In echinoids, most species adopt one of two developmental modes. Indirect developers make numerous small eggs, which develop into larvae that require a feeding period before metamorphosis. In contrast, direct developers make few large eggs. These large eggs then develop into larvae that do not feed, reducing the time in the water column before metamorphosis (Emlet et al.,1987; Raff,1987). Indirect development via a planktotrophic pluteus larva is thought to be the ancestral mode of echinoid development, and direct development with a non-feeding lecithorophic larva is thought to have evolved independently in several lineages (Strathmann,1978; Raff,1987; Emlet,1990).
Peronella japonica is a direct-type developing sand dollar, first characterized by Mortensen (1921). Its eggs are ∼300 μm in diameter, making them the smallest known among direct-developing echinoids (Okazaki and Dan,1954; Wray and Raff,1991). The larva may also represent an intermediate form in the evolution from indirect to typical direct development (Raff,1987; Amemiya and Arakawa,1996; Yajima,2007; Iijima et al.,2009). Indeed, the zygote forms micromeres at the 16-cell stage (Fig. 1A), and the descendants ingress into the blastocoel as the primary mesenchyme cells (PMCs) before hatching (Fig. 1B) to eventually differentiate to skeletogenic cells (Okazaki,1975; Amemiya and Arakawa,1996; Yajima,2007; Iijima et al.,2009). The embryo develops into an abbreviated pluteus larva with a pair of the postoral arms (Fig. 1E) and then metamorphoses without feeding on day three (Fig. 1F; Okazaki and Dan,1954; Okazaki,1975). PMCs contribute exclusively to the formation of larval skeletal elements, whereas late mesenchyme cells, similar to the secondary mesenchyme cells (SMCs) of typical indirect developers, are involved in adult skeletogenesis (Yajima,2007; Iijima et al.,2009).
P. japonica exhibits unique adult rudiment formation (Mortensen,1921; Okazaki and Dan,1954; Okazaki,1975). Gastrular invagination begins with the migration of late mesenchyme cells (Fig. 1C). Within a few hours, another invagination begins in the ectoderm in the center of the flattened oral field, eventually developing into a stomodeum-like invagination (Fig. 1D, G). This invagination extends along the dorsal side of the endomesoderm to the posterior end of the larva, forming the vestibule (Fig. 1E, H, I). The mouth does not open, and the blastopore closes, resulting in a blind sac (Fig. 1G). At ∼24 h, the hydrocoel begins to differentiate in a nearly median position from the left or right coelom, whichever lies close to the ventral side of the larva (Okazaki and Dan,1954; Fig. 1H, I). The location of the hydrocoel, together with the unusual median position of the vestibule, is strikingly different from the corresponding structures in other echinoids. However, the processes underlying formation of coelomic compartments, particularly the origin of the hydrocoel, remain undefined. After the enlargement of the hydrocoel, five lobes are pushed out and arranged in a bilaterally symmetrical fashion with regard to the midline of the larva. Metamorphosis then begins with the protrusion of rudimentary spines and tube feet from the dorsal side of the larva (Okazaki and Dan,1954; Okazaki,1975).
In this study, we examined the formation of coelomic compartments, which are enclosed by the water vascular system or which become the main body cavities in P. japonica. To do this, we reconstructed three-dimensional (3D) images from serial sections of larvae. We show that the left coelom developed by both schizocoely and enterocoely from the archenteron tip. In contrast, the hydrocoel and right coelom sequentially formed from the archenteron by enterocoely. Furthermore, we defined each ambulacrum of P. japonica according to Lovén's (1874) system by raising juveniles until they had a mouth and anus.
P. japonica Formed an Adult Anus at the Anterior End of the Larva
Similarly to Okazaki and Dan (1954; Fig. 1G), we defined the site of the vestibular opening (stomodeum-like structure in typical indirect developers) as the anterior side of the larva. We define the ventral side as the side containing the blastopore. The blastopore closes during the prism stage but remains as a pit for several hours. The vestibule, therefore, invaginates along the dorsal side of the larva along the anteroposterior (AP) axis (Fig. 1H, I).
