Hemichordate Nervous System
Summary and Keywords
Hemichordates are marine invertebrates consisting of two distinct groups: the solitary enteropneusts and the colonial pterobranchs. Hemichordates are phylogenetically a sister group to echinoderm composing Ambulacraria. The adult morphology of hemichordates shares some features with chordates. For that reason, hemichordates have been considered key organisms to understand the evolution of deuterostomes and the origin of the chordate body plan. The nervous system of hemichordates is also important in the discussion of the origin of centralized nervous systems. However, unlike other deuterostomes, such as echinoderms and chordates, information on the nervous system of hemichordates is limited. Recent improvements in the accessibility of embryos, development of functional tools, and genomic resources from several model organisms have provided essential information on the nervous system organization and neurogenesis in hemichordates. The comparison of the nervous system between hemichordates and other bilaterians helps to elucidate the origin of the chordate central nervous system.
Extant hemichordates are divided into two groups: enteropneusts and pterobranchs. The nervous system of adult enteropneusts consists of nerve cords and the basiepidermal nerve net. The two nerve cords run along the dorsal and ventral midlines. The dorsal nerve cord forms a tubular structure in the collar region. The two nerve cords are connected through the prebranchial nerve ring. The larval nervous system of enteropneusts develops along the ciliary band and there is a ganglion at the anterior end of the body called the apical ganglion. A pair of pigmented eyespots is situated at the lateral side of the apical ganglion. The adult nervous system of pterobranchs is basiepidermal and there are several condensations of plexuses. The most prominent one is the brain, located at the base of the tentaculated arms. From the brain, small fibers radiate and enter tentaculated arms to form a tentacle nerve in each. There is a basiepidermal nerve cord in the ventral midline of the trunk.
Hemichordates are a phylum belonging to the deuterostomes, together with echinoderms and chordates. Hemichordates and echinoderms comprise the Ambulacraria, which is the sister group of chordates (Cannon et al., 2016). Compared with the highly specialized radial form of the extant echinoderms, the bilateral body of hemichordates may retain more characteristics from the ancestral deuterostome form. In addition, hemichordates have some features shared with chordates, such as pharyngeal gill slits. Thus, hemichordates are key organisms for elucidating early deuterostome evolution and the origin of our own phylum, Chordata. In the context of the nervous system evolution, hemichordates also occupy an important position. Traditionally, hemichordate nervous system have been described by histological section and electron microscopy (reviewed in Bullock, 1965; and Horst, 1939). Based on these neuroanatomical studies, the nervous system of hemichordates has been considered to be diffused, without a distinct brain and ganglia (Bullock, 1965; Knight-Jones, 1952). Recent molecular neuroanatomical studies have shown that hemichordates have some domains of neurons that are somewhat concentrated (Nomaksteinsky et al., 2009). In addition, hemichordates have a region where a nerve cord forms an epithelial tube called the collar cord, which has been compared with the neural tube of chordates (Kaul & Stach, 2010; Miyamoto & Wada, 2013; Morgan, 1894). For these reasons, hemichordates occupy a crucial phylogenetic position in understanding the centralization of the nervous system and the origin of the neural tube.
