Ascidian Eggs Research Articles

Mitochondria Required for Second Calcium Waves

Ascidian oocytes are a model system used to study the signaling events associated with fertilization and completion of meiosis. The sperm triggers a first set of calcium oscillations by the first pacemaker (PM1) upon fertilization, and then the polar body is released. A second set of calcium waves is initiated by pacemaker 2 (PM2) from the region in the vegetal pole where there is a dense network of endoplasmic reticulum (ER) and mitochondria. Pacemakers are defined as the site of the source of the calcium oscillation. Dumollard et al . showed that during the calcium oscillations, mitochondrial respiration was increased. Treatment of the eggs with mitochondrial respiration inhibitors or uncouplers showed that PM2 activity required adenosine-triphosphate (ATP)-generating mitochondria that could accumulate calcium. The PM2-initiated calcium waves could be partially restored by release of caged ATP to restore ATP levels when the mitochondria were inhibited. The authors propose that there is an intimate connection physically and biochemically between the ER and mitochondria in ascidian eggs that controls the calcium oscillations produced by PM2. R. Dumollard, K. Hammar, M. Porterfield, P. J. Smith, C. Cibert, C. Rouvière, C. Sardet, Mitochondrial respiration and Ca 2+ waves are linked during fertilization and meiosis completion. Development 130 , 683-692 (2003). [Abstract] [Full Text]

Spermosin, a trypsin‐like protease from ascidian sperm

We have previously reported that two trypsin-like enzymes, acrosin and spermosin, play key roles in sperm penetration through the vitelline coat of the ascidian (Urochordata) Halocynthia roretzi [Sawada et al. (1984), J. Biol. Chem. 259, 2900-2904; Sawada et al. (1984), Dev. Biol. 105, 246-249]. Here, we show the amino-acid sequence of the ascidian preprospermosin, which is deduced from the nucleotide sequence of the isolated cDNA clone. The isolated ascidian preprospermosin cDNA consisted of 1740 nucleotides, and an open reading frame encoding 388 amino acids, which corresponds to a molecular mass of 41 896 Da. By sequence alignment, it was suggested that His178, Asp230 and Ser324 make up a catalytic triad and that ascidian spermosin be classified as a novel trypsin family member. The mRNA of preprospermosin is specifically expressed in ascidian gonads but not in other tissues. Purified spermosin consists of 33- and 40-kDa bands as determined by SDS/PAGE under nonreducing conditions. The 40-kDa spermosin consists of a heavy chain (residues 130-388) and a long light chain designated L1 (residues 23-129), whereas the 33-kDa spermosin includes the same heavy chain and a shorter light chain designated L2 (residues 97-129). The L1 chain contains a proline-rich region, designated L1(DeltaL2) which is lacking in L2. Investigation with the glutathione-S-transferase (GST)-spermosin-light-chain fusion proteins, including GST-L1, GST-L2, and GST-L1(DeltaL2), revealed that the proline-rich region in the L1 chain binds to the vitelline coat of ascidian eggs. Thus, we propose that sperm spermosin is a novel trypsin-like protease that binds to the vitelline coat and also plays a part in penetration of sperm through the vitelline coat during ascidian fertilization.

Sperm surface proteases in ascidian fertilization.

Ascidian eggs are surrounded by a noncellular layer and two cellular layers, which are penetrated by sperm. Three sperm surface proteases are essential for fertilization of eggs from the stolidobranch ascidian Halocynthia: spermosin, acrosin, and the proteasome. In the phlebobranch Ciona, a chymotrypsin-like protease and the proteasome are essential in fertilization. Sperm from the phlebobranch ascidians Phallusia mammillata, Ascidia (=Phallusia) nigra, and Ascidia columbiana, all express spermosin, acrosin, and the proteasomal chymotrypsin activities on their surfaces. Chymostatin blocks cleavage in phlebobranchs, but inhibitors of spermosin and acrosin only delay it by several minutes. Protease inhibitors have little effect upon sperm binding in Phallusia but strongly affect the rate of sperm passage through the vitelline coat. Peptide substrates and inhibitors to spermosin and acrosin cause a significant decline in the number of eggs undergoing pre-meiotic contractions at 3 min after fertilization. Thus while chymotrypsin activity is essential for penetration of the vitelline coat, spermosin and acrosin both function to increase the rate of fertilization. A crucial step in the divergence of the phlebobranchs and stolidobranchs may have been the conversion of spermosin and acrosin to essential proteases in the stolidobranchs, or, perhaps, their essential function was lost in the evolution of phlebobranchs. Aplousobranch ascidians are all colonial with very small zooids. Sperm from Aplidium californicum, Aplidium solidum (Polyclinidae), and Distaplia occidentalis (Holozoidae) have acrosin and chymotrypsin activities but lack spermosin activity. This enzyme is also missing from sperm of colonial phlebobranch and stolidobranch ascidians, suggesting that spermosin is not necessary for small zooids with internal fertilization.

