Animal and gametes

The sea urchin Peronella japonica were collected near the Noto Marine Laboratory, Kanazawa University. Gametes were obtained by intracoelomic injection of 0.5 M KCl. Jamarine U (JSW; Jamarine Laboratory) was used as artificial seawater throughout experiments. After insemination, fertilized eggs were washed with JSW three times and cultured at 25°C at a concentration of 50 embryos/ml in Petri dishes without agitation.

Nucleic acid extraction

Genomic DNA was extracted from sperm of P. japonica. Dry sperm was suspended in 20 vol of calcium-magnesium-free sea water, and to the suspension 10 vol of DNA extraction buffer (0.15 M NaCl/10 mM Tris-HCl, pH 8.0/10 mM EDTA/0.1% SDS) containing 100 μg/ml Proteinase K was added. The mixture was incubated at 55°C for 2 hr then 37°C for 16 hr, and followed by extraction with phenol and then phenol/chloroform. DNA was precipitated by adding 2 vol of ethanol to the mixture, and dissolved in TE. Total RNA was extracted from eggs or larvae with LiCl/urea method of Auffray and Rougeon (1980). Poly (A)⁺ RNA was isolated with Oligotex-dT30 (Roche) from total RNA.

Synthesis of cDNA

Complementary DNA was synthesized using 3′ RACE System (Gibco BRL) from a mixture of poly(A)⁺ RNAs that were extracted from all the larval stages at 6 hr-intervals from the unfertilized egg to the metamorphosis (0–72 hr after fertilization).

PCR, RT-PCR, and 3′ RACE

Four degenerated primers, F0, F, R1, and R2, were used to amplify Hox fragments of P. japonica. F0 and F, forward primers, corresponded to partially overlapping amino acid sequences, QLTELEK and LELEKEF, in the first helix of the homeodomain of the Hox genes, respectively. On the other hand, R1 and R2, reverse primers, located to the sequences, FQNRRMK and KIWFQNR, in the third helix, respectively. Sequences of the primers were as follows. F0; 5′-CARYTNACNGARYTNGARAA-3′, F; 5′-YTNGARYTNGARAARGARTT-3′, R1; 5′-TTCATNCKNCKRTTYTGRAA-3′, R2; 5′-CKRTTYTGRAACCADATYTT-3′. To amplify the engrailed gene fragment, we designed another primer, EN-F (5′-GAYGARAARMGNCCNMG-3′), which located at N-terminal of the homeodomain, and used with R2 primer. We amplified the 3′ end of Pj3 cDNA using 3′ RACE System (Giboco BRL) and Pj3-specific primer (5′-CGATATCTCACCCGACG-3′).

For each 25 μl reaction, 100 ng of genomic DNA or cDNA synthesized from10 ng of poly (A)⁺ RNA was used as a template. Reaction mixtures contained 10 μM of each primer, 200 μM of deoxynucleotides, 1 unit of Taq DNA polymerase (Toyobo), and 1 unit of anti-Taq antibody (Toyobo) in a buffer supplied by the manufacturer. PCR cycles for genomic PCR and RT-PCR were as follows: 2 min at 94°C, 33 cycles (30 sec at 94°C, 2 min at 50°C, 30 sec at 72°C), 20 min at 72°C. In the 3′ RACE, we elongated the extension time from 30 sec to 2 min. After PCR, the reaction mixture was applied to an agarose gel, and electrophoresed in TAE buffer. Gels were stained with ethidium bromide (0.5 μg/ml) to detect PCR products.

Sequencing PCR products

PCR products from excised agarose gel bands were purified using MERmaid Kit (Bio 101) or Prep-A-Gene DNA Purification Systems (BioRad), and ligated into pT7/T-vector (Novagen). Plasmid DNAs were purified using Plasmid Miniprep Kit (BioRad) and subjected to sequencing reactions (Amersham). Sequences were determined with an automated sequencer LI-COR 4200.

Sequence analysis

Derived amino acid sequences of P. japonica Hox-type genes were compared to those of Tripneustes gratilla (Dolecki et al., 1986; Dolecki et al., 1988; Wang et al., 1990), Paracentrotus lividus (Di Bernardo et al., 1994), Lytechinus variegatus (Ruddle et al., 1994), Heliocidaris erythrogramma (Popodi et al. 1996), Hemicentrotus pulcherrimus (Ishii et al., 1999), Strongylocentrotus purpuratus (Martinez et al., 1999), Asterina minor (Mito and Endo, 1997), Oxycomanthus (Comanthus) japonicus, and Stegophiura sladeni (Mito and Endo, 2000). Multiple alignments and neighbor-joining analyses were performed using CLUSTAL W (Thompson et al., 1994).