Figure 2 shows a P. japonica imago 3 weeks after metamorphosis, which had been fed the diatom Chaetoceros gracilis. It developed five sets of dental elements, adult six-rayed spines, and 15 tube feet (out of focus in this photo). It also retained two larval postoral rods (black and white arrowheads in Fig. 2), which are traces of the anterior side of the larva. In our culture condition, several percent of the imagoes (n > 100) retained the larval rods. We observed the digestive tract by diatom chlorophyll fluorescence and found that it started at the masticatory apparatus in the oral center of the imago, involuted counter-clockwise (when viewed from the aboral side) approximately three quarters around, reached the former anterior side of the larva and twisted nearly once around, and ended at the anus (red arrowhead in Fig. 2) between the postoral rods. This means that the AP axes of the larva and juvenile are parallel but opposite in direction. Lovén (1874) developed a numbering system for the ambulacra of echinoids based on the AP axis of Irregularia species. According to Lovén's system, we defined each ambulacrum of P. japonica (see below).
The Left Coelom Developed by Both Schizocoely and Enterocoely From the Archenteron Tip
To examine the formation of the coeloms in P. japonica larvae, we reconstructed 3D images from serial sections of larvae (2.5–3 μm thick) using DeltaViewer.
We examined gastrulae at 16 h, which had developed the vestibule in the oral field (Fig. 3A), and found that the archenteron leaned toward the left side. We also observed a number of mesenchyme cells, similar to the SMCs of indirect developers, migrating out of the archenteron tip (Fig. 3B). Figure 4A–D shows four of 70 horizontal serial sections (3 μm thick) along the dorsoventral (DV) axis of an early prism larva at 18 h, in which the ectodermal layer, a mass of mesenchyme cells, and archenteron-derived epithelia are colored green, red, and yellow, respectively. In the coloring process, we have eliminated the mesenchyme cells scattered in the blastocoel, including the PMCs and the blastocoelar cells. Figure 4E–H indicates reconstructed 3D images of the exterior and internal endomesodermal structures viewed from the right-posterior and slightly dorsal side of the larva, respectively. The exterior image (Fig. 4E) is shown in a reduced scale compared to the internal structures (Fig. 4F–H). From the archenteron, a coelomic pouch, marked in yellow, elongated counter-clockwise to the anterior and then dorsal direction; the tip of the coelomic pouch reached the anterodorsal side of the larva, which was adjacent to the vestibular floor (arrowheads in Fig. 4D, G, H). The mesenchyme, marked in red, covered the left side of the coelomic pouch (Fig. 4A–D, G). We should note that the boundary between the mesenchyme and enterocoelic epithelia was obscured in portions just below the tip of the coelomic pouch (blue asterisks in Fig. 4C, G, H; arrowheads in Supp. Fig. S1, which is available online). This lobe, marked in yellow, appeared to participate in the formation of the left coelom probably by enterocoely since it disappeared from the enterocoelic epithelium by the next stage, and the portion of the left coelomic cavity was encircled by an epithelial layer in the next stage (Fig. 5; arrowheads in Supp. Fig. S2).
Figure 5 shows six of 84 horizontal serial sections (3 μm thick) of an early pluteus larva at 24 h, and Figure 6 shows reconstructed 3D images of the exterior and internal endomesoderm structures, viewed from the right-posterior or left-anterior side of the larva, both with a slightly dorsal view. Color code is the same as in Figure 4, except for red. Red shows the left coelom developed from the mesenchyme plus an epithelial lobe formally marked in yellow (asterisks in Fig. 4). By this stage, the mesenchyme and lobe completely separated from the enterocoelic epithelium and started to form coelomic cavities (Fig. 5; Supp. Fig. S2). Additionally, the coelom expanded to the anterior side of the larva on either side of the enterocoelic structure. The left side of the coelom extended ventrally while the right side extended dorsally (Figs. 5B–F, 6C, G). Together with the original left side location, the C-shaped coelom dominantly covered the enterocoelic structures on the left side, with a ventral tilt of the left side (Fig. 6C, G).