Large-scale cDNA analysis of the maternal genetic information in the egg of Halocynthia roretzi for a gene expression catalog of ascidian development.

The ascidian egg is a well-known mosaic egg. In order to investigate the molecular nature of the maternal genetic information stored in the egg, we have prepared cDNAs from the mRNAs in the fertilized eggs of the ascidian, Halocynthia roretzi. The cDNAs of the ascidian embryo were sequenced, and the localization of individual mRNA was examined in staged embryos by whole-mount in situ hybridization. The data obtained were stored in the database MAGEST (http://www.genome.ad.jp/magest) and further analyzed. A total of 4240 cDNA clones were found to represent 2221 gene transcripts, including at least 934 different protein-coding sequences. The mRNA population of the egg consisted of a low prevalence, high complexity sequence set. The majority of the clones were of the rare sequence class, and of these, 42% of the clones showed significant matches with known peptides, mainly consisting of proteins with housekeeping functions such as metabolism and cell division. In addition, we found cDNAs encoding components involved in different signal transduction pathways and cDNAs encoding nucleotide-binding proteins. Large-scale analyses of the distribution of the RNA corresponding to each cDNA in the eight-cell, 110-cell and early tailbud embryos were simultaneously carried out. These analyses revealed that a small fraction of the maternal RNAs were localized in the eight-cell embryo, and that 7.9% of the clones were exclusively maternal, while 40.6% of the maternal clones showed expression in the later stages. This study provides global insights about the genes expressed during early development.

Three different calcium wave pacemakers in ascidian eggs

Calcium wave pacemakers in fertilized eggs of ascidians and mouse are associated with accumulations of cortical endoplasmic reticulum in the vegetal hemisphere. In ascidians, two distinct pacemakers (PM1 and PM2) generate two series of calcium waves necessary to drive meiosis I and II. Pacemaker PM2 is stably localized in a cortical ER accumulation situated in the vegetal contraction pole. We now find that pacemaker PM1 is situated in a cortical ER-rich domain that forms around the sperm aster and moves with it during the calcium-dependant cortical contraction triggered by the fertilizing sperm. Global elevations of inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) levels produced by caged Ins(1,4,5)P3 or caged glycero-myo-PtdIns(4,5)P2 photolysis reveal that the cortex of the animal hemisphere, also rich in ER-clusters, is the cellular region most sensitive to Ins(1,4,5)P3 and acts as a third type of pacemaker (PM3). Surprisingly, the artificial pacemaker PM3 predominates over the natural pacemaker PM2, located at the opposite pole. Microtubule depolymerization does not alter the activity nor the location of the three pacemakers. By contrast, blocking the acto-myosin driven cortical contraction with cytochalasin B prevents PM1 migration and inhibits PM2 activity. PM3, however, is insensitive to cytochalasin B. Our experiments suggest that the three distinct calcium wave pacemakers are probably regulated by different spatiotemporal variations in Ins(1,4,5)P3 concentration. In particular, the activity of the natural calcium wave pacemakers PM1 and PM2 depends on the apposition of a cortical ER-rich domain to a source of Ins(1,4,5)P3 production in the cortex. Movies available on-line

Macho-1 encodes a localized mRNA in ascidian eggs that specifies muscle fate during embryogenesis.

