Biol. Bull. 216: 322-334. (June 2009)
© 2009 Marine Biological Laboratory
Nervous System Development in Feeding and Nonfeeding Asteroid Larvae and the Early Juvenile
Laura Elia*,
Paulina Selvakumaraswamy and
Maria Byrne
Discipline of Anatomy and Histology, Bosch Institute, F13, University of Sydney, NSW 2006, Australia
* To whom correspondence should be addressed. E-mail: elia{at}anatomy.usyd.edu.au
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Abstract
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Larval and juvenile nervous systems (NS) of three asterinid sea stars with contrasting feeding and nonfeeding modes of development were characterized using the echinoderm-specific synaptotagmin antibody. In the feeding bipinnaria and brachiolaria larvae of Patiriella regularis, the species with ancestral-type development, an extensive NS was associated with the ciliary bands (CBs) and attachment complex. Lecithotrophic planktonic (Meridastra calcar) and benthic (Parvulastra exigua) brachiolariae lacked CBs and the associated NS, but had an extensive NS in the attachment complex. The similarity in the distribution and morphology of synaptotagmin immunoreactive neurons and the anatomy of the NS in the attachment complex of these closely related sea stars suggests conservation of neurogenesis in settlement-stage larvae regardless of larval feeding mode. Nerve cells were prominent on the brachia of all three species. In advanced brachiolariae the larval nervous system was localized to the adhesive disc as the larval body resorbed during metamorphosis. The structures and tissues that contained larval neurons degenerated during metamorphosis. There was no evidence that the larval NS persists through metamorphosis. In juvenile development, synaptotagmin IR was first evident in the NS of the tube feet. As the central nervous system developed, synaptotagmin IR reflected the histological organization of the adult NS. The juvenile NS formed de novo with a temporal lapse between histogenesis and synaptotagmin IR. We evaluated the ontogeny of NS organization in the change in body plan from the bilateral larva to the radial juvenile.
Abbreviations: CB, ciliary band • IR, immunoreactivity • LNS, larval nervous system • NS, nervous system
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Introduction
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The anatomy of the echinoderm larval nervous system (LNS) has been the subject of numerous investigations based on histochemistry, electron microscopy, and immunocytochemistry (reviews: Cobb, 1987; Nakajima et al., 2004a; Byrne et al., 2007). The most widely used and successful approach to investigate the anatomy of the LNS has involved immunocytochemistry using antibodies to a range of neurochemicals. In particular, antibodies to serotonin (5-HT), dopamine, GABA, and the echinoderm-specific neuropeptide GFNSALMFamide 1 (S1) have been used to visualize the LNS of planktotrophic larvae (Burke et al., 2006; Chia et al., 1986; Bisgrove and Burke, 1987; Nakajima, 1987, 1988; Bisgrove and Raff, 1989; Nakajima et al., 1993; Moss et al., 1994; Byrne et al., 1998,2001, 2007; Chee and Byrne, 1999a, b; Cisternas et al., 2001; Falugi et al., 2002; Byrne and Cisternas, 2002; Cisternas and Byrne, 2003; Yaguchi and Katow, 2003; Katow et al., 2009). Recently, the pan-neural synaptotagmin B monoclonal antibody generated from the sea star radial nerve cord has been used to great effect in studies of the LNS of echinoids, holothuroids, and asteroids (Nakajima et al., 2004a, b; Burke et al., 2006; Nakano et al., 2006; Bishop and Burke, 2007; Katow et al., 2009) and the nervous system (NS) of adult asteroids (Saha et al., 2006; Murabe et al., 2008). Synaptotagmin is a calcium-binding protein localized in synaptic vesicles (Sudhof, 2002).