The Hydrocoel and Right Coelom Sequentially Developed by Enterocoely From the Archenteron
In an early prism larva at 18 h, the tip of the coelomic pouch reached the anterodorsal side of the larva (arrowheads in Fig. 4), forming the putative right coelom. During the next 6 h of hydrocoel formation, the tip of the coelomic pouch elongated in the posterior direction until nearly reaching the dorsal center of the larva. At the end of elongation, the newly formed hydrocoel was inserted into the C-shaped putative left coelom (Figs. 5F, 6C, G). The putative right coelom corresponded to a turning portion of the coelomic pouch, from the anterior to the dorsal direction (Figs. 5D,E, 6D, H). This portion of the coelomic pouch became coelom-like in shape with a relatively large cavity in the pluteus larva at 28 h (Figs. 7B, C, 8D).
The Hydrocoel, Left Somatocoel, and Right Somatocoel Were Arranged Along the DV Axis of the Larva
Figure 7 shows six of 65 horizontal serial sections (2.5 μm thick) of a pluteus larva at 28 h that were used to reconstruct the 3D images of the exterior and internal structures (Fig. 8). Color code is the same as in Figure 5. By the pluteus larva stage, the formerly C-shaped left coelom had fused at the anterior side of the larva to encircle the hydrocoel (Fig. 8F, G). The hydrocoel started branching lobes to form the podium primordia. According to Lovén's system, we defined the presumptive ambulacra I–V, although the branches of the hydrocoel in ambulacra I and V were immature compared to those in ambulacra II, III, and IV (Fig. 8D, H). P. japonica juveniles have a triad of podia in each ambulacrum after metamorphosis (Okazaki and Dan,1954). At 28 h, the primordia of a triad of podia and the radial canal developed exclusively in the ambulacrum III (blue asterisks and ra, respectively, in Fig. 8D, H) on the future anterior side of adult sand dollars. On the other hand, the left coelom generated projections against the vestibular floor that covered the dorsal side of the hydrocoel and left coelom (Fig. 8B, F). This projection was evident in presumptive interambulacra 2 and 3 (Fig. 8C, G). We believe that these projections are rudiments of the dental sac because their development is similar to the formation of dental sacs from the left somatocoel in indirect developers (MacBride,1903; von Übish,1913; Smith et al.,2008).
Figure 9 shows six of 71 horizontal serial sections (2.5 μm thick) of a pluteus larva at 32 h that were used to reconstruct the 3D images of the exterior and internal structures viewed from either the dorsal side or the right-posterior (and slightly dorsal) side of the larva (Fig. 10). Color code is the same as in Figure 5. By 32 h, the five lobes of the hydrocoel were evident, although closure of the hydrocoel crescent resulting in a closed ring canal had not yet occurred between ambulacra IV and V (arrow in Fig. 10D). This is consistent with what is observed in echinoids (Hotchkiss,1995). Although the primary lobe was largely bilateral along Lovén's axis, which passes through both the ambulacrum III and the interambulacrum 5 (ra3–ds5 in Fig. 10C), it did not lie on the midline of the larva, but on the left side (see location of the future ring canal; arrow in Fig. 10D). Along with the location, the left coelom tilted leftward (see positions of dental sacs in Fig. 10B, F). By this stage, five dental sacs developed from the left coelom and interdigitated with five lobes of the hydrocoel (ds1–5 in Fig. 10C, G). This observation indicates that dental sacs in the posterior side of the larva (ds2, 3 in Fig. 8B, F) developed before the anterior ones, and suggests that the left coelom should actually be designated the left somatocoel, like that found in the larval anatomy of indirect developers (Smith et al.,2008).