Maternal information stored in particular regions of the egg cytoplasm has an important function in the determination of developmental fate during early animal development. Ascidians show mosaic development; such autonomous development has been taken as evidence that prelocalized ooplasmic factors specify tissue precursor cells during embryogenesis. Interest has been concentrated on the mechanisms underlying the formation of muscle cells in the tail, as yellow-coloured myoplasm in eggs is preferentially segregated into muscle-lineage blastomeres. Here we show that maternal messenger RNA of the macho-1 gene is a determinant of muscle fate in the ascidian Halocynthia roretzi. The macho-1 mRNA encodes a zinc-finger protein, and the mRNA is localized to the myoplasm of eggs. Depletion of the mRNA specifically resulted in the loss of primary muscle cells in the tail, as shown by the expression of muscle-specific molecular markers. The myoplasm of macho-1-deficient eggs lost its ability to promote muscle formation. Injection of synthesized macho-1 mRNA caused ectopic muscle formation in non-muscle-lineage cells. Our results indicate that macho-1 maybe both required and sufficient for specification of muscle fate, and that the mRNA is a genuine, localized muscle determinant.

CDNA Cloning and Functional Analysis of Ascidian Sperm Proacrosin

cDNA cloning and functional analysis of proacrosin from the ascidian Halocynthia roretzi were undertaken. The isolated cDNA of the ascidian preproacrosin consists of 2367 nucleotides, and an open reading frame encodes 505 amino acids, which corresponds to the molecular mass of 55,003 Da. The mRNA of proacrosin was found to be specifically expressed in the gonad by Northern blotting and in the spermatocytes or spermatids by in situ hybridization. The amino acid sequences around His(76), Asp(132), and Ser(227), which make up a catalytic triad, showed high homology to those of the trypsin family. Ascidian acrosin has paired basic residues (Lys(56)-His(57)) in the N-terminal region, which is one of the most characteristic features of mammalian acrosin. This region seems to play a key role in the binding of (pro)acrosin to the vitelline coat, because the peptide containing the paired basic residues, but not the peptide substituted with Ala, was capable of binding to the vitelline coat. Unlike mammalian proacrosin, ascidian proacrosin contains two CUB domains in the C-terminal region, in which CUB domain 1 seems to be involved in its binding to the vitelline coat. Four components of the vitelline coat that are capable of binding to CUB domain 1 in proacrosin were identified. In response to sperm activation, acrosin was released from sperm into the surrounding seawater, suggesting that ascidian acrosin plays a key role in sperm penetration through the coat. These results indicate that ascidian sperm contains a mammalian acrosin homologue, a multi-functional protein working in fertilization.

The Follicle Cells of Styela Plicata (Ascidiacea, Tunicata): A Sem Study

The morphological aspect of the follicle cells of Styela plicata eggs is described by means of scanning electron microscope investigations. The follicular layer is made of spaced, cylindrical box-like cells which are arranged hexagonally. They adhere to the egg through a complex network of membrane extensions making an overall thin layer on the vitelline coat. The walls of the follicle cells are plentifully provided with microvilli, filopodia and lamellipodia, which allow a connection among the cells. At their apical end lies a large vacuole containing a granule, probably involved in secretion. At insemination the majority of spermatozoa is distributed on the apical membrane of the follicle cells. The membrane often breaks after sperm-egg impact and the granule is therefore displaced. By means of the present investigations it is once again suggested a role played by follicle cells in ascidian eggs at fertilization.

Cell cycle-dependent repetitive Ca(2+ )waves induced by a cytosolic sperm extract in mature ascidian eggs mimic those observed at fertilization.

