The Hox gene cluster controls spatial patterning mechanisms along the anterior/posterior axis of bilateral metazoans. There exists a co-linearity between the order of the Hox genes in the genome and the spatial order of their domains of expression during development. Echinoderms have pentamerous radial body plans that are different from those of other deuterostomes. Thus expression patterns of echinoderm Hox genes in the adult rudiment might illuminate their evolutionary transformations from bilateral to radial structures. Since Peronella japonica is a direct-type developer that metamorphoses in 3 days without feeding, it provides a good system in which to analyse Hox gene expression in the rudiment.

We PCR-amplified the Hox-type sequences of P. japonica using genome DNA and cDNA prepared from larval RNAs as a template. We used two pairs of degenerated primers that corresponded to the first and third helices of the homeodomain of the Hox genes. As a result we obtained 13 Hox-type sequences, named Pj1–Pj13 in order of their determination. All the sequences were detected by RT-PCR, suggesting that they are being expressed in larvae (Table 1).

Spicule formation in the sea urchin is one of the conspicuous cellular processes occurring in early embryo-genesis, in which stereotyped spicules form through deposition of minerals onto the spicule matrix protein scaffold. This process requires many genes to be functional: the spicule matrix alone needs more than 50 different genes. Until now, however, only a few skeletogenic genes have been known. Recently SM37, a new putative spicule matrix protein gene, was cloned and found to be linked to SM50 (Lee et al., 1999). The structure of the new gene raised the possibility of the presence of a gene family involved in skeletogenesis which consists of SM50, SM37 and LSM34 (a homologue of SM50) (Benson et al., 1987; Livingston et al., 1991). Characteristics of the gene family include: (1) skeletongenic mesenchyme-specific expression, (2) onset of gene expression as early as the mesenchyme blastula, (3) presence of glycine, proline and glutamine-rich repeats in the middle of the proteins. Another feature of the family is the presence of conserved sequence motifs at both the amino-terminal and carboxyl-terminal regions of the proteins – SCYR(A/Y)F and PNPXXXRPRM(L/Y)QE, respectively – which we speculate play a role in protein guidance.

Spicule matrix proteins of sea urchin embryo are the specific products of the micromere / primary mesenchyme cell (PMC) lineage, and are considered to be involved in spicule formation (Wilt, 1999). One of these proteins, SM50, has been described for three species: Strongylocentrotus purpuratus (SP), Lytechinus pictus (Lp) and Hemicentrotus pulcherrimus (Hp) (for references see Wilt, 1999). The nucleotide and amino acid sequences are well conserved in these species. SM50 proteins of these species have repetitive amino acid sequences in the carboxyl-terminal half of the proteins. Therefore, examination of SM50 sequences, especially the repetitive sequence region, in various species will help an understanding of the process of sea urchin ontogeny and evolution. In this study we tried to amplify, by PCR, the SM50 sequences of species for which no sequence data are reported.

Total DNA was extracted from the sperm of sea urchins by standard procedures. The purified DNA was subjected to PCR to amplify the repetitive amino acid region and its upstream region. The primers were designed based on the highly conserved sequences in the reported SM50 as Consensus-Degenerate Hybrid Oligonucleotide Primers (Rose et al., 1997). The amplified products were gel-purified, and sequenced using ABI PRISM 310 Genetic Analyzer using PCR primers. The determined nucleotide sequences were translated into amino acid sequences and compared among species with a phylogenetic tree constructed by the neighbour-joining method. For indirect immunofluorescent staining, embryos were fixed with 70% methanol and reacted with rabbit antiserum against recombinant SM50 protein.

It has been reported that the number of cells per embryo increases from the cleavage stage to the pluteus stage. Also, it has been reported that the number of cells per embryo from the early gastrula stage to the mid-gastrula stage increases very slightly (Mizoguchi, 1999). A detailed analysis of cell proliferation during this period would thus seem to be necessary.

On the other hand, Roccheri et al. (1997) reported spontaneous apoptosis at the early pluteus stage, especially in the regions of arm and intestine. However, it is unknown whether apoptosis occurs before the pluteus stage.