Extant hemichordates are exclusively marine organisms. They are divided into two major groups: the enteropneusts, which are solitary and vermiform; and the pterobranchs, which are colonial and tube-dwelling. Although enteropneusts and pterobranchs are morphologically distinct, they share some important features. Their bodies consist of three regions: the prosome, mesosome, and metasome. In enteropneusts, these regions are referred to as the proboscis, collar, and trunk, respectively; and in pterobranchs, they are correspondingly known as the cephalic shield, neck, and trunk. This tripartite body plan directly corresponds to the tripartite coeloms of the larvae, which have also been observed in echinoderm larvae. Enteropneusts have two types of development: indirect, with pelagic larvae called tornaria larvae that metamorphose into benthic adults; and direct development, without a larval stage. Because of the similarity between hemichordate and echinoderm larvae, indirect development is considered an ancestral state in ambulacrarians (hemichordates and echinoderms). Pterobranchs have lecithotrophic larvae, which are swimming but nonfeeding larvae and depend on the yolk for nutrition. The absence of feeding larvae in pterobranchs supports a molecular phylogenetic analysis, demonstrating that pterobranchs are a sister group to harrimaniid enteropneusts, which show direct development (Cannon, Rychel, Eccleston, Halanych, & Swalla, 2009). Recent phylogenetic analyses using transcriptome or microRNA data, however, have supported that both enteropneusts and pterobranchs are monophyletic groups (Cannon et al., 2014; Peterson, Su, Arnone, Swalla, & King, 2013). Thus, the ancestral state of hemichordates is still unclear. In this article, we overview the neuroanatomy of enteropneusts and pterobranchs, and introduce recent discussions on the origin of the chordate central nervous system.
Nervous System of Enteropneust Larva
Two groups of enteropneusts, ptychoderids, and spengelids, have a planktonic larval stage called tornaria. The larva is oval shaped, transparent, and has four ciliary bands: the circumoral band or neotroch, the ventral band or neurotroch, the telotroch or opisthotroch, and the perianal ciliary ring or secondary teletroch (Figure 2). The nervous system of tornaria larvae develops along the ciliary bands and there is a ganglion at the anterior end of the body called apical ganglion (Figure 2C and 2D). The apical ganglion consists of elongated and columnar epithelial cells in contrast to the adjacent flattened epidermis (Braun, Kaul-Strehlow, Ullrich-Lüter, & Stach, 2015; Nakajima, Humphreys, Kaneko, & Tagawa, 2004; Nezlin & Yushin, 2004). A pair of pigmented eyespots is situated at the lateral side of the apical ganglion. The development and organization of the larval nervous system has been studied in several species using immunohistochemistry against antibodies for neuron markers (Dautov & Nezlin, 1992; Hay-Schmidt, 2000; Miyamoto, Nakajima, Wada, & Saito, 2010; Nakajima et al., 2004; Nezlin & Yushin, 2004; Nielsen & Hay-Schmidt, 2007), transmission electron microscope (TEM) (Brandenburger, Woollacott, & Eakin, 1973; Lacalli & Gilmour, 2001), and gene expression patterns (Gonzalez, Uhlinger, & Lowe, 2017; Tagawa, Humphreys, & Satoh, 2000; Tagawa, Satoh, & Humphreys, 2001; Taguchi et al., 2000; Taguchi, Tagawa, Humphreys, & Satoh, 2002; Takacs, Moy, & Peterson, 2002). Neurogenesis of entire developmental stages was observed in Balanoglossus simodensis (Miyamoto et al., 2010). The first sign of the neurogenesis appears at late-gastrula stage when the protocoel forms from the tip of the archenteron (Miyamoto et al., 2010). A few neurons that are immunoreactive to anti-synaptotagmin antibody, and anti-serotonin antibody appear at the anterior end of the larvae forming the apical ganglion (Miyamoto et al., 2010). As larvae develop, the larval nervous system forms along the ciliary bands. The circumoral nervous system, which includes the apical ganglion, develops along the circumoral band (Miyamoto et al., 2010; Nakajima et al., 2004; Nezlin & Yushin, 2004; Nielsen & Hay-Schmidt, 2007). The cell bodies of the neurons are associated with the circumoral band, and these cells project axons along the ciliary band and toward the oral field forming the nerve net (Miyamoto et al., 2010). Among the circumoral nervous system, the nerve net around the mouth and esophagus is prominent, and these neurons innervate the circular muscles surrounding it (Miyamoto et al., 2010). Some genes, which are expressed in the chordate brain, are expressed in the apical region (Gonzalez, Uhlinger, & Lowe, 2017; Tagawa et al., 2000). The other domain of the larval nervous system is the perianal nerve ring developing along the telotroch (Miyamoto et al., 2010). The cell bodies of the neurons are associated with the telotroch. While these neurons project axons mainly along the telotroch, a few axons project to the circumoral nervous system (Miyamoto et al., 2010). The larval nervous system probably degenerates during the metamorphosis and does not contribute to the newly formed adult nervous system.