At the same developmental time point, the right coelom, which had been largely segregated from the enteric sac (Fig. 9A–E), narrowed in the anterodorsal portion (arrow in Fig. 9D) and divided the coelom into anterodorsal and posteroventral sacs. The anterodorsal sac was connected to the hydrocoel at the base of the presumptive ambulacrum II via an epithelial duct (arrowhead in Fig. 9E; st in Fig. 10H). Although further analysis is required for definitive identification, we believe the duct is a presumptive stone canal. In indirect-developing echinoids, the stone canal that is associated with the left axocoel (ampula) and the hydroporic canal connects the ring canal to the madreporite on genital plate 2 (MacBride,1903; Gordon,1929; Smith et al.,2008). This presumptive stone canal suggests that the anterodorsal and posteroventral sacs derived from the right coelom may be the axocoel and right somatocoel, respectively. This classification is consistent with coelomic stacking, the echinoderm-characteristic arrangement of coeloms where the hydrocoel, left somatocoel, and right somatocoel are stacked along the oral-aboral axis of adults (David and Mooi,1998; Peterson et al.,2000). Our data show that the hydrocoel, left somatocoel, and putative right somatocoel were arranged along the DV axis of the larva with a leftward tilt in P. japonica (Figs. 9, 10).
Unusual Coelom Formation in P. japonica
Our observations indicate that P. japonica development represents an example of an extremely modified coelom formation in echinoids. The left coelom developed from mesenchyme cells that had migrated out of the archenteron tip and an epithelial lobe that had projected from the archenteron, whereas the hydrocoel and right coelom sequentially formed from the archenteron by enterocoely (Figs. 4, 5; Supp. Figs. S1, S2). Although enterocoely is the typical coelom formation strategy employed in most echinoderms, schizocoely has been described in several direct-developing species, including the spatangoid Abatus cordatus (Schatt and Féral,1996), the ophiuroid Amphipholis squamata (Fell,1946), and the crinoid Oxycomanthus japonica (Kubota,1988).
The coelom formation of P. japonica is unique in two regards. The first is that P. japonica uses two means of coelom formation: both schizocoely and enterocoely for the left somatocoel, and enterocoely for formation of the rest of the coelomic compartments, including the hydrocoel, stone canal, axocoel, and right somatocoel. The second unique feature is that P. japonica uses sequential coelom formation from the archenteron tip. In indirect developers, the left and right coelomic pouches pinch off from the respective sides of the archenteron near the end of gastrulation. Then, during the eight-arm pluteus stage, each coelom divides into three compartments, the axocoel, hydrocoel, and somatocoel, along either side of the esophagus and stomach (Smith et al.,2008). Even in the direct developers Asthenosoma iijimai and Heliocidaris erythrogramma, the left and right coeloms form independently from the archenteron tip, and then the left coelom divides into the hydrocoel and left somatocoel (Amemiya and Emlet,1992; Ferkowicz and Raff,2001). Thus, unlike both the indirect and the direct developers, the P. japonica archenteron tip sequentially generates coelomic compartments: first, mesenchyme cells plus an epithelial lobe that give rise to the future left somatocoel, and then the future hydrocoel, axocoel, and right somatocoel by enterocoely. By skipping the initial bilateral phases of coelom formation, sequential coelom formation results in direct arrangement of the coelomic compartments along the adult oral-aboral axis in both stacking order and connection via the stone canal. Additionally, P. japonica probably skips formation of the hydroporic canal and hydropore. In indirect developers, these structures develop from the left coelom prior to its differentiation into the left axocoel, hydrocoel, and somatocoel (Smith et al.,2008). However, we did not observe these types of tubular structures or an opening in either the serial sections or the 3D images of P. japonica larvae from the early prism (18 h) to pluteus larva (32 h) stages. In fact, the dorsal and ventrolateral sides of the endomesoderm were covered with the vestibular floor consisting of stratified epithelia and a gap-less larval ectodermal layer with a blastopore, respectively.
P. japonica does not form larval mouth (stomodeum), but instead develops a vestibule in the region (Okazaki and Dan,1954; Kitazawa and Amemiya,1997). In addition to the direct arrangement of coelomic compartments along the adult oral-aboral axis, this precocious formation of the vestibule may contribute to the rapid adult rudiment formation in P. japonica.
