Animals and embryos

Mature adult Ptychodera flava were collected during December at a sand bar in Kaneohe Bay, Oahu, Hawaii (Hadfield, 1975). Natural spawning was induced as described (Tagawa et al., 1998a). Fertilized eggs were allowed to develop to the desired developmental stages at room temperature for a week.

In Japan, the sea cucumber Stichopus japonicus is cultivated for marketing. Collection of adult sea cucumbers from Mutsu Bay, induction of spawning by temperature shift, artificial fertilization and cultivation of embryos were performed by the staff of Aomori City Fisheries Technology Center, Aomori, Japan. These larvae were provided to us, and we cultured them at room temperature by feeding them algae in glass beakers containing seawater supplemented with 50 mg/l of streptomycin, in our laboratory at Kyoto University. Auricularia larvae began to change into doliolaria larvae as judged morphologically about 10 days after fertilization. They metamorphosed to juveniles about 3 weeks after fertilization (Shoguchi et al., 2000).

Molecular cloning

We first isolated cDNA clones for hemichordate BMP-2/4 (Pfbmp2/4) and then those for sea cucumber BMP-2/4 (Sj-bmp2/4). PCR amplification was carried out using a P. flava gastrula cDNA library (Tagawa et al., 1998b) as template DNAs. The primer sequences were as follows: BMP-F: 5′-TGGGAYGAYTGGATHGTNGC-3′, BMP-R: 5′-GTYTGNACDATNGCRTGRTT-3′, (where D = not C, H = not G, N = any, R = A or G, and Y = C or T), which correspond to the amino acid sequences, WDDWIVA and NHAIVQT, respectively. We screened the same cDNA library by probing with the thus-obtained PCR fragments labelled with [³²P]dCTP. Several cDNA clones were isolated. The phage cDNA clones were converted into plasmids. Isolated cDNA clones were sequenced on both strands using an automated DNA sequencer (ABI PRISM, Perkin Elmer).

Total RNA was extracted from 24-hr gastrulae of S. japonicus by the AGPC method (Chomczynski and Sacci, 1987). Poly(A)⁺ RNAs were purified by use of Oligotex dT30 beads (Roche Japan). A cDNA library was constructed using a kit (ZAP-cDNA synthesis kit, Stratagene) following the supplier's instructions. cDNA clones for Sj-bmp2/4 were isolated as in the case of those for Pf-bmp2/4.

Sequence comparison and molecular phylogenetic analysis

The putative amino acid sequences of the Pf-bmp2/4 and Sjbmp2/4 proteins were deduced from their nucleotide sequences and aligned with other TGF-β superfamily gene product sequences. Their molecular phylogenetic relationships were analyzed by means of the neighbor-joining method using PHYLIP ver. 3.5 (Felsenstein, 1993).

Genomic Southern hybridization

High-molecular weight genomic DNA of P. flava was exhaustively digested with EcoRI, HindIII or PstI and fractionated by electrophoresis. The DNA fragments blotted onto Hybond-N+ nylon membranes (Amersham) were hybridized with [³²P]-dCTP-labelled DNA probes and washed under specific conditions: hybridization, 0.5 M Na₂HPO₄, pH 7.2, 1 mM EDTA, 7% SDS at 65°C; washing, 2 x SSC, 0.1% SDS at 65°C. The probes were 0.9 kb in length containing the C-terminal conserved region of Pf-bmp2/4.

On the other hand, high-molecular weight genomic DNA of S. japonicus was exhaustively digested with BglII, EcoT22I, EcoRV or PvuII, fractionated by electrophoresis, hybridized with [³²P]-dCTP-labelled DNA probes, and washed under the same conditions as mentioned above. The probes were 1.0 kb in length containing the C-terminal conserved region of Sj-bmp2/4.

