Open Access
How to translate text using browser tools
27 January 2004 Development and Neural Organization of the Tornaria Larva of the Hawaiian Hemichordate, Ptychodera flava
Yoko Nakajima, Tom Humphreys, Hiroyuki Kaneko, Kunifumi Tagawa
Author Affiliations +

We report scanning and transmission electron microscopic studies of the early development of the Hawaiian acorn worm, Ptychodera flava. In addition, we provide an immunohistochemical identification of the larval nervous system. Development occurs and is constrained within the stout chorion and fertilization envelope that forms upon the release of the cortical granules in the cytoplasm of the egg. The blastula consists of tall columnar blastomeres encircling a small blastocoel. Typical gastrulation occurs and a definitive tornaria is formed compressed within the fertilization envelope. The young tornaria hatches at 44 hr and begins to expand. The major circumoral ciliary band that crosses the dorsal surface and passes preorally and postorally is well developed. In addition, we find a nascent telotroch, as well as a midventral ciliary band that is already clearly developed. The epithelium of tornaria is a mosaic of monociliated and multiciliated cells. Immunohistochemistry with a novel neural marker, monoclonal antibody 1E11, first detects nerve cells at the gastrula stage. In tornaria, 1E11 staining nerve cells occur throughout the length of the ciliary bands, in the apical organ, in a circle around the mouth, in the esophageal epithelium and in circumpylorus regions. Axon(s) and apical processes extend from the nerve cell bodies and run in tracks along the ciliary bands. Axons extending from the preoral and postoral bands extend into the oral field and form a network. The tornaria nervous system with ciliary bands and an apical organ is rather similar to the echinoderm bipinnaria larvae.


The advances of molecular biology in the last two decades have added a new dimension to phylogeny. Hemichordates are again highlighted as key organisms to study the evolutionary diversification of body plans between the chordates and the invertebrates (Tagawa et al., 1998a; Tagawa et al., 2001). Hemichordates have been closely associated by their adult morphology with chordates as they exhibit gill slits and a dorsal hollow nerve cord formed by neurulation (Bateson, 1885; Morgan, 1891). The postulated homology of the stomochord of hemichordates with the notochord of chordates (Bateson, 1885) now seems unlikely (Balser and Ruppert, 1990; Peterson et al., 1999). The planktonic ciliated larval form of enteropneusts (acorn worms), the tornaria, resembles the echinoderm asteroid bipinnaria larva or the holothuroid auricularia larva establishing a strong phylogenetic connection between hemichordates and echinoderms (Bateson, 1884; Willmer, 1990; Nielsen, 1995).

One of the intriguing subjects in phylogenetic analysis has been the origin of the dorsal nervous system of chordates from ancestral deuterostome forms (Lacalli, 1996). Molecular studies in tornaria have shown the expression of orthologues of a number of vertebrate brain-specific regulatory genes occurs in larval neural structures (T-brain, Tagawa et al., 2000; Otx, Harada et al., 2000; Distalless, Harada et al., 2001; Sox B, Taguchi et al., 2002; Nk2.1, Takacs et al., 2002). The expression of some of these genes have also been studied in echinoderm larvae (Gan et al., 1995; Lowe and Wray, 1997; Shoguchi et al., 2000; Fuchikami et al., 2002). The failure of many genes to conserve their evolutionary function in the echinoderms with highly derived body organization (Lowe et al., 2002) warns that attention must be paid to the structure and cell diversity of each animal when making generalizations about the relationships between gene expression patterns and gene function.

In spite of a great deal of attention to the phylogenetic position of hemichordates, most of our knowledge of their development and structure was defined a number of years ago (Hyman, 1959) and there has been a paucity of recent work (Hadfield, 1975; Dautov and Nezlin, 1992; Benito and Pardos, 1997). To provide further structural information on the development of enteropneust tornaria larva, we undertook ultrastructural and immunohistochemical observations of early development and formation of the larval nervous system of P. flava.


Adult acorn worms P. flava were collected from shallow sand bars at Kaneohe Bay or coral reefs at Paiko, Oahu Island, Hawaii. Mature eggs and sperms were obtained by induction of spawning using a shift of seawater temperature (Tagawa et al., 1998b). Fertilized eggs and embryos were reared at room temperature of 20 to 22°C.

For electron microscopy, eggs and embryos were fixed with 2.5% glutaraldehyde, 0.35M sucrose in 0.1M sodium cacodylate buffer, pH 7.4 for 1 hr at room temperature, postfixed with 1% OsO4 in 0.1M cacodylate buffer and dehydrated through ethanol series. For the scanning electron microscopy (SEM), dehydrated samples were critical point dried, and then sputter coated with gold. Specimens were examined with a Hitachi S510 scanning electron microscope at 25kV. For transmission electron microscopy (TEM), ultrathin sections of epoxy resin embedded specimens were stained with uranyl acetate and lead citrate, and observed in a JEOL JEM 1001 electron microscope at 80kV. Four week old tornaria larvae were prepared by rapid freezing in liquid propane using a Leica KF80 apparatus and freeze substitution with 4% OsO4 in acetone at −85°C.