The tornaria larva possesses a pair of pigmented eyespots in the apical organ. The first detailed description of the photoreceptor cells of tornaria larvae was performed in Ptychodera flava, and it was argued that the photoreceptor cells showed an intermediate state between the ciliary and rhabdomeric types, which included microvilli as well as a modified ciliary membrane (Brandenburger et al., 1973). Arendt and Wittbrodt (2001), however, regarded the photoreceptor cells of tornaria larvae as the rhabdomeric type citing the study by Brandenburger et al. (1973). Recently, the larval eye of Glossobalanus marginatus was studied in detail using TEM and 3D reconstruction (Braun et al., 2015). This study revealed that the eye structure of G. marginatus is similar to that of P. flava and is a rhabdomeric type (Braun et al., 2015). The tornaria eye is composed of two types of cells: photoreceptor and pigmented cells (Brandenburger et al., 1973; Braun et al., 2015). In G. marginatus, the eye consists of 13 cells of each type and these cells are part of the epidermis (Braun et al., 2015). The13 pigmented cells form the eyecup. The rhabdomeres are oriented toward the concavity of the pigment cells and this cellular arrangement resembles that of larval eyes in trochophore larvae (Braun et al., 2015). The eyespot must be related to phototaxis of tornaria larvae. However, behavior and regulation of tornaria larvae are poorly understood. While light plays a minor role in the behavior in some species (Ritter & Davis, 1904), positive phototactic behavior has been reported in P. flava and in Balanoglossus clavigerus (Brandenburger et al., 1973; Stiasny, 1914). In G. marginatus, the late stage tornaria larvae show negative phototactic behavior (Braun et al., 2015). The phototactic behavior probably shifts during ontogeny. Namely, early planktonic stage larvae are positively phototactic and late stage larvae are negatively phototactic to prepare for a benthic lifestyle. The eyespots also degenerate during the metamorphosis.
Nervous System of Enteropneust Adult
The nervous system of the adult enteropneusts consists of basiepidermal nerve net, two nerve cords (the dorsal and ventral nerve cords), and the prebranchial nerve ring (Figure 3A). The dorsal nerve cord runs along the body length from the tip of the proboscis to the end of the trunk (Figure 3A). The ventral nerve cord is only present in the trunk (Figure 3A). These two nerve cords are connected through the prebranchial nerve ring located at the anterior end of the trunk (Figure 3A). These nerve cords and the nerve ring are basiepithelial except for the collar region, where the nerve cord forms a tubular structure called the collar cord. Traditionally, the nervous system of the enteropneust had been considered a non-centralized and diffused nervous system without any centralization (Bullock, 1965; Knight-Jones, 1952). Recent studies, however, have shown that there are some domains: the proboscis stem, collar cord, and dorsal and ventral nerve cords, where the cell bodies of neurons are somewhat concentrated (Nomaksteinsky et al., 2009). It is still unclear whether these domains are functionally centralized to integrate the information from the peripheral sensory neurons.
In direct developers, the adult nervous system develops during embryonic stages. Neurogenesis of direct developers has been studied in Saccoglossus kowalevskii in detail (Cunningham & Casey, 2014; Kaul & Stach, 2010; Kaul-Strehlow, Urata, Minokawa, Stach, & Wanninger, 2015; Lowe et al., 2003). At the gastrula stage, serotonergic neurons form throughout the future proboscis and project axons posteriorly (Cunningham & Casey, 2014). At this stage, cell bodies and axons are not concentrated at either the dorsal or the ventral midlines (Cunningham & Casey, 2014). At this stage, it is difficult to discern the presence of axons by TEM observation (Kaul & Stach, 2010). As the embryogenesis progresses, the axons develop, and at hatching stage, a considerable basiepidermal nerve net develops throughout the embryo (Kaul & Stach, 2010). The collar cord formation occurs before hatching (Kaul & Stach, 2010). At the juvenile stage, although a circumferential basiepidermal nerve net is present in the proboscis and collar, most axons seem to run within the dorsal and ventral nerve cords (Kaul & Stach, 2010).