Sperm-triggered Ca(2+) oscillations occur throughout the animal kingdom. The mechanism sperm use to trigger Ca(2+) oscillations at fertilization has not been resolved in any egg. The temporal, spatial and regulatory characteristics of the Ca(2+) oscillations during fertilization in ascidians offer a unique advantage over other systems for determining the mechanism of fertilization. For example, sperm trigger two phases of Ca(2+) oscillations that are all waves in ascidians. The first of these Ca(2+) waves begins at the point of sperm-egg fusion while a second phase of Ca(2+) waves originates at a vegetal protrusion termed the contraction pole. In addition, cyclin B1-dependent kinase activity provides a form of positive feedback, maintaining the second phase of Ca(2+) waves during meiosis and thereby ensuring meiotic exit. We therefore prepared cytosolic ascidian sperm extracts or MonoQ-fractionated ascidian sperm extracts from this urochordate to investigate if a Ca(2+)-releasing sperm-borne factor was responsible for egg activation. Spatially, ascidian sperm extract induced repetitive Ca(2+) waves that mimicked the spatial pattern displayed during fertilization: all the second-phase Ca(2+) waves originated at a vegetal protrusion termed the contraction pole (thus mimicking fertilisation). We also demonstrated that ascidian sperm extract-induced Ca(2+) oscillations were maintained when CDK activity was elevated and MAP kinase activity was low, as found previously for sperm-triggered Ca(2+) oscillations. As would be predicted, large doses of ascidian sperm extract injected into prophase-stage oocytes, lacking CDK activity, failed to induce any Ca(2+) release even though they responded to microinjection of the Ca(2+)-releasing second messenger inositol 1,4,5-trisphosphate. Finally, since the Ca(2+)-releasing activity from Mono-Q fractionated ascidian sperm extract eluted predominantly as one fraction, this may imply that one factor is responsible for the Ca(2+)-releasing activity. These data support a model of egg activation whereby the sperm introduces a Ca(2+)-releasing cytosolic factor into the egg. We demonstrated that ascidian sperm contain a protein factor(s) that is regulated by the egg CDK activity and can trigger all the Ca(2+ )waves observed at fertilization with a spatial pattern that mimics those initiated by sperm.

Calmodulin and Immunophilin Are Required as Functional Partners of a Ryanodine Receptor in Ascidian Oocytes at Fertilization

Fertilization of oocytes incites numerous changes relying on Ca2+ signaling. In inseminated ascidian eggs, an increase in the egg surface membrane, monitored by a change in electrical capacitance, is recorded at the onset of meiosis resumption. This membrane addition to the cell surface is controlled by calcium release through a ryanodine receptor (RyR), sensitive to cyclic ADP-ribose. Using confocal microscopy analysis of ascidian oocytes immunostained with anti-RyR antibody, we show here that this calcium channel is asymmetrically located in the vegetal cortical zone. Interestingly, the increase in cell capacitance occurring at fertilization is correlated with a fluorescent signal, imaged by the marker of vesicle trafficking FM 1-43, located close to the RyR region. Two putative partners of RyR, namely an FKBP-like protein and a calmodulin, are identified in these oocyte extracts by detection of enzyme activity and PCR amplification. Both are necessary to sustain ryanodine receptor activity in these oocytes since the membrane insertion triggered by fertilization is inhibited by the FKBP ligand rapamycin and by a calmodulin antagonist peptide. These findings suggest that exocytosis in ascidian eggs is triggered at fertilization by a functional Ca2+ release unit operating as a complex of several proteins, including a calmodulin and an immunophilin, around the intracellular calcium channel itself.

Sperm extract injection into ascidian eggs signals Ca(2+) release by the same pathway as fertilization.

Injection of eggs of various species with an extract of sperm cytoplasm stimulates intracellular Ca(2+) release that is spatially and temporally like that occurring at fertilization, suggesting that Ca(2+) release at fertilization may be initiated by a soluble factor from the sperm. Here we investigate whether the signalling pathway that leads to Ca(2+) release in response to sperm extract injection requires the same signal transduction molecules as are required at fertilization. Eggs of the ascidian Ciona intestinalis were injected with the Src-homology 2 domains of phospholipase C gamma or of the Src family kinase Fyn (which act as specific dominant negative inhibitors of the activation of these enzymes), and the effects on Ca(2+) release at fertilization or in response to injection of a sperm extract were compared. Our findings indicate that both fertilization and sperm extract injection initiate Ca(2+) release by a pathway requiring phospholipase C gamma and a Src family kinase. These results support the hypothesis that, in ascidians, a soluble factor from the sperm cytoplasm initiates Ca(2+) release at fertilization, and indicate that the activating factor from the sperm may be a regulator, directly or indirectly, of a Src family kinase in the egg.