Using the Tumor, Neuro and/or Cardio TACS in situ apoptosis detection kit (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling: TUNEL method, Trevigen, USA), the Comet assay kit (Trevigen, USA) and DAPI (4,6-diamidino-2-phenylinodole dihydrochloride) staining, we investigated the distribution of apoptosis-like cells in sea urchins during early embryogenesis to clarify the relationship between cell death and morphogenesis.

The embryos of the sea urchin Hemicentrotus pulcherrimus were used in the present study. Three methods of detection of apoptosis signals in the embryos were performed: (1) defects in chromosomes and punctured nuclear envelopes of cells were detected by DAPI staining; (2) apoptosis-like cells were stained a brown colour by the TUNEL method; (3) some clear tails which detected the fragmentation of DNA were found by the Comet assay. These signals indicate apoptosis-like cells.

Larvae of marine invertebrates undergo metamorphosis in response to environmental cues (Chia & Burke, 1978). In sea urchins, free fatty acids (Kitamura et al., 1993), dibromomethane (Taniguchi et al., 1994), pheromonal peptides (Burke, 1984) and L-glutamine (Yazaki & Harashima, 1994; Yazaki, 1995) have been known as metamorphosis-inducing substances. The mechanisms by which cells respond to these cues and how the larval tissues are absorbed have not been clear, however. In the present study, we used L-glutamine (Gln) and a natural cue, green algae (Ulvella sp.), to induce metamorphosis of Hemicentrotus pulcherrimus and Anthocidaris crassispina, and investigated the intracellular changes during metamorphosis.

After being subjected to 10−5–10−3 M Gln for 10–24 h, larvae cease swimming, settle, begin to retract their larval arms, extrude the primary podia and finally evert their echinus rudiment (ER). In H. pulcherrimus, larvae retracted their arms from 6 h to 24 h after the start of Gln treatment and then everted the ER. A. crassispina larvae underwent similar processes to those of H. pulcherrimus. The larval surface is composed of squamous epithelium and columnar epithelium. The epithelium of the ciliary bands or epaulets is columnar.

In the squamous epithelium, the nuclear chromatin in the larval arms and body, and in the oesophagus, markedly condensed after treatment with Gln for 24 h. Electron microscopy revealed swelling of both nuclei and mitochondria, while their membranes seemed to be intact. In the cytoplasm, lipid-like structures and electron-dense substances appeared. A further 24 h after Gln treatment, the chromatin condensation had progressed. Most nuclei in which chromatin had condensed were positive to the TUNEL assay, which detects DNA fragmentation. These results suggest that cell death in the squamous epithelium is apoptotic rather than necrotic.

Peronella japonica, a direct developer, exhibits certain peculiar features during development, particularly heterochrony, a change in the relative timing of expression among tissues and organs. One of the important heterochronical changes in the species was found in the development of the amniotic cavity, a component of an adult rudiment. In indirect developers the amniotic cavity is formed on the left side of the larval body in the late pluteus stage. In P. japonica the organ is formed at the gastrula stage in the region located on the midline of the larval body.

In the present study, the ability of partial embryos isolated from 8- or 16-cell stage embryos of P. japonica to differentiate an amniotic cavity was investigated to assess the regulative potential of a direct developer.

The embryos were dissected at 8-cell stage with a glass needle to obtain half embryos. Some of the half embryos were further divided into four blastomeres to obtain mesomere pairs. Each half embryo and blastomere that did not form micromeres but divided equally during the next cleavage was identified as an animal cap and presumptive mesomere pair. Isolated animal caps and mesomere pairs were cultured, and differentiation of the amniotic cavity was examined at 24 and 48 h after fertilisation, when the organ in the normal embryos had already completed differentiation.

Echinoid embryos of indirect developers comprise three tiers at the 16-cell stage: eight mesomeres, four macromeres and four micromeres. Micromeres differentiate autonomously into skeletogenic mesenchyme cells and induce adjoining macromeres to form a vegetal plate, resulting in archenteron invagination (Ransick & Davidson, 1995). It has been shown that micromeres have the potential to induce archenteron differentiation of presumptive ectoderm (Hörstadius, 1939).