Asteroids with planktotrophic development have two larval stages: the feeding bipinnaria and the settlement-stage brachiolaria. Possession of the feeding bipinnaria larva is considered to represent the ancestral developmental mode for modern Asteroidea (review, Strathmann, 1975; Byrne, 2006; Raff and Byrne, 2006). The bipinnaria was lost in the evolution of lecithotrophic development (Byrne et al., 2001). Application of the neural markers, synaptotagmin and S1, reveals an extensive network of neurites and cell bodies in the ciliary band (CB) nerves, oral region, and digestive tract in the bipinnaria and in the attachment complex of the brachiolaria (Moss et al., 1994; Byrne and Cisternas, 2002; Nakajima et al., 2004a; Murabe et al., 2008). Here we used the synaptotagmin marker in a comparative study of the LNS of asterinid sea stars with planktotrophic and lecithotrophic modes of development. Development of Patiriella regularis through feeding bipinnaria and brachiolaria larvae represents the ancestral mode of development, while Meridastra calcar and Parvulastra exigua have lecithotrophic planktonic and benthic brachiolaria, respectively (Byrne, 2006). With an estimated divergence time of less than 10 Mya (Hart et al., 1997), the evolution of enhanced maternal provisioning in M. calcar and P. exigua has resulted in loss of the bipinnaria and associated feeding structures (Byrne et al., 2001; Byrne and Cisternas, 2002; Byrne, 2006; Prowse et al., 2008). In association with this loss, there is no trace of the extensive network of CB nerves characteristic of the bipinnaria (Byrne et al., 2001). However species with feeding and nonfeeding larvae all have the settlement- and metamorphic-stage larva, the brachiolaria. The brachiolaria has morphological specializations for benthic settlement—three brachia and the adhesive disc (Byrne et al., 2001; Byrne, 2006). The anatomy of the attachment complex in the planktonic larvae of P. regularis and M. calcar is similar in the presence of a long median brachium flanked by two small ones and a central adhesive disc. In contrast, the benthic brachiolaria of P. exigua has a hypertrophic attachment complex that creates the tripod larval form with three large muscular brachia equal in length and a large adhesive disc (Byrne, 2005). These are adaptations for permanent benthic attachment.
Here we document the distribution of synaptotagmin immunoreactive cells and the organization and morphology of the NS in the brachiolaria and juveniles of asterinids with different modes of development. This is the first use of this marker with lecithotrophic asteroid larvae. The primary objectives were to identify modifications of the LNS associated with evolution of lecithotrophic development in M. calcar and P. exigua within a robust phylogenetic context and to document the fate of the LNS through metamorphosis and development of the juvenile NS. A recent study of neurogenesis in echinoids revealed that the larval NS is anatomically connected to that of the juvenile in both feeding and nonfeeding larvae prior to metamorphosis, after which there is no trace of the LNS (Katow et al., 2009). Here we focused on the larval stage shared by asterinid species with feeding and nonfeeding larvae, the brachiolaria. Although the LNS of asteroid bipinnariae is well characterized, this stage in P. regularis is included here for comparative purposes as the second bipinnaria examined with the synaptotagmin marker.
In all animals, development of the body plan is closely tied to development of the nervous system (Holland, 2003). For echinoderms, which arose from a bilateral ancestor, the evolution of pentamery and the fivefold NS is not well understood. It is also not known how the bilateral LNS of echinoderms relates to the pentamerous central nervous system (CNS) of the adult. The change in distribution of IR through metamorphosis from the bilateral LNS to the radial CNS is documented to assess the relationship between these two systems in the Asteroidea.
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Materials and Methods
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Specimen collection and spawning
Adult individuals of Patiriella regularis (Verrill) were collected near Hobart, Tasmania (November, December 2004–2006), and adults of Parvulastra exigua (Lamark) and Meridastra calcar (Lamark) were collected near Sydney, New South Wales (May–November 2004–2007). Spawning was induced by placing the sea stars in filtered seawater (FSW: 1 µm, Millipore) with 10-5 mol l–1 1-methyladenine. The gametes were released within 3–4 h and used for fertilization to establish larval cultures. For P. regularis, cultures were fed with Pavlova lutheri, Isochrysis galbana, orChaetoceros muelleri at a cell concentration of 30000 l-1 every second day. The FSW was changed by reverse filtration prior to feeding. Patiriella regularis larvae were reared to the juvenile stage (3 mon) at 20 °C in 25-l containers with aeration. The nonfeeding lecithotrophic larvae of P. exigua and M. calcar were reared to the juvenile stage in petri dishes.
Immunocytochemistry
Larvae from P. regularis, P. exigua, and M. calcar were used for synaptotagmin immunocytochemistry using the 1E11 antibody (Nakajima et al., 2004a). The larvae were placed in baskets (1-ml) made from BEEM capsules capped with a 60-µm-mesh lid. Larvae or juveniles (n = 30–50) were placed in each basket in 24-well culture dishes and rinsed with 0.22-µm FSW (Millipore). After 15 min (M. calcar, P. exigua) or 45 min (P. regularis) in 7% MgCl2 to reduce muscle contraction, the specimens were fixed in 4% paraformaldehyde in FSW for 10 min at room temperature (RT). After fixation, specimens were immediately processed for immunocytochemistry, and all steps (except where specified) were carried out at RT on a rocker to create a gentle agitation of the solutions. After fixation, the specimens were placed in cold 100% methanol at RT for 2 min following Nakajima et al. (2004a). The samples were then rinsed three times (10 min) in 0.1 mol l–1 phosphate buffered saline (PBS) pH 8.2–8.3 to eliminate excess fixative. After pre-incubation for 1 h in 10% normal goat serum (Vector laboratories) diluted in PBS with 0.1% Tween 20 (PBST), the samples were rinsed three times (10 min) in PBST. Specimens were incubated in primary antibody (1E11) overnight at 4 °C. After three washes in PBST, the specimens were incubated in secondary antibody anti-mouse IgG goat tagged with Alexa fluor 594 (1:500 in PBST) for 2 h at RT, followed by a final rinse in PBST (3 x 10 min). After dehydration through an ethanol series to 100%, the specimens were cleared and stored in benzyl benzoate/benzyl alcohol (Sigma-Aldrich) 2:1 (v/v). Controls consisted of omitting the primary antibody and omitting the secondary antibody. The presence of background autofluorescence was also checked. Samples were stored in glass dishes in the dark at 4 °C to preserve the fluorescent signal. Specimens stored for several months showed no sign of fading when examined.
