Biol. Bull. 216: 293-306. (June 2009)
© 2009 Marine Biological Laboratory
Shaping the Things to Come: Ontogeny of Lophotrochozoan Neuromuscular Systems and the Tetraneuralia Concept
Andreas Wanninger
University of Copenhagen, Department of Biology, Research Group for Comparative Zoology, Universitetsparken 15, DK-2100 Copenhagen, Denmark
E-mail: awanninger{at}bio.ku.dk
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Abstract
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Despite the large variation in adult bodyplan phenotypes, a worm-shaped morphology is considered plesiomorphic for both Lophotrochozoa and Bilateria. Although almost all larval and adult lophotrochozoan worms have serially arranged ring muscles in their body wall, a comparison of their ontogeny reveals no less than six different developmental pathways that lead to this homogenous arrangement of ring muscles. However, in all taxa, with the exception of chaetodermomorph molluscs and the segmented annelids, ring muscle development starts with synchronous formation of certain pioneer myocytes, which is thus considered basal for Lophotrochozoa. Recent studies on spiralian neurogenesis revealed remnants of ancestral segmentation in echiurans and sipunculans, thus confirming molecular phylogenetic studies that propose a close relationship of these three taxa. Larval entoprocts exhibit a mosaic of larval and adult molluscan characters and, among other apomorphies, share with polyplacophoran Mollusca a complex larval apical organ and a tetraneurous nervous system, strongly suggesting a monophyletic assemblage of Entoprocta and Mollusca. The term Tetraneuralia is proposed herein for this lophotrochozoan clade. Overall, formation of the lophotrochozoan neuromuscular bodyplan appears as a highly dynamic process on both the ontogenetic and the evolutionary timescales, highlighting the importance of insights into these processes for reconstructing ancestral bodyplan features and phylogenetic relationships.
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Introduction
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Lophotrochozoan invertebrate animals—that is, taxa that commonly have a ciliated larval stage with an anteriorly placed apical organ and one pair of protonephridia as part of their life cycle—exhibit an overwhelming diversity of adult bodyplan phenotypes. Among these are worm-shaped clades such as Plathelminthes or Nemertea, tentacle-bearing phyla such as Entoprocta and Lophophorata (Ectoprocta, Brachiopoda, and Phoronida), or highly mobile pelagic taxa such as cephalopod molluscs. Comparing adult morphologies of these diverse phyla inevitably leads to a "lophotrochozoan bodyplan paradox" because —despite a wide morphological disparity within the respective phyla—some phenotypes are reoccurring in a number of distantly related clades. As a result, gross anatomical features of certain taxa belonging to different phyla often resemble each other much more closely than those of taxa within a given phylum. Most strikingly, this is exemplified in the Mollusca, where the most basal forms, the aplacophoran taxa Neomeniomorpha and Chaetodermomorpha, constitute a vermiform appearance, while all other molluscan subgroups derive from benthic or pelagic shell-bearing taxa (Ponder and Lindberg, 2008). Interestingly, the shell may have been secondarily lost independently in some lineages (e.g., in octopod cephalopods or in marine and terrestrial slugs), while the worm-like appearance has been reacquired secondarily in some groups (e.g., in shipworm bivalves).
While today it is generally considered that many of these gross morphological similarities between phyla are based on functional and ecological constraints rather than on shared evolutionary ancestry (see Salvini-Plawen, 1973), they have often been used as a basis for far-reaching phylogenetic inferences (e.g., Nielsen, 2001). As a prominent example, the tentacle-bearing Entoprocta and Ectoprocta have for decades been considered sister groups and were united in the clade "Bryozoa" within the Spiralia (Nielsen, 1971, 2001). However, molecular phylogenetic and developmental analyses have shown that only Entoprocta nests within Spiralia, while ectoprocts often cluster with the phoronids, the brachiopods, or both, although a monophyletic Lophophorata is normally not recognized in recent molecular phylogenies (for discussion, see Halanych, 1996; Mackey et al., 1996; Giribet et al., 2000; Peterson and Eernisse, 2001; Passamaneck and Halanych, 2004, 2006; Philippe et al., 2005; Helmkampf et al., 2008).