For immunolocalization studies, embryos and larvae were fixed with 4% paraformaldehyde in sea water (SW) for 15 min at room temperature, washed with phosphate buffered saline (PBS, 0.1M, pH7.4) several times and briefly post-fixed with acetone at −20°C. Samples were stored in PBS containing 0.1% NaN3. The specimens were incubated in culture supernatant of 1E11 hybridoma cells and/or rabbit anti-serotonin polyclonal antibody (diluted 1/ 1,000 in PBS, DiaSorin, USA) overnight in a refrigerator or 2 hr at room temperature. After several rinsing with PBST (PBS containing 0.1% Tween 20), samples were treated with secondary antibodies labeled with Alexa 488 and/or Texas Red (Molecular Probes, Oregon, USA). Samples were observed with a confocal laser scanning microscope (Olympus Fluoview V300).

The monoclonal antibody, 1E11, was developed in mice immunized with an extract of radial nerve of the starfish, Asterina pectinifera. Fused hybridoma cell supernatants were screened for staining of the known nervous system of the bipinnaria larva of the same species. The 1E11 monoclonal antibody recognizes unreported adult and larval echinoderm nerve tracks as well as those that have been previously described (Nakajima et al., in press).


Ultrastructure of early development

SEM examination shows the egg is invested in a fibrous vitelline envelope (Fig. 1a, b). When this is removed the outer surface of P. flava unfertilized eggs is covered with dense, spiky microvilli, 1 to 2 μm in length (Fig. 1b). TEM micrographs of sections of unfertilized eggs show a 1 to 2 μm deep cytoplasmic cortical layer at the surface of the egg with 0.3 μm electron-dense cortical granules arrayed just beneath the plasma membrane (Fig. 1c). Yolk granules (YGs) occupy most of the endoplasm of the egg below the cortical layer. The YGs have an electron dense region and a relatively electron-lucent region of fine meshwork. Other than YGs, the cytoplasmic organelles are poorly developed and only scattered mitochondria, electron-dense lysosomal granules, and free ribosomes are observed.

Fig. 1

SEM and TEM figures of early development of Ptychodera flava. a. SEM photograph of the surface of an exposed unfertilized egg with a remnant of the opened egg envelope (arrow). (Scale bar: 10 μm) b. Close up of the unfertilized egg surface shown in a. The surface of the unfertilized egg is covered with dense microvilli that do not appear to attach to or enter the egg envelope. The egg envelope (asterisk) consists of matted fibrous materials as viewed by SEM techniques. (Scale bar: 2 μm) c. TEM image of the cortical zone of an unfertilized egg. The cortical layer (CL) as delimited by an “I” consists of electron-dense cortical granules (CG) aligned underneath the cell membrane and a 2 μm wide area of cortical cytoplasm clear of yolk granules. Fine microvilli are evident at the surface of the egg. The major part of the cytoplasm includes electron-dense yolk granules (YG) of 1.5 to 2 μm in diameter. The yolk granules consist of a uniformly electron-dense component and a finely reticulated more electron-lucent component. (Scale bar: 2 μm) d. TEM image of a fertilized egg 2 hr after fertilization. The fertilization envelope (FE) consists of an electron-dense outer layer lined with a thicker inner layer of uniformly moderately electron-dense substances. The perivitelline space is filled with finely granular lightly staining materials. The cortical granules are no longer observed in the cytoplasm at this stage. (Scale bar: 2 μm). e. SEM of the furrow at first cleavage 3 hr after fertilization. Microvilli cover the surface of the embryo. (Scale bar: 5 μm) f. TEM of the ectodermal cells of a gastrula at 24 hr after fertilization. The yolk granules have now moved into the apical region of the cells and aligned against the cell membrane at the outer surface of the embryo. The nuclei (N) have localized beneath the yolk granule layer. FE: fertilization envelope. (Scale bar: 5 μm)


Upon fertilization, the fertilization envelope forms, meiosis occurs and the egg cleaves into 2 equal blastomeres at 3 hr (Fig. 1e). In the TEM, the fertilization envelope consists of a thin electron-dense outer surface layer and a thicker 0.2 to 0.3 μm underlying amorphous layer (Fig. 1d). A similar, but more electron-lucent substance fills the perivitelline space. The cortical layer at the surface of the unfertilized eggs is no longer present in fertilized eggs. Dense microvilli cover the surface of the blastomeres just as they covered the surface of the unfertilized egg (Fig. 1e).

