeared at the time of ingression of primary mesenchyme cells (PMC's) in vivo , several hours earlier than the onset of spicule formation. The synthesis of 32–kDa protein was paralleled to active spicule formation and the uptake of Ca²⁺. Cell-free translation products directed by poly (A)⁺ RNAs isolated from descendant cells of micromeres and meso-, macromeres were compared by two-dimensional gel electrophoresis. Several spots specific to the micromere lineage were detected. However, none of them comigrated with the proteins synthesized specifically by the cultured micromeres. The results suggest that the expression of these proteins specific to differentiating micromeres may involve post-translational modification.     

The potency of the first two cleavage cells in echinoderm development: the experiments of Driesch revisited

A hundred years have passed since Driesch performed the classical experiment of separating sea urchin blastomeres from a two-cell-stage embryo, finding that each developed into a complete though smaller larva. The earlier studies of Roux using frogs showed that inactivating one of the two blastomeres by a heated needle resulted, during the early stages of development, in the formation of a half embryo. In this type of experiment, in which the two blastomeres are not separated, the live blastomere continues its development while it is still attached to an inactivated neighbour. In the work reported here, Roux's experimental design was used on two-cell-stage embryos of sea urchins. In contrast to the findings of Roux using amphibians, it was found (as claimed by Driesch) that the living blastomere developed as in the case of separated blastomeres.     

Role of cell adhesion in the specification of pigment cell lineage in embryos of the sea urchin, Hemicentrotus pulcherrimus

To clarify the role of cell adhesion in the specification of pigment cell lineage in sea urchin embryos, cell contacts were inhibited by Ca²⁺-free artificial seawater (ASW) treatment, and the number of differentiated pigment cells was examined by the method devised for the present study. Obtained results showed that inhibition of cell contacts during mid-to-late blastula stage greatly affects the number of pigment cells. Treatment with Ca²⁺-free ASW during 7.5–10.5h of development drastically decreased the number of pigment cells, indicating that cell adhesion during this period is indispensable for the specification of pigment cell lineage. On the other hand, the number of pigment cells were increased by the treatment during 9.5–12.5 h of development. It was suggested that this increase was caused by excess divisions of the precursor cells, that is, the division schedule of the precursor cells was altered by inhibition of cell contacts at this period. Interestingly, the number of pigment cells was a multiple of four in a majority of embryos in which pigment cells were drastically decreased in number. These findings suggest that the founder blastomeres of the pigment cell lineage are specified during 7–10 h of development, and that these blastomeres divide twice before they differentiate into pigment cells.     

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.     

Type 2 and type 3 inositol 1,4,5-trisphosphate (IP3) receptors promote the differentiation of granule cell precursors in the postnatal cerebellum

During postnatal development of the cerebellum, granule cell precursors (GCPs) proliferate in the external granular layer (EGL), exit the cell cycle, differentiate, and migrate from the EGL to the internal granular layer. In the present study, we report that type 2 and 3 inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R2 and IP₃R3) regulate the differentiation of GCPs after postnatal day 12 (P12). 5-Bromodeoxyuridine labeling experiments revealed that in mutant mice lacking both of these receptors (double mutants) a greater number of GCPs remain undifferentiated after P12. Consequently, the EGL of the double mutants is thicker than that of control mice at this age and thereafter. In addition, granule cells remain in the EGL of the double mutants at P21, an age when migration has concluded in wild-type mice. Whereas differentiation of GCPs was reduced in the double mutants, the absence of IP₃R2 and IP₃R3 did not affect the doubling time of GCPs. We conclude that intracellular calcium release via IP₃R2s and IP₃R3s promotes the differentiation of GCPs within a specific interval of postnatal development in the cerebellum.     

Gastrulation in the sea urchin embryo: a model system for analyzing the morphogenesis of a monolayered epithelium

Processes of gastrulation in the sea urchin embryo have been intensively studied to reveal the mechanisms involved in the invagination of a monolayered epithelium. It is widely accepted that the invagination proceeds in two steps (primary and secondary invagination) until the archenteron reaches the apical plate, and that the constituent cells of the resulting archenteron are exclusively derived from the veg2 tier of blastomeres formed at the 60-cell stage. However, recent studies have shown that the recruitment of the archenteron cells lasts as late as the late prism stage, and some descendants of veg1 blastomeres are also recruited into the archenteron. In this review, we first illustrate the current outline of sea urchin gastrulation. Second, several factors, such as cytoskeletons, cell contact and extracellular matrix, will be discussed in relation to the cellular and mechanical basis of gastrulation. Third, differences in the manner of gastrulation among sea urchin species will be described; in some species, the archenteron does not elongate stepwise but continuously. In those embryos, bottle cells are scarcely observed, and the archenteron cells are not rearranged during invagination unlike in typical sea urchins. Attention will be also paid to some other factors, such as the turgor pressure of blastocoele and the force generated by blastocoele wall. These factors, in spite of their significance, have been neglected in the analysis of sea urchin gastrulation. Lastly, we will discuss how behavior of pigment cells defines the manner of gastrulation, because pigment cells recently turned out to be the bottle cells that trigger the initial inward bending of the vegetal plate.     