Nicotinic Acid Adenine Dinucleotide Phosphate-induced Ca2+ Release: INTERACTIONS AMONG DISTINCT Ca2+ MOBILIZING MECHANISMS IN STARFISH OOCYTES

An intracellular mechanism activated by nicotinic acid adenine dinucleotide phosphate (NAADP(+)) contributes to intracellular Ca(2+) release alongside inositol 1,4,5-trisphosphate (Ins-P(3)) and ryanodine receptors. The NAADP(+)-sensitive mechanism has been shown to be operative in sea urchin eggs, ascidian eggs, and pancreatic acinar cells. Furthermore, most mammalian cell types can synthesize NAADP(+), with nicotinic acid and NADP(+) as precursors. In this contribution, NAADP(+)-induced Ca(2+) release has been investigated in starfish oocytes. Uncaging of injected NAADP(+) induced Ca(2+) mobilization in both immature oocytes and in oocytes matured by the hormone 1-methyladenine (1-MA). The role of extracellular Ca(2+) in NAADP(+)-induced Ca(2+) mobilization, which was minor in immature oocytes, was instead essential in mature oocytes. Thus, the NAADP(+)-sensitive Ca(2+) pool, which is known to be distinct from those sensitive to inositol 1,4,5-trisphosphate or cyclic ADPribose, apparently migrated closer to (or became part of) the plasma membrane during the maturation process. Inhibition of both Ins-P(3) and ryanodine receptors, but not of either alone, substantially inhibited NAADP(+)-induced Ca(2+) mobilization in both immature and mature oocytes. The data also suggest that NAADP(+)-induced Ca(2+) mobilization acted as a trigger for Ca(2+) release via Ins-P(3) and ryanodine receptors.

Spatiotemporal Analysis of Ca2+ Waves in Relation to the Sperm Entry Site and Animal–Vegetal Axis during Ca2+ Oscillations in Fertilized Mouse Eggs

Fertilized mouse eggs exhibit repetitive rises in intracellular Ca2+ concentration ([Ca2+]i) necessary for egg activation. Precise spatiotemporal dynamics of each [Ca2+]i rise were investigated by high-speed Ca2+ imaging during early development of monospermic eggs. Every [Ca2+]i rise involved a Ca2+ wave. In the first Ca2+ transient, [Ca2+]i increased in two steps separated by a “shoulder” point, suggesting two distinct Ca2+ release mechanisms. The first step was a Ca2+ wave that propagated from the sperm-fusion site to its antipode in 4–5 s (velocity, ∼20 μm/s in most eggs). The second step from the shoulder to the peak was a nearly uniform [Ca2+]i rise of 12–15 s. A slight cytoplasmic movement followed the Ca2+ wave in the same direction and recovered in 25–35 s. These characteristics changed as follows, as Ca2+ oscillations progressed during the second meiosis up to their cessation at the stage of pronuclei formation (∼3 h after fertilization). (1) The duration of Ca2+ transients became shorter. (2) The shoulder point shifted to higher levels and the first step occupied most of the rising phase. (3) The rate of [Ca2+]i rise became greater and wave speeds increased up to 80–100 μm/s or more. (4) The transient cytoplasmic movement always resulted from the Ca2+ wave, although its displacement became smaller. (5) The Ca2+ wave initiation site was freed from the sperm-fusion or -entry site and eventually localized in the cortex of the vegetal hemisphere. Since the shift of the wave initiation site to the vegetal cortex is observed in fertilized eggs of nemertean worms and ascidians, this might be an evolutionarily conserved feature.

Germ-cell warfare in ascidians: sperm from one species can interfere with the fertilization of a second species.