Specimen preparation for microscopy
For examination with an upright fluorescence microscope, immunostained specimens were placed on a slide in a drop of benzyl benzoate/benzyl alcohol, and the coverslips were mounted on small plasticine feet. For examination using an inverted microscope, the larvae were placed in a chamber made by sealing a coverslip to the underside of a hole cut in a glass slide.
Specimens were viewed using confocal or deconvolution microscopy. For confocal microscopy, an FV 1000 confocal system on a IX 81 inverted confocal microscope (Olympus, Japan) was used. The 488-nm line of the argon laser was used with a filter block to visualize paraformaldehyde-induced autofluorence. The 543-nm line of the HeNe laser was used to excite the Alexa fluor 594. Some specimens were examined with a Zeiss LSM 510 Meta inverted microscope. Excitation was achieved using the 488-nm diode laser (for autofluorescence) and 561-nm diode laser (for Alexa fluor 594). For deconvolution microscopy, a Zeiss Axioplan 2 upright automated microscope (Carl Zeiss Germany) fitted with FITC (EX 480/40, 505LP, EM 535/50) and Texas Red (EX 560/55, 595LP, EM 645/75) filter sets were used.
Images captured on the Olympus confocal microscope were analyzed with Fluoview ver. 1.5. Images taken using the Zeiss were acquired with AxioVision ver. 4.0 image acquisition software. These software packages were used to construct extended focus projections of all serial sections.
For scanning electron microscopy, larvae were fixed for 1 h at RT in 2.5% glutaraldehyde in 0.22-µm FSW, then rinsed in 2.5% NaHCO3 (pH 7.2). The conventional secondary fixation step in osmium was deleted as in previous studies of larval structure (e.g., Nunes and Jangoux, 2007). The specimens were rinsed in distilled water, dehydrated in graded ethanols, critical-point-dried, mounted on stubs, sputter-coated, and viewed with a JOEL 35C scanning electron microscope.
For wax histology, P. exigua juveniles were fixed and decalcified in Bouin' fluid for 24 h, rinsed in distilled water, dehydrated in graded ethanols, and embedded in paraffin. Serial sections (7 µm thick) stained with haematoxylin and eosin were photographed.
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Results
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Synaptotagmin immunoreactivity in the bipinnaria of Patiriella regularis
In the bipinnaria of P. regularis, synaptotagmin IR revealed an extensive larval nervous system (LNS) comprising an array of cell bodies and neurites along the length of the ciliary band (CB) nerve (Fig. 1A, C). The most prominent IR was localized along the pre- and post-oral CB sections (Fig. 1A, C). In the oral region, two clusters of cells corresponded to the adoral CB nerve and ganglion (Fig. 1A, B). The esophagus had also immunoreactive neurites (Fig. 1B). Immunoreactive cell bodies and neurites were positioned in the CB epithelium (Fig. 1C–H). In the preoral CB nerve, the cell bodies gave rise to processes that connected with the adjacent network of neurites in the pre-oral hood (Fig. 1C). The CB neurons were closely aligned along the basal region of the epithelium and were joined by basal processes (Fig. 1D). The cell bodies of neurons of the preoral CB nerve varied in shape. These cells included triangular cells with the basal region at the base of the CB epithelium, and these gave rise to an apical process that connected with the epithelial surface and basal processes that merged with the CB nerve (Fig. 1E). Round cells at the basal region of the epithelium did not have an immunoreactive apical region (Fig. 1F). The cell body of bipolar spindle-shaped cells was positioned in the mid-epithelial region. These spindle-shaped cells had a narrow apical region and gave rise to a basal neurite (Fig. 1G). In the post-oral CB nerve, some cell bodies were in a mid-epithelial position, had a round profile, and connected with the network of fibers adjacent to the CB (Fig. 1C). Some of these cells gave rise to several neurites and appeared multipolar (Fig. 1G).
