Comparative Lophotrochozoan Neurogenesis and Larval Neuroanatomy: Recent Advances from Previously Neglected Taxa*

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Acta Biologica Hungarica 59 (Suppl.), pp. 127–136 (2008) DOI: 10.1556/ABiol.59.2008.Suppl.21

COMPARATIVE LOPHOTROCHOZOAN NEUROGENESIS AND LARVAL NEUROANATOMY: RECENT ADVANCES FROM PREVIOUSLY NEGLECTED TAXA*

A. WANNINGER**

Research Group of Comparative Zoology, Department of Biology, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen, Denmark

(Received: October 22, 2007; accepted: December 7, 2007)

Recently, a number of neurodevelopmental studies of hitherto neglected taxa have become available, contributing to questions relating to the evolution of the nervous system of Lophotrochozoa (Spiralia + Lophophorata). As an example, neurogenesis of echiurans showed that these worm-shaped spiralians, which as adults do not exhibit any signs of segmentation, do show such traits during ontogeny, e.g. by segmentally arranged perikarya and commissures. Similarly, sipunculan worms, which have a single ventral nerve cord in the adult stage, develop this nerve cord by gradual fusion of a paired larval nerve during metamorphosis, and show transitional stages of segmentation. These findings indicate that echiurans, annelids and sipunculans stem from a segmented ancestor. By contrast, no traces of body segmentation are present during neurogenesis of basal molluscs. However, a tetraneurous condition (i.e. one pair of ventral and one pair of lateral nerve cords), as is typical for Mollusca, and a serotonergic larval apical organ that matches the complexity of polyplacophoran apical organs, were found in larval entoprocts, thus strongly supporting a mollusc-entoproct clade. Within the Lophophorata (Ectoprocta + Phoronida + Brachiopoda), data on nervous system development for any of the 3 lophophorate phyla are as of yet too scarce for profound phylogenetic inferences. Taking into account the most recent advances in molecular phylogenetics and developmental neurobiology, a scenario emerges that proposes a clade comprising Sipuncula + Annelida (including Echiura) on the one hand and a monophyletic assemblage of Entoprocta + Mollusca on the other.

Keywords: Nervous system – invertebrate – evolution – phylogeny – confocal microscopy

* Presented during the 11th ISIN Symposium on Invertebrate Neurobiology, 25–29 August, 2007, Tihany, Hungary ** E-mail: [email protected]

INTRODUCTION

Although current (molecular) phylogenetic analyses commonly recognise the proto- stomian superclades Ecdysozoa (animals with a cuticle that is molted during their life cycle) and Lophotrochozoa (animals with a ciliated larva), the sister group relationships within these clades are still heavily debated [6, 22]. Accordingly, comparative developmental zoology has recently received considerable attention, resulting in an increased neuroanatomical database for little known, non-model system species. While traditional studies had been limited to light and electron microscopic analyses of semi- and ultrathin sections [e.g., 12, 17], the last decade saw significant method- ological innovations mainly involving fluorescence labelling, confocal microscopy, and 3D reconstruction software. This allows high-throughput data generation, analysis, and 3D depiction of neurological bodyplans of larval and adult stages of microscopic metazoans and thus enables documentation of the dynamics of neurogenesis over time [28]. Lophotrochoza comprises phyla with a large variety of bodyplan morphologies, e.g., unsegmented and segmented worms (phoronids, nemertines, platyhelminths, sipunculans, annelids), the sessile or semi-sessile tentacle-bearing entoprocts and ectoprocts, as well as molluscs, brachiopods, and the enigmatic cycliophorans. A peculiar feeding organ, the lophophore, morphologically unites Ectoprocta, Phoronida, and Brachiopoda to form the Lophophorata, the proposed sister group of Spiralia. In the following, I will review the current state of knowledge on the larval nervous system and neurogenesis of spiralian and lophophorate phyla with respect to the progress that has been made in the last decade. I will particularly focus on previously widely neglected taxa such as Sipuncula, Entoprocta (Kamptozoa), Ectoprocta (Bryozoa), or Cycliophora, and will discuss the evolutionary and phylogenetic rele- vance of these data in the light of the currently widely accepted “Lophotrochozoa- Ecdysozoa-Hypothesis”.