Whole-mount in situ hybridization

Whole-mount specimens were hybridized in situ basically as described by Tagawa et al. (1998b) and Shoguchi et al. (2000). Embryos were fixed in 4% paraformaldehyde in 0.5 M NaCl, 0.1 M MOPS, pH 7.5, on ice overnight. After a thorough wash with PBST (phosphate-buffered saline containing 0.1% Tween 20), the specimens were partially digested with 2 μg/ml proteinase K (Sigma) in PBST for 20 min at 37°C and post-fixed with 4% paraformaldehyde in PBST for 1 hr at room temperature. After a 1-hr prehybridization at 42°C, the specimens were hybridized with digoxigenin (DIG)-labeled antisense probes for about 16 hr at 42°C. The probe was synthesized following the instructions supplied with the kit (Boehringer Mannheim DIG RNA Labeling kit), and the hybridized fragments were reduced to about 200-500 nucleotides by alkaline hydrolysis. The probes were used at 0.5 μg/ml in the hybridization buffer. After hybridization, the specimens were washed and then digested with 20 μg/ml RNase A. After further washing, a signal was detected following the supplier's instructions.

Isolation and characterization of Pf-bmp2/4 and Sj-bmp2/4 cDNAs

cDNA clones for Pf-bmp2/4 (acorn worm P. flava -BMP2/4) and those for Sj-bmp2/4 (sea cucumber S. japonicus - BMP2/4) were isolated by PCR amplification and by library screening. The insert of the longest clone for Pfbmp2/4 was 2,349 bp (DDBJ/GenBank/EMBL database accession number,  AB028219), and encoded a predicted polypeptide of 405 amino acids. The insert of the longest clone for Sj-bmp2/4 was 2,403 bp (DDBJ/GenBank/EMBL database accession number,  AB057451), and encoded a predicted polypeptide of 422 amino acids.

Fig. 1A shows an alignment of the amino acid sequences of the BMP-2/4 subclass gene products within the conserved carboxyl-half. The five asterisks underneath the alignment show residues specifically conserved among the BMP-2/4 subclass members. Although Sj-bmp2/4 contained a relatively high proportion of amino acid substitutions, both Sj-bmp2/4 and Pf-bmp2/4 contained the BMP-2/4 subclass-specific residues.

Fig. 1

(A) An alignment of the amino acid sequences of the C-terminal half of Sj-bmp2/4 and Pf-bmp2/4 (underlined) with those of other BMP-2/4 subclass members. High-lighted residues are identical among all members shown here. The five asterisks underneath the alignment show typical BMP-2/4 subclass-specific residues within the highly conserved C-terminal region, where other BMPs contain other amino acids. Arrows indicate sequences used for the degenerate oligonucleotide primers in the PCR cloning. The region used for the molecular phylogenetic analysis is shaded. (B) Molecular phylogenetic analysis of relationships among TGF-β superfamily members as deduced from the neighbor-joining method. Both Sj-bmp2/4 and Pf-bmp2/4 are grouped with the Dpp subclass (Drosophila Dpp and BMP-2/4 gene products). Branch lengths are proportional to evolutionary distance corrected for multiple substitutions with the scale denoting 0.1 amino acid substitutions per site. The numbers indicate the relative robustness of each node as assessed by bootstrap analysis (100 replications).

We constructed a molecular phylogenetic tree for these gene products with other related TGF-β superfamily members (Fig. 1B). Both Pf-bmp2/4 and Sj-bmp2/4 were included in a group of the BMP-2/4 subclass, the clade of which was supported by a 74% bootstrap value. Taken together, these findings led us to conclude that these genes are orthologs of BMP-2/4.

The copy number of the genes in the genome

To determine the copy numbers of Pf-bmp2/4, a genomic Southern hybridization analysis was conducted. Fig. 2A shows a Southern blot probed with a DNA fragment of Pf-bmp2/4 including a C-terminal conserved region. The hybridization condition in Fig. 2A showed low stringency which allowed a single cross-hybridized band in the lane of HindIII-digested Halocynthia roretzi (ascidian) genomic DNA (data not shown). Single bands were observed in the lanes from digestion by EcoRI or HindIII, while two hybridization bands were observed in the PstI lane. The Pf-bmp2/4 DNA fragment used for this assay contained an internal PstI site. Therefore, this pattern of genomic Southern hybridization strongly suggests that Pf-bmp2/4 is the only member of Dpp subclass of the TGF-β family in the P. flava genome.

Fig. 2

(A) Low-stringency genomic Southern blot analysis of DNA from a single individual of P. flava. A Southern blot probed with a 0.9 kb DNA fragment of Pf-bmp2/4 including a C-terminal conserved region. Each lane was loaded with 5 μg of restriction-digested P. flava genomic DNA. ER, EcoRI; Hi, HindIII; Ps, PstI. (B) Low-stringency genomic Southern blot analysis of DNA from a single individual of S. japonicus. A Southern blot probed with a 1.0 kb DNA fragment of Sj-bmp2/4 including a C-terminal conserved region. Each lane was loaded with 10 μg of restriction-digested S. japonicus genomic DNA. Bg, BglII; ET, EcoT22I; EV, EcoRV; Pv, PvuII.