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Figure 1. Deconvolution (A) and confocal (B–H) reconstructions showing synaptotagmin IR in the bipinnaria larva of Patiriella regularis (11 days old) in ventral view. (A) The ciliary band (CB) nerve is strongly fluorescent, especially in the oral region associated with the pre- and post-oral CBs. (B) Oral region showing IR in the adoral ganglion and esophagus. (C) Neurons of the CB nerves give rise to processes (arrows) that connect with the adjacent network (arrowheads). (D–G) Detail of cells in the pre-oral CB nerve. (D) Bipolar spindle-shaped cells in the mid-epithelial region (arrowheads) with a narrow apical region and a basal neurite (arrow). (E) Triangular cell with an apical projection (arrow) that connected with the epithelial surface and basal processes (arrowheads) that merged with the CB nerve. (F) Round cells (arrows) positioned at the basal region of the epithelium. (G) Cell bodies closely aligned along the basal region of the epithelium and joined by basal processes (arrowheads). (H) Multipolar neurons in the post-oral CB nerve. ag, adoral ganglia; cbe, ciliary band epithelium; cbn, ciliary band nerve; e, esophagus; n, nucleus; pocb, pre-oral ciliary bands; ptcb, post-oral ciliary bands;. Scales: A = 100 µm; B = 50 µm; C = 10 µm; D–H = 5 µm.
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Synaptotagmin immunoreactivity in the brachiolaria of Patiriella regularis
The brachiolaria of P. regularis has an attachment complex comprising the three brachia and the adhesive disc (Fig. 2A). The median brachium was covered by prominent adhesive papillae (Fig. 2B). Elongate spindle-shaped neurons extended through the epithelium of the adhesive papillae (Fig. 2F). At their base these cells gave rise to a process that merged with the basi-epithelial nerve plexus (Fig. 2F). Synaptotagmin IR was evident in the CB nerves that followed the contours of the bands in a manner similar to that we observed in the bipinnaria. Two lines of IR in the anterior projection coincided with the position of the CB on the dorsal and ventral sides (Fig. 2C, E). Neurites projecting between these two regions anatomically connected the ventral and dorsal sides in an anastomosing network of cell bodies and processes (Fig. 2E). The dorsal side of the brachium had neurites and a few cell bodies (Fig. 2E). Immmunoreacitivity was also evident in the esophagus (Fig. 2C).
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Figure 2. Light (A), scanning electron microscope (B), and confocal (C–J) images of the brachiolaria larva of Patiriella regularis: (A–F) 2-mon-old larvae; (G–J) 3-mon-old larvae that had attached in preparation for metamorphosis; (A–B, G–J) ventral view; (C–F) dorsal view. (A–B) The median brachium is covered by adhesive papillae, and contractile bands (CBs) traverse the anterior projection. (C) Synaptotagmin IR in the anterior projection along the CBs. Arrowheads show the IR in the esophagus. (D) immunoreactive cells (arrows) dot the surface of the adhesive papillae (boxed area, see F). (E) CB nerves on the ventral and dorsal sides of the anterior projection and connecting network. Multipolar neurons (arrow) give rise to processes that connect the dorsal and ventral sides. Boxed area in D surrounds adhesive papillae. (F) High magnification of D showing two adhesive papillae with elongate spindle-shaped neurons that span the width of the papilla and connect with the basi-epithelial nerve plexus (arrowheads). (G) In attached brachiolaria the adhesive disc is strongly immunoreactive. Some IR is also evident on the surface of the central brachium (arrowhead). (H) Network of immunoreactive neurites in the adhesive disc. (I) Transverse optical section of the adhesive disc showing tripolar and flask-shaped neurons (arrowheads) along the base of the epithelium. Flocculent material extending from the disc is composed of adhesive secretions and debris (arrows) adhering to the disc after the larvae were detached from the surface. (J) High magnification of the cell marked by arrowheads in I. These cells are positioned in the mid (left cell) or basal (right epithelial cell) region. ad, adhesive disc; ap, adhesive papilla; apj, anterior projection; b, brachium; cb, ciliary band; D, dorsal side; e, esophagus; jr, juvenile rudiment; M, mouth; n, nucleus; pocb, pre-oral ciliary bands; ptcb, post-oral ciliary bands; V, ventral side. Scales: A–C, G = 100 µm; D–E = 25 µm; F = 10 µm; H = 50 µm; I = 20 µm; J = 10 µm.
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As the larval body was resorbed during metamorphosis, the LNS degenerated. In advanced brachiolaria, IR became focused on the adhesive disc, forming an extensive nerve plexus (Fig. 2G–H). Transverse optical section of the adhesive disc revealed cell bodies located along the base of the disc (Fig. 2I–J).