Neurogenesis during metamorphosis of indirect developers has been studied in P. flava, B. simodensis, and Balanoglossus misakiensis (Kaul-Strehlow et al., 2015; Miyamoto et al., 2010; Nielsen & Hay-Schmidt, 2007). In indirect developers, the adult nervous system starts to develop at the Krohn stage, the late larval stage. At the Krohn stage, immunoreactivity to isynaptotagmin appears at the dorsal midline from the apical organ to the telotroch (Miyamoto et al., 2010). The basiepidermal nerve net of the proboscis stem also starts to form from the dorsal midline. At the Spengel stage, the early metamorphosis stage, the dorsal nerve cord extends to the anus (Miyamoto et al., 2010). The proboscis stem nerve net extends to the lateral side. The ventral nerve cord forms from the telotroch to the anus at this stage (Miyamoto et al., 2010). At Agassiz stage larva, just before settlement, the prebranchial nerve ring forms, and the ventral nerve cord extends to the prebranchial nerve ring (Miyamoto et al., 2010). The larval nervous system along the circumoral band and the telotroch disappear at this stage in B. simodensis, whereas serotonin immunoreactive cells of the telotroch nervous system are still present at this stage in B. misakiensis (Kaul-Strehlow et al., 2015). After settlement, a boundary is established between the collar and the trunk; the prebranchial nerve ring is positioned at the boundary. From the Agassiz stage, the collar cord, mentioned later, is formed (Miyamoto & Wada, 2013). As metamorphosis progresses, the basiepidermal nerve net becomes prominent, especially in the proboscis stem and in the collar (Kaul-Strehlow et al., 2015).
Molecular Patterning of Enteropneust Neuroectoderm
Molecular developmental studies related to the neurogenesis and patterning of the nervous system have been carried out in the direct developers S. kowalevskii and P. flava and in the B. simodensis (Aronowicz & Lowe, 2006; Cunningham & Casey, 2014; Lowe et al., 2006; Lowe et al., 2003; Miyamoto & Wada, 2013; Nomaksteinsky et al., 2009; Pani et al., 2012). More recently, the spengelid enteropneust Schizocardium californicum has been studied as another example of an indirect developing organism (Gonzalez et al., 2017). Because hemichordates occupy a crucial phylogenetic position and have a unique organization of the nervous system with dorsal and ventral nerve cords, the expression and function of genes related to the nervous system of other metazoans, such as vertebrates and insects, have been well studied. Neurogenesis progresses in the following steps: (1) induction of neuroectoderm; (2) specification of neuroectoderm; (3) immature neuron formation; and (4) terminal differentiation of mature neurons (Cunningham & Casey, 2014).