Direct development, in which the larval stages are more or less abbreviated, has evolved in several phylogenetic lineages of echinoids. In most of them, the fourth cleavage of the embryos produces 16 blastomeres of almost the same size (Raff, 1987). Because of their indistinguishability, the inductive potential of vegetal-most blastomeres of direct developers remained to be studied.

The 16-cell stage embryos of the direct-developing sand dollar, Peronella japonica, are an exception and produce three tiers, the same as indirect developers. The embryos develop into two-armed pluteus-like larvae and metamorphose within 3 days after fertilisation without feeding. The micromeres of the species had been shown to differentiate into the skeletogenic mesenchyme cells (Amemiya & Arakawa, 1996). In the present study, we investigated the potential of the micromeres to induce or promote archenteron invagination in Peronella japonica.

Micromere signalling is a key to understanding the developmental mechanisms underlying endomesoderm differentiation along the A-V axis in sea urchin embryos. A recent study has shown that micromere activity in inducing endo-mesoderm differentiation of mesomere descendants is, unexpectedly, maximal at the hatching blastula (HB) stage in the echinoids Scaphechinus mirabilis and Hemicentrotus pulcherrimus. This study focused mainly on the timing of emission of the inductive signal by the micromere descendants. The timing of animal cap competent to react to the inductive signal from micromeres was not specifically investigated.

In the present study, we examined the competence of mesomere descendants at the HB stage to react to the inductive signal of micromere descendants by producing recombinant embryos of the descendants of mesomeres and micromeres.

Adults of the sand dollar Scaphechinus mirabilis and the sea urchin Hemicentrotus pulcherrimus were collected along the shore of Shiraishi Island, Okayama Prefecture and in the vicinity of the Misaki Marine Biological Station, Kanagawa Prefecture, respectively. The fertilised eggs were separated into two groups immediately after removal of the fertilisation membranes. One group was cultured in normal artificial seawater (ASW), and the other in ASW containing 50 mg/ml of rhodamine B isothiocyanate (RITC). At the early 16-cell stage unlabelled embryos were transferred to calcium-free seawater (CFSW) and dissected in the equatorial plane with a glass needle to isolate animal caps consisting of eight mesomeres. Embryos labeled with RITC were also transferred to CFSW at the same stage and dissected to isolate four micromeres. An isolated animal cap and a quartet of micromeres were cultured separately in ASW and recombined at the stage corresponding to the HB of control (undisturbed) embryos.

At the late pluteus stage, sea urchin larvae form an adult rudiment left–right (LR) asymmetrically, on only the left side. Little is known about how the LR asymmetry of the adult rudiment is established in earlier stages during which the larval body is basically LR symmetric. To investigate how the different regions of the embryo function to establish LR asymmetry, we removed different regions at the gastrula stage and assessed the effects of the operation on the establishment of LR asymmetry of the adult rudiment.

Surgery was performed on mid- to late-gastrula embryos of two indirect developing species: Scaphechinus mirabilis and Hemicentrotus pulcherrimus. The embryos were placed under a dissecting microscope (SMZ8, Leica) and ectodermal epithelium together with underlying mesenchyme cells was dissected out with a glass needle. The region along the midline of the embryo at the width of the archenteron was designated the ‘midline region’, and the region lateral to the ‘midline region’ was designated the ‘lateral region’. When the left and/or the right side was excised, the whole lateral region was precisely removed on the animal side, but the plane of incision deviated more laterally on the vegetal side to avoid the ventro-lateral cluster of primary mesenchyme cells, so that a part of the defined ‘lateral region’ was left on the vegetal side. The vertical excision was made by cutting mid-gastrula embryos vertically to the archenteron at a level just superior to the tip of the archenteron. In the sham operation, embryos were pressed with the needle as in the vertical excision, but the incision was stopped slightly before the animal and vegetal halves of the embryos were completely divided from each other. The operated embryos were examined through an optical microscope (Optiphoto, Nikon) to confirm that the excision was correct, and they were then cultured with a food supply (diatoms, Chaetoceros gracilis). The handedness of the adult rudiment was examined at the six-armed or eight-armed pluteus stage through an optical microscope (Table 1).

