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Immunocytochemical studies reveal novel neural structures in nemertean pilidium larvae and provide evidence for incorporation of larval components into the juvenile nervous system
© Hindinger et al.; licensee BioMed Central Ltd. 2013
Received: 27 February 2013
Accepted: 13 May 2013
Published: 23 May 2013
Nemertea is one of the least studied lophotrochozoan phyla concerning neurogenesis. The sparse data available do not unambiguously allow for answering questions with respect to the neural groundplan of the phylum or the fate of larval neural structures during metamorphosis. In order to contribute to this issue, we studied neurotransmitter distribution during development of the pilidiophoran Lineus albocinctus Verrill, 1900.
Two serotonin-like immunoreactive (lir) neurons are present in the anterior part of the apical plate. They send numerous processes into the four lobes of the pilidium larva, where they form a complex subepithelial nerve net. All four larval lobes are underlain by a marginal neurite bundle, which is associated with numerous serotonin-lir monociliated perikarya. A serotonin-lir oral nerve ring encircles the stomach sphincter and is associated with few serotonin-lir conical cells. Two suboral neurites descend from the oral nerve ring and merge with the marginal neurite bundle. The oral nerve ring and the suboral neurites contain the mollusk-specific VD1/RPD2 α-neuropeptide. The lateral lobes of the larva have three and the anterior and the posterior lobes two VD1/RPD2-lir marginal neurite bundles. The lobar FMRFamide-lir plexus of Lineus albocinctus is much more complex than previously described for any pilidium larva. It includes a circumesophageal neurite that descends along each side of the larval esophagus and together with the inner marginal neurite bundle gives rise to the lobar plexus of the lateral lobes. An outer FMRFamide-lir marginal neurite bundle with numerous associated FMRFamide-lir marginal sensory cells surrounds all four lobes. FMRFamide-lir structures are absent in the larval apical region. The oral nerve ring and the two suboral serotonin-lir neurites are incorporated into the juvenile nervous system.
Our study confirms the presence of serotonin-lir components in the apical region of the pilidium larva of Lineus albocinctus and thus contradicts an earlier study on the same species. We show that the nervous system of pilidium larvae, especially the FMRFamide-lir components, is much more complex than previously assumed. The presence of the VD1/RPD2-α-neuropeptide indicates that this compound may have been part of the lophotrochozoan neural groundplan.
Adult Nemertea, also known as ribbon worms, are benthic predators and are predominantly found in marine habitats, but freshwater and terrestrial species also exist [1, 2]. They are bilaterally symmetric, unsegmented and dorsoventrally flattened. One apomorphic character for the phylum is the eversible and sometimes stylet-bearing epidermal proboscis [1, 3]. It lies within a fluid-filled cavity of mesodermal origin, the so-called rhynchocoel. Unlike in many other protostome invertebrates, the nemertean brain encircles the proboscis and the two frontal blood vessels, but not the foregut [1, 4, 5].
The development of the nemertean larval nervous system has been studied by various methods in the hoplonemertean Quasitetrastemma stimpsoni and in different species of Pilidiophora. Transmission electron microscopy (TEM) investigations revealed the presence of a marginal nerve that runs along the four larval lobes in undetermined pilidium larvae and in the larva of Lineus albocinctus[8, 16]. In addition, two cell types, each with a single cilium surrounded by a microvilli collar, are present in an undetermined pilidium larva. These cells are always associated with the marginal nerve . In an undetermined pilidium and in Lineus albocinctus an oral nerve ring encircles the sphincter between esophagus and stomach. Two suboral nerves descend from the oral nerve in both species. They merge with the marginal nerve at the transition between the posterior and the lateral lobes in an undetermined pilidium . Ultrastructural investigations showed that in the larva of Lineus albocinctus the suboral nerve splits into an anterior and a posterior portion; both merge with the marginal nerve at the transition between the lobes. An additional pair of so-called “lateral helmet nerves” descends from the anterior part of the anterior lobe to the junction of the anterior and the lateral lobes, where it merges with the marginal nerve . Interestingly, no neurons associated with the apical plate could hitherto be found by TEM analysis in any pilidiophoran larva [8, 16].