P. japonica Retains Traits of Indirect-Developing Sand Dollars
Unlike in Echinacea species (so-called regular urchins; Smith,1984), the bilateral symmetry of adult skeletal elements in Echinarachnius parma, an indirect-developing sand dollar, is marked along Lovén's axis rather than von Übisch's axis (Gordon,1929). Furthermore, Gordon (1929) showed E. parma-characteristic features: the ambulacrum III develops more rapidly than the others, and the skeletal plates in interambulacra 2 and 3 are larger and more numerous than those in the three posterior areas of adults. In P. japonica, we observed bilateral symmetry of the primary lobe along Lovén's axis (Figs. 8, 10) and precocious formation of both the triad of podia in ambulacrum III and the dental sacs in interambulacra 2 and 3 (Figs. 8, 10). Thus, P. japonica appears to conserve traits of indirect-developing sand dollars, except for adult rudiment location.
Kitazawa et al. (2004) discovered two asymmetric traits along the left-right axis in P. japonica larvae: shifts toward the left dorsal side of both the vestibular opening and the ciliary band. Together with subsequent enlargement of the vestibular opening and dorsal expansion of the oral field, Kitazawa et al. (2004) observed part of the adult rudiment, such as the podia, through the vestibular opening by scanning electron microscopy. This external observation is consistent with our internal observations that there is a leftward tilt of the left somatocoel encircling the hydrocoel and a concomitant leftward shift of the primary lobe (Figs. 8, 10). These observations indicate that P. japonica retains ancestral asymmetry along the left-right axis and, furthermore, that the location of the adult rudiment in P. japonica is not as exceptional as previously thought.
Animals, Embryos, and Larvae
Adult P. japonica were collected in Matsushima Beach, Noto Island, Ishikawa, Japan. Gametes were obtained by intracoelomic injection of 0.5 M KCl. Embryos, larvae, and juveniles were cultured in plastic dishes at 24°C in Marine Art SF-1 artificial seawater (Tomita Pharmaceutical, Tokushima, Japan). To culture juveniles, the seawater was changed every other day, and a new suspension of the diatom Chaetoceros gracilis was added.
Reconstruction of Three-Dimensional (3D) Images
For anatomical observations, embryos and larvae were fixed with 4% paraformaldehyde in artificial seawater (van't Hoff,1903), dehydrated in an ethanol series and acetone, embedded in Technovit 8100 (Heraeus Kulzur, Hanau, Germany), and cut serially into 2.5–3-μm-thick sections on a microtome LEICA RM 2255 (Leica, Nussloch, Germany). Sections were observed with a fluorescence microscope BZ-9000 (KEYENCE, Osaka, Japan). Because P. japonica embryos and larvae emit autofluorescence, we recorded fluorescence images of unstained sections. To reconstruct 3D images, the fluorescence images were converted to black and white and false-colored by hand with Adobe Photoshop CS5 (Adobe Systems, Inc., San Jose, CA). To examine the formation of coelomic compartments, the ectodermal layer, a mass of mesenchyme cells (the left coelom), and archenteron-derived epithelia were colored green, red, and yellow, respectively. In the coloring process, mesenchyme cells scattered in the blastocoel, including the blastocoelar and skeletogenic cells, were eliminated. Three-dimensional images were reconstructed from colored section images using DeltaViewer (DeltaViewer Project, http://vivaldi.ics.nara-wu.ac.jp/∼wada/Delta-Viewer/).
We especially thank Dr. Shonan Amemiya and Dr. Takuya Minokawa for their technical advice and valuable comments regarding this study. We thank Dr. Atsuko Yamazaki for helpful discussions. We also thank Dr. Daisuke Kurokawa, Dr. Akihito Omori, and Ms. Natsu Katayama for technical advice on the microtomy, and Dr. Yuichi Sasayama for allowing us to use the fluorescence microscope, BZ-9000. We thank the reviewers and editor whose thoughtful suggestions helped us to improve the manuscript. This study was supported by JSPS KAKENHI grant-in-aids to J.T. and M.Y.