After first cleavage, cell divisions continue about every 30 min to produce blastulae within 12 hr. The first 3 cleavages occur equally, the fourth cleavages appear unequal as previously described by Tagawa et al. (1998b). However, by blastula stage, the blastomeres look relatively similar in size as seen in an SEM image of a blastula still enclosed in the fertilization envelope (Fig. 2a). Eggs and embryos are opaque with yolk which makes discerning the internal details difficult with light microscope observations. To investigate the internal structure, fixed embryos were split with a tungsten needle and observed by SEM as shown in Fig. 2. The blastomeres of blastula are tall columnar cells 35 to 45 μm in height and 5 to 15 μm in width. The blastocoel is very small; it is only about 20 μm in diameter (Fig. 2b).

Fig. 2

SEM observations of early developmental stages. a. Blastula 14.5 hr after insemination. Similar sized blastomeres are evident arrayed underneath the egg envelope. b. An inner view of the blastula of the same stage as in a. The blastula consists of tall columnar cells about 35 to 45 μm in height and tapering from 5 to 15 μm in width arrayed around a narrow blastocoel of about 20 μm. c. Early gastrula 22 hr after fertilization. The ectodermal cells have shortened and the vegetal plate cells have elongated. Arrow indicates egg envelope. d. Gastrula 38 hr after insemination. The invaginated archenteron has expanded to fill the blastocoel. Arrow: chorion, double arrow: fertilization envelope. e and f. Young tornaria just after the hatching at 44 hr after insemination. Ciliary bands (arrowheads) and future telotroch (arrow) are already differentiated. (Scale bars: 10 μm)


SEM observations of split beginning gastrulae show the ectodermal cells flattened to about 20 μm in height while the vegetal plate cells are elongated into the blastocoel to a height of about 50 μm (Fig. 2c) almost filling the blastocoel with the archenteron cells (Fig. 2d). Gastrulation appears to be a continuation of this process, apparently adapted to morphogenesis in the tight chorion, with the elongating archenteron cells filling the blastocoel and ultimately forming a tight cleft, which is the gut opening. TEM images of the gastrula stage ectodermal cells show that the yolk granules align in the apical region of the cells and nuclei localize just beneath this yolk granule layer (Fig. 1f). The cytoplasm is filled with in a range of electron-lucent to moderately electron-dense granules.

SEM observation of tornaria larvae

The embryos remain constricted in the tight chorion and fertilization membrane until they hatch as an early tornaria larva at 44 to 45 hr. Just after hatching, the larva is dense and ovoid (Fig. 2e, f) with the major ciliary bands and the nascent telotroch of the definitive tornaria already visible on the surface. By 5 days, three days after hatching, the larva has increased considerably in size due to expansion of blastocoel space. During this expansion the epithelial cells flatten, and the tornaria becomes more angular in shape (compare Fig. 2e with Fig. 3a) and optically transparent (Tagawa et al., 1998b). The major ciliary surface features of the tornaria can best be discerned and will be described in context of SEM images of the 5 day swimming tornaria (Fig. 3), but these features are all evident in the just-hatched tornaria (Fig. 2e, f).

Fig. 3

SEM figures of 5 day tornaria. a. Overview of a tornaria. The major circumoral ciliary band loops around the larva. A preoral and a postoral portion pass, respectively, anterior and posterior to the mouth. These are connected on each side by loops that run from the preoral band to the anterior dorsal apical organ and then extend along the sides of the larva before turning ventrally and connecting to the postoral band. The apical tuft is evident at the anterior end of the larva. The oral surface is thickly covered with cilia. A mid ventral ciliary band is evident as marked by the arrow. AP: apical tuft, PO: postoral ciliary band, PR: preoral ciliary band. b. A dorsal view of a tornaria larva. The dorsal epidermis is a mosaic of cells with a single cilium or multiple cilia. The nascent telotroch (arrow) is evident as a circle of cilia at the posterior end of the larva. D: dorsal segment of major ciliary band, PR: preoral ciliary band. c. A close-up of the oral field with a tuft of cilia from the perioral ciliary band marking the mouth opening. H: ectoderm of oral hood, M: mouth, PR: preoral ciliary band. d. An apical view of a tornaria. The flattened apical organ is evident with the convergence at the four corners of the circumoral ciliary bands. An arrow marks the bundle of long apical tuft cilia emerging from the apical organ. Arrowheads indicate grooves of the eye spots in the apical organ. e. A posterior ventral view. Multiciliated cells surround the anus (A). Arrowheads mark two dimples near the anus. A ciliary band runs from the anal field along the ventral midline to the postoral ciliary band (PO). As it approaches the postoral band it trifurcates to demark two small circular areas of non-ciliated cells (arrow). f. An SEM photograph of the matted basal lamina on the inner surface of the ectoderm of an opened tornaria larva. A mesenchyme cell (MC) on the basal lamina (BL) associates with fine fibrous extracellular matrix in the blastocoel (arrow). Scale bars are 10 μm (a to e) and 2 μm (f).