Because neurogenesis of S. kowalevskii has been well studied among enteropneusts, here, we highlight this species. Maternal expression of marker genes of neuron progenitors, SoxB1 and msi in S. kowalevskii suggests that neuroectoderm is specified by maternal factors, similar to a sea urchin (Cunningham & Casey, 2014; Lowe et al., 2003). At the blastula stage, a marker gene of post-mitotic differentiating neurons, elav is expressed ubiquitously in the ectoderm (Cunningham & Casey, 2014; Lowe et al., 2003). Another post-mitotic neuron marker gene, prox, starts to express at the early gastrula stage suggesting that neuronal differentiation is initiated at this stage (Cunningham & Casey, 2014). The expression of elav becomes restricted to the puncta at the late-gastrula stage and other marker genes, bruA and dclk start to express at this stage in the same manner (Cunningham & Casey, 2014). At the early neurula stage, soon after punctate elav, bruA, and dclk expression, a mature neuron marker gene, syt1, which encodes a transmembrane calcium sensor located at presynaptic vesicles and necessary for neurotransmitter release, starts to express (Cunningham & Casey, 2014). The syt1 expression indicates the presence of terminally differentiated neurons at this stage (Cunningham & Casey, 2014). The ubiquitous expression of soxB1, msi, and elav at early stages and spotted expression of neural marker genes at the following stages suggest that the entire neuroectoderm is competent for neurogenesis, which is restricted to a selected few cells (Cunningham & Casey, 2014). At the neurula stage, embryos start to elongate and the dorsal and ventral nerve cord formation and collar cord invagination occur. At this stage, expression of the neuron marker genes, which are detected broadly at the previous stage, shifts to the dorsal and ventral midlines (Cunningham & Casey, 2014). The expression of soxB1 and msi are not concentrated in the midline cells because these genes are likely to be involved in the peripheral nervous system that forms throughout the trunk ectoderm (Cunningham & Casey, 2014). The restricted expression of neuron marker genes in the dorsal and ventral nerve cords are also detected in adults of S. kowalevskii and P. flava (Nomaksteinsky et al., 2009).
Expression and function of genes whose orthologs are involved in the patterning of the central nervous system of chordates have been also investigated. In vertebrates, the anterior neural plate and later in the developing brain are patterned anteroposteriorly by the three signaling centers: the anterior neural ridge (ANZ), zona limitans intrathalamica (ZLI), and isthmic organizer (IsO). At the gastrula stage of S. kowalevskii, three distinct organizers are established at the anterior proboscis, the boundary between the proboscis and collar, and the boundary between the collar and trunk (Figure 3B, Pani et al., 2012). Cells of these three regions express combinations of gene encoding signaling molecules: fgf8/17/18, sfrp1/5, and hedgehog in the anterior proboscis; hedgehog in the proboscis-collar boundary; and fgf8/17/19 and wnt1 in the collar-trunk boundary (Pani et al., 2012).
The expression profile of these three signaling centers is quite similar to that of vertebrate signaling centers. Moreover, the functions of three signaling centers that regulate specific sets of downstream transcription factors, whose orthologous genes determine the identity of neuronal cell types in vertebrates, are also similar to those of vertebrates (Figure 3C, Pani et al., 2012). The posterior region of vertebrate central nervous system, the spinal cord, is patterned by the combination of hox gene expression. In S. kowalevskii, hox genes are expressed in ectoderm posterior to the first gill slits in the same order as vertebrates (Figure 3C, Aronowicz & Lowe, 2006; Lowe et al., 2003). These extensive similarities in expression pattern and function of genes encoding signaling molecules and transcription factors suggest that the origin of the genetic program patterning the anteroposterior axis dates back to a deuterostome ancestor (Pani et al., 2012).
A striking difference in the expression pattern of these genes between vertebrates and hemichordates is that hemichordates present circumferential expression whereas this is restricted in the nervous system of vertebrates (Figure 3C, Aronowicz & Lowe, 2006; Lowe et al., 2003; Pani et al., 2012). This difference suggests that the ancestral role of these signaling centers was in general body plan regionalization rather than the patterning of the central nervous system (Pani et al., 2012). Recently, the expression of these anteroposterior patterning genes was investigated in the indirect developer S. californicum (Gonzalez et al., 2017). Some genes, such as en and pax6, express circumferentially in the same manner as in S. kowalevskii (Gonzalez et al., 2017). Hox genes, however, are not expressed in the circumferential manner. Hox1, hox4, hox5, and hox6 are expressed in the dorsal and ventral midline where the nerve cords are present (Gonzalez et al., 2017). Although the expression domain of hox7 is also restricted, hox7 is expressed in a punctate pattern in the ventral trunk ectoderm, except for the ventral midline (Gonzalez et al., 2017).