Ascidians (invertebrate chordates) are very abundant in many marine subtidal areas. They often live in dense multispecies clumps; thus, interspecific competition for space may be intense. Although most noncolonial species are broadcast spawners, their eggs can be fertilized only by sperm of the same species (1). Multiple fertilization is lethal and all animals have evolved blocks to polyspermy. Ascidian eggs block polyspermy by enzymatic (2) and electrical mechanisms (3). Sperm bind to N-acetylglucosamine groups on the vitelline coat (4, 5, 6, 7). Follice cells surrounding the vitelline coat release N-acetylglucosaminidase during egg activation (8), preventing the binding of all sperm but a few (2). I show here that this interaction is not species-specific; sperm from one species can cause glycosidase release from follicle cells of a second species. Furthermore, once glycosidase release has been induced, the subsequent addition of sperm from the egg-producing species fails to fertilize a substantial proportion of these eggs. This leads to the hypothesis that sperm from one species of ascidian can interfere with fertilization of a second species. While intraspecific sperm competition has been well documented in several taxa (9, 10), this is the first record of sperm competition between species, or interspecific sperm competition.

Maternal information and localized maternal mRNAs in eggs and early embryos of the ascidian Halocynthia roretzi

Summary The mosaic behavior of blastomeres isolated from ascidian embryos has been taken as evidence that localized ooplasmic factors (cytoplasmic determinants) specify tissue precursor cells during embryogenesis. Experiments involving the transfer of egg cytoplasm have revealed the presence and localization of various kinds of cytoplasmic determinants in eggs of Halocynthia roretzi. Three cell fates, epidermis, muscle and endoderm, are fixed by cytoplasmic determinants. The three kinds of tissue determinants move in different directions during ooplasmic segregation. Prior to the onset of the first cleavage the three kinds of determinants reside in egg regions that correspond to the future fate map of the embryo and then they are differentially partitioned into specific blastomeres. In addition to tissue-specific determinants, there is evidence suggesting that ascidian eggs contain localized cytoplasmic factors that are responsible for controlling the cleavage pattern and morphogenetic movements. Transplantation of posterior-vegetal egg cytoplasm to an anterior-vegetal position causes a reversal of the anterior-posterior polarity of the cleavage pattern. Localized cytoplasmic factors in the posterior-vegetal region are involved in the generation of a unique cleavage pattern. When vegetal pole cytoplasm is transplanted to the animal pole or equatorial position of the egg, ectopic gastrulation occurs at the site of transplantation. This finding supports the idea that vegetal pole cytoplasm specifies the site of gastrulation. Recently, we started a cDNA project to analyze maternal mRNAs. An arrayed cDNA library of fertilized eggs of H. roretzi was constructed, and more than 2000 clones have been partially sequenced so far. To estimate the proportion of the maternal mRNAs that are localized in the egg and embryo, 150 randomly selected clones were examined by in situ hybridization. We found eight mRNAs that are localized in the eight-cell embryo, of which three were localized to the myoplasm (a specific region of the egg cytoplasm that is partitioned into muscle-lineage blastomeres) of the egg, and then to the postplasm of cleavage-stage embryos. These results indicate that the proportion of localized messages is much higher than we expected. These localized maternal messages may be involved in the regulation of various developmental processes.

Posterior end mark 3 (pem-3), an Ascidian Maternally Expressed Gene with Localized mRNA Encodes a Protein with Caenorhabditis elegans MEX-3-like KH Domains

Maternal factors localized in the posterior-vegetal cytoplasm of an ascidian egg are essential for cell specification and pattern formation of the embryo. The molecular identification of the localized factors and the elucidation of the machinery associated with the localization are therefore key research subjects. I report here the isolation and characterization of a novel maternally expressed gene, posterior end mark 3 (pem-3). The pem-3 cDNA was obtained from a cDNA library of fertilized egg mRNAs subtracted with gastrula mRNAs of Ciona savignyi. As in the case of pem (Yoshida et al., 1996, Development 122, 2005–2012), the pem-3 maternal transcript was gradually concentrated after fertilization in the posterior-vegetal cytoplasm of the egg, and it later marked the posterior end of developing embryos. The PEM-3 protein was also detected in the posterior end of early embryos. The nucleotide sequence predicted that pem-3 encodes a probable RNA-binding protein with two KH domains that have an extensive similarity with those of Caenorhabditis elegans MEX-3. MEX-3 is also localized in nematode embryos (Draper et al., 1996, Cell 87, 205–216), suggesting that PEM-3 is a candidate homologue of MEX-3. In addition to maternal expression, a zygotic transcript of pem-3 and its gene product were detected in cells of the neural plate, mesenchyme, and epidermis of embryos after the neural-plate stage. Inhibition of zygotic expression using an antisense oligonucleotide resulted in the development of abnormal larvae without sensory pigment cells, suggesting that the zygotic PEM-3 plays a role in the differentiation of the brain of the ascidian larva.

