Sea urchin pluteus larvae contain dozens of pigment cells in their ectoderm. These pigment cells are the descendants of the veg2 blastomeres of the 60-cell stage embryo. According to the fate map made by Ruffins and Ettensohn, the prospective pigment cells occupy the central region of the vegetal plate. Most of these prospective pigment cells exclusively give rise to pigment cells. Therefore, specification of the pigment cell lineage should occur at some point between the 60-cell and mesenchyme blastula stage. However, the detailed process of the specification of the pigment lineage is unknown.

When are pigment cells specified? Are cell interactions necessary for the specification? Do founder cells exist? To answer these questions, I treated embryos with Ca2+-free seawater during the cleavage stage and examined the number of pigment cells observed in pluteus larvae. Treatment at 5.5–8.5 h and especially 7.5–10.5 h postfertilisation markedly reduced the number of pigment cells. The decrease was statistically significant. On the other hand, the treatment at 3.5–6.5 h or 9.5–12.5 h never reduced the number of pigment cells. By examining the frequency of the appearance of embryos whose numbers of pigment cells were less than 20, it was also found that the numbers of pigment cells were frequently in multiples of 4. Embryos having 4, 8, 12, 16 and 20 pigment cells were more frequently observed. Statistics indicated that the frequency of appearance was not random. These results indicated that cell contacts are necessary for the specification of pigment cells and that the specification occurs from 7 to 10 h postfertilisation. The results also suggest that the founder cells, if they exist, divide twice before they differentiate into pigment cells.

Echinoid development progresses through embryonic and larval stages to metamorphosis and the adult form. Despite vast differences in embryos and larval forms, including bilaterally symmetric echinopluteus larvae, ovoid non-feeding larvae and brooded embryos, all metamorphose into juvenile sea urchins with pentaradial symmetry. The adult sea urchin body plan is initiated as the juvenile rudiment. The rudiment has been called the phylotypic stage for the class Echinoidea, a designation that implies little variation at this midpoint in development (e.g. Raff et al., 1991; Richardson, 1995; Raff, 1996). However, right at metamorphosis (upon eversion of the juvenile rudiment), variations in test symmetry, shape and number of spines, and number of skeletal plates, podia and pedicellariae are present in juveniles. This variation suggests either that there is no phylotypic stage or that such a stage occurs earlier in rudiment formation. To distinguish between these possibilities, I explored the patterns by which the juvenile rudiment is formed as well as the variation among juveniles approximately 1 day after metamorphosis in 19 echinoid taxa covering a broad taxonomic range including cidaroids, diadematids, irregular echinoids (spatangoids and clypeasteroids), arbaciids, temnopleurids, echinometrids and strongylocentrotids. Most of the material for analysis of juveniles was obtained by the author. Additional information was gathered from classical studies of metamorphosis. Data were collected on the number and shape of dorsal pedicellariae, juvenile and adult spines, primary and secondary podia, and juvenile test shape. When possible multiple individuals within a species were examined, revealing no or only minor trait variation. These data were mapped on a well-resolved phylogeny established from adult characters.

Metamorphosis of sea urchin larvae is initiated by one or more cues from the environment. The cues can be from bacterial films (Cameron & Hinegardner, 1974), algae (Kitamura et al., 1993) or sand and seawater from adult habitats (Highsmith, 1982). The substances from sand are peptides (Burke, 1984), and those from red algae are free fatty acids (Kitamura et al., 1993) and dibromomethane (Taniguchi et al., 1994). Burke (1983a) suggested that chemical and physical stimuli were received by sensory receptors, probably podia of the adult rudiment, and transmitted to effectors of metamorphosis such as larval and adult tissues. Morpho-genetic, histolytic and histogenic processes progress during metamorphosis to create a juvenile, though direct evidence for the mechanism of induction has not been shown.

Glutamine (Gin) induces metamorphosis in larvae of many sea urchin species (Strongylocentrotus intermedius: Naidenko, 1991; Pseudocentrotus depressus: Yazaki & Harashima, 1994; Hemicentrotus pulcherrimus: Yazaki, 1995). We have analysed the metamorphosis of sea urchin larvae using Gln, neurotransmitters and a natural cue (green algae).