Synaptotagmin immunoreactivity in the brachiolaria of Meridastra calcar and Parvulastra exigua
The brachia of the lecithotrophic brachiolaria of M. calcar and P. exigua do not have adhesive papillae as seen in P. regularis. In these species, neurons were scattered fairly uniformly over the brachial surface, and IR was most prominent in the attachment complex (Fig. 3A, D–F). The neurons varied in shape and included spindle-shaped cells (Fig. 3F). Most of the cells had an elongate profile with an apical projection and a basal neurite that merged with the basi-epithelial nerve plexus along the basal lamina of the epidermis (Fig. 3F, H–I). This network of fibers spanned between the three brachia so the nervous tissue of these structures were connected (Fig. 3A, D–E). Immunoreactive cells were also present scattered across the general epithelium of the larval body (Fig. 3G). A few immunoreactive cells were also present in the adhesive disc (Fig. 3E).
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Figure 3. Confocal (A, D–G, I) and deconvolution (H) reconstructions of synaptotagmin IR and light microscopy images (B–C) of the brachiolaria larva of Meridastra calcar (A–B, E, G–H) and Parvulastra exigua (C–D, F, I). (A, D–E) IR is evident in the attachment complex, particularly in the brachia. (B–C) Side views of larvae showing the position of brachia and adhesive disc. (E) Neurons are scattered over the surface of the brachia, and few immunoreactive cells are present in the adhesive disc. (F) Neurons with apical projections (arrows) scattered on the surface of the brachia. (G) General IR in epithelium of the larval body. (H–I) Neurons in the brachia give rise to a basal neurite (arrowheads) that connects with the basi-epithelial plexus. ad, adhesive disc; b, brachium; be, basi-epithelial plexus. Scales: A–D = 100 µm; E, G = 50 µm; F, H = 25 µm; I = 10 µm.
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As the brachiolariae developed, IR in the adhesive disc increased (Fig. 4A–B). In advanced larvae competent to metamorphose, the adhesive disc contained prominent elongate spindle-shaped neurons, similar to those seen in the brachial epithelium (Fig. 4B–C). Neurons in the adhesive disc and brachia merged with the basi-epithelial plexus underlying the entire attachment complex (Fig. 4C–D).
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Figure 4. Deconvolution (A) and confocal (B–D) reconstructions of synaptotagmin IR in the brachiolaria larva of Meridastra calcar(A) and Parvulastra exigua (B–D). (A–C) Elongate spindle-shaped cells (arrowheads) span the epithelium of the adhesive disc and connect with the basi-epithelial plexus. (D) Neurites in the basi-epithelial plexus connect the adhesive disc and brachia. ad, adhesive disc; b, brachium; be, basi-epithelial plexus. Scales: A, C = 25 µm; B, D = 50 µm.
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Synaptotagmin immunoreactivity in juveniles
Development of the juvenile NS was similar in the three species examined. In early juveniles, IR was first observed in the NS of the tube feet and more faintly in the circumoral nerve ring and radial nerve cords of the CNS (Fig. 5A). A network of neurites lined the podial epithelium (Fig. 5A–B). At the base of the tube foot where the podial NS joined with the radial nerve cords, IR decreased and merged with that in the developing nerve cord (Fig. 5D). Neurons were scattered across the surface of the tip of the tube feet and connected with a plexus at the base of the epithelium (Fig. 5E–F). Confocal sections revealed the presence of additional immunoreactive cells in a mid-epithelial position that gave rise to neurites that projected apically and basally (Fig. 5G). The processes from these cells connected with the basal plexus (Fig. 5C, G).
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Figure 5. Confocal (A, C–G) and deconvolution (B) reconstructions of synaptotagmin IR in early juveniles of Patiriella regularis in oral view. (A–B) Fluorescence is evident in the tube feet and is faint in the circumoral nerve ring (conr) and radial nerve cords (rnc). (C, E) Basi-epithelial network of neurites lining the tube foot epithelium. (D) Merging of podial immunoreactive neurites with the rnc. (F) Distribution of immunoreactive cell bodies on the tip of the tube feet. (G) High magnification of the boxed area in E, showing the immunoreactive cells (arrowheads) and neurites (arrows) in the tube foot epithelium. be, basi-epithelial plexus; conr, circumoral nerve ring; L, lumen; rnc, radial nerve cord; tf, tube foot. Scales: A–B = 100 µm; C–F = 25 µm; G = 10 µm.