Behavior and differentiation process of pigment cells in a tropical sea urchin Echinometra mathaei

The behavior and differentiation processes of pigment cells were studied in embryos of a tropical sea urchin Echinometra mathaei, whose egg volume was one half of those of well-known sea urchin species. Owing to earlier accumulation of pigments, pigment cells could be detected in the vegetal plate even before the onset of gastrulation, distributed dorsally in a hemi-circle near the center of the vegetal plate. Although some pigment cells left the archenteron during gastrulation, most of them remained at the archenteron tip. At the end of gastrulation, pigment cells left the archenteron and migrated into the blastocoele. Unlike pigment cells in typical sea urchins, however, they did not enter the ectoderm, and stayed in the blastocoele even at the pluteus stage. It is of interest that the majority of pigment cells were distributed in the vicinity of the larval skeleton. Aphidicolin treatment revealed that eight blastomeres were specific to pigment cell lineage after the eighth cleavage, one cell cycle earlier than that in well-known sea urchins. The pigment founder cells divided twice, and the number of pigment cells was around 32 at the pluteus stage. It was also found that the differentiation of pigment cells was blocked with Ni2+, whereas the treatment was effective only during the first division cycle of the founder cells.     

Timing of early developmental events in embryos of a tropical sea urchin Echinometra mathaei

Egg volume of a tropical sea urchin Echinometra mathaei is about one half that of other well-known species. We asked whether such a small size of eggs affected the timings of early developmental events or not. Cleavages became asynchronous from the 7th cleavage onward, and embryos hatched out before completion of the 9th cleavage. These timings were one cell cycle earlier than those in well-known sea urchins, raising the possibility that much earlier events, such as the increase in adhesiveness of blastomeres or the specification of dorso-ventral axis (DV-axis), would also occur earlier by one cell cycle. By examining the pseudopodia formation in dissociated blastomeres, it was elucidated that blastomeres in meso- and macromere lineages became adhesive after the 4th and 5th cleavages, respectively. From cell trace experiments, it was found that the first or second cleavage plane was preferentially employed as the median plane of embryo; the DV-axis was specified mainly at the 16-cell stage. Timings of these events were also one cell cycle earlier than those in Hemicentrotus pulcherrimus. The obtained results suggest that most of the early developmental events in sea urchin embryos do not depend on cleavage cycles, but on other factors, such as the nucleo-cytoplasmic ratio.     

Co-option of an oral-aboral patterning mechanism to control left-right differentiation: the direct-developing sea urchin Heliocidaris erythrogramma is sinistralized, not ventralized, by NiCl2

Larval dorsoventral (DV) and left-right (LR) axial patterning unfold progressively in sea urchin development, leading to commitment of the major embryonic regions by the gastrula stage. The direct-developing sea urchin Heliocidaris erythrogramma has lost oral-aboral differentiation along the DV axis but has accelerated vestibular ectoderm development on the left side. NiCl(2) radializes indirect-developing sea urchins by shifting cells toward a ventral fate (oral ectoderm). We treated embryos of H. erythrogramma and the indirect-developing H. tuberculata with NiCl(2). H. tuberculata was ventralized exactly like other indirect developers, establishing that basic patterning mechanisms are conserved in this genus. H. erythrogramma was also radialized; timing, dosage response, and some morphological features were similar to those in other sea urchins. Ectodermal explant and recombination experiments demonstrate that the effect of nickel is autonomous to the ectoderm, another feature in common with indirect developers. However, H. erythrogramma is distinctly sinistralized rather than ventralized, its cells shifting toward a left-side fate (vestibular ectoderm). This geometric contrast in the midst of pervasive functional similarity suggests that nickel-sensitive processes in H. erythrogramma axial patterning, homologous to those in indirect developers, have been redeployed, and hence co-opted, from their ancestral role in DV axis determination to a new role in LR axis determination. We discuss DV and LR axial patterning and their evolutionary transformation.     

POLYSOMES OF THE SEA URCHIN EMBRYO: AN IMPROVED METHOD FOR EXTRACTION OF INTEGRATED POLYSOMES

Suitable conditions for extracting integrated polysomes from embryos of the sea urchins, Hemicentrotus pulcherrimus and Pseudocentrotus depressus were investigated.
Integrated polysomes could not be extracted under the conditions reported by other investigators. It was found, however, that use of 5 m m MgCl₂, 0.30 m KCl, 0.5 m m EDTA and 2 m m cycloheximide was effective for maintaining the integrity of polysomes. At higher concentrations of Mg²⁺, and even at higher concentrations of K⁺, monosomes and polysomes aggregated to form polysome-like particles which had sedimentation patterns with a small amount of nascent peptide. Thus, a medium consisting of 0.05 m Tris-HCl buffer, pH 7.5, 0.30 m KCl. 5 m m MgCl₂, 0.5 m m EDTA-2K, 2 m m cycloheximide, 5 m m mercaptoethanol and 0.5% (v/v) Nonidet P-40 is concluded to be the most suitable for extraction of sea urchin polysomes. Under the conditions used EDTA did not suppress polysome degradation completely and their degradation was linear with time.     

Involvement of Tcf/Lef in establishing cell types along the animal-vegetal axis of sea urchins

Members of the Tcf/Lef family interact with β-catenin to activate programs of gene expression during development. Recently β-catenin was shown to be essential for establishing cell fate along the animal-vegetal axis of the sea urchin embryo. To examine the role of Tcf/Lef in sea urchins we cloned a Strongylocentrotus purpuratus Tcf/Lef homolog. Expression of SpTcf/Lef was maximal when β-catenin became localized to nuclei of vegetal blastomeres, consistent with its acting in combination with β-catenin to specify vegetal cell fates. Expression of a dominant-negative SpTcf/Lef inhibited primary and secondary mesenchyma, endoderm, and aboral ectoderm formation in a manner similar to that observed when nuclear accumulation of β-catenin was prevented. Our results suggest that SpTcf/Lef functions by interacting with β-catenin to specify cell fates along the sea urchin animal-vegetal axis. Received: 6 July 1999 / Accepted: 27 August 1999