Immunocytochemical investigations of the pilidiophorans Lineus albocinctus and Micrura alaskensis revealed a serotonin-lir marginal nerve that underlies the ciliary band of the four larval lobes. An extensive subepithelial serotonin-lir nerve net with numerous interconnecting serotonin-lir neurons and a serotonin-lir oral nerve ring is present in the larvae of Lineus albocinctus and Micrura alaskensis, but apical serotonin-lir structures were only found in the pilidium larva of Micrura alaskensis[9, 16]. Juveniles of both species show a pair of serotonin-lir lateral nerve cords that emerges from the cephalic discs and runs ventro-laterally. In addition, several longitudinal serotonin-lir proboscis neurites are present [9, 16]. To date, data on FMRFamide-lir structures are available only for one pilidiophoran species, namely Lineus albocinctus, where one to four FMRFamide-lir cells were found in the larval episphere. They send their processes to the anterior region of the apical plate and to the junction between the anterior and the lateral lobes. In the juvenile of Lineus albocinctus no FMRFamide-lir structures were found .
Despite these data on larval neuroanatomy, development of immunoreactive compounds in Nemertea, especially Pilidiophora, remains largely unknown, as is the fate of the larval nervous system and the question as to what degree larval components are incorporated into the juvenile neural bodyplan. In order to address these issues and to provide novel data on the neural architecture of nemertean pilidium larvae, we analyzed neurogenesis of Lineus albocinctus using antibodies directed against the common neurotransmitters serotonin and FMRFamide as well as the VD1/RPD2 α-neuropeptide, which was originally described from the mud snail, Lymnaea stagnalis, but seems to be present in non-molluscan invertebrates as well [17–19].
Several neuroanatomical terms, such as “nerve” and “nerve cord”, are used in different ways for various invertebrate taxa without any consensus on their exact definitions. A recently published glossary on neuroanatomical terminology defines a “nerve” as a structure of condensed axons free of cell bodies . Whole-mount immunocytochemical methods, however, usually neither allow for in toto visualization of entire nerves (including their cell bodies, entity of axons and dendrites) nor for a clear distinction between axons and dendrites. Therefore, terms such as “nerve” and “nerve cord” are only used herein if they were unambiguously identified by transmission electron microscopy (TEM). In accordance with a recent paper  we use “neurite” and “neurite bundle” if the data were generated by immunocytochemistry only.
Development of serotonin-lir structures from early larva to the juvenile worm
Dissected juveniles, one with almost fused discs (“torus stage”, see ) and one with the discs and anlagen that have entirely fused, show, that the oral nerve ring and the suboral neurites are incorporated into the juvenile nervous system (Figures 3E, F and 4C-E). The suboral neurites now descend into the trunk disc portion of the lateral neurite bundles of the juvenile and are no longer connected to the larval marginal neurite bundle (Figures 3F and 4C-E). Additionally, several neurites that originate from the oral nerve ring and run around the stomach along the anterior-posterior axis of the juvenile are formed (Figures 3E and 4C-E). Two prominent lateral neurite bundles are present along the ventral side of the juvenile worm (Figures 3D-F and 4C-E). They emerge from the posterior part of the already fused cephalic discs, transverse the cerebral organ disc region, continue laterally where they transverse the trunk discs, and project into the very posterior region of the juvenile (Figure 3E, F). The cephalic discs region is traversed by numerous serotonin-lir neurites connected to the lateral neurite bundles (Figure 3E, F). However, this region shows no serotonin-lir cell bodies associated with the lateral neurite bundle. The proboscis anlage exhibits several serotonin-lir neurites along its anterior-posterior axis. In the region of the cerebral organ the lateral neurite bundles express a high density of associated serotonin-lir cells (Figure 3F). Along the trunk discs and in the posterior region of the juvenile such cells are also present, but are lower in number (Figures 3F and 4D, E; for comparison with the adult neural condition see Figure 4F).
Development of FMRFamide-lir structures from the early larva to the juvenile worm
VD1/RPD2 α-neuropeptide-lir structures in the early pilidium larva
Development of the serotonin-lir nervous system in larval and juvenile nemerteans
Recently, two serotonin-lir apical cells as well as two serotonin-lir cells which lie beneath the apical cells were described for the hoplonemertean Quasitetrastemma stimpsoni. These cells are all flask-shaped and most likely homologous to similar cells found in the apical organ of many lophotrochozoan larvae [21, 22]. In Pilidiophora, neurogenesis has been investigated in several species using TEM and immunocytochemical staining. We found that a serotonin-lir marginal neurite bundle that underlies the ciliary band is present in all four lobes of Lineus albocinctus, which corroborates previous studies on the same species as well as on Micrura alaskensis[9, 16]. The marginal neurite bundle in Lineus albocinctus was reported to show numerous associated serotonin-lir cells which form contact with the marginal neurite bundle via two processes . These associated serotonin-lir cells in Lineus albocinctus were also found in our study, while a connection to the marginal neurite bundle via two processes could not be confirmed. Interestingly, in Lineus albocinctus, every serotonin-lir cell associated with the marginal neurite bundle bears a single cilium. These findings on serotonin-lir structures are partly in accordance with data on Micrura alaskensis, which likewise exhibits a marginal neurite bundle with numerous associated serotonin-lir cells . However, the latter work provides no information about the innervation of these associated serotonin-lir cells, nor does it report any ciliary structures that project from these cells.