It is well known that sea urchin embryos treated with lithium chloride (LiC1) develop to abnormally into vegetalised embryos, in which differentiation of ectodermal cells is inhibited. When embryos of the sea urchins, Hemicentrotus pulcherrimus and Anthocidaris crassispina were treated with 20 mM LiC1 from the 8-cell stage to the corresponding early gastrula stage, they developed to vegetalised embryos with a large exogut 45 h after fertilisation. In these vegetalised embryos, high activity of alkaline phosphatase (AP) was detected histochemically at the end of the exogut where it is attached to the embryo body. High activity of AP is known to be detected specifically in the gut of sea urchin pluteus larvae by the same procedure as used in this study. Hence, we concluded that this part of the exogut is composed of the cells which develop into the cells of the gut in normal development.

When exogut isolated from vegetalised embryos was cultured in the extract obtained from eggs or embryos, the end composed of the cells in which high AP activity was detected, expanded during culture and formed a large spherical structure about 24 h after the initiation of culture. The minimum concentration of extract to cause expansion of isolated exogut was 5 × 103 egg or embryo equivalent/ml ASW (artificial seawater). The extract boiled at 95 °C for 1 h also caused expansion of isolated exogut at the same concentrations as non-boiled extract. On the other hand, the extract obtained from eggs or embryos by chloroform–methanol extraction did not cause any expansion of exogut, but the aqueous phase, heat-dried and dissolved in ASW, induced expansion of isolated exogut.

Classical neurotransmitters such as acetylcholine (ACh), biogenic amines and ϒ-aminobutyric acid (GABA) have been shown to mediate various forms of intercellular signalling in marine invertebrates including sea urchin larvae (Buznikov et al., 1996). The presence of amines and GABA in the nervous system of sea urchin larvae has been demonstrated by a cytochemical method at the gastrula stage (Bisgrove & Burke, 1987). However, few quantitative analyses of neurotransmitters have been attempted in the embryonic sea urchin. To accomplish this during the period of embryogenesis, in the present study we attempted to make homogenate samples adequate for HPLC analysis and to determine the levels of neurotransmitter in their extracts.

We established an HPLC protocol using a batch of four-armed sea urchin larvae, and detected neurotransmitters (per milligram wet weight) in the homogenate of pluteus larvae of Hemicentrotus pulcherrimus (1.5–3.5 pmol dopamine, 0.22–0.51 pmol serotonin, 4.4–40 pmol ACh, 0.28–0.36 nmol glutamate, 20–23 nmol glycine) (Figs. 1, 2). GABA was not detected (detection limit, 10 pmol/10 μl injection). We also observed glyoxylic-acid-induced yellowish-green monoamine fluorescence in the larval nervous system. Serotonin has been a leading candidate neurotransmitter in sea urchin larvae (Nakajima et al., 1993).

Two closely related species in the camarodont sea urchin family Strongylocentrotidae, Strongylocentrotus pallidus and Strongylocentrotus droebachiensis, have circumarctic ranges with overlapping depth distributions and spawning seasons. Both species show genetic differences between Europe and the Pacific in the nuclear sperm bindin gene, and S. droebachiensis also differs in mitochondrial DNA. Gamete recognition systems of allopatric populations may or may not have drifted apart, but are unlikely to have diverged by reinforcing selection. Geographic divergence in gamete compatibility may illustrate mutational biases and variability and hence possible initial stages of reproductive isolation.

We carried out fertilisation experiments with both species' gametes from Norway and from the Eastern Pacific Ocean. Serially diluted sperm were added to egg suspensions in factorial designs, and fertilisation success was assessed by counting the proportion of cleaving embryos. To separate the specificity of the acrosome reaction from subsequent sperm–egg binding steps, a subsample of sperm was pre-activated by performing the last dilution step in diluted egg jelly from conspecific females. Fertilisation successes were compared by logistic regression ANCOVAs over five, 4-fold sperm dilutions for each cross. The graphs shown here illustrate fertilisation success for one sperm concentration only.