Since adult morphologies alone fail to explain lophotrochozoan bodyplan diversity, investigators are currently increasingly focusing on comparative analyses of embryonic and larval stages, using state-of-the-art tools such as confocal laserscanning microscopy and 3D reconstruction software (e.g., Wanninger et al., 1999, 2005a, b, 2007; Friedrich et al., 2002; Hessling, 2002, 2003; Hessling and Westheide, 2002; Page, 2002; Santagata, 2002, 2008a, b; Santagata and Zimmer, 2002; Voronezhskaya et al., 2002, 2003; Wanninger and Haszprunar, 2002a, b, 2003; Wanninger, 2005, 2007, 2008; McDougall et al., 2006; Brinkmann and Wanninger, 2008; Fuchs and Wanninger, 2008; Gruhl, 2008; Kristof et al., 2008; Neves et al., 2009). In particular, a wealth of data has become available on the anatomical features of the trochophore-like larva, an intermediate key stage in the ontogeny of many lophotrochozoans. These larvae commonly comprise a ring of ciliated cells (called the prototroch in spiralian larvae) as the prime swimming (and sometimes also feeding) organ, an apical organ with a ciliary tuft, and one pair of protonephridia (Hatschek, 1878; Rouse, 1999; Nielsen, 2001; Fig. 1). Micro-anatomical analyses have focused primarily on the structure and formation of the trochophore nervous system (Friedrich et al., 2002; Hessling, 2002, 2003; Hessling and Westheide, 2002; Voronezhskaya et al., 2002, 2003; Wanninger and Haszprunar, 2003; Wanninger, 2005; Brinkmann and Wanninger, 2008; Fuchs and Wanninger, 2008; Kristof et al., 2008), but other organ systems such as the musculature are increasingly considered (Wanninger et al., 1999, 2005a, b; Wanninger and Haszprunar, 2002a, b; Bergter et al., 2008; Neves et al., 2009).
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Figure 1. Diversity of lophotrochozoan larvae, scanning electron micrographs. Scale bars equal 50 µm in A, F, H; 25 µm in B–E and G. Anterior is up in all aspects. (A) Lateral left view of a Müller-type larva of the plathelminth Pseudoceros canadensis, covered with epidermal cilia (ci) and showing several lobe-like protrusions (asterisks). Image courtesy of H. Semmler (University of Copenhagen). (B) Ventral view of the chordoid larva of the cycliophoran Symbion pandora with anterior ciliary field (ac), ventral ciliary field (vc), and posterior ciliary field (pc). (C) Dorsal view of the chaetodermomorph mollusc Chaetoderma with anteriorly positioned prototroch (pt) and posterior telotroch (tt). (D) Dorsal view of the three-lobed larva of the articulate brachiopod Argyrotheca cordata with ciliated anterior lobe (alc) and well-developed setae bundles (se). (E) Dorsal view of an early-stage trochophore larva of the sipunculan Phascolion strombus with prototroch (pt) and apical tuft (at). (F) Anterolateral left view of a late-stage metatrochophore of the polychaete annelid Spirorbis with prominent apical tuft (at) and narrow prototroch (pt). (G) Ventral view of a creeping-type larva of the entoproct Loxosomella murmanica with frontal cilia (fc), prototroch (pt), and ciliated foot (ft). (H) Dorsal view of the corona-type larva of the gymnolaemate ectoproct Triphyllozoon mucronatum with heavily ciliated corona (co) and apical plate (ap).
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Compared to common light or electron microscopy applications, confocal microscopy facilitates acquisition of data on various life-cycle stages of an organism. Accordingly, the processes underlying the often dramatic bodyplan changes involved in the metamorphosis of the larva into the juvenile can be documented in great detail (Hessling, 2002, 2003; Hessling and Westheide, 2002; Wanninger 2007, 2008; Wanninger et al., 2005a, b; Brinkmann and Wanninger, 2008; Kristof et al., 2008). As a consequence, a whole new pool of datasets dealing with the ontogenetic dynamics that shape given morphological phenotypes has become available (Wanninger, 2007; Wanninger et al., 2008). This has led to significant insights not only on lophotrochozoan interrelationships but also on the evolutionary history of lophotrochozoan key innovations such as body segmentation or central nervous system (CNS) evolution (Hessling, 2002, 2003; Hessling and Westheide, 2002; Brinkmann and Wanninger, 2008; Kristof et al., 2008; Wollesen et al., 2008; Wanninger et al., 2009). Thus, at the dawn of the 21st century, comparative developmental morphology has reached the 4th dimension: documenting and analyzing animal morphogenesis in space and time.
In this article, I summarize recent results on lophotrochozoan larval myo- and neuroanatomy, specifically focusing on the dynamics that lead to their respective phenotypes. I discuss these data in the light of the newly emerging picture of animal interrelationships and highlight the importance of the data on myo- and neurogenesis for our understanding of the evolution of significant bodyplan features such as segmentation, the nervous system, and the body wall musculature of vermiform lophotrochozoans, the proposed ancestral gross morphological phenotype of the last common ancestor of Lophotrochozoa and Bilateria (Salvini-Plawen, 1978; Valentine, 2004).