Metamorphosis is a remarkable process in echinoderms, transforming a bilaterally symmetrical planktonic larva into a radially symmetrical benthic adult. This shift in habitat involves functional and anatomical changes in virtually every organ system (Bury, 1895; MacBride, 1914; Okazaki, 1975). Although metamorphosis is a crucial process in echinoderm development, we know relatively little about it. Furthermore, most of what we do know concerns sea urchins, and even less information is available about metamorphosis in other echinoderms. We have examined the expression of regulatory and structural genes during metamorphosis in several different echinoderm species (Lowe & Wray, 1997 and unpublished results). These data, together with those from several recent studies concerning additional genes (reviewed in Wray & Lowe, 2000), are beginning to shed new light on this complex and important process in echinoderm development.

The overt transformation from swimming larva to settled juvenile is quite rapid in echinoderms (Cameron & Hinegardner, 1978), requiring less than half an hour in many species. The complete process of metamorphosis takes much longer, however (Okazaki, 1975; Gosselin & Jangoux, 1998). Extensive preparations begin several days to weeks before settlement (MacBride, 1914; Okazaki, 1975), depending upon the species and upon environmental conditions (Strathmann et al., 1992). During this preparatory phase, initially small populations of ectodermal and mesodermal cells fated to become the adult proliferate, differentiate and undergo complex morphogenetic movements to form the imaginal rudiment (MacBride, 1914; Okazaki, 1975). The rudiment is more complex than the larva in several important ways: it contains a greater number of cell types, it is the first place where true tissues form, and it contains the first well-organised nervous system (Okazaki, 1975; Chia & Burke, 1978).

In echinoderm development, conversion of the oral-aboral axis takes place during metamorphosis, and the left and right sides of larvae become the oral and aboral sides of juveniles, respectively. Since the rudiments of the adult organs are formed before metamorphosis, late brachiolaria larvae of Asterina pectinifera show distinct bilateral asymmetry. On the other hand, early bipinnaria larvae look almost symmetric bilaterally, except for hydropore and posterior coelomic pouch formation which take place only on the left side. The relationship between the axis of the adult rudiment and the bilateral asymmetry in the coeloms of early stage larvae was examined by (1) bisecting larvae, (2) discarding left or right anterior coeloms (lac, rac) and/or left posterior coelom (1pc) and (3) replacing lac or rac with the counterpart.

In normal brachiolaria larvae, adult organs such as hydrolobes and adult spicules are formed in the posterior part of the larval body. In the left half of the adult rudiment, a row of five adult arm-type spicules are aligned along the median plane. Hydrolobes, which form the hydrovascular system in the juvenile, were observed on the left of the arm-type spicules. The right half of the rudiment was covered with adult disc-type spicules (Fig. 1). The left half derived from larvae older than 40 h (mouth formation stage) lacked adult disctype spicules, and two rows of arm-type spicules existed on both side of the median plane (L-type; Fig. 2a).

Algae were supplied continuously to four-armed pluteus larvae of the sea urchin Hemicentrotus pulcherrimus until the adult rudiment reached stage g, which is the initial stage of rudiment formation (Chino et al., 1994) After stage g, one group of larvae was reared without the addition of algae for comparison of the development of the adult rudiment with that in larvae given algae. Three days later, only 9.1 ± 0.3% of larvae without algae had reached stage j, while 76.8 ± 0.6% of larvae with algae had reached a stage beyond j, which indicates the formation of the complete adult rudiment.

When larvae at rudiment stage g were reared in a medium supplemented with T4 or its derivatives, such as T3, 3,3′,5′-L-triiodothyronine (rT3) or triiodothyropropionine (Tp3), in place of algae, the adult rudiment developed in a dose-dependent manner. T4 was the most effective and induced formation of the adult rudiment in more than 70% of specimens at 1 nM and in almost 100% at 100 nM. T3 was one-tenth as effective as T4. Other derivatives were still less effective. On the other hand, casein, ovalbumin, tyrosine and a mixture of 20 amino acids had no effect on the development of larvae and adult rudiments, suggesting that they were not available as nutrients and sources of thyroid hormones.