Spiralian neurogenesis and larval neuroanatomy Annelida (incl. Echiura)

Despite the long tradition of annelids as model system animals, recent studies employing fluorescent antibody staining of neural markers remain scarce. In all larval polychaetes investigated so far, a serotonergic nerve ring underlying the prototroch is found [3, 14, 25]. An apical organ with a ciliated tuft is usually present. The number of associated (flask-shaped?) serotonergic and FMRFamidergic cells varies from none to 4, respectively. In most cases, a paired ventral nerve cord with interconnecting commissures is established early in development, which subsequently grows along the anterior-posterior axis, hereby adding commissures in strict anterior-posterior direction (Fig. 1). Anteriorly, the ventral nerve cords connect to the

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Fig. 1. Fluorescence labelling of the serotonergic (A) and the FMRFamidergic (B) nervous system of larvae of the polychaete annelid Filograna implexa. Anterior faces upwards and scale bars equal 25 μm. A. The prototroch nerve (pn), parts of the (adult) cerebral ganglion (cg), and the paired ventral nerve cord (arrows) stain positive for serotonin. The anlage of the first commissure (arrowhead) is being formed. B. Slightly older stage than the specimen shown in A, depicting the subsequent formation of the segmental commissures. The most anterior commissure stains more intensely than the second, while the third commissure is still in the process of being formed (arrowheads)

cerebral ganglion (“brain”). In late larval and adult stages the number of ventral nerve cords may vary from 1 to 5 and circumoral and preoral nerve loops may be found, thus indicating the plasticity of the adult polychaete neural architecture [16]. While adult echiurans show no signs of segmentation in their gross morphology, neurogenesis revealed a strict anterior-posterior formation of perikarya and ganglia associated with the ventral nervous system [9, 10], thus corroborating recent molecular phylogenetic analyses that place the echiurans within or closely related to Annelida [1, 2, 11, 15, 24]. Moreover, it could be shown that the adult single echiuran ventral nerve cord results from a fusion process of an originally paired nerve [9, 10]. However, fluorescence labelling failed to detect immunoreactive cells in the apical organ or a serotonergic prototroch nerve ring, which may be due to secondary loss of these features in the short-lived planktonic echiuran larvae. Accordingly, the LCA of the annelid-echiuran assemblage most likely was a segmented animal with a paired ventral nerve cord with associated ganglia and commissures, a larval serotonergic nerve ring, and few serotonergic cells in the larval apical organ.

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Sipuncula

Sipunculans are worm-shaped spiralians that develop via a trochophore-like larva, which in some species transforms into a second larval type, the pelagosphera, before eventually metamorphosing into the juvenile worm. While adult sipunculans have a single ventral nerve cord without ganglia, immunostaining of the larval stages of the species Phascolion strombus against serotonin and FMRFamide revealed a paired ventral nerve cord, with the serotonergic axons gradually fusing during development [30]. The FMRFamidergic nervous system of late larval stages show 3 ventral commissures that disappear during metamorphosis. Instead, a median nerve, which is sit- uated between the 2 initial nerve cords, is found [30]. Unpublished observations indicate a segmental formation mode of the ventral nervous system in the species Phascolosoma agassizii. A serotonergic prototroch nerve ring and serotonergic cell bodies in the apical organ are present in P. agassizii but not in P. strombus. However, 2–3 minute FMRFamidergic cells are found in this sensory organ in early larval stages of both species.

Mollusca

Molluscs show a wide variety of larvae. Since the focus of this review is on the bearing of recent neurodevelopmental data on lophotrochozoan phyletic interrelation- ships, I will restrict the discussion to the most basal molluscan clade for which such data are available, the Polyplacophora (chitons). Polyplacophoran larvae develop via a lecithotrophic trochophore. The apical organ contains 8 centrally positioned serotonergic and some FMRFamidergic flask- shaped cells in both species investigated to date, Mopalia muscosa and Ischnochiton hakodadensis [4, 26]. In addition, Ischnochiton shows one pair of peripheral cells on each side of the flask cells [26; Fig. 2A]. Subsequently, the paired ventral (pedal) and lateral (visceral) nerve cords are formed, thus establishing the typical molluscan tetraneurous condition. Several sets of serotonergic (and in Ischnochiton also FMRFamidergic) perikarya are found associated with the pedal nerve cord. The commissures that link the pedal nerve cords to each other form “randomly”, i.e. not in strict anterior-posterior direction as in annelids, thus directly contradicting segmented molluscan ancestry. Underneath the prototroch there is a dense serotonergic nerve net that is considered homologous to net- or ring-like prototroch nerves found in other lophotrochozoan larvae. A set of flask-shaped FMRFamidergic and (weakly stained) serotonergic cells with a densely ciliated lumen on both sides of the epi- sphere forms a sensory organ (“ampullary system”) that is connected to the cerebral commissure and may constitute a polyplacophoran apomorphy [7]. All larval neural features such as the apical organ, the ampullary system, and the prototroch nerve net are lost during metamorphosis.