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Shaping Spiralian Worms: Different Myogenetic Pathways Leading to Similar Results
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Worm-like taxa are found in the vast majority of lophotrochozoan phyla, and a (flat)worm-like, planuloid, benthic species is often proposed as the last common ancestor of all Bilateria (see Valentine, 2004). Indeed, even the phylum that exhibits the widest range of bodyplans within Lophotrochozoa, the Mollusca, has vermiform taxa as its earliest extant offshoots (Ponder and Lindberg, 2008). Most of these worms have a bodywall comprising outer ring and inner longitudinal muscles, with some clades exhibiting additional intermediate diagonal muscles (e.g., the basal molluscan taxa Neomeniomorpha and Chaetodermomorpha; see Haszprunar and Wanninger, 2000). In addition, a number of taxa show sets of dorsoventral muscles along the anterior-posterior body axis. Both the dorsoventral muscles and the ring muscles of the bodywall typically appear as serially repetitive structures, although the former may be restricted to certain body regions (e.g., to the anterior part in chaetodermomorph molluscs).
Despite the relatively uniform and homogeneous arrangement of these serial muscles in adult worms, a detailed look at their embryonic and larval formation modes shows an astonishing degree of plasticity in how these muscular bodyplans are established during ontogeny (Figs. 2, 3). No less than six myogenetic pathways can be identified.
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Figure 2. Myogenesis in the polychaete annelid Filograna implexa (A, B), the sipunculan Phascolion strombus (C), and the polyplacophoran mollusc Mopalia muscosa (D, E). Confocal micrographs (A–D) and 3D reconstruction (E). Scale bars equal 25 µm in all aspects except for the inset, where it equals 5 µm. Anterior is up in A–D and toward the viewer in E. (A) Early polychaete myogenesis starts with the establishment of a prototroch muscle ring (pr) and paired sets of longitudinal muscles (lm). Subsequently, the metamerically arranged ring muscles (arrows) start to form. Note the gradual decrease of the fluorescence signal from the ring muscles from anterior to posterior, reflecting the decreasing differentiation gradient of these muscles in anterior-posterior direction. (B) Later larva with fully developed ring muscles (arrows) and early formation of the dorsoventral musculature (asterisks). Again, the gradual decrease of signal and thus the decreasing level of dorsoventral muscle differentiation from anterior to posterior is obvious. (C) Differentiation of the bodywall ring muscles overlying the longitudinal muscles (lm) in the juvenile sipunculan occurs along the entire anterior-posterior axis by fission of existing myocytes (arrows). Inset is a higher magnification of the boxed area. (D) Myoanatomy of a late-stage polyplacophoran larva shows homogeneous distribution of numerous myocytes of the dorsoventral musculature (boxed area) along the entire post-trochal body axis behind the prototroch muscle ring (pr) and the apical muscle grid (agr). (E) 3D reconstruction of the boxed area depicted in D showing the multiple seriality of the polyplacophoran larval dorsoventral musculature. Concentration into the seven pairs of shell muscles occurs after metamorphosis, with addition of the eighth pair in later juveniles.
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Figure 3. Schematic representation of the key steps of early myogenesis in vermiform lophotrochozoans. Increase in brightness of blue color indicates higher degree of differentiation of the respective muscle. Indicated is the formation of the serially arranged ring muscles (transversal muscles in Polyplacophora) by stripes, and of the dorsoventral muscles (in Annelida and Polyplacophora) by circles or squares. Six modes of muscle differentiation are recognized: the segmental-type (annelid-type) with subsequent formation of ring and dorsoventral muscles in anterior-posterior progression; the unordered random-type (chaetodermomorph-type); the synchronous-concentration-type (polyplacophoran-type) with synchronous formation of the first rudiments of the transversal and dorsoventral muscles, which is followed by concentration of the latter into seven and later eight paired shell muscles; the synchronous-fission-type (sipunculan-type) with synchronous formation of primary myocytes which then multiply by fission; the intermediate-type (Hoploplana-type) starting with an anterior and a posterior founder myocyte which is followed by the formation of one pair of ring muscles just posterior to the anterior myocyte and eventual formation of a homogenous ring muscle arrangement along the longitudinal body axis; and the cluster-type (Macrostomum-type), which starts with randomly distributed primary ring muscle clusters between which new myocytes are added subsequently.