In starfish, oocytes in the ripe ovary do not develop beyond the end of the first prophase stage of meiosis. Such immature oocytes are not fertilisable. The resumption of meiosis in these oocytes and release from the ovary are induced by the maturation-indueing hormone 1-methyladenine (1-MeAde) (Kanatani et al., 1969). 1-MeAde is produced by ovarian follicle cells after stimulation by a gonad-stimulating hormone (GSS) secreted from radial nerves. The action of GSS on 1-MeAde production in follicle cells appears to be mediated by its receptor, G-proteins and adenylyl cyclase (Mita & Nagahama, 1991). It has been shown that upon incubation of follicle cells with GSS, there is a dose-related increase in cyclic AMP production, coincident with an increase in 1-MeAde production (Mita et al., 1989). Thus, it was suggested that a G-protein coupled (seven transmembrane type) receptor is involved in GSS signal transduction in a similar manner to the pituitary–gonadal axis in vertebrates. To elucidate the mechanism of starfish oocyte maturation, we focused on a receptor protein for GSS on the ovarian follicle cell surface. In this study, we cloned the cDNA with glycoprotein hormone receptor homology in a transmembrane region from ovaries of the starfish Asterina pectinifera.

Using consensus sequences from glycoprotein hormone receptors, a 3953 bp cDNA was cloned from mRNA of starfish A. pectinifera ovaries. The region from nucleotide 72 to 3109 represents the open-reading frame. The seven transmembrane spanning domains were found in the region from nucleotide 1745 to 2533. The lengths of specific domains in the cDNA were 1671 bp, 789 bp and 579 bp for extracellular (ECD), transmembrane (TMD) and intracellular domains (ICD), respectively. Similar to other glycoprotein hormone receptors, the cDNA from starfish ovaries had a large ECD region. With respect to the predicted amino acid sequence, the ECD, TMD and ICD were composed of 557, 263 and 192 amino acids, respectively.

The sea urchin Echinometra mathaei (Blainville) which belongs to the order Echinoida, occurs in abundance on shallow reefs throughout the tropical to warm Indo-Pacific region. Though Okinawan E. mathaei had been considered as a single species showing extensive morphological variation in test shape and spine colour, it was recently shown that the genus Echinomtra consists of four independent species (Uehara & Shingaki, 1984, 1985; Arakaki et al., 1998). However, the scientific names of these four complex species found in Okinawa are still unclear. These Echinometra are tentatively described here as Echinometra sp. A, B, C and D. Echinometra sp. A is characterised by white-tipped spines, a definite bright milled ring and dark skin on the peristome. Echinometra sp. B is characterised by spines with no white tip, with very faded milled rings and dark skin on the peristome. Echinometra sp. D is characterised by spines without a white tip but with a definite bright milled ring and bright skin on the peristome. In the field, these two Echinometra sp. exhibit a richly coloured variation in spines. Echinometra sp. D is characterised by deep black spines with well-defined milled rings and dark skin around the peristome. Only black-spined individuals have been found so far. In addition, these species show different distribution patterns, habitat preference and agonistic behaviour. It has also been shown that these two species are not possible to cross-fertilise under sperm at limiting concentration.

Previously, it has been reported that NADH cytochrome c reductase and succinate cytochrome c reductase, in which the redox reaction in cytochrome b is involved, are activated by light irradiation with peaks of photo-activation at wavelengths of 430, 530 and 570 nm corresponding to those in the absorption spectrum of reduced cytochrome b in mitochondria isolated from sperm of echiuroid, oyster and sea urchin (Tazawa et al., 1996). In sperm of these species, augmentation of respiration due to photo-activation of the cytochrome b reaction is observed only when the electron transport in this span of the mitochondrial respiratory chain is inhibited by carbon monoxide (Fujiwara et al., 1991; Yasumasu et al., 1991) or by a decrease in the amount of cytochrome b due to sperm ageing. In sperm cultured for a long time, the respiratory rate was very low and almost all sperm became immotile. In these sperm, respiration was reactivated by light irradiation at the wavelengths of 430, 530 and 570 nm (Fujiwara et al., 1999).