  1. circumoral ciliary band

    The major ciliary band of the larva loops twice around the larval body (Fig. 3a, c) crossing at the apical organ on the anterior dorsal surface (Fig. 3d) and connecting ventrally via preoral and postoral sections that pass, respectively, anterior and posterior of the mouth.

  2. dorsal anterior apical organ and apical tuft

    The apical organ forms a flattened plate on the anterior apex of the tornaria (Fig. 3d.). The circumoral ciliary bands looping around the larval body join at it four corners. The apical tuft of long cilia emerges from its center with the eyespots evident as grooves to the left and right.

  3. perioral ciliary band

    The mouth opening is filled with cilia from the perioral ciliary band that tightly encircles the opening of the mouth (Fig. 3c). As will be shown below, the ring of nerve tracks associated with the perioral band is a significant component of the larval nervous system.

  4. midventral ciliary band

    A midventral ciliary band extends from a dense zone of cilia surrounding the anus and connects anteriorly with the transverse postoral ciliary band (Fig. 3e). Among the zone of cilia around the anus, there are two distinct round depressions that have not previously been noticed and are to the right and left and slightly dorsal of the anus (Fig. 3e). As the midventral ciliary band reaches the postoral ciliary band, it branches to the right and the left. All three branches join the post oral band and delimit two round areas of non-ciliated cells (Fig. 3e).

  5. telotroch

    A nascent telotroch is present on the earliest larvae, forming a dorsally projecting circle around the anus at the posterior end of the larva (Fig. 3b).

In other features of note, the dorsal ectoderm is a mosaic of cells, some with a single cilium and some with multiple cilia (Fig. 3c). The cells on the inner surface of stomach and intestine are also multiciliated (data not shown).

Looking into the inside of a tornaria that has been broken open, there is a matted basal lamina on the blastocoelar surface of the ectodermal cells (Fig. 3f). The blastocoelar space contains a fine fibrous matrix with a few mesenchyme cells associated here and there.

The nervous system of tornaria larvae

The nervous system of P. flava has not been studied well immunohistochemically. The staining with anti-serotonin antibodies of the neurons in the apical organ of the tornaria larva in this Hawaiian species is the only reported study (Tagawa et al., 2001). We examined the histochemistry with a novel neural marker, monoclonal antibody 1E11, developed with echinoderm neural tissue as antigen (Nakajima et al., in press). As shown on the Fig. 4a, cells immunoreactive to 1E11 are present in the apical organ and along the ciliary bands. Prominent immunoreactive nerve tracks are evident at the base of the ciliary bands. The staining of the nerve track around the perioral ciliary band is prominent (Fig. 4a, double arrows). In the major circumoral ciliary bands, the axons that extend along the base of the ciliary bands, the nerve bodies and the apical processes of the nerve cells that extended along the outer surface of the ciliary bands (Nakajima, 1986b) are all immunoreactive. Consequently, this ciliary band appears as ladder-like structures of two lines connected periodically with nerve cell bodies (Fig. 4a). Ultrastructurally, the ciliary band consists of epidermal cells that are characterized by a conspicuous nucleus with dense heterochromatin, electron-dense granules and vacuoles. In the basal part of the ciliary band, there is a bundle of nerve track consisting of 20 to 30 axons (Fig. 5b).

Fig. 4

Immunohistochemical study of the tornaria nervous system. a. Conforcal stacked image of immunostaining in a 7-day larva using the monoclonal antibody, 1E11, specific for echinoderm neural tissue. The apical organ, marked by an arrow, and the ciliary bands running around the body, marked by an arrowhead, represent the major immunostaining structures. The double lines of staining of the ciliary bands represent the connections of nerve tracks with apical processes of ectodermal nerve cell bodies in the ciliary band. A fine network of nerve tracks is evident in the ectoderm between the preoral and post oral ciliary bands. A perioral circle of nerve tracks, marked by double arrows, is also immunoreactive. Asterisk: primordial telotroch. b and c. Confocal stacked images of lateral views of a 7-day larva stained with both 1E11 (b) or antiserotonin antibody (c). Serotonergic nerve cells stain with 1E11 and are associated with the apical organ. d. Confocal optical section of the apical organ of a double stained 7 day tornaria showing many green, 1E11 staining cells and several reddish yellow cells indicating reactivity to both 1E11 and anti-serotonin antibodies. An arrow marks the basal nerve plexus of the apical organ that stains strongly with 1E11. Anterior is upper left. e. A higher power optical section through the ciliary band and oral epidermis of a 7-day tornaria stained with 1E11 merged with a DIC image of the same optical section. Ectodermal ciliary band cell bodies with apical processes and bundles of axons that run along the base of the ciliary band are stained. The arrow marks a fine nerve process derived from a 1E11 reactive cell body in the preoral ciliary band and extending posteriorly into the ectoderm. f. An optical slice near the midline of a 7 day larva stained with 1E11 merged with a DIC image of the same optical section. In addition to locations already noted, nerve cells are observed in the stomodeal ectoderm and the esophageal epithelium (PE) as well as around the pylorus as marked with an arrow. I: intestine, S: stomach. g. Immunoreactivity to 1E11 of a 40 hr late gastrula. Nerve cells reacting with 1E11 fist appear at this stage. h. Double staining to 1E11 and anti-serotonin of the same larva shown in g. One of two serotonergic cells expresses 1E11 distinctly. i. DIC image of the same larva. A: archenteron, PC: protocoel.