Similarly we determine the copy numbers of Sj-bmp2/4 by a genomic Southern hybridization analysis. As shown in Fig. 2B, single bands were observed in the lanes from digestion by BglII, EcoRV or PvuII, while several hybridization bands were observed in the EcoT22I lane. Therefore, it is likely that Sj-bmp2/4 is the only member of Dpp subclass of the TGF-β family in the S. japonicus genome.

Developmental expression of the acorn worm Pf-bmp2/4

Cleavage of P. flava is holoblastic and radial, with the embryo developing into a hollow blastula (Hadfield, 1975; Tagawa et al., 1998a). Gastrulation begins approximately 18 hr after fertilization with the appearance of a cleft at the vegetal plate. The cleft rapidly extends to form the archenteron. Approximately 25 hr after fertilization, the enterocoelic pouch appears from the anterior swelling of the archenteron. Later, the pouch develops into a protocoel. At about 40 hr of development, an anterior prolongation of the protocoel occurs to make a connection with the apical plate and apical strand. The tubular evagination of the protocoel makes contact with the aboral (dorsal) wall of the embryo, opening exteriorly to form a proboscis pore or coelomopore (Fig. 3A, B). The tip of the archenteron contacts the thickened stomodeum on the oral (ventral) ectoderm, and eventually opens to form a mouth (Fig. 3A, C).

Fig. 3

(A–D) Spatial expression pattern of Pf-bmp2/4, as revealed by whole-mount in situ hybridization with a DIG-labeled antisense probe. (A, B) An early tornaria larva 48 hr after fertilization, (A) lateral view and (B) aboral view. A region of proboscis coelom contacting the aboral ectoderm shows a Pf-bmp2/4 signal (arrowhead). bp, blastopore; pc, proboscis coelom; pp, proboscis pore; sd, stomodeum. Scale bar, 50 μm. (C, D) A four-day-old tornaria larva, (C) left lateral view and (D) posterior view. m, mouth; st, stomach. Signal in the proboscis canal persists (arrowhead). (E–M) Spatial expression pattern of Sj-bmp2/4, as revealed by whole-mount in situ hybridization with a DIG-labeled antisense probe. (E) An auricularia larva (48 hr), left lateral view. (F–H) An auricularia (94 hr), (F) dorsal view and (G, H) 4x further magnified images. (H) shows a negative control hybridized with a sense probe. The signals in the hydroporic canal were detected in the specimens with the antisense probe (F, G; arrowhead). (I) A doliolaria larva, left lateral view. The expression in the hydroporic canal continues (arrowhead). (J) A hydrocoel of an auricularia larva before metamorphosis. (K) A hydrocoel of a ciliary band remodeling larva during metamorphosis. Additional expression appears in the evagination regions of the hydrocoel (red arrowheads), anlage formation for the buccal podia, radial vessels, and polian vesicle. (L, M) A doliolaria larva, ventral view (L) and right lateral view (M). Signals were observed in the hydroporic canal (white arrowhead), mid-ventral radial vessel (arrow), and buccal podia (red arrowheads). The abbreviations are: bup, buccal podium; es, esophagus; hc, hydrocoel; hp, hydropore; hpc, hydroporic canal; m, mouth; mvrv, mid-ventral radial vessel. Scale bar in (E): 100 μm for (E), (F), (I), (L), (M); 50 μm for (J), (K); and 25 μm for (G), (H).

Pf-bmp2/4 mRNA was not detected above background level before the late gastrula stage (data not shown). In the late gastrula or early tornaria larva, distinct expression of Pfbmp2/4 began in the region where the protocoel (proboscis canal) attached to the dorsal ectoderm (Fig. 3A, B; arrowhead). This proboscis-canal-specific expression lasted into the later stage of tornaria (Fig. 3C, D; arrowhead). No Pfbmp2/4 expression was observed during embryogenesis, suggesting that it is not involved in axis formation.