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As the juveniles developed, strong IR was evident in the circumoral nerve ring and radial nerve cords, and the pattern of synaptotagmin localization reflected the histological organization of the asteroid CNS into the ectoneural and hyponeural tissue layers (Fig. 6A–E). Histological sections show the ectoneural system formed by an outer somatic layer of cell bodies positioned along the body wall epithelium and the inner axonal layer (Fig. 6A–B). Synaptotagmin-positive cell bodies were scattered along the outer somatic layer, indicating that not all cells in this tissue layer express this marker (Fig. 6D–E). Immunoreactivity in the axonal layer appeared fibrous and is likely to be localized to axons (Fig. 6D–E). A network of cells and neurites present in the inter-ambulacral region corresponded to the general body nerve plexus (Fig. 6F). The cells were irregularly shaped and gave rise to several neurites (Fig. 6G).
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Figure 6. (A–B) Light microscopic image of sections of a juvenile Parvulastra exigua showing the radial nerve cords (rnc). The ectoneural region has an outer layer of cell bodies and an inner axonal layer. (C–G) Confocal reconstructions of synaptotagmin IR in juvenile Patiriella regularis (C) and P. exigua (D–G). (C) IR in circumoral nerve ring (conr) and rnc. (D, E) immunoreactive cell bodies are scattered in the somatic layer of the CNS, and the axonal layer is strongly IR. (F) Neuronal network in the inter-ambulacral region. (G) Details of a cell body (arrow) with neurites (arrowheads) in the inter-ambulacral region. al, axonal layer; conr, circumoral nerve ring; hs, hyponeural nervous system; rnc, radial nerve cord; sl, somatic layer; tf, tube foot. Scales: A = 1 mm; B = 50 µm; C = 100 µm; D–F = 25 µm; G = 5 µm.
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Discussion
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Neurogenesis in asterinids with feeding larvae is divided into two distinct modules: the bipinnaria stage with its well-developed ciliary band (CB) nerves and the brachiolaria where a new set of neurons form in association with development of the attachment complex (Byrne et al., 2001). Evolution of nonfeeding development is associated with loss of the bipinnaria stage and all traces of the CB nerves (Byrne et al., 2001; Byrne and Cisternas, 2002). Similarly, evolution of nonfeeding development in sea urchins has resulted in loss of the CBs and associated nervous system (NS) (Byrne et al., 2007; Katow et al., 2009). The asterinid species investigated here all have the brachiolaria stages, which are morphologically similar among species with or without the preceding bipinnaria. This shows the conservation of the brachiolaria stage, which serves in benthic attachment, an obligatory phase in the transition from the planktonic to the benthic environments.
Synaptotagmin IR in the bipinnaria was seen in the CB nerves, adoral ganglia, and digestive tract, similar to that seen with peptidergic IR (S1) in Patiriella regularis and other asteroids and to synaptotagmin IR in Asterina pectinifera bipinnaria (Cisternas et al., 2001; Cisternas and Byrne, 2003; Nakajima et al., 2004a; Murabe et al., 2008). As seen here for P. regularis, the extensive plexus of neurites associated with the CB system and digestive tract is characteristic of echinoderm feeding larvae (Nakajima et al., 2004a, b; Nakano et al., 2006; Hirokawa et al., 2008). The distribution of the LNS in planktotrophic larvae is suggested to reflect its role in modulation of feeding and swimming (Lacalli, 1996; Katow et al., 2004, 2007). The cilia of the adoral CB carry food particles into the esophagus, which is followed by esophageal muscle contraction (Strathmann, 1975). The connection of neuronal cells to the CB nerve and adjacent neuronal network seen here with P. regularis is suggested to be important in coordination of ciliary activity for swimming and feeding (Chee and Byrne, 1999a). In P. regularis, cells of the CB nerve gave rise to neurites that connected to the adjacent neuronal network in the pre- and post-oral region; this arrangement was similar to that in A. pectinifera (Nakajima et al., 2004a). It is not known if these processes are axonal projections to effector cells or whether they are dendritic projections to sensory cells, as is generally the case for echinoderm LNS (Nakajima et al., 2004a; Bishop and Burke, 2007). The cell bodies of the immunoreactive neurons of the CB nerve varied in shape, a common feature of asteroid LNSs (Chee and Byrne, 1999a; Nakajima et al., 2004a). This variation is also reported for the cells of the serotonergic system of the larva of P. regularis (Chee and Byrne, 1999a). It is not known if these differences in cell shape represent different populations of neurons. In the anterior projection of the brachiolaria of P. regularis, both the distribution of neurites in the CB sectors and the neuronal network between the dorsal and ventral sides are similar to those described for A. pectinifera (Murabe et al., 2008). The 1E11 immunoreactive paired lateral ganglia that compose part of the asteroid larval apical organ in A. pectinifera (Murabe et al., 2008) were not immunoreactive in the present study in P. regularis.