During larval development of Lineus albocinctus the number of serotonin-lir cells associated with the marginal neurite bundle increases. The previously described serotonin-lir subepithelial nerve net with its interconnected multipolar interneurons as well as the increase in complexity of the nerve net during subsequent development is confirmed herein (see [9, 16]). A serotonin-lir oral nerve ring has been described for Lineus albocinctus and Micrura alaskensis, albeit depiction is lacking for the latter species [9, 16]. So far, the presence of two monociliated serotonin-lir apical neurons had been demonstrated for Micrura alaskensis only . In our study, two serotonin-lir apical neurons are shown in the apical plate of Lineus albocinctus for the first time, although they are not flask-shaped, thus rendering homology to the apical cells reported for the hoplonemertean Quasitetrastemma (see above) and other, apical organ-bearing lophotrochozoans, unlikely.
The apical neurons of Lineus albocinctus send their processes into each of the four lobes. An apical neurite plexus that underlies the apical plate is formed by the processes of the apical neurons. These findings contradict those of the previous study, where no neural structures in the apical plate of Lineus albocinctus were found .
During postlarval development of Lineus albocinctus the serotonin-lir oral nerve ring and the serotonin-lir suboral neurite are incorporated into the juvenile serotonin-lir nervous system. This and the fusion of the suboral neurites with the lateral neurite bundles of the juvenile clearly demonstrates that larval structures form parts of the juvenile nervous system. Confirming an earlier study on Lineus albocinctus, two serotonin-lir lateral neurite bundles are present in the ventro-lateral region of the juvenile worm . In Micrura alaskensis two neural structures have been interpreted as lateral neurite bundles of the juvenile, but these structures were identified by phalloidin, a stain specific for filamentous actin rather than defined neural structures . The cell progenies of the cephalic discs and the cerebral organ discs of Micrura alaskensis have been proposed to contribute to the development of the lateral neurite bundles . This is confirmed by the results of our study, although it has to be mentioned that former methodical approaches by use of phalloidin for visualizing neural, in particular serotonin-lir structures , renders these previous data doubtful. As in Micrura alaskensis, longitudinal serotonin-lir neurites run through the proboscis anlage of Lineus albocinctus.
Development of the FMRFamide-lir neural structures in larval and juvenile nemerteans
The data presented herein show a much higher grade of complexity of the larval FMRFamide-lir system in Lineus albocinctus than reported by an earlier study . A circumesophageal neurite loops around the apical part of the esophagus and descends into the abapical region of the episphere. The circumesophageal neurite is associated with few FMRFamide-lir cell bodies. Due to its position within the larva and its FMRFamide-like immunoreactivity, this circumesophageal neurite may constitute the very process previously termed “lateral helmet process” . The present study shows that this neurite splits into several peripheral lobar neurites in the region of the lobe junctions in the Lineus albocintus pilidium. A complex lobar nerve net with numerous multipolar interconnected interneurons originates from the peripheral lobar neurites. In addition, the peripheral lobar neurites contribute to the inner marginal neurite bundle of the lateral lobes. The neurites of the lobar nerve net are always connected to the inner marginal neurite bundle of the lateral lobes via inner marginal interneurons. An outer marginal neurite bundle, which is associated with FMRFamide-lir marginal sensory cells, runs along the four lobes. Due to the presence of a single cilium, the microvilli collar and the connection to the marginal neurite bundle, these cells may correspond to a cell type shown previously by TEM studies of an unknown pilidiophoran species .
Throughout the different developmental stages investigated no essential changes in the FMRFamide-lir nervous system of the larva were found. No FMRFamide-lir structures were found in the region of the apical plate.
Recently, an apical organ that includes four flask-shaped serotonin-lir apical neurons was found in the hoplonemertean Quasitetrastemma stimpsoni. This was the first work that unequivocally showed the existence of an apical organ that contains distinct sensory cells in any nemertean larva and suggests that such a morphological feature may have been part of the hoplonemertean groundplan. In Pilidiophora, the serotonin-lir apical neurons in Micrura alaskensis show a single cilium, but are not flask-shaped and lack the connection to the apical cilia, and those in Lineus albocinctus are neither flask-shaped, nor do they appear to bear a single cilium. Thus, these cells are not indicative of a rudimentary apical organ. Since many lophotrochozoans, such as Mollusca, polychaete annelids, Phoronida and Entoprocta, exhibit an apical organ, it is more parsimonious to assume that such a structure was also present in the LCA of Lophotrochozoa, and was secondarily lost in several subclades, including Pilidiophora. Our data also demonstrate the previously overlooked high complexity of the larval FMRFamide-lir nervous system in Lineus albocinctus and adds the mollusk-specific VD1/RPD2 α-neuropeptide to the suite of nemertean (larval) neural components. This finding calls for further investigations into other lophotrochozoan and bilaterian taxa to assess its degree of conservation in the respective clades. Accordingly, large-scale comparative immunocytochemical studies using the antibody against this peptide could reveal an important set of novel data that may prove highly useful for (neuro)phylogenetic studies.