Fig. 5

Ultrastructure (TEM) of the apical organ and ciliary band of a 4-week tornaria. a. The apical organ consists of nerve cells with large nuclei (N) and axon bundles (AB). (Scale bar: 2 μm) Inset: The ectoderm includes nerve cells connected to other cells by septate junctional complexes. (Scale bar: 0.1 μm) b. A cross section of a ciliary band. The main components of ciliary bands are cells with large nuclei, cells with 1 μm diameter electron dense granules and axon bundles (AB). The large vacuoles are not characterized. HL: hyaline layer with microvilli. (Scale bar: 1 μm) c. Higher power view of the axon bundles of the apical organ. Axons are filled with a variety of synaptic vesicle-like bodies including ones appearing as open, cored, or very dense. (Scale bar: 0.5 μm)


Fine immunoreactive nerve tracks project into the oral field from cells in the ciliary band (Fig. 4a, e) creating a neural network in the oral field. Some cells in the stomodeal and esophageal epithelial cells and cells around the pyloric sphincter also exhibit 1E11 reactivity (Fig. 4f). Immunofluorescence is also seen in the nascent telotroch (Fig. 4a).

Cells in the apical organ are highly reactive to 1E11. We examined double staining with anti-serotonin and 1E11 antibodies of neural cells in the apical organ. Staining is first detected in the apical epidermis of the late gastrula around 40 hr of development. At this stage one or two cells stain and often they are both serotonergic and 1E11-reactive (Fig. 4g, h, i). In later tornaria, serotonergic cells and cell processes appear in the apical plate and in a restricted region of the adjacent anterior ciliary bands (Fig. 4c). These cells plus other are immunoreactive with 1E11 (Fig. 4b, c). Numerous nerve cells in the apical organ react to 1E11 (Fig. 4b), a sub-set of these also expresses serotonin (Fig. 4c, d).

We examined the ultrastructure of the apical organ by TEM. It consists of a cluster of tall nerve cells, supporting epithelial cells and a large mass of numerous axon tracks (Fig. 5a, c). Epidermal cells and nerve cells are connected to each other by septate junctions at their apical ends (Fig. 5, inset). The nuclei of nerve cells are less dense and larger than those of epidermal cells. The axons in the apical organ accumulate a large number of 30 to 50 nm diameter synaptic vesicle-like inclusions with a variety of profiles that may be characterized as dense cored, cored, or open. Patent synaptic endings are not observed.


Early development of P. flava

The SEM and TEM observations of early development of P. flava reported in this paper confirm, increase and revise our knowledge from previous studies of tornaria (Colwin and Colwin 1953; Hyman, 1957; Hadfield; 1975; Tagawa et al., 1998b) that were mostly based on the light microscopic observations.

The details of fertilization processes of Saccoglossus kowalevskii, a direct developing acorn worm were reported by Colwin and Colwin (1963a, b). As in S. kowalevskii, the fertilization envelope of P. flava is derived from an inner layer of the unfertilized egg envelope with the addition of a fertilization envelop apparently derived from the release of the cortical granule contents upon egg activation. Although a cortical layer has not been previously observed in the unfertilized egg of P. flava (Hadfield, 1975; Tagawa et al., 1998b), the TEM images show clearly the existence of a distinct cortical layer with unique electron dense cortical granules. This specialized layer with its granules disappears upon fertilization. The outer and inner envelopes of the unfertilized egg remain as chorion and fertilization envelope respectively during cleavage, blastula, gastrula and early larval stages (Tagawa et al., 1998b). Unlike echinoderm embryos, which hatch and start swimming as early embryos, the P. flava embryo remains enclosed in the very tight chorion until it is a fully developed tornaria. The diameter of blastula stage is at most 10% larger than that of unfertilized egg allowing for only a very limited blastocoel. The topology of gastrulation with the differentiation of thinner ectodermal cells and elongated vegetal plate cells to achieve gastrulation also appears to be an adaptation to morphogenesis to this restricted space. Even when the tornaria hatches it is still compressed and dense and only expands after escape from the tight chorion. It has been reported, based on light microscopic observation, that P. flava hatches at late gastrula stage (Tagawa et al., 1998b). Our results with SEM show that the larva just after hatching is a definitive tornaria with its specific ciliary band arrangement.