PCR-amplification of Hox genes

In order to survey the Hox genes of P. japonica, we PCR-amplified the Hox-type sequences using genomic DNA and cDNA as templates. Complementary DNA was synthesized from poly (A)⁺ RNA that was prepared from all the larval stages from the unfertilized egg to the metamorphosis. We first used two degenerated primers, F and R1, corresponding to the first and the third helices of the homeodomain, which are highly conserved among the Hox genes (Bürglin, 1994). As the result, 11 Hox-type sequences of 85 nucleotides long were identified, and named Pj1-Pj11 in order of the determination. For their sequence determination, at least two clones of identical sequence were independently obtained to exclude the possibility of mutations introduced by PCR. Still a variation was observed on Pj7 sequence, where T was replaced by C at the nucleotide 53. It is reported that the DNA of S. purpuratus displays 4–5% intraspecific sequence polymorphism (Britten et al., 1993). Since the substitution was synonymous in the amino acid sequence, we interpreted that the variation was due to a polymorphic allele.

We next used other primer sets, F/R2 for genomic PCR and F0/R1 for RT-PCR, and use of the latter set resulted in identification of two more Hox-type sequences, Pj12 and Pj13. Fig. 1 shows the sequences of Pj1–Pj13 (homeobox positions 60–144), the derived amino acid sequences (homeodomain positions 21–48), and also the number of clones obtained by genomic PCR and RT-PCR.

Fig. 1

(A) P. japonica Hox-type sequences at positions 60–144 of the homeobox, (B) the derived amino acid sequences at positions 21–48 of the homeodomain, and (C) the number of clones obtained by genomic PCR and RT-PCR.

Phylogenetic analysis of Hox genes

So far eight Hox genes as well as other types of the homeobox genes have been isolated from several sea urchin species, and they have been termed Hbox1-12 in order of the isolation. Recently Martinez et al. (1999) showed the organization of the Hox gene cluster in the S. purpuratus genome, and renamed the Hox genes as SpHox 1-11/13b on the basis of both their order in the cluster and their paralogous affinities with the vertebrate Hox genes. In order to infer paralog (cognate) groups to which the P. japonica Hox-type genes belong, we compared sequences of Pj1–Pj13 with those of the Hox genes of S. purpuratus and other sea urchins. Fig. 2 shows a neighbor-joining tree based on 25 amino acids of the homeodomains from P. japonica and 6 other species of sea urchins. This tree showed the corresponding relationship as follows. Pj3, Pj6, and Pj10 were identical with Hox4/5, Hox6, and Hox7 of S. purpuratus, respectively. Pj1, Pj2, Pj4, Pj5, Pj7, Pj10, Pj11, and Pj12 were strongly suggested to be orthologous to Hox11/13a, Hox8, Hox2, Hox11/13b, Hox6, Hox9/10, Hox7, Hox3, and Hox1, respectively, with high percentages of support (82-99%). Pj8, Pj9, and Pj13, however, had no counterparts in other sea urchin Hox genes reported so far. As is shown in Fig. 2, our PCR survey of the P. japonica Hox genes succeeded in identification of all the orthologs of sea urchin Hox genes. Pj8, 9, and 13 isolated in this study were considered to be novel Hox-type genes in sea urchins.

Fig. 2

Neighbor-joining tree based on 25 amino acids of the homeodomains from P. japonica (Pj), S. purpuratus (Sp), H. erythrogramma (He), T. gratilla (Tg), L. variegatus (Lv), P. lividus (Pl), and H. pulcherrimus (Hp). The tree is rooted with corresponding sequence of P. japonica engrailed (QQSNYLTEQRRRTLAKELTLSESQI). Percentage of support (50% or higher) in 1000 bootstrap searches is shown on the branches. SpHox is a new name of sea urchin Hox genes based on its order in the cluster and its affinities with vertebrate Hox genes. Numerals of Pj are in order of the determination in the present study, while those of other sea urchins except Sp indicate Hbox numbers (previous names of sea urchin Hox genes).

Database searches revealed that Pj8 and Pj9 were 93% and 100% identical in the amino acid sequence with the mouse Evx-1/2 and the Amphioxus Xlox, respectively. The Evx-1/2 are homologs of the Drosophila even-skipped gene that controls segmentation along the anterior/posterior axis. They are not only similar to the Hox genes in their homeodomain sequences, but each linked to the 5′ end of Hox gene clusters (Bürglin, 1994). A cnidarian Evx-like gene also locates adjacent to a Hox-like gene (Miller and Miles, 1993). Therefore the sequence similarity between the Evx and Hox genes reflects tandem duplication. This explains the reason the primers designed for the Hox genes amplified not only the Hox genes but also the Evx-type gene (Pj8) in the present study. Similarly, it is also the case with isolation of the Xlox-type gene (Pj9). The Xlox was first isolated as a Hox-type gene that does not map to the Hox cluster, and was later shown to be a member of the ParaHox gene cluster, a duplicated sister of the Hox gene cluster (Brooke et al., 1998). This is the first report to show the Evx-type gene in the echinoderm, while Xlox-type sequences have been isolated in the PCR survey of the Hox genes of other classes of echinoderms (Mito and Endo, 1997, 2000).