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1. The segmental-type (annelid-type)
The segmental- (or annelid-)type of serial ring and dorsoventral muscle formation is found in taxa that express a segmented bodyplan. Accordingly, polychaete and oligochaete annelids form their metameric muscle units in a strict anterior-posterior progression (Hill and Boyer, 2001; Seaver et al., 2005; Bergter and Paululat, 2007; Bergter et al., 2008; Figs. 2A, B; 3). This is typically considered to be the result of an evolutionary innovation within Annelida, namely the possession of a posterior growth zone from which all except for the (three?) larval segments are budded off (Weisblat et al., 1988; Davis and Patel, 1999; De Rosa et al., 2005). Owing to this process, anterior segments appear to be older than posterior ones, and this is also reflected in the degree of differentiation of the respective organ systems associated with these segments. Thus, the segmental dorsoventral and ring muscles in developmental stages of annelids show gradual increase of their degree of differentiation from posterior to anterior (Fig. 2A, B). However, formation of the dorsoventral and the ring musculatures may be chronologically dissociated from each other, resulting in, for example, appearance of the first anlagen of the ring muscles prior to the onset of dorsoventral muscle formation (Fig. 2A, B). Interestingly, in some species such as the sessile, brooding polychaete Filograna implexa (Fig. 2A, B), myogenesis follows this strict anterior-posterior formation gradient even in the first three segments which—according to common textbook knowledge —should arise synchronously. This demonstrates the dynamics and plasticity of ontogenetic segment formation in annelids, which are yet far from being understood (see below and Brinkmann and Wanninger, 2008). Moreover, this finding hints at a shared evolutionary ancestry of larval and adult segments with a heterochronic shift that results in the synchronous formation of the first segments in some polychaete taxa.
Recently, increasing evidence has become available that echiurans are ingroup polychaetes, a notion that is confirmed both by gene sequence data (Struck et al., 2007; Dunn et al., 2008) and by their segmental mode of neurogenesis (Hessling, 2002, 2003; Hessling and Westheide, 2002; see below). Unfortunately, data on echiuran myogenesis are still lacking, and thus the question of whether myogenesis follows the segmental-type or rather the synchronous-fission-(i.e., sipunculan-type (see below) remains unresolved.
2. The random-type (chaetodermomorph-type)
A worm-shaped gross morphology as exemplified in the Neomeniomorpha and Chaetodermomorpha is considered basal (plesiomorphic) for Mollusca (Haszprunar et al., 2008). While no data exist on larval myogenesis of the presumably most basal clade, the neomeniomorphs, some information has recently become available for the second extant molluscan offshoot, the Chaetodermomorpha (Nielsen et al., 2007). This information indicates that formation of the first rudiments of the bodywall ring musculature does not follow a distinct pattern, resulting in larvae that have ring muscles with different degrees of differentiation distributed randomly along the anterior-posterior axis (Fig. 3). Whether the dorsoventral musculature, which is restricted to the anterior body region in chaetodermomorph molluscs, follows the same pattern is not known. Although detailed analyses are lacking, the few data available indicate that the random-type of muscle formation may also occur in nemertines (Maslakova et al., 2004).
3. The synchronous-concentration-type (polyplacophoran-type)
Along the molluscan line leading from the aplacophoran to the conchiferan clades (all molluscs that arose from a shelled ancestor), the Polyplacophora (chitons) occupies an intermediate position. The serial arrangement of eight shell plates and the corresponding sets of dorsoventral and transversal muscles have traditionally been used to argue for a segmented origin of the entire phylum (Götting, 1980; Ghiselin, 1988; Lake, 1990). However, myogenesis revealed that the dorsoventral musculature in chitons forms as a meshwork of numerous serially repeated muscle fibers that are evenly distributed along the length of the post-trochal larval body (Wanninger and Haszprunar, 2002a; Fig. 2D, E). This multiple set of fibers forms synchronously (i.e., all muscle fibers arise and develop simultaneously) and thus show the same degree of differentiation (Fig. 2D, E). Concentration of this multiple fiber meshwork into the seven paired dorsoventral shell muscle sets is a slow, gradual process that occurs only after the completion of metamorphosis and is thus considered a secondary condition (Wanninger and Haszprunar, 2002a). It should be mentioned that establishment of the adult condition with an eighth set of dorsoventral and transversal muscles and their associated shell plate is completed considerably later during development, thus illustrating the dynamic processes underlying chiton myogenesis (Wanninger and Haszprunar, 2002a; Fig. 3).
Multiple seriality of the dorsoventral musculature, as exemplified in the polyplacophoran larva, resembles the situation found in the adult basal aplacophoran clades. Accordingly, the chiton trochophore larva ontogenetically recapitulates the condition that is basal for the entire phylum—namely, mutiseriality of the dorsoventral musculature—thus differing significantly from the segmental-type of annelid myogenesis (Fig. 3). Likewise, the bands of transversal muscles, which are located on the dorsal side of the animal, form synchronously and confirm once more the nonsegmented ancestry of polyplacophorans (Wanninger and Haszprunar, 2002a; Fig. 3).
The pre-trochal region of the chiton larva, which is devoid of dorsoventral muscles, shows a fine muscular grid that resembles the bodywall musculature of adult aplacophorans (see Haszprunar and Wanninger, 2000; Wanninger and Haszprunar, 2002a; Fig. 2D)—again a recapitulative event in the life history of the polyplacophoran larva. This apical muscle grid is lost during metamorphosis.