The multiciliated cell cluster and cell alignment at the posterior region of the young P. flava tornaria appear to precursors of telotroch and midventral ciliary band, respectively, as they appear to be in Balanoglossus biminiensis (Lacalli and Gilmour, 2001). These structures have not been recorded in P. flava by prior light microscopic observations. Although Lacalli and Gilmour (2001) report that a well developed nerve cord exists under the telotroch of B. biminiensis tornaria, we did not detect any significant immunohistochemical staining with 1E11 that might appear to be a nerve track under the telotroch of the P. flava tornaria for at least the first 7 days of development.

Nervous system

One of the interesting questions concerning tornaria morphology is the transition of the larval nervous system to the adult nervous system, especially because the transition from tornaria larva to juvenile acorn worm does not involve the loss of larval structures or cells (Agassiz, 1873). There are several previous reports concerning the ultrastructure of the nervous system and related structures of tornaria (Brandenburger et al., 1973; Nielsen, 1987; Dautov and Nezlin, 1992; Lacalli and West, 1993; Lacalli and Gilmour, 2001). Anti-serotonin antibody, which has been the most broadly applied for showing larval nervous system structures, reacts to the apical organ with nerve tracks extending into oral hood (Fig. 4c, Tagawa et al., 2001). Ultrastructural observations that show nerve cell bodies along the whole length of ciliary bands and axons running along the basal part of ciliary bands do not stain with anti-serotonin (Dautov and Nezlin, 1992; Lacalli and West, 1993; Lacalli and Gilmour, 2001). Using the novel neural marker 1E11, we can visualize a much more complete complement of the nervous system of young tornaria. 1E11 reveals the neural structures already reported by anti-serotonin and electron microscopy as well as previously undescribed neural structures.

The details we have been able to add to the description of the nervous system of tornaria show that it even more resembles that of asteroid bipinnaria larva (Nakajima et al., in press). As has been noted previously (Lacalli and West, 1993; Lacalli, 1996), a number of features are very similar. The flask shaped nerve cells evenly spaced along the ciliary bands, with axons extending from the base of the nerve cells and apical processes extending from the apex of these cells along the ciliary bands appear identical. Fine nerve filaments derived form nerve cells in the ciliary bands extend into oral field and form a network in both sets of larvae. They both exhibit a nerve plexus in esophageal epithelium.

There are some contrasting features of the apical organ/apical ganglion between the two groups. The tornaria has an apical organ with eyespots at the apical tip of the body and contains both serotonergic and 1E11 positive nerve cells. In the bipinnaria the ganglia containing serotonergic, anti-GFNSALMFamide (S1)- and 1E11-positive cells are bilateral and are in the more proximal ciliary band of the oral hood (Nakajima, 1988, Moss et al., 1994, Nakajima et al., in press). In the echinoid pluteus, the apical ganglion containing serotonergic, S1- and 1E11- positive cells appears at the apical tip between the anterolateral arms (Nakajima, 1986a; Bisgrove and Burke, 1987; Yaguchi et al., 2000; Beer et al., 2001; Nakajima et al., in press). The similarities and shifts in the location of apical neural structures between hemichordate, echinoid and asteroid larvae are intriguing. The future study of the apical ganglion in the holothuroid auricularia larva, which is more similar to the tornaria in morphology, will provide important information.

Our results here provide the evidence that hemichordate and echinoderm larval nervous systems resemble each other and support current molecular phylogeny that these two phyla are sister groups. In spite of these larval similarities, the later development of these two phyla is quite distinct. The bilateral body plan of the enteropneust hemi-chordate larvae is maintained as the larva transitions into the adult, while echinoderm larvae totally reorganize from their bilateral larval form into the radial adult form. The comparative study of the nervous system during metamorphosis of these animals remains as a very interesting issue and its further study will contribute to our understanding of the evolution of the nervous system in deuterostomes. The new neural marker 1E11 promises to contribute significantly to such studies. Assuming larval nervous systems of hemichordates and echinoderms share the common ancestral feature(s) with the central nervous system of chordates, it will be especially interesting to use this antibody to study the developmental stages of chordates, the third phylum of deuterostomes.


YN was supported by Keio Gijuku Academic Development Funds and KT was supported by American Cancer Society Institutional Grant 436328. TH was supported in part by a Special Visiting Professor MEXT Award of the Japanese government through Tokyo Medical and Dental University. We express our thanks to Dr. T. C. Lacalli for important advice and comments on the manuscript and to Dr. R. D. Burke for critical reading and comments of the manuscript.