Order Echinoida in Japan is now classified into the following five families: Temnopleuridae, Toxo-pneustidae, Strongylocentrotidae, Echinometridae and Parasaleniidae. With the exception of the Parasaleniidae, four of the families are commonly found in shallow waters in Japan. Interrelationships among these four families have been extensively studied by many workers using morphological criteria or at the biochemical and molecular levels, and four different theories have been proposed (Jensen, 1981; Smith, 1984; Mortensen, 1928–1951; Shigei, 1986; Matsuoka, 1988). To examine these theories, I attempted to obtain hybrids using 14 species (4 species of the family Temnopleuridae: Temnopleurus toreumaticus, Temnopleurus hardwickii, Temnopleurus reeversii and Mespilia globulus; 3 species of the family Toxopneustidae: Toxopneustes pileolus, Tripneustes gratilla and Pseudoboletia maculata; 4 species of the family Strongylocentrotidae: Strongylocentrotus intermedius, Strongylocentrotus nudus, Hemicentrotus pulcherrimus and Pseudocentrotus depressus; and 3 species of the family Echinometridae: Anthocidaris crassispina, Echinostrephus aciculatus and Echinometra mathaei).

Recent morphological, biochemical, ecological and reproductive studies have revealed that there are four sympatric biological species of sea urchins in Echinometra from Okinawa, which are currently referred to as Echinometra species A, B, C and D (e.g. Uehara et al., 1990,1991; Arakaki & Uehara, 1991; Matsuoka & Hatanaka, 1991; Palumbi & Metz, 1991; Nishihira et al., 1991). Of these four species, E. sp. A (Ea) and E. sp. C (Ec), while being most closely related to each other genetically (Matsuoka & Hatanaka, 1991), can also be distinguished from each other by differences in adult morphology and microhabitat preference. No studies have addressed the mechanisms that maintain the independence of these species despite their close affinity and sympatry.

Experimental hybridisation conducted between two genetically very divergent species of Okinawan Echinometra, E. sp. A (Ea) and E. sp. D (Ed), revealed the presence of prezygotic isolation (Asian & Uehara, 1997). To test whether the closely related Okinawan Echinometra spp. are isolated by pre- or postzygotic mechanisms, we carried out experimental hybridisation by using much closer species, Ea and Ec, through a series of cross-fertilisation experiments and rearing of the resulting hybrids. The percentage of successful heterogametic fertilisation was high when eggs of Ec and sperms of Ea were involved, whereas it was considerably lower with eggs of Ea and sperms of Ec. This reduction, at least among some heterogametic crosses, indicates the presence of a gamete recognition protein binding system, as reported by Metz et al. (1994) and Metz & Palumbi (1996), which might eventually lead to gametic incompatibility and reproductive isolation.

Ran is an abundant small G protein that has a central role in nuclear transport during interphase. Ran function depends on distinct pools of RanGTP in the nucleus and RanGDP in the cytoplasm, which are maintained by compartmentalisation of the nucleotide exchange and GTPase activating proteins for Ran. RCC1 (Regulator of Chromosome Condensation) is the only known guanine nucleotide exchange factor for Ran and is associated with chromatin. RanGAPl together with RanBPl are the primary GTPase activator proteins and are located in the cytoplasm.

Ran function in nuclear transport is clearly inactivated after the nuclear envelope has disassembled during mitosis. There is now evidence that Ran plays a role in organising the spindle. Experiments using well-characterised cell-free extracts from meiotic metaphase II arrested Xenopus eggs suggest that Ran regulates microtubules involved in spindle assembly (Carazo-Salas et al., 1999; Kalab et al., 1999; Ohba et al., 1999; Wilde & Zheng, 1999). These experiments demonstrate that high levels of RanGTP or GTP-locked forms of Ran result in aster formation and can cause formation of bipolar spindles in the absence of centrosomes or chromosomes. In contrast, reduction of RanGTP blocks centriole- and chromatin-dependent aster formation.