4. The synchronous-fission-type (sipunculan-type)
Currently, Sipuncula is one of the most hotly debated lophotrochozoan assemblages: in some recent molecular phylogenetic analyses it even loses its status as a phylum and is downgraded to a subtaxon within (polychaete) annelids (Struck et al., 2007; Dunn et al., 2008). Although this issue is not yet settled, data on neurogenesis indeed show remnants of an ancestral segmented bodyplan in the sipunculan larva (Kristof et al., 2008; Wanninger et al., 2009; see below), thus confirming previous studies that already argued for an annelid-sipunculan sister relationship (McHugh, 1997; Boore and Staton, 2002; Halanych et al., 2002; Wanninger et al., 2005a). With respect to myogenesis, however, sipunculans show a different, nonsegmental formation mode that cannot be explained by the existence of a posterior growth zone. The first rudiments of numerous bodywall ring muscles form synchronously in the early trochophore larva. Their number stays constant for some time until the onset of body elongation (Wanninger et al., 2005a). As growth along the anterior-posterior axis proceeds, the ring muscles split, giving rise to more fibers (Wanninger et al., 2005a; Figs. 2C; 3). This process occurs along the entire anterior-posterior axis of the animal, which thus can be regarded as a homogeneous muscle formation zone. Although such a two-step process of ring muscle formation appears to be unique to Sipuncula, generation of new fibers by fission of existing fibers seems to be an evolutionarily conserved mechanism that also occurs in Acoela, presumably the most basal bilaterian animals (Semmler et al., 2008).
5. The intermediate-type (Hoploplana-type)
Indirect-developing plathelminths often have a so-called Müller-type larva that remains planktonic for several days or even weeks (Smith et al., 2002). Early myogenesis events before the actual larva is established are very characteristic for this group of flatworms and start with the synchronous formation of an anterior and a posterior pioneer ring muscle (Fig. 3). Slightly later, two pairs of additional, secondary ring muscles form synchronously posterior to the anterior founder muscle in Hoploplana inquilina, resulting in a heterogeneous distribution of six ring muscles in the early embryo (Reiter et al., 1996; Fig. 3). As development proceeds, two more fibers emerge posterior to the secondary ring muscles. These processes eventually give rise to an evenly distributed arrangement along the anterior-posterior axis of the embryo (Reiter et al., 1996; Fig. 3). The detailed remodeling mechanisms that lead to the establishment of the complex muscular architecture of the Müller's larva are still unknown, as are the events involved in shaping the juvenile/adult musculature during metamorphosis. However, preliminary data suggest fission or duplication of existing muscle fibers as a driving force for ring muscle growth in the Müller's larva of Pseudoceros canadensis (Semmler and Wanninger, unpubl.).
6. The cluster-type (Macrostomum-type)
The adult bodywall musculature of direct-developing turbellarian-like plathelminths as well as parasitic forms is highly complex and includes interwoven sets of ring, diagonal, and longitudinal muscles (Rieger et al., 1991; Cebrià et al., 1997; Mair et al., 1998). In early embryos, diffuse clusters of ring muscles are formed synchronously, but with no particular order, along the anterior-posterior body axis (Reiter et al., 1996; Fig. 3). Subsequently, additional muscles are added in a random manner between these pioneer ring muscle clusters, giving rise to the homogeneous ring muscle arrangement of the juvenile worm (Fig. 3). Whether these intermediate secondary fibers form in synchronous patterns and whether they arise by fission of the pioneer muscle fibers or from newly developing myoblasts remains unknown.
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Spiralian Neurogenesis and the Tetraneuralia Concept
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Spiralian larvae are characterized by subsets of neural structures that appear in most clades (Table 1). The most common of these are specific, often flask-like cells associated with the apical organ, which in most cases show serotonergic immunoreactivity but may also contain the neuropeptide FMRFamide (Fig. 4). Most basal clades of spiralian phyla exhibit few (often four) such flask cells, which have a circular arrangement and directly underlie the apical ciliary tuft, although secondary reduction of the entire apical organ has occurred independently in several lineages (Wanninger, 2008). An exception to this are the trochophores of polyplacophoran molluscs and the creeping-type larva of entoprocts, which both have a highly complex apical organ with 8–10 central flask cells surrounded by numerous peripheral cells, a condition that seems to occur only in these two clades (Friedrich et al., 2002; Voronezhskaya et al., 2002; Wanninger et al., 2007; Wanninger, 2008; Fig. 4E). Since this larval type and not the planktotrophic swimming larva is considered basal for Entoprocta (Nielsen, 1971), this complex architecture of the apical organ is considered apomorphic for an entoproct-mollusc clade, although data for the larvae of the most basal molluscs, the aplacophorans, are still lacking (Wanninger et al., 2007). In addition, the entoproct creeping larva shows a typical tetraneurous condition, which previously had been considered diagnostic for Mollusca alone (Wanninger et al., 2007). Hereby, molluscan-entoproct tetraneury is not merely restricted to the existence of four longitudinal nerve cords, but rather is defined by a nervous system that exhibits one pair of ventral (pedal) nerve cords with associated serotonergic perikarya and commissures, as well as one pair of lateral (visceral) nerve cords that do not run in the same plane as the pedal cords but more dorsal to them (Fig. 4E). The present data suggest that such a condition is apomorphic for a mollusc-entoproct clade, which I accordingly name Tetraneuralia (Fig. 5).