A. Agassiz 1873. The history of Balanoglossus and Tornaria. Mem. Amer. Acad. Arts & Sciences 9:421–436. Google Scholar


E. J. Balser and E. E. Ruppert . 1990. Structure, ultrastructure, and function of the preoral heart-kidney in Saccoglossus kowakevskii (Hemichordata, Enteropneusta) including new data on the stomochord. Acta Zoologica 72:235–249. Google Scholar


W. Bateson 1884. The early stages in the development of Balanoglossus. Quart J Micr Sci 24:208–237. Google Scholar


W. Bateson 1885. The later stages in the development of B. kowalevskii, with a suggestion as to the affinities of the Enteropneusta. Quart J Micr Sci 25:Suppl81–122. Google Scholar


A. J. Beer, C. Moss, and M. Thorndyke . 2001. Development of serotonin-like and SALMFamide-like immunoreactivity in the nervous system of the sea urchin Psammechinus miliaris. Biol Bull 200:268–280. Google Scholar


J. Benito and F. Pardos . 1997. Hemichordata. In “Microscopic Anatomy of Invertebrates, Vol. 15: Hemichordata, Chaetognatha, and the Invertebrate Chordates”. Ed by F. W. Harrison and E. E. Ruppert , editors. Wiley-Liss. New York. pp. 15–101. Google Scholar


B. W. Bisgrove and R. D. Burke . 1987. Development of the nervous system of the pluteus larva of Strongylocentrotus droebachensis. Cell Tissue Res 248:335–343. Google Scholar


L. L. Brandenburger, R. M. Woolacott, and R. M. Eakin . 1973. Fine structure of eyespots in tornarian larvae (Phylum:Hemichordata). Z Zell-forch 142:89–102. Google Scholar


A. L. Colwin and L. H. Colwin . 1953. The normal embryology of Saccoglossus kowalevskii (Enteropneusta). J Morph 92:401–453. Google Scholar


A. L. Colwin and L. H. Colwin . 1963a. Role of the gamete membranes in fertilization in Saccoglossus kowalevskii (Enteropneusta) I. The acrosomal region and its changes in early stages of fertilization. J Cell Biol 19:477–500. Google Scholar


L. H. Colwin and A. L. Colwin . 1963b. Role of the gamete membranes in fertilization in Saccoglossus kowalevskii (Enteropneusta) II. Zygote formation by gamete membrane fusion. J Cell Biol 19:501–518. Google Scholar


S. S. Dautov and L. P. Nezlin . 1992. Nervous system of the tornaria larva (hemichordate: Enteropneusta). A histochemical and ultrastructural study. Biol Bull 183:463–475. Google Scholar


L. Gan, C. A. Mao, A. Wikramanayake, L. M. Angerer, R. C. Angerer, and W. H. Klein . 1995. An orthodenticle-related protein from Strongylocentrotus purupuratus. Dev Boil 167:517–528. Google Scholar


T. Fuchikami, K. Mitsunaga-Nakatsubo, S. Amemiya, T. Hosomi, T. Watanabe, D. Kurokawa, M. Kataoka, Y. Harada, N. Satoh, S. Kusunoki, K. Takata, T. Shimotori, T. Yamamoto, N. Sakamoto, H. Shimada, and K. Akasaka . 2002. T-brain homologue (HpTb) is involved in the archenteron induction signals of micromere descendant cells in the sea urchin embryo. Development 129:5205–5216. Google Scholar


M. G. Hadfield 1975. Hemichordata. In “Reproduction of Marine Invetebrates, Vol 2”. Ed by A. C. Guese and J. S. Pearse , editors. Academic Press. New York. pp. 185–240. Google Scholar


Y. Harada, N. Okai, S. Taguchi, K. Tagawa, T. Humphreys, and N. Satoh . 2000. Developmental expression of the hemichordate otx ortholog. Mech Dev 91:337–339. Google Scholar


Y. Harada, N. Okai, S. Taguchi, E. Shoguchi, K. Tagawa, T. Humphreys, and N. Satoh . 2001. Embryonic expression of a hemichordate distalless gene. Zool Sci 18:57–61. Google Scholar


L. Hyman 1959. The Invertebrates. Vol 5. Smaller Coelomate Groups McGrow-Hill. New York. Google Scholar


T. C. Lacalli 1996. Mesodermal pattern and pattern repeats in the starfish bipinnaria larva, and related patterns in other deuterostome larvae and chordates. Phil Trans R Soc Lond B 351:1737–1758. Google Scholar


T. C. Lacalli and T. H. J. Gilmour . 2001. Locomotory and feeding effectors of the tornaria larva of Balanoglossus biminiensis. Acta Zool (Stockh) 82:117–126. Google Scholar


T. C. Lacalli and J. E. West . 1993. A distinctive nerve cell type common to diverse deuterostome larvae: Comparative data from echinoderms, hemichordates and amphioxus. Acta Zool (Stockh) 74:1–8. Google Scholar