Developmental expression of the sea cucumber Sj-bmp2/4

S. japonicus embryos undergo an indirect development via the auricularia larval stage, and their early development is that of a type of deuterostome embryos (Imai et al., 1950; also see Smiley et al., 1991). The cleavage is radial, equal and holoblastic. The embryo develops into a coeloblastula approximately 10 hr after fertilization, and then hatches before gastrulation. Gastrulation begins approximately 17 hr after fertilization, as an involution from the vegetal plate. As the archenteron reached about halfway across the blastocoel, it made a right angle bend and grew out toward the blastoderm ventrally, and then fused with the stomodeum to open a mouth (data not shown). The invaginating enterocoel is single in S. japonicus, whereas it is paired in sea stars and sea urchins (Smiley et al., 1991). It then made contact with the aboral (dorsal) ectoderm to open a hydropore (dorsal pore or coelomopore). The hydropore was located just to the left of the mid-dorsal line of the aboral (dorsal) ectoderm (Fig. 3F, J).

After gastrulation, the embryo became an auricularia larva with a looped circum-oral ciliated band. The arch-enteron differentiated into three regions, esophagus, stomach and rectum. The enterocoel also divided into an anterior portion, i.e., the primordium of the left hydrocoel, and a posterior portion, i.e., the primordia of the left and right somatocoels. The left hydrocoel was linked to the opening of the hydropore by a hydroporic canal. In this species, the left axocoel was not clearly identifiable (in echinoderms, the left axocoel and left hydrocoel usually do not separate). No right axocoel or hydrocoel was formed (cf., Smiley et al., 1991). Fig. 3E shows a 48-hr early auricularia larva. In the early auricularia, Sj-bmp2/4 expression commenced in the region of the hydroporic canal (Fig. 3E; arrowhead). This expression in the hydroporic canal continued in later auricularia larvae (94 hr; Fig. 3F, G; arrowhead). Fig. 3G shows the expression in the hydroporic canal under higher magnification (arrowhead), whereas Fig. 3H shows a sense-strand hybridization control.

Subsequently, the auricularia larva metamorphosed into a barrel-shaped doliolaria larva. Fig. 3I, M shows doliolaria larvae. The expression in the hydroporic canal persisted in this stage (Fig. 3I, M). The metamorphic morphogenesis was accompanied by shrinking of the body, traveling of the oral-anal axis, dynamic remodeling of the ciliated bands from a single continuous perioral loop into five serial latitudinal loops, and rapid growth of the hydrocoel (compare Fig. 3K with Fig. 3J) (Smiley, 1986). At this stage, new expression appeared in the evagination regions of the hydrocoel (Fig. 3K; red arrowheads). The hydrocoel grew around the esophagus to form a water vascular ring. The hydrocoel also produced pentametric buccal podia, pentametric radial vessels, and a single Polian lobe, as evaginations. The buccal podia grew anteriorly, whereas the radial vessels grew posteriorly between the evaginations of the buccal podia. The anterior tips of buccal podia seemed to express Sj-bmp2/4 weakly (Fig. 3L, M; red arrowheads). Among the five radial vessels, the mid-ventral one grew faster than the other four (Smiley, 1986). The posterior tip of the growing mid-ventral radial vessel also seemed to express Sj-bmp2/4 (Fig. 3L, M; arrow).

Conserved expression of nonchordate deuterostome BMP-2/4 in the coelomic duct

The complex of coelomopore and pore-canal is the most characteristic structure among the nonchordate deuterostome larvae (Ruppert and Balser, 1986; Nielsen, 1995). It consists of a duct leading from a coelomic cavity to an external pore situated asymmetrically to the left of the dorsal midline. In S. japonicus, the coelomic evagination is formed from the archenteron during gastrulation, and it then curves dorsally, and makes contact with the dorsal ectoderm to form a hydropore (Smiley et al., 1991). Sj-bmp2/4 is expressed in this hydroporic canal. In P. flava, the protocoel is pouched out from the archenteron, and then a tubular evagination extends from the protocoel dorsally, which opens as a proboscis pore at the midline of the dorsal ectoderm (Tagawa et al., 1998a). Pf-bmp2/4 expression was observed only in this proboscis canal in the present study. We thus concluded that the BMP-2/4 gene is expressed in the anterior coelomic canal in both the larvae of the sea cucumber and acorn worm. This suggests that not only the larvae in other echinoderms may also express the BMP-2/4 gene in the same region, but also that all the dipleurula larvae may share this expression.