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Figure 4. Neurogenesis and larval neuroanatomy of the polychaete annelid Filograna implexa (A–C; confocal micrographs of FMRFamidergic immunoreactivity); the sipunculan Phascolosoma agassizii (D; confocal micrograph of serotonergic immunoreactivity); the creeping-type entoproct larva of Loxosomella murmanica (E; 3D reconstruction of the serotonergic nervous system); and the chordoid larva of Symbion pandora (F; 3D reconstruction of the serotonergic nervous system). Anterior is up in all aspects. A and B are ventrolateral left views, C, E, and F are ventral views, and D is a lateral right view. Scale bars equal 25 µm in A–C, 50 µm in D, and 12.5 µm in E–F. (A) Early polychate neurogenesis starts with the establishment of two ventral nerve cords (vn), the first cells of the apical organ (arrows), and the rudiment of the adult cerebral ganglion (cg). (B) The first commissure (arrowhead) has been established. (C) Late-stage larva with completely reduced larval apical organ, two ventral commissures of which the first appears thicker than the second one, and the beginning of the formation of the third commissure (arrowheads), demonstrating the typical segmental mode of annelid neurogenesis. (D) Sipunculan pelagosphera larva with four flask cells in the apical organ (arrows), prototroch nerve ring (pn), and metamerically arranged perikarya (boxed area) along the ventral nerve cord (vn). Image courtesy of A. Kristof (University of Copenhagen). (E) The serotonergic nervous system of the entoproct creeping-type larva exhibits a mosaic of larval and adult molluscan characters and includes a complex larval apical organ (light green) with an underlying neuropil (light blue), which laterally connects to the prototroch nerve ring (dark blue). In addition, the ventral (pedal) nerve cords (yellow) with four pairs of associated perikarya (magenta), the first commissure (dark green), the lateral (visceral) nerve cords (red), and the anterior nerve loop (brown) are visible. (F) The serotonergic nervous system of the chordoid larva of the enigmatic Cycliophora consists of a relatively large brain (orange), a paired lateral nerve with a posterior commissure (red), and an inner, paired median nerve (yellow). Note that the median nerves fuse with the lateral nerves in the posterior third of the body. All four nerves are located in the same plane on the ventral side of the larva and thus do not correspond to the four longitudinal nerve cords of the Tetraneuralia. Image courtesy of R. Neves (University of Aveiro and University of Copenhagen).
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Figure 5. Current view of spiralian phylogeny based on neuro-developmental characters. Key character states are listed for each node. Loss of characters is indicated in italics. Tetraneury and a complex larval apical organ is considered apomorphic for Entoprocta + Mollusca, which accordingly form the monophyletic assemblage Tetraneuralia. Although some recent molecular phylogenetic studies suggest that sipunculans may be ingroup annelids, their status as a distinct phylum is maintained herein until further independent confirmation of this hypothesis becomes available.
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It is important to stress that it is not the tetraneurous condition and the complexity of the apical organ alone that morphologically defines this clade. Detailed ultrastructural investigation of the entoproct creeping larva revealed a number of shared characters with the molluscan groundpattern, including a distinct creeping foot with a ciliated gliding sole (see Fig. 1G) and with epidermal mucous cells, a large pedal gland, and anteriorly placed cirri, as well as a ventrally intercrossing dorsoventral musculature, which forms a basket-like structure inside of which all major visceral organs are placed (Haszprunar and Wanninger, 2008). One might argue that a ciliated foot with associated mucous cells and glands might have evolved convergently in molluscs and entoprocts to serve similar functional locomotive needs. However, the high number of shared characters between polyplacophoran larvae and the entoproct creeping-type larva that involve different organ systems clearly make a scenario of a common ancestry of these features more parsimonious than assuming independent evolution of each of these traits in both phyla.
Although a monophyletic mollusc-entoproct clade is not commonly recognized in current molecular phylogenies, which may be due to the paucity of entoproct species for which adequate sequence data are available, some former morphology-based cladistic analyses have already recognized such an assemblage, classifying it as "Lacunifera" or "Sinusoida" (Bartolomaeus, 1993a,b; Haszprunar, 1996, 2000; Ax, 1999). However, the term suggested herein—Tetraneuralia—is in my view more suitable as it casts the spotlight on the striking apomorphies of this clade with respect to their neural architecture.