The Collar Cord
Because of the tubular form of the collar cord, it has been compared to the neural tube of chordates (Bateson, 1884, 1885; Brown, Prendergast, & Swalla, 2008; Kaul & Stach, 2010, Luttrell, Konikoff, Byrne, Bengtsson, & Swalla, 2012; Miyamoto & Wada, 2013; Morgan, 1894). The morphogenesis of the collar cord formation is also similar to that of the neural tube and sometimes referred to as hemichordate neurulation (Figure 3A and 3B, Kaul & Stach, 2010; Miyamoto & Wada, 2013). The hemichordate neurulation has been described in both direct and indirect developers. The collar cord develops during embryonic stages in direct developers, whereas it develops during metamorphosis in indirect developers. Although there is a difference between direct and indirect developers in the number of cells related to neurulation, the processes are almost the same between the two types of development. Ultrastructure of neurulation was studied in the direct developer S. kowalevskii and gene expression pattern during neurulation was studied in the indirect developer B. simodensis (Kaul & Stach, 2010; Miyamoto & Wada, 2013). The dorsal midline cells of the collar precursor are positive for neuron marker gene, such as elav, and form the neural plate (Figure 4Ai, Miyamoto & Wada, 2013). In S. kowalevskii, cells of the neural plate can be distinguished from other epidermal cells by the dense packing of their nuclei (Kaul & Stach, 2010). The boundary cells between the neural plate and epidermis express pax3/7 and soxE genes, which are expressed in the neural plate border in chordates (Figure 4Ai, Holland, 2009; Miyamoto & Wada, 2013). The collar cord forms by a sinking in of the neural plate and subsequent fusion of epidermal cells at the dorsal midline (Figure 4Aii–iv). As neurulation progresses, the neurites develop in the ventral side of the collar cord and about a half of the collar cord is occupied by the neurites forming the ventral neuropil of the collar cord (Kaul & Stach, 2010). Within the collar cord, some genes show dorsoventrally restricted expression patterns similar to that of chordates (Miyamoto & Wada, 2013). In chordates, signals from the notochord play essential roles in the induction of neurulation and dorsoventral patterning of the neural tube (Echelard et al., 1993). During metamorphosis, the neural plate and the collar cord are located just dorsally in the anterodorsal endoderm including the stomochord, which is the endodermal diverticulum. Based on the similarity of the stomochord and the notochord, Bateson (1886) coined the term Hemichordata and positioned it as a group of the phylum Chordata. The anterior endodermal cells express genes encoding paracrine factors, such as hedgehog and dkk, which are expressed in the notochord (Gerhart, Lowe, & Kirschner, 2005; Miyamoto & Wada, 2013). Additionally, the most midline cells of the neural plate and collar cord express patched gene, which encodes a receptor of the hedgehog signal (Miyamoto & Wada, 2013). In addition to the ventral signals from the endoderm, the most dorsal cells of the collar cord express bmp2/4, which plays an important role in the dorsoventral patterning of the neural tube (Miyamoto & Wada, 2013). These results suggest that there are some genetic mechanisms shared between the chordate and hemichordate neurulations and the origin of the tubular nervous system predates the divergence of chordates and ambulacrarians (Miyamoto & Wada, 2013). However, if the dorsoventral axis inversion (which is believed to have occurred in the chordate lineage) (Lowe et al., 2006) is taken into account, the dorsally situated collar cord and the neural tube are not homologous. In contrast to the chordate neurulation, there are few reports on the hemichordate neurulation.