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Entoprocta (Kamptozoa)

Species of the tentacle-bearing clade Entoprocta show 2 strikingly different types of larvae. While most representatives exhibit a planktotrophic swimming larva, others have a creeping-type larva that is considered basal for the phylum and alternates between short swimming and creeping periods [18]. The swimming-type larva has a prominent apical organ, from which 2 lateral nerves emerge, that split halfway along the anterior-posterior axis with one branch connecting to a prototroch nerve ring, while the other branch curves and innervates a paired frontal sensory organ. A dis- tinct neuropil is found beneath and connected to the larval apical organ [17]. Serotonin-labelling of swimming-type larvae recognised the paired lateral nerves, the neuropil underlying the apical organ, the prototroch nerve ring, and labelled 4 flask-

Fig. 2. Graphic representation of the serotonergic portion of the larval apical organ of polyplacophoran molluscs (A), the creeping-type larva of the entoproct Loxosomella murmanica (B), and a 3D reconstruction of the entire serotonergic nervous system of Loxosomella. Anterior faces upwards and scale bars equal 10 μm in A, B, and 25 μm in C. A. Polyplacophoran larvae have an apical organ with 8 central flask-shaped cells (black) and few peripheral cells (grey), which are all in contact with an underlying neuropil (np). The 2 white cell bodies are probably apomorphic for Polyplacophora. B. Cell types homologous to those in A are indicated with the same colour. C. The serotonergic nervous system of Loxosomella exhibits a complex apical organ (ao), an anterior nerve ring (an), a paired pedal nerve cord (arrows) with associated perikarya (asterisks), a paired visceral nerve cord (double arrow), and a prototroch nerve ring (pn)

Acta Biologica Hungarica 59, 2008 132 A. WANNINGER shaped cells in the apical organ [5, 8]. The architecture of the serotonergic nervous system of the creeping-type larva is strikingly different [29]. An elaborated neuroanatomy, including a prototroch nerve ring as well as anterior and circumoral nerve rings, a buccal nervous system, and a highly complex apical organ comprising 6–8 central serotonergic flask cells and 8 peripheral bipolar cells are present (Fig. 2B, C). This condition resembles the situation found in polyplacophoran larvae (see above) and thus renders the apical organs of Loxosomella and polyplacophoran molluscs the most complex within basal Spiralia. In addition, the larva of Loxosomella shows true tetraneury, i.e., one pair of lateral (visceral) and one pair of ventral (pedal) nerve cords, a feature that had previously been considered diagnostic for Mollusca. Moreover, 4 pairs of serotonergic perikarya associated with the pedal nerve cords and several pedal commissures are present. Accordingly, the entoproct creeping-type larva exhibits a mosaic of characters found in larval and adult basal molluscs, respectively, thus clearly supporting a monophyletic asemblage of Mollusca and Entoprocta.

Cycliophora

Cycliophorans are characterised by a very complex life cycle which includes a free swimming “chordoid” larva that exhibits ciliated regions on the ventral side and in the anterior part, which has been used to interpret this larva as a modified trochophore. Immunocytochemical studies of this larva, however, show little congruen- cies with other trochozoans. While an apical organ is missing, the chordoid larva has a large bilobed brain and a ventral nervous system that comprises 2 FMRFamideric and 4 serotonergic longitudinal cords without ganglia or commissures [27]. The fate of the larval nervous system during metamorphosis remains unknown.

Platyhelminthes

Adult platyhelminths express an “orthogonal” nervous system which comprises an apical “brain” and 2–8 longitudinal nerve cords that are connected to each other via numerous commissures. While a relatively large larval “apical ganglion”, from which several longitudinal nerves emerge, has been described on the ultrastructural level, the scarce immunocytochemical data on the platyhelminth Müller’s larva revealed 2 serotonergic cells in the apical organ from which one axon per cell emerges and runs in posterior direction. There, they form contact with a serotonergic nerve ring, which is connected to 6 serotonergic cell bodies that are associated with 6 ciliated lobes of the larva [8]. While the larval lobes are reduced during metamorphosis, the fate of the larval serotonergic nervous system is yet uncertain.

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Nemertea

Nemerteans exhibit two different modes of development, with the “direct” developing species probably representing the ancestral condition [13]. No immunocytochemical data of this larval type are currently available. Serotonin staining of pilidi- um larvae of indirect developing taxa showed no immunoreactivity in the apical organ but a nerve ring and bipolar nerve cells associated with the ciliated, prototroch- like structure of this larval type [8].

Lophophorate neurogenesis and larval neuroanatomy Brachiopoda

Almost no immunocytochemical data on the developing brachiopod nervous system are currently available. Planktotrophic juveniles of the direct developing genus Glottidia have a serotonergic cluster of numerous cells in the anterior pole, which may or may not correspond to the spiralian apical organ. From these cells, serotonergic axons emerge, which underlie the ciliated finger-like structures of the adult feeding organ, the lophophore. Later in ontogeny, a serotonergic cell cluster arises ventrally in close proximity to the esophagus [8]. No data on larvae of indirect developing species are currently available.