Comparing the nervous system anatomies of the entoproct creeping larva and basal molluscs, it becomes obvious that the former expresses a mosaic of larval and adult tetraneuralian characters. Accordingly, the complex apical organ and the prototroch nerve ring are typical larval subsets of the nervous system, while tetraneury together with the buccal nerves and the anterior nerve loop are part of the adult bodyplan (Wanninger et al., 2007). The dramatic remodeling of the free-living larva into the sessile, tentacle-bearing adult constitutes an entoproct apomorphy and obscures the shared characters with Mollusca, thus impeding the immediate recognition of Tetraneuralia. This once more illustrates the importance of larval characters for evolutionary and phylogenetic deductions.
Considering entire developmental sequences in comparative morphological research is even more important for inferences concerning bodyplan features that are defined by their developmental mode. A prominent example is the above-mentioned segmental or metameric arrangement of organs, which morphologically can only be proven by the subsequent formation of these organs, while serial repetition alone is not diagnostic for a segmented body (Weisblat et al., 1988; Davis and Patel, 1999; De Rosa et al., 2005). Accordingly, in annelids the metameric ventral commissures are formed in an anterior-posterior progression similar to those of the bodywall ring muscles and the dorsoventral musculature (Fig. 4A–C). A similar mode of CNS patterning has also been shown for echiurans (Hessling, 2002, 2003; Hessling and Westheide, 2002), thus confirming recent molecular phylogenetic studies that recognize them as polychaete annelids (McHugh, 1997; Struck et al., 2007; Dunn et al., 2008).
A recent study on sipunculan neurogenesis revealed that the larvae of this taxon undergo cryptic segmentation by forming a paired ventral nerve cord with associated, subsequently appearing, segmental commissures and four pairs of perikarya (Kristof et al., 2008; Wanninger et al., 2009; Fig. 4D). However, while the metameric organization of the nervous system is retained in the adult stage of most annelids and echiurans (although the ventral nerve cords may fuse), the sipunculans lose this arrangement during development by migration of the perikarya. This eventually results in two distinct cell clusters instead of a repetitive perikaryal arrangement in the late sipunculan larva (Kristof et al., 2008; Wanninger et al., 2009). In sipunculans with a shorter larval phase, segmentation of the nervous system is even less pronounced—only three segmental commissures are present and the associated perikarya are lacking altogether (Wanninger et al., 2005a). If viewed in an overall developmental context, the dual nature of sipunculan ontogeny becomes obvious: whereas myogenesis proceeds in a typical nonsegmental mode along the entire longitudinal body axis, early neurogenesis shows the characteristic annelid-like anterior-posterior progression, demonstrating the segmented ancestry of sipunculans (see Figs. 2C; 4D). The recognition of a segmented ancestry is in accordance with the current view of molecular phylogenetic analyses, which consider Sipuncula either as an annelid sister taxon or as ingroup annelids (McHugh, 1997; Boore and Staton, 2002; Struck et al., 2007; Dunn et al., 2008). However, it should be noted that the ontogenetic pathways of segmentation itself are probably much more plastic than commonly assumed, as demonstrated in a recent study that revealed different neurogenetic pathways that shape the segmental ventral and peripheral nervous systems, respectively, in the polychaete annelid Sabellaria alveolata (Brinkmann and Wanninger, 2008).
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Toward a Reconstruction of the Ancestral Lophotrochozoan Neuromuscular Bodyplan
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As outlined above, the apical organs of most spiralians except for the polyplacophoran trochophore and the entoproct creeping larva are simple, with only about four serotonergic flask cells; this condition is thus regarded as basal for Spiralia. This situation differs sharply from the one in lophophorates, where numerous such cells were found in the apical organ of phoronids, although peripheral cells comparable to those of the tetraneuralians have not been clearly identified (Santagata, 2002; Santagata and Zimmer, 2002). Interestingly, this is also true for the planktonic stage of the brachiopod Glottidia, which already exhibits several adult traits such as a shell and tentacles (Hay-Schmidt, 2000). It is thus tempting to speculate that the last common ancestor of the lophophorates also had an apical organ composed of numerous serotonergic flask cells (Santagata, 2008b). However, ectoproct coronate and cyphonautes larvae show some interphyletic variety and may either have one pair of serotonergic cells in the apical organ or a nerve ring, or both (Pires and Woollacott, 1997; Shimizu et al., 2000; Wanninger et al., 2005b; Wanninger, 2008; Table 1). The general morphological plasticity of lophophorate larvae is also reflected in their neural and muscular anatomy and hampers solid conclusions about the ancestral neuromuscular lophophorate bodyplan. It seems reasonable to assume, however, that a serotonergic nerve ring or nerve net underlying a ciliated swimming organ is part of the lophophorate groundpattern, because such a neural structure seems to be lacking only in brachiopod larvae (see Table 1).