The oral nerve ring and the two suboral neurites are incorporated into the juvenile nervous system of Lineus albocinctus. The two lateral neurite bundles develop inside the juvenile imaginal discs and most likely form the future ventral nerve cords of the adult nemertean. Based on comparative juvenile and adult neuroanatomy, it appears most likely that the incorporated oral nerve ring develops into (parts of) the adult brain commissures. This requires, however, that the juvenile mouth develops secondarily, because only then the oral nerve ring would encircle the proboscis but not the esophagus, corresponding to the situation found in adult Nemertea. This scenario would also provide an explanation for the formation of the adult brain by concentration (fusion?) of the oral nerve ring and the anterior connection of the lateral neurite bundles. However, data on neurogenesis from early juveniles through adulthood are needed to further assess this issue.
Materials and methods
Animal collection, determination and fixation
In pilidium larvae of Lineus albocinctus the anterior lobe is more prominent than the posterior one. The helmet as well as the anterior and the posterior lobes show a concave outline. The apical tuft can be as long as the entire height of the larva, and the apical plate, formed by epidermal cells, is relatively large.
Larvae of different developmental stages were collected in August 2011 in the Gullmarsfjord, Swedish west coast by plankton tows (58°15′7″N, 11°26′30″E; 58°15′02″N, 11° 27′23″E). Animals were relaxed in a 3.5% MgCl2 solution and were fixed for approximately 1.5 to 2 hours at room temperature (RT) in 4% paraformaldehyde in 0.1 M PBS (phosphate buffered saline, pH 7.4) with 10% sucrose added. Afterwards, animals were rinsed in 0.1 M PBS with 0.1% NaN3 and stored in the same solution at 4°C.
Immunocytochemistry and confocal laserscanning microscopy
For confocal microscopy larval tissue was permeabilized for 60 min in PBT (0.1 M PBS with 0.1% NaN3 and 4% Triton X-100). Non-specific binding sites were blocked in PBT with 6% normal goat serum (Jackson ImmunoResearch, West Grove, PA, USA) at RT overnight. Subsequently, the larvae were incubated in either of the following primary antibodies: 5-HT antibody (serotonin) raised in rabbit, diluted 1:800 (ImmunoStar, Hudson, WI, USA); FMRFamide antibody raised in rabbit, diluted 1:500 (Biotrend, Cologne, Germany); a mollusk-specific α-neuropeptide antibody, diluted 1:350 (CASLO ApS, Lyngby, Denmark), directed against the VD1/RPD2 system of the pond snail Lymnaea stagnalis raised in rabbit; acetylated-α-tubulin antibody raised in mouse, diluted 1:400 (Sigma-Aldrich, St. Louis, MO, USA) with blockPBT (PBT with 6% goat serum) for 24 hours at RT. Specimens were rinsed four times in blockPBT for at least 6 hours. Then, they were incubated for 24 hours at RT in Alexa Fluor 568 anti-rabbit (Invitrogen, Molecular Probes, Eugene, OR, USA) or Alexa Fluor 633 anti-mouse (Invitrogen) secondary antibody, respectively, both at a dilution of 1:300 in blockPBT. DAPI (Sigma) was added at a 1:400 dilution for staining of the cell nuclei. Stained larvae were washed four times in PBS for at least six hours and then mounted in Fluoromount-G (Southern Biotech, Birmingham, AL, USA). Cover glasses were provided with clay feet to prevent squashing of specimens. Slides were stored at 4°C.
Samples were examined with a Leica SP5 II confocal microscope (Leica Microsystems, Wetzlar, Germany) and stacks of optical sections of 0.3-0.6 μm Z-thickness were generated. Further digital image processing was done with Adobe Photoshop CS (Adobe, San Jose, CA, USA). Adobe Illustrator CS (Adobe) was used for creating the line drawings.
The authors thank Tim Wollesen and Alen Kristof (both Dept. of Integrative Zoology, University of Vienna) for constructive discussions. AW is grateful for generous support by the Faculty of Life Sciences, University of Vienna.
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