Our results shown here support the idea that echinoderms and hemichordates share a common body plan at the larval stage. However, we must note that we could find a similarity only between the fully-organized larval forms, but their developmental processes involved in organizing this final morphology may rather include a good deal more evolutionary divergence than previously thought. The diversity in this process may represent an aspect of the property of development known as a “developmental hourglass”, also referred to in other phyla (Slack et al., 1993; Richardson, 1999), as vertebrate embryos show strikingly similar morphology only at the phylogenetic stage. If that is the case, the dipleurula larva represents a ‘phylotypic’ stage of the nonchordate deuterostomes. With respect to characterization of developmental regulatory genes, the data available about sea urchin embryos is the most detailed for all the nonchordate deuterostomes (e.g. Davidson et al., 1998). To construct a molecular map of the phylotypes of the nonchor-date deuterostomes, comprehensive analyses of multiple taxa of nonchordate deuterostomes will provide us further evolutionary insights.

Insight into the chordate evolution

We discussed above the possibility that the echinoderm and hemichordate larvae may share BMP-2/4 expression in the coelomopore complex. Interestingly, the amphioxus BMP-2/4 ortholog, AmphiBMP2/4, is expressed in the developing paired anterior diverticula, although the expression in the left diverticulum disappears in early larva, and thus the organized preoral pit does not express BMP-2/4 (Panopoulou et al., 1998; Table 1).

Table 1

The anterior coelomopore complex (left protocoel + ectodermal invagination) in deuterostome embryos and expression domains of BMP-2/4

Regarding the coelomopore complex, its putative presence in chordates has long been argued (Goodrich, 1917; Nielsen, 2001; Table 1). The coelomopore complex consists of the left protocoel and the ectodermal invagination forming the coelomopore. In amphioxus, a pair of coelomic sacs are given off as lateral pouches at the anterior end of the archenteron. The right one expands to form the head cavity of the larva, whereas the left one makes an opening exteriorly in a preoral pit. Later in adults, Hatschek's pit is retained on the left side of the roof of the mouth (Goodrich, 1917). As for vertebrate head development, it has been reported that Rathke's pouch first establishes a connection with premandibular somites, based on observations in some cartilaginous fishes. This connection is transient: as development proceeds, it is lost (Goodrich, 1917; Ruppert, 1990). Therefore, the components of the coelomopore complex, i.e. the left protocoel plus the coelomopore, are regarded as follows: in amphioxus, the left anterior head coelom plus the preoral pit (Hatschek's pit in adult); in vertebrates, the premandibular somite (eye muscle in adults) plus Rathke's pouch (anterior pituitary in adults) (see Ruppert, 1990; Table 1).

Development of the vertebrate Rathke's pouch has been a good model system in which to investigate organo-genesis with multiple inductive regulatory mechanisms. BMP-2/4 genes carry a major part of the inductive signals for Rathke's pouch development. Recent studies suggest that Rathke's pouch is initially induced by the BMP-4 signal from the neighboring ventral-diencephalon, whereas BMP-2 plays an intrinsic role in the developing pituitary anlage (Takuma et al., 1998; Treier et al., 1998). Although the BMP-2/4 expression in the putative chordate counterparts of the coelomopore is interesting, for now we cannot exclude the possibility that this similarity is due to a chordate-specific recruitment of BMP-2/4, since BMP-2/4 possesses a wide range of developmental roles, even including roles in head development (Jones et al., 1991; Francis-West et al., 1994).

Rathke's pouch development is a well-characterized process, and a number of developmental regulatory genes have been shown to play specific developmental roles in it (Treier et al., 1998). Accordingly, analyses of the developmental roles of Sj-bmp2/4 and Pf-bmp2/4 in the coelomopore formation in the respective organisms, in relation to the roles of the homologs of such genes, may be important topics for further comparative studies. In addition, since the earlier observations were reported, Rathke's pouch development has not been described in much detail in relation to the mesoderm (Gleiberman et al., 1999), and therefore we have little information about this structure as an enterocoelic pouch. Therefore, it may also be interesting to examine the expression of BMP-2/4 or other regulatory genes expression in the head development of lampreys or cartilaginous fishes, because they are thought to preserve ancestral conditions better than do higher vertebrates (see Northcutt, 1993; Kuratani et al., 1999).