As in previous studies of nervous system development in nonfeeding asteroid larvae (Byrne et al., 2001; Byrne and Cisternas, 2002), Meridastra calcar and Parvulastra exigua did not have any trace of the LNS seen in the bipinnaria. Echinoids with nonfeeding larvae also lack the CB and the associated NS of the echinopluteus (Byrne et al., 2007; Katow et al., 2009). Our results suggest that development of the brachiolaria stage of P. regularis initiated a new neurogenic program as the attachment complex formed.
Regardless of the presence or absence of a feeding-stage larva, the synaptotagmin IR of the brachiolaria of the three species was strikingly similar. This provides insights into the aspects of the NS essential for the attachment stage in sea star development.
The distribution of synaptotagmin documented here and the peptidergic IR from other studies (Byrne et al., 2001; Byrne and Cisternas, 2002) shows that the organization of neuronal cells and the neural anatomy of the attachment complex are very similar in these closely related asteroids. This similarity supports the notion of evolutionary conservation of the complex and its function regardless of developmental mode; it also suggests conservation of neurogenesis in attachment-stage larvae. An investigation of neural development earlier in embryogenesis in both M. calcar and P. exigua and, in a more detailed fashion, in P. regularis is needed to strengthen the hypothesis of neurogenic conservation.
In P. regularis, the apical region of the synaptotagmin immunoreactive neurons in the adhesive papillae connected to the surface of the epithelium, as noted for serotonergic cells in this larva (Chee and Byrne, 1999b). As in A. pectinifera, the 1E11 immunoreactive cells in the adhesive papillae of P. regularis are spindle-shaped and span the width of the papillae, with apical projections that extend to the surface (Murabe et al., 2008). These projections have been taken to suggest that these neurons have a sensory role in reception and transduction of chemical stimuli originating from the substrata, potentially communicating with the basal bundle of fibers (Murabe et al., 2008). Behavioral and ablation studies of settling larvae indicate that the adhesive papillae play a sensory role in substrate selection during metamorphosis (Barker, 1978; Murabe et al., 2007). After microsurgical removal of the papillae, brachiolaria fail to recognize settlement substrata (Murabe et al., 2007). The brachia of the three species investigated here have an abundance of serotonergic (Chee and Byrne, 1999b) and 1E11 immunoreactive cells. In the larvae of P. regularis, these cells give rise to sensory-like apical projections (Byrne et al., 2001, 2007), as is also the case for the larvae of A. pectinifera (Murabe et al., 2008). Scanning electron microscopy revealed the presence of short sensory-like cilia on the surface of the brachia of P. regularis (Byrne et al., 2001). The brachia play an active role in substrate selection and benthic attachment, and so the sensory-like cilia may serve as surface receptors in association with this function (Byrne et al., 2001). Similar cells that likely serve a similar function have been observed in the primary podia of juvenile echinoids (Burke, 1980). Prior to settlement, the larvae of P. regularis and M. calcar move around on the substrate until the appropriate settlement signal is recognized. In response to the appropriate cue, the adhesive disc creates a stable attachment by secretion of adhesive material from secretory cells (Barker, 1978; Byrne et al., 2001).
The pattern of NS development in brachiolaria larvae of the three species reflected differences in the onset and length of benthic attachment. In P. exigua, the adhesive disc of early larvae had a well-developed NS on hatching. In P. regularis and M. calcar, by contrast, the NS of the adhesive disc takes some days to develop. This difference reflects the fact that the brachiolaria of M. calcar and P. regularis are initially planktonic, whereas the brachiolaria of P. exigua is holobenthic. The adhesive disc of P. exigua exhibits heterochronic and hypertrophic development to be functional on hatching for permanent benthic attachment (Byrne, 2005). Here we present the first images of immunoreactive neurons in attachment-stage larvae undergoing metamorphosis. Interestingly, the NS of the adhesive disc of attached larvae of P. regularis at the final metamorphic stage was virtually identical to that seen in P. exigua. In both species, the adhesive disc of metamorphosing brachiolaria had immunoreactive cells at the base of the epithelium, and some extended from the base of the epithelium distally toward the apical surface. The nerve cells in the adhesive disc formed a plexus of fibers within the disc. During metamorphosis, fluorescence disappeared from the rest of the larval body as the LNS degenerated.