Phoronida

Phoronids are benthic, worm-shaped lophophorates that often live in tubes. The actinotroch larvae of the species investigated so far exhibit an apical organ that contains numerous serotonergic cell bodies [8, 20, 21]. In at least some of the species, 2 different types of serotonergic cells are found in the apical organ, namely bipolar sensory cells as well as non-sensory cells. In addition, the apical organ is lined with an outer ring of catecholaminergic cell bodies or nerve fibers in some species [21]. A both catecholaminergic and serotonergic nerve ring connects the larval apical organ to the juvenile ventral nervous system. Parts of the larval nervous system may be integrated with the juvenile neuroarchitecture [20, 21]. In certain species, paired serotonergic axons emerging from the apical organ have been shown to project in posterior direction, eventually forming contact with a serotonergic nerve underlying a ciliary band [8]. Due to their unique morphology that includes larval and adult ten- tacles and an anteriorly placed “hood sense organ”, phoronid larvae show several apomorphic features in their neural bauplan such as hood and tentacle nerves and a secondary anterior neuropil that is believed to be part of the hood sense organ [20, 21].

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Ectoprocta (Bryozoa)

Ectoprocta exhibit 2 different larval types, a long-lived planktotrophic cyphonautes larva and a short-lived lecithotrophic corona larva. Both larval types undergo “cata- strophic” metamorphosis during which all major larval organs are lost, while the adult bodyplan forms de novo [17]. The coronate larva of Triphyllozoon mucronatum has a relatively elaborated nervous system with a serotonergic apical ring from which 2 lateral neurons arise which probably innervate a paired light sensory organ. In addition, a serotonergic nerve net underlying the ciliated corona, one pair of axons that innervate a frontal (“pyriform”) organ, and an internal nerve net are present [31]. The FMRFamidergic nervous system is less prominent and is restricted to the apical nerve ring and the lateral nerves innervating the eyes. All these neural components are lost during metamorphosis. Serotonin staining of the corona larva of Bugula neritina failed to recognise the internal nerve net and the corona nerves, but does show the lateral nerves and the apical nerve ring. In addition, Bugula exhibits one pair of serotonergic apical cells that are connected to this nerve ring [19, 23]. Larvae of the – most likely derived – cyphonates larva of Membranipora lack most of the structures found in the coronate larvae except for the paired serotonergic axon innervating the pyriform organ. By contrast, they contain 2 apical serotonergic cells from which a paired longitudinal nerve emerges that forms contact to a serotonergic nerve ring underlying a ciliary band [8].

CONCLUSIONS AND OUTLOOK

Combined application of fluorescence labelling, confocal microscopy, and 3D reconstruction software has been proven extremely valuable to acquire novel microanatomical data [28]. Considering the most recent data on lophotrochozoan larval neuroanatomy and neurogenesis in the light of recent molecular phylogenetic analyses, the following scenario appears to be the most probable: (1) The LCA of Lophotrochozoa was unsegmented with a simple CNS without ganglia and 2 or more ventral nerve cords. A serotonergic nerve ring underlying the prototroch was present, together with an apical organ with few (at the most 4) serotonergic (flask-shaped?) and possibly also some FMRFamidergic cells. (2) Echiurans are (polychaete) annelids. Annelida is the only lophotrochozoan phylum that expresses a segmented adult bodyplan with a paired ventral nerve cord and ganglia. (3) Annelida and Sipuncula form a monophyletic taxon. Cryptic segmentation during sipunculan neurogenesis argues for a segmented LCA of the annelid-sipunculan clade. (4) Mollusca + Entoprocta are monophyletic, expressing a tetraneurous nervous system with 2 pedal nerve cords with commissures and 2 visceral nerves. In addition, a complex apical organ showing numerous flask-shaped central cells and several

Acta Biologica Hungarica 59, 2008 Recent advances in lophotrochozoan neurobiology 135 peripheral cells, as well as an apical and an oral nerve ring and a buccal nervous system are present. (5) The phylogenetic relationships of Platyhelminthes, Nemertea, and Cyclio- phora remains dubious, as does their possible basal neuroarchitecture. This is mainly due to their abberant life cycles, lack of neurodevelopmental data, and the absence of congruent signals from molecular phylogenetic analyses. The same is true for the lophophorate clades Phoronida, Brachiopoda, and Ectoprocta. In summary, continued acquisition of neurodevelopmental data on still widely neglected taxa in combination with more robust phylogenetic hypotheses should eventually allow reconstruction of the basal traits of the respective lophotrochozoan phyla and the last common ancestor of Lophotrochozoa itself.

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