In the collar cord, there is a type of larger neuron, called a giant neuron. Traditionally, it had been considered that giant neurons were present in species belonging to the family Ptychoderidae showing indirect development (Brown et al., 2008); however, recent ultrastructural study showed the presence of giant neurons in S. kowalevskii, a direct developer (Kaul & Stach, 2010). The giant neurons project their axons across the midline and posteriorly to the contralateral sides of the trunk (Kaul & Stach, 2010). Because there are muscle fibers in the ventrolateral side of the trunk for the rapid contraction of enteropneusts, giant cells have been proposed to be involved in escape responses (Kaul & Stach, 2010). Giant cells connect each other through two types of cell junctions (Kaul & Stach, 2010). One is spot desmosomes and another is a unique type of the cell connection, whose intracellular cleft is filled with electron dense material and the cytoplasmic side is associated with an extension of the rough endoplasmic reticulum, so-called sub-synaptic cisternae (Kaul & Stach, 2010). The presence of cell junctions supports the hypothesis that the collar cord functions as a centralized nervous system to integrate information (Kaul & Stach, 2010). Further neurophysiological studies will unveil the function of the collar cord and will provide important information on the evolution of the tubular centralized nervous system.
Pterobranchs are a very small group, which is composed of approximately 25 species (Figure 5A). Because of their small size and limited distribution, little is known about the biology of this group. As with other biological aspects, knowledge on the nervous system of pterobranchs is limited. Histological studies have described the brain of pterobranchs in the mid-dorsal part of the neck (Dilly, 1975; Schepotieff, 1907). These authors called the domain of the nervous system brain; however, they considered the brain of pterobranchs to be primitive due to its basiepithelial nature. Recently, a comprehensive morphological study of the pterobranch neuroanatomy was performed using TEM, immunohistochemistry, and 3D reconstructions based on complete serial histological sections in Cephalodiscus gracilis (Figure 5B, Stach et al., 2012). In their nervous system, there are several condensations of plexuses. The most prominent domain where the plexus is condensed is the brain located at the base of the tentaculated arms in the neck (Stach et al., 2012). Cells immunoreactive against serotonin antibody are concentrated in the anterior portion of the brain (Stach et al., 2012). These serotonergic cells project axons posteriorly. Some of these serotonergic cells seem to be bipolar with a longer and a shorter fiber (Stach et al., 2012). The plexus of the brain extends anteriorly to the dorsal epidermis of the cephalic shield and becomes thinner (Stach et al., 2012). From the brain, two prominent nerves project posteriorly to the mesocoel and branchial pores (Stach et al., 2012). The branchial nerves run along the lateral side to the ventral of the trunk becoming thinner and finally merging with the basiepidermal plexus (Stach et al., 2012). The branchial nerves are well-stained by anti-acetylated tubulin antibody. In the posterior part of the trunk, two nerves appear from the plexus on the ventrolateral side (Stach et al., 2012). These two nerves fuse at the ventral midline and run into the posterior stalk forming the ventral stalk nerve (Stach et al., 2012; Stach, 2014). From the brain, small fibers leave the brain and enter each tentaculated arm forming a tentacle nerve (Stach et al., 2012). Serotonergic cells in the brain project axons to the tentaculated arms and the axons run along the dorsal sides of the arms. In the lateral lips of the neck, there is a bilaterally symmetrical pair of plexus (Stach et al., 2012). In addition to these domains, a thin plexus is present throughout the epidermis and serotonergic cells are scattered throughout the epidermis (Stach et al., 2012). There is little information of the neurogenesis of pterobranchs because it is quite difficult to obtain embryos of this group of animals. Stach (2013) performed ultrastructural observation of an embryo of C. gracilis and found no concentration of nerve cells and no sensory organs in the epidermis.