Organization of early development by calcium patterns.

This survey focuses on early or primitive developmental phenomena for which the location of a steady high calcium region or the direction of a calcium wave is critical and calcium is more than a trigger. It starts with the long studied roles of calcium in fucoid eggs and in Dictyostelium and progresses to newer work on high calcium regions in medaka fish, zebrafish, and Drosophila eggs. It then proposes that propagated, ultraslow developmental waves in six diverse systems indicate a new and important class of calcium waves. These include the morphogenetic furrow in Drosophila eye discs, floret formation in sunflowers, DNA replication waves in protozoan macronuclei, growth-cone like waves in hippocampal neurons, and two others. It then considers the possible organizing roles of slow calcium waves. Here, it emphasizes surface contractile waves during primary neural induction and elsewhere as well as the possibility of cellular peristalsis. Finally, it reviews the organizing roles of fast calcium waves in ascidian eggs.

Isolation and characterization of a highly sulfated heparan sulfate from ascidian test cells

Several sulfated polysaccharides have been isolated from the test cells of the ascidian Styela plicata. The preponderant polysaccharide is a highly sulfated heparan sulfate with the following disaccharide composition: (1) UA(2SO 4)-1→4 GlcN(SO 4)(6SO 4), 53%; (2) UA(2SO 4)-1→4-GlcN(SO 4), 22%; (3) UA-1→4-GlcNAc(6SO 4), 14% and (4) UA-1→4-GlcN(SO 4), 11%. Two others unidentified sulfated polysaccharides and a glycogen polymer are also present in the ascidian eggs. Histochemistry with the cationic dye 1,9-dimethyl-methylene blue and biochemical analysis of the 35S-sulfate incorporation into the eggs reveal that the sulfated glycans are present exclusively in the test cells. Possibly these sulfated polysaccharides are involved in important functions of these cells, such as to confer an external and hydrophilic layer which protect the eggs and the larvae of ascidians.

Interactions between cytoskeletal components during myoplasm rearrangement in ascidian eggs.

Ooplasmic segregation in ascidian eggs consists of two phases of cytoplasmic movement, the first phase is mediated by the microfilament system and the second is mediated by the microtubule system. Recently, two novel proteins, p58 and myoplasmin-C1, which are localized to the myoplasm, were suggested to have important roles in muscle differentiation. In order to analyze the molecular mechanisms underlying ooplasmic segregation, the interactions between actin, tubulin, p58 and myoplasmin-C1 were examined. During the first segregation, microtubule meshwork in the unfertilized egg disappeared. At the second segregation, a novel structure of the microtubules that extended from the sperm aster and localized in the cortical region of the myoplasm was found. Moreover, uniform distribution of the cortical actin filament was observed at the second segregation. During the course of myoplasm rearrangement, p58 and myoplasmin-C1 are colocalized and can form a molecular complex in vitro. This complex of p58 and myoplasmin-C1 is a good candidate for a cytoskeletal component of the myoplasm, and is likely to be involved in the correct distribution of cytoplasmic determinants.

Mechanisms of Specification in Ascidian Embryos

Ascidians (subphylum Urochordata, class Ascidiacea) are ubiquitous marine animals. Since the work of Chabry (1) in 1887, which described the first blastomere destruction experiments in the history of embryology, ascidian eggs and embryos have served as an experimental system in developmental biology. The fertilized egg develops quickly into a tadpole larva (about 2600 component cells) consisting of a small number of tissues including epidermis, central nervous system with two sensory organs, nerve cord, endoderm, mesenchyme, notochord, and muscle (2). The lineage of these embryonic cells is described almost completely. In addition, the recent isolation of cDNA clones of various tissue-specific genes by our laboratory provides molecular probes with which to monitor the differentiation of each type of tissue (3). The advantageous features of the embryo, together with tissue-specific molecular probes, allow us to study the mechanisms underlying the specification and subsequent differentiation of embryonic cells (2, 3).