In the present study, photo-reactivation of movement in these somewhat degenerated sperm incubated for a long time was also found to occur, with peaks at the above-mentioned wavelengths. We concluded that photo-reactivation of movement in these sperm, in which the cytochrome b reaction probably became rate-limiting in the respiration chain, is supported by reactivation of respiration by light irradiation. On the other hand, though the ATP level decreased to a rather low level at about 30 min after the initiation of incubation in sperm of all species examined, sperm swam for more than 10 h, by which time the ATP level was quite low. Light irradiation induced reactivation of movement but did not alter the rather low ATP level in these sperm. Thus the ATP level does not seem to be responsible for making sperm immotile. In sperm treated with Triton X-100, movement was induced by adding ATP and Mg2+, even when many sperm had become immotile after a long incubation. Hence, capacity for movement does not seem to be reduced by a long incubation time. The movement of sperm treated with Triton X-100 was not activated bv light irradiation.

Protein phosphorylation is highly coupled with sperm motility activation in several animal species. The micro-tubule based flagellar motor protein, dynein, is a candidate for a phosphoprotein related to sperm activation in many animal species (Morisawa & Hayashi, 1985; Hayashi et al., 1987; Dey & Brokaw, 1991; Stephens & Prior, 1992; Inaba et al., 1998, 1999). Sperm motility of the ascidians Ciona intestinalis and C. savignyi is activated by a factor derived from unfertilised eggs named sperm activating and attracting factor (SAAF). SAAF elevates the intracellular cyclic AMP (cAMP) level by a mechanism dependent on membrane hyperpolarisation and extracellular Ca2+ (Yoshida et al., 1994; Izumi et al., 1999). Experiments using demembranated Ciona sperm showed that cAMP is required prior to ATP for the activation of axonemal movement (Opreska & Brokaw, 1983; Morisawa et al., 1984; Brokaw, 1985; Dey & Brokaw, 1991; Chaudhry et al., 1995) and that many sperm flagellar proteins including dynein light chain are phosphorylated during incubation of demembranated sperm with ATP and cAMP (Dey & Brokaw, 1991). However, there is no evidence of which proteins are phosphorylated during the SAAF-dependent activation of Ciona sperm motility.

The vitelline coat (VC) lysin of Tegula, a marine molluscan genus, is released from the acrosome of sperm during fertilisation and can lyse the VC of only the same species. The lytic action of this lysin against the VC is not an enzymatic reaction, but a stoichiometric and irreversible one (Haino-Fukushima, 1974).

The VC of Tegula pfeifferi consists of glycoproteins containing sulphated polysaccharides, which account for roughly two-thirds of the entire weight of the VC. The presence of a large quantity of polysaccharides in the VC had prevented rapid progress in the analysis of its protein components. Last year, we succeeded in a complete solubilisation of the VC by boiling for a long time in 1% SDS solution, and determined the cDNA sequence coding for a mature 41 kDa glycoprotein, which appears to be the major component of the VC from the results of SDS-polyacrylamide gel electrophoresis (PAGE). The cDNA, referred to as vcp41, comprises 1072 base pairs and contains one open reading frame with a sequence for 319 amino acids containing 19 amino acids of a signal peptide. The deduced amino acid sequence has five N-glycosylation sites and ten cysteine residues. It seems that almost 7 kDa in this 41kDa glycoprotein is polysaccharide constituents (Fan & Haino-Fukushima, 1998).