At metamorphosis, larval structures and tissues were absorbed in parallel with morphogenesis of the adult rudiment, and we noticed a temporal lapse in expression of synaptotagmin between the degeneration of the LNS and histogenesis of the juvenile NS. Investigation of peptidergic NS through metamorphosis in P. regularis also indicated a temporal lapse in the appearance of nerve-specific IR in CNS development (Byrne and Cisternas, 2002). Neither 1E11 nor S1 recognize undifferentiated nerve cells (Byrne and Cisternas, 2002; Murabe et al., 2008). The same "discontinuity" in neurogenesis and neuronal differentiation in development of the juvenile CNS was seen in in situ hybridization with the neurogenic gene engrailed where gene expression follows CNS histogenesis (Byrne et al., 2005). It appears that generation of the histological and anatomical architecture of the juvenile CNS precedes expression of neurochemicals (e.g., synaptotagmin and peptidergic neurochemicals) and neurogenic genes (engrailed) in differentiated neurons. The discontinuity in the presence of synaptotagmin immunoreactive cells at the metamorphosis observed herein suggests that the adult asteroid NS develops independently from the LNS, at least in the sense of material contribution. This is consistent with the hypothesis of independent evolution of the larval and adult NS in the last common ancestor of echinoderms (Beer et al., 2001; Nakano et al., 2006; Hirokawa et al., 2008). In juvenile development of A. pectinifera, the 1F9 antibody, which recognizes a START (steroidogenic acute regulatory protein-related) lipid-transfer domain, localizes to an extensive network of fibers that may be neurons (Murabe et al., 2008). These fibers are suggested to contribute to the adult NS, but it is not clear if they are neurons.
From our findings in the developing juvenile, synaptotagmin IR was first strongly expressed in the NS of the tube feet, followed by IR in the CNS, similar to that seen with engrailed expression (Byrne et al., 2005). This may reflect the early role for the tube feet in benthic attachment after the adhesive disc is resorbed as the CNS differentiates post-settlement. The immunoreactive cell bodies seen here on the tip of the tube feet are likely to play a sensory role, as suggested for similar cells in echinoid podia (Burke, 1980; Burke et al., 2006). The cells give rise to a neurite that connects with the dense bundle of processes in the basi-epithelial plexus. We found that the tube feet and CNS of asteroid juveniles, similar to those of newly metamophosed echinoids (Burke et al., 2006), are strongly 1E11 immunoreactive. In echinoids, however, there is no difference in the timing of onset of 1E11-IR in the tube feet and CNS (Burke et al., 2006), which differs from what we observed with juvenile sea stars.
As the juvenile developed, synaptotagmin IR in the three species investigated here was similar to that seen in the adult CNS (Saha et al., 2006). In both juveniles and adults, synaptotagmin localizes to the circumoral nerve ring and radial nerve cords, and its distribution reflects the histological organization of the sea star CNS. The distribution of synaptotagmin IR is virtually identical to the pattern seen with peptidergic IR (Byrne and Cisternas, 2002). The axonal tissue layer of the CNS was strongly immunoreactive to 1E11. Along the periphery of the nerves in the cell body layers, only a few cells were immunoreactive.
Development of the three asterinid species investigated here converged in the formation of the juvenile. The structures and tissues supporting the LNS degenerated during metamorphosis and were resorbed, so it is likely that the neurons also degenerate. It appears that none of the LNS was incorporated into the adult NS, which conforms to observations with peptidergic and serotonergic markers (Byrne et al., 2001, 2007). Similar results were reported for echinoid and holothuroid metamorphosis (Chia and Burke, 1978; Burke et al., 2006; Nakano et al., 2006; Katow et al., 2009). Although it could be considered that the larval neurons that lose their 1E11 epitope re-differentiate later as juvenile neurons, this would require individual cells to migrate out of the degenerating larval domain into that of the developing juvenile.
Among the Bilateria, the change from a bilateral nervous system to a radial one in the Echinodermata represents an extreme case, and none of the larval nervous system appears to persist through metamorphosis. This contrasts with other Bilateria. In molluscs and insects, a subset or most of the larval nervous system persists through metamorphosis to serve as a scaffold for the juvenile nervous system or is remodeled as the brain is reorganized (Technau and Heinsenberg, 1982; Marois and Carew, 1990). Further studies of nervous system development during the critical metamorphic transition in other echinoderm larvae will be important in understanding evolution of the unusual pentameral body plan of these bilaterians.
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Acknowledgments
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Yoko Nakajima (Keio University, Yokohama, Japan) kindly provided the 1E11 antibody. We thank Thomas Prowse and Drs. Louise Page and Devarajen Vaitilingon for assistance in collection of material and for provision of algal cultures. The microscopy facilities were provided by the Bosch Institute. The work was funded by a grant from the Australian Research Council (MB). We thank Dr. Svetlana Maslakova, Dr. Cory Bishop, and an anonymous reviewer for helpful comments.
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Footnotes
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Received 31 October; accepted 16 April 2009.
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Abstract
Introduction