C. J. Lowe, L. Issel-Tarver, and G. A. Wray . 2002. Gene expression and larval evolution: changing roles of distal-less and orthodenticle in echinoderm larvae. Evo Dev 4:111–123. Google Scholar


C. J. Lowe and G. A. Wray . 1997. Radical alterations in the roles of homeobox genes during echinoderm evolution. Nature 389:718–721. Google Scholar


T. H. Morgan 1891. The growth and Metamorphosis of Tornaria. J Morph 5:407–458. Google Scholar


C. Moss, R. D. Burke, and M. C. Thorndyke . 1994. Immunocytochemical localization of the neuropeptide S1 and serotonin in the larva of the starfish Pisaster ochraceus and Asterias rubens. J Mar Biol Assoc UK 74:61–71. Google Scholar


Y. Nakajima 1986a. Presence of a ciliary patch in preoral epithelium of sea urchin plutei. Dev Growth Differ 28:243–249. Google Scholar


Y. Nakajima 1986b. Development of the nervous system of sea urchin embryos: formation of ciliary bands and the appearance of two types of ectoneural cells in the pluteus. Dev Growth Differ 28:531–542. Google Scholar


Y. Nakajima 1988. Serotonergic nerve cells of starfish larvae. In “Echinoderm Biology”. Ed by R. D. Burke, et al , editor. Balkema. Rotterdam. pp. 235–239. Google Scholar


Y. Nakajima, H. Kaneko, G. Murray, and R. D. Burke . 2004. Divergent patterns of neural development in larval echinoderm and asteroids. Evo Dev in press. Google Scholar


C. Nielsen 1987. Structure and function of metazoan ciliary bands and their phylogenetic significance. Acta Zool (Stockh) 68:205–262. Google Scholar


C. Nielsen 1995. Animal evolution. Oxford Univ Press. Oxford. Google Scholar


K. J. Peterson, R. A. Cameron, K. Tagawa, N. Satoh, and E. H. Davidson . 1999. A comparative molecular approach to mesodermal patterning in basal deuterostomes: the expression pattern of Brachyury in the enteropneust hemichordate Ptychodera flava. Development 126:85–95. Google Scholar


E. Shoguchi, N. Satoh, and Y. K. Maruyama . 2000. A starfish homolog of mouse T-brain-1 is expressed in the archenteron of Asterina pectinifera embryos: possible involvement of two T-box genes in starfish gastrulation. Dev Growth Differ 42:61–68. Google Scholar


K. Tagawa, T. Humphreys, and N. Satoh . 1998a. Novel pattern of Brachyury gene expression in hemichordate embryos. Mech Dev 75:139–143. Google Scholar


K. Tagawa, T. Humphreys, and N. Satoh . 2000. T-Brain expression in the apical organ of hemichordate tornaria larvae suggests its evolutionary link to the vertebrate forebrain. J Exp Zool 288:23–31. Google Scholar


K. Tagawa, A. Nishino, T. Humphreys, and N. Satoh . 1998b. The spawning and early development of the Hawaiian acorn warm (Hemichordate), Ptychodera flava. Zool Sci 15:85–91. Google Scholar


K. Tagawa, N. Satoh, and T. Humphreys . 2001. Molecular studies of hemichordate development: a key to understanding the evolution of bilateral animals and chordates. Evo Dev 3:443–454. Google Scholar


S. Taguchi, K. Tagawa, T. Humphreys, and N. Satoh . 2002. Group B sox genes that contribute to specification of the vertebrate brain are expressed in the apical organ and ciliary bands of hemichordate larvae. Zool Sci 19:57–66. Google Scholar


C. M. Takacs, V. N. Moy, and K. J. Peterson . 2002. Testing putative hemichordate homologues of the chordate dorsal nervous system and endostyle: expression of NK2.1 (TTF-1) in the acorn worm Ptychodera flava (Hemichordata, Ptychoderidae). Evo Dev 4:405–417. Google Scholar


P. Willmer 1990. Invertebrate Relationships. Cambridge University Press. Cambridge. Google Scholar


S. Yaguchi, K. Kanoh, S. Amemiya, and H. Katow . 2000. Initial analysis of immunochemical cell surface properties, location and formation of the serotonergic apical ganglion in sea urchin embryos. Dev Growth Differ 42:479–488. Google Scholar
Yoko Nakajima, Tom Humphreys, Hiroyuki Kaneko, and Kunifumi Tagawa "Development and Neural Organization of the Tornaria Larva of the Hawaiian Hemichordate, Ptychodera flava," Zoological Science 21(1), 69-78, (27 January 2004).[69:DANOOT]2.0.CO;2
Received: 9 September 2003; Accepted: 1 September 2003; Published: 27 January 2004
larval nervous system
neural marker
Back to Top