Animal eggs are generally encased in extracellular investments. These structures are not simply a protective barrier against infectious microbes, parasites and various small predators: in starfish, three components of the egg jelly, the outermost egg investment, are responsible for triggering the acrosome reaction. These components are a highly sulphated glycoprotein of an extremely large molecular size named acrosome reaction-inducing substance (ARIS), a steroid saponin named Co-ARIS, and asteroidal sperm-activating peptides (asterosaps) (Matsui et al., 1986a, b; Nishigaki et al., 1996). ARIS can induce the acrosome reaction in homologous spermatozoa with asterosaps or Co-ARIS in normal seawater. Specificity at the genus or order level was found for sperm activation by asterosaps, whereas the acrosome reaction by jelly components was species-specific. The main sugar saccharide chain of ARIS, composed of the pentasaccharide repeating units [Xyl-Gal-Fuc(SO3−)-Fuc(3−)-Fuc-], has been observed to induce the acrosome reaction in starfish sperm at high calcium concentrations (Koyota et al., 1997). Recently, we cloned cDNAs encoding asterosaps and elucidated their nucleotide sequences (Matsumoto et al., 1999). The mRNA encoding asterosaps was transcribed only in the oocytes but not in the follicle cells, and the length was 3.7 kb. The cDNA clones contained multiple isoforms of asterosaps. We assume that asterosap cursors are large prepolypeptide chains with an unusual ‘rosary-type’ structure composed of 10 successive similar stretches of 51–55 residues. Each stretch ends with a ‘spacer’ of 17–21 residues immediately followed by the sequence of one asterosap isoform. The amino terminal of this precursor has 19–21 successive glutamine-rich repeating units.

It has been shown that lipid raft, a microdomain of plasma membrane, is a hot spot of signal transduction in somatic cells, because it contains several transducer proteins as well as various receptor molecules. The lipid raft is characterised by its low-density detergent-insoluble nature and by enrichment of glycosphingolipids (GSLs). We hypothesised that lipid raft was also on the gamete surface, and might function as a sperm–egg adhesion site as well as in signal transduction during fertilisation. To test this hypothesis, we have initiated studies using sea urchin gametes. Recently we have demonstrated the presence of the lipid raft in sperm of three sea urchin species as the first example in gametic cells (Ohta et al., 1999). Here we show several lines of evidence for the functional importance of the lipid raft in sperm–egg interaction as well as in subsequent signal transduction.

In sea urchin sperm, lipid rafts were able to be prepared as a low-density detergent-insoluble membrane (LD-DIM) fraction and were rich in GSLs including gangliosides and sulphatide, containing more than 50% of the total amount of GSL present in sperm. On the other hand, cholesterol and sphingomyelin were not so enriched, which contrasted with the LD-DIM from MDCK cells, where these lipids were reported to be abundant (Brown & Rose, 1992).

Our goal is to identify and understand points of regulation in sperm–egg recognition as well as in steps of early development. These processes are species-specific and are the key to understanding speciation. In both vertebrates and invertebrates, the interaction of the sperm and egg displays a wide range of species-specificity. The questions we would like to answer are: What kinds of molecules determine the specificity and control the fertilisation process? Are early steps in development regulated in a species-specific manner?

As an approach to identifying genes that determine species-specificity, in two different species, S. purpuratus (S. p.) and S. franciscanus (S. f.), we used a new subtractive hybridisation method known as RDA (representational difference analysis) (Lisitsyn et al., 1993; Hubank et al., 1994). Several species-specific clones were isolated from S. f. ovary mRNA by this method using mRNA from another species but the same genus of sea urchin, S. p. Four different clones were obtained and the species-specificity of the sequence was confirmed by hybridisation. One of them has four tandem EGF repeats and is homologous to the S. p. EGF-II gene (Yang et al., 1989) and A. crassispina EGIP (exogastrula inducing protein) gene (Ishihara et al., 1982). The first three EGF repeats (EGF 1–3) have 60% similarity among these species, but the fourth EGF domain (EGF 4) is highly divergent. The EGF-II protein is believed to be involved in the signalling events of early development, because purified EGF causes exogastrulation when it is added to the seawater prior to gastrulation (Ishihara et al., 1982). Recombinant EGF 3 from S. f. induces exogastrulation in both S. f. and S. p.

In the early cleavage stages of animal embryos, blas-tomeres lack devices to connect to each other. It is well known that in sea urchin embryos the hyaline layer plays an important role in maintaining the position of individual blastomeres, and provides a scaffold for morphogenesis to the embryo. In starfish embryos, however, the presence of the hyaline layer is not certain in the early cleavage stages, although it has been observed at the gastrula stage (Dan-Sohkawa et al., 1986). In the present study we have investigated the devices corresponding to the hyaline layer of sea urchin embryos in the early cleavage stages, where blastomeres lack such devices for cell adhesion as desmosomes.