For the Spiralia, the situation concerning basal neuromuscular features is much clearer. Despite a high degree of variation in the number of longitudinal nerve cords in annelids (including echiurans) and sipunculans, neurodevelopmental studies have shown that a paired ventral nerve always forms the first anlage of the future ventral nervous system, even in those annelid taxa that as adults have multiple ventral cords. Two ventral cords are also expressed in adult nemertines, while plathelminths may have between two and eight cords (Table 1). The enigmatic cycliophoran chordoid larva has one pair of FMRFamidergic and two pairs of serotonergic nerves (Wanninger, 2005; Fig. 4F). Taken together, these data suggest that one pair of ventral nerve cords with commissures was probably present in the last common spiralian (and possibly also lophotrochozoan) ancestor, although a higher number cannot be completely ruled out. Moreover, this ancestor most likely was unsegmented and had no ganglia associated with the ventral nerve cords, but did have a concentrated CNS in the anterior region ("brain") (Fig. 5). As mentioned above, most basal spiralians have not more than four flask cells in the apical organ (see Hay-Schmidt, 2000), thus arguing for a simple apical organ with up to four serotonergic (and possibly also FMRFamidergic) flask cells and a nerve ring underlying the ciliated prototroch in the larva of the last common spiralian ancestor (Fig. 5).
Neuroanatomical evolution appears to be a highly dynamic process that has resulted in a number of morphological modifications and secondary losses of characters in various lineages. The most important of these are the evolution of segmentation at the base of the annelid-echiuran-sipunculan line, multiplication and fusion of the ventral nerve cords in several annelid/echiuran taxa as well as in the sipunculans, the secondary loss of segmentation in Sipuncula, and the evolution of tetraneury and a complex apical organ with eight to ten central flask cells and a number of peripheral cells in the Tetraneuralia (entoprocts and molluscs) (Fig. 5). In lophotrochozoans, ventral nerve cords with segmentally arranged ganglia are a key innovation of Annelida.
The bodywall musculature of the spiralian last common ancestor probably contained outer ring, inner longitudinal, and maybe also intermediate diagonal muscles, similar to the condition found in the most basal extant bilaterians, the Acoela (Ladurner and Rieger, 2000; Semmler et al., 2008). Since most spiralian worms with the exception of Annelida and chaetodermomorph molluscs form the first ring muscle rudiments synchronously, similar to the Acoela (Ladurner and Rieger, 2000; Semmler et al., 2008), this probably constitutes the ancestral mode of lophotrochozoan and bilaterian ring muscle differentiation.
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Invertebrate Morphogenesis: Quo Vadis?
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Innovations in microscopic and computer applications enable detailed, time-efficient three-dimensional reconstructions of lophotrochozoan neuromuscular anatomy and development, and have resulted in a wealth of recent data. Adding information on the dynamics of bodyplan morphogenesis provides in-depth insights as to how animal phenotypes might have evolved. This is especially the case in a large, comparative, and integrative framework that combines various research disciplines including morphogenesis, gene expression analysis, and molecular phylogeny. Synergies emerging from these combined approaches should eventually lead to a better understanding of animal interrelationships and the evolution of bodyplan diversity. For example, while molecular phylogenetics revealed a close relationship between the segmented annelids and the unsegmented sipunculans, studies on the neurogenesis showed that the sipunculans indeed stem from a segmented ancestor and that the segmental body architecture is still rudimentarily present during larval development, thus providing evidence for the first time that large, free-living animals may lose a segmented body organization.
With gene expression analysis available as a routine research tool, it is now possible to infer the molecular basis of animal morphogenesis. The recent boom in this research area has resulted in the somewhat grotesque scenario that for some taxa more data are available on gene activity during ontogeny than on the ontogeny of the organs in which these genes are presumably expressed, rendering sound interpretations of gene expression data sometimes difficult. Making sense of gene expression patterns therefore requires expert knowledge of anatomy and the morphological dynamics that take place during ontogeny. Accordingly, research combining comparative morphogenesis and molecular techniques is a highly promising approach to uncovering the full story that animal ontogeny is able to tell us about metazoan evolution since the Early Cambrian some 540 Myrs ago.
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Acknowledgments
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First and foremost I thank my teacher Gerhard Haszprunar (Munich), who introduced me to the wonderful world of marine invertebrate larvae more than a decade ago. Continuous discussion with him has significantly shaped my approach to evolutionary questions. Furthermore, I thank the members of my lab for their dedication to the subject and the productive and inspiring atmosphere in our group. I am also indebted to various funding bodies for past and present support of the research in my lab, namely the German Science Foundation, the Danish Research Council, the Carlsberg Foundation, the Faculty of Science at the University of Copenhagen, and the European Commission. The latter helped, through funding of our EU Research Training Network MOLMORPH, to establish a dense net of international collaborators.
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Footnotes
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Received 10 October 2008; accepted 11 March 2009.
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Abstract
Introduction