In vertebrates, five paralogous groups of posterior genes, PG9-13, have been identified. Phylogenetic analysis of the homeodomain sequences suggests that two ancestral posterior genes, one for PG9-10 and another for PG11-13, had already existed before divergence of protostomes and deuterostomes (Zhang and Nei, 1996). Tandem duplication of the ancestral genes is thought to have occurred in the deuterostome lineage to increase posterior genes in number. Amphioxus has at least four posterior genes, two of which are orthologous to the vertebrate PG9-10, but the rest two genes are uncertain in the relationship to the vertebrate paralog groups (Garcia-Fernàndez and Holland, 1996). In sea urchins, three posterior genes, Hox9/10, Hox11/13a, and Hox11/13b, have been isolated. Although Hox9/10 is very similar to the chordate PG9-10 genes, there is no specific orthologous relationship between Hox11/13 and the chordate PG11-13 genes (Martinez et al., 1999). In the present study, four posterior genes were identified from P. japonica: three orthologs of the known posterior genes and a novel one (Pj13). Pj13 was suggested the most similar to the posterior Hox genes by database searches as well as phylogenetic analysis (Fig. 2). Recently Mito and Endo (2000) have reported that at least four Hox Genes of Peronella japonica cognate groups are recognizable among the echinoderm posterior genes, and designated this new group HboxP9. Figure 3 shows a neighbor-joining tree of the echinoderm posterior genes including HboxP9. It suggested, however, that Pj13 might be rather related to Hox11-13b than to HboxP9 since the C. japonicus Hbox7 is considered to be an ortholog of S. purpuratus Hox11-13b (Mito and Endo, 2000). These observations imply that some of these posterior gene duplications may have occurred independently in different lineage within a phylum or even a class.

Fig. 3

Neighbor-joining tree of the echinoderm posterior Hox genes. Percentage of support (50% or higher) in 1000 bootstrap searches is shown on the branches. Pj13, a novel sea urchin posterior gene, may be rather related to Hox11-13b than to HboxP9, the fourth posterior group identified in the sea star A. minor (AM), the feather star C. japonicus (CJ), and the brittle star S. sladeni (SS). Numerals of AM, CJ, and SS indicate Hbox numbers (see Fig. 2).

Putative Hox gene cluster

Based on the estimation of orthology to the known Hox genes mainly from S. purpuratus, the present results revealed the putative Hox gene cluster of P. japonica (Fig. 4). The putative cluster contains three anterior, four medial, and four posterior Hox genes plus the Evx, although their physical linkage remains to be elucidated. They represent all the paralog groups of vertebrate Hox clusters except for a single gene of Hox4-5 types. The feature that the cluster lacks either Hox4 or Hox5 has been also suggested in other classes of echinoderms including sea stars, feather stars, and brittle stars (Mito and Endo, 1997, 2000). On the other hand hemichordates contain both Hox4 and Hox5 (Pendleton et al., 1993). Since the hemichordate is suggested to be a sister group of the echinoderm (Wada and Satoh, 1994), loss of either Hox4 or Hox5 may be characteristic of the echinoderm Hox cluster, a synapomorphy of echinoderms.

Fig. 4

Organization of the vertebrate Hox cluster, S. purpuratus Hox cluster, Amphioxus ParaHox cluster, and the putative Hox cluster of P. japonica. Each box indicates a Hox gene, and the number in the box shows the vertebrate paralog group. The vertebrate cluster is based on Bürglin (1994), and the S. purpuratus cluster is on Martinez et al. (1999). The Amphioxus ParaHox cluster is by Brooke et al. (1998).

Paralog groups of the vertebrate Hox clusters can be correlated with some of the Hox genes of Amphioxus, sea urchins, and even Drosophila. This fact indicates that the paralogous relationships have still remained distinctive over hundreds million of years. Sharkey et al. (1997) have identified characteristic residues that define the different paralog groups of the Hox genes. Although Hox4 and Hox5 are almost identical in their homeodomain sequences, both conserve several paralog-characteristic residues outside the homeodomain. In order to infer a paralog group to which Pj3 (Hox4/5) belongs, we isolated the 3′ end of Pj3 cDNA by 3′ RACE and compared the derived amino acid sequence with those of Hox4 and Hox5. Fig. 5 shows the conserved paralog-characteristic residues in the C-terminal amino acid sequences outside the homeodomain of Hox4 and Hox5, and the corresponding sequence of Pj3. Four continuous residues of ‘PNTK’ are characteristic to Hox4, which are conserved in Drosophila as well as all vertebrate members. On the other hand, Hox5 conserves two characteristic lysine residues among vertebrates. Pj3 sequence included one of the Hox5-characteristic lysine residues, but it did not match widely conserved Hox4-characteristic residues at all. We inferred from the sequence that Pj3 may be Hox4 rather than Hox5.

Fig. 5

Conserved and paralog characteristic residues of Hox4 and Hox5 in the C-terminal side of the homeodomain, and the corresponding amino acid sequence of Pj3. Amino acids represented in bold are conserved by all vertebrate members of the paralog group, while those in white are conserved by Drosophila as well as vertebrates (Sharkey et al. 1997). Note that Pj3 (Hox4/5) sequence includes one of the Hox5-characterisitic lysine residues, but does not match the Hox4-characteristic residues at all.