We examined how blastomeres keep their position in the early cleavage stages of starfish embryos using Asterina pectinifera and Astropecten scoparius. By neither electron microscopy nor immunofluorescent staining with an antibody against the hyaline layer of sea urchin embryos (Yazaki, 1968) could we detect the hyaline layer, at least up to 6 h after fertilisation.

When the fertilisation envelope (FE) was dilated larger by urea treatment, blastomeres increasingly came apart with the expansion of the FE, resulting in the formation of plural small blastulae due to the failure of the blastomeres to come together into a single mass. In urea-treated embryos, blastomeres were observed closely apposed to the inner surface of the FE. These observations suggest that blastomeres are fixed by some means to the FE. Differential interference-contrast microscopy revealed numerous projections between the cell surface of the blastomeres and the FE. Probably, the FE and the projections are involved in maintaining the three-dimensional position of each blastomere in an embryo.

An animal egg such as amphibian, mammalian or sea urchin egg receives only a single sperm at fertilisation. After binding of the first sperm, the egg is prevented from allowing the entry of additional sperm. In fact, polyspermy results in aborted development of the zygote. It has been generally accepted that a molecule(s) released from cortical granules participates in the block to polyspermy. As one such molecule, a cortical granule lectin has been isolated from unfertilised Xenopus eggs (Xenopus cortical granule lectin; XCGL). XCGL is released into the perivitelline space after fertilisation, and forms a complex with J1 jelly molecules to form an F layer, resulting in a block to additional sperm penetration.

A lectin molecule has also been purified from the eggs of several species of fish. The fish egg lectin is located in the cortical alveoli and is released from them after fertilisation. However, its biological function is unclear. We isolated cortical alveolar lectin from unfertilised eggs of Chinook salmon through affinity column chromatography (salmon egg lectin; SEL). The lectin activity was estimated by haemagglutination. The activity of the purified SEL was most strongly inhibited by L-rhamnose and D-galactose, but not by EDTA. Further analysis by C4 reverse-phase column chromatography using HPLC revealed that the lectin was composed of three subunit proteins: 24K, 26Ka and 26Kb proteins. In addition, we cloned cDNAs for them by RT-PCR. The deduced amino acid sequence of the 26Ka protein was homologous with that of the 26Kb protein (identity, 96.4%). Identities of the 24K with the 26Ka and the 26Kb proteins were 55.9% and 66.7%, respectively. A database search revealed that a lectin molecule similar to the SEL had been identified in Anthocidaris crassispina egg (sea urchin egg lectin; SUEL). The SUEL is composed of 105 amino acids, and is similar to both amino-terminal and carboxyl-terminal halves of the SELs. Thus, the SEL molecule is composed of two repeats of such SUEL-like domains, suggesting that the SEL gene was produced by gene duplication.

Cell division and motility are generated by the cytoskeletal structures contained in networks of actin filaments and microtubules. Among the Ca2+-activated protein kinases, protein kinase C (PKC) can be activated with tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA) (Kikkawa et al., 1983), which induces alterations in the cytoskeleton of cultured cells. To study the regulation of the cytoskeleton by PKC, we investigated the formation of actin filaments, microtubules, changes in cell shape and Ca2+ signalling after treatment with TPA in unfertilised eggs of the sea urchin Hemicentrotus pulcherrimus.

Formation of actin filaments and microtubles was induced in the eggs by the TPA treatment. When the eggs were treated with an inactive TPA, 4α-PMA, these cytoskeletal changes did not occur. Moreover, elongation of actin filaments was inhibited by the Ca2+ chelating agent BAPTA, but microtubule organisation was unaffected by intracellular Ca2+ chelation in the TPA-treated egg. TPA is known to induce alkalisation in sea urchin eggs (Swann & Whitaker, 1985) and when the eggs are treated with TPA in Na+-free seawater, the rise in intracellular pH (pHi) is depressed. However, elongation of actin filaments and microtubules occurred under these conditions. An increase in pHi is considered to be essential for triggering the bundle of actin filaments (Begg et al., 1982) and microtubule assembly (Schatten et al., 1992). These results show that actin and microtubule assemblies require activation of PKC, and that the formation of actin and microtubules may be related to the type of PKC isoform: Ca2+-dependent or -independent. Moreover, this mechanism is essentially independent of cytoplasmic alkalisation.

Serine/threonine protein phosphatases expected to participate in the process of signal transduction, cell movement such as cell division and gene expression (Kinoshita et al., 1990; Healy et al., 1991; Mayer-Jaekel et al., 1993; Mumby & Walter, 1993), are classified into type 1 (PP1), type 2A (PP2A), type 2B and type 2C in mammalian cells. PP1 and PP2A are known to be strongly inhibited by okadaic acid (OA) (Tachibana et al., 1981; Bialojan Takai, 1988), a polyether fatty acid isolated from the marine sponge Halicondria okadai (Haystead et al., 1989). OA is also known to inhibit PP2A at lower concentrations than that to block PP1 in mammalian cells, but does not inhibit the activities of other phosphatase species (Ishihara et al., 1989).

The p-nitrophenyl phosphate (pNPP) splitting activity in the extract obtained from eggs of the sea urchin Hemicentrotus pulcherrimus was found to be inhibited by OA and calyculin A (CLA), potent inhibitors of PP1 and PP2A. OA-sensitive phosphatases are known to catalyse pNPP splitting (Takai & Mieskes, 1991), in the same manner as other OA-insensitive phosphatases.

Four peaks of the pNPP splitting activity were obtained by QAE-Toyopearl chromatography in the extract of sea urchin eggs. In two of these four peaks, pNPP splitting reactions were strongly inhibited by OA and CLA at quite low concentration. High sensitivities of the pNPP splitting reaction to OA and CLA in these two peaks suggest that pNPP splitting results from the reaction catalysed by PP2A. The molecular masses of proteins exhibiting OA-sensitive pNPP splitting activities in these two peaks were found to be about 160 kDa by Superdex 200HR, and were similar to that of mammalian PP2A trimeric holoenzyme. By immunoblot analyses with anti-human PP2A catalytic subunit antibody, an immunoreactive 36 kDa protein was found by SDS-PAGE in a peak of OA-sensitive pNPP splitting activity obtained by QAE-Toyopearl chromatography. Sea urchin eggs have at least two PP2A-like enzymes with similar molecular masses to that of mammalian PP2A, and one of them contains human-type catalytic subunit.

The starfish is an advantageous organism in which to investigate developmental modes. It is widely known that maternal substances accumulated in the course of oogenesis affect various developmental phenomena. Vitellogen is the most abundant maternal substance in the egg and has been studied in various species including sea urchins. Vitellogen and the vitellogenin gene have been analysed with regard to their relevance to developmental modes in two Heliocidaris species (Byrne et al., 1999) and Japanese sea urchins (Yokota & Amemiya, 1998). In starfish, however, relatively little is known about the yolk and yolk protein.

A characteristic cysteine-rich motif, LIM domain, was first detected in three different transcription factors: lin-11, Islet-1 and mec-3. A feature shared by these genes is the presence of two LIM domains linked to a DNA-binding homeodomain (Sánchez-García et al., 1994). LIM homeodomain (LHX) proteins have been reported to be implicated in a variety of developmental processes (Dawid et al., 1998).

Expression patterns of LHX genes have been analysed in a wide variety of organisms and reported to be cell-type specific (Dawid et al., 1998). In vertebrates, they are expressed in organiser equivalent regions at the gastrula stage, suggesting their involvement in mesoderm induction (Taira et al., 1992; Barnes et al., 1994; Toyama et al., 1995). Hrlim, an ascidian Lim3, zygotically expresses in the endoderm lineage before gastrulation, suggesting that it is involved in the endoderm determination (Wada et al., 1995).

Here, cDNA cloning of the Lim1-related homeobox gene (HpLim1) of the sea urchin, Hemicentrotus pulcherrimus, is described together with the spatially as well as temporally regulated expression of HpLim1 during sea urchin development. A possible role of HpLiml in sea urchin development is also discussed based on its spatial pattern of expression and on the result of an over-expression study.