There are some differences between the pterobranch and enteropneust nervous systems. For example, the brain of pterobranchs is located in the dorsal midline of the neck, which is homologous to the collar of enteropneusts, but does not have a tubular structure (Stach et al., 2012). The dorsal nerve cord and prebranchial nerve ring have not been reported. Homology between the ventral stalk nerve of pterobranchs and the ventral nerve cord of enteropneusts is unclear (Stach et al., 2012). The knowledge on the pterobranch nervous system is still limited and further studies using molecular and physiological techniques will be required. Comparisons between the pterobranch and enteropneust nervous systems based on comprehensive research will provide essential information on the evolution and ancestral state of hemichordate nervous system. The information should shed new light in the evolution of the central nervous system and the origin of our brain.
Hemichordate Nervous System and the Origin of the Central Nervous System
The origin of the central nervous system of our own phylum, Chordata, is a long-standing question in the field of evolutionary biology, which dates back to the mid-19th century. Since the first comparative studies of hemichordates and chordates by Bateson (1886), hemichordates have been considered as key organisms to elucidate the evolution of chordates (Brown et al., 2008; Garstang, 1928; Gee, 1996; Gerhart et al., 2005; Lacalli, 2005, Lowe et al., 2015; Luttrell et al., 2012; Miyamoto & Wada, 2013; Nielsen, 1999; Satoh, 2008). Bateson (1886) suggested that the stomochord of hemichordates is homologous to the notochord of chordates, coined the name of hemichordates, and placed them as a group of chordates. Morgan (1894) also pointed out the similarity between the hemichordate collar cord formation and the chordate neurulation. However, the homology between the collar cord and the neural tube was questioned because the nervous system of hemichordates started to be considered as a basiepithelial diffused nervous system based on histological studies (Bullock, 1965; Knight-Jones, 1952). Another scenario about the origin of chordates arose in the late 19th century. Garstang (1928) proposed that a common ancestor of deuterostomes was a sedentary animal like modern pterobranchs with pelagic larvae similar to dipleurula-type larvae, named auricularia. In Garstang’s theory, the neural tube of chordates evolved from the larval nervous system associated with the circumoral ciliary band (Garstang, 1928). Namely, the ciliary bands of the larvae rolled up and fused at the dorsal midline forming the neural tube. In this theory, the sedentary pterobranch-like deuterostome ancestor with auricularia larva first evolved into a tunicate-like chordate with a tadpole-like larva (Garstang, 1928). The tadpole-like larvae of ancestral chordates already possessed the dorsal hollow nervous system like the modern tunicate larvae. Therefore, in Garstang’s theory, centralization and evolution of the tubular nervous system occurred in this stage. Garstang’s theory has been modified by some biologists. In the 1990s, molecular developmental studies using model animals, such as vertebrates and insects, shed new light on the evolution of bilaterians. One of the most striking findings is that molecular mechanisms determining a dorsoventral axis are inverted between vertebrates and insects (De Robertis & Sasai, 1996). Both the dorsoventral axes of vertebrates and insects are determined by antagonistic action of secreted proteins BMP/Dpp and Chordin/Sog (De Robertis & Sasai, 1996). In vertebrates, the BMP side becomes the ventral (mouth) side and the Chordin side becomes the dorsal side. In contrast, the Dpp (an insect ortholog of BMP) side becomes dorsal and the Sog (an insect ortholog of Chordin) side becomes ventral. In that respect, the dorsal side of vertebrates corresponds to the ventral side of insects and vice versa. Interestingly, the dorsoventral axis formation of hemichordates is the same as in protostomes rather than vertebrates, dorsal BMP, and ventral Chordin (Lowe et al., 2006). This finding indicates that the dorsoventral axis inversion occurred in the chordate lineage. When the dorsoventral axis inversion is taken into account, the dorsally rolling up of the ciliary band in Garstang’s theory becomes incompatible with the dorsally situated neural tube of chordates. To adjust to the dorsoventral axis inversion hypothesis, Nielsen (1999) modified Garstang’s theory as follows: (1) the chordate central nervous system evolved from fusion of the posterior part of the ciliary band, and (2) the remaining part of the ciliary band degenerated.
The dorsoventral axis inversion hypothesis gave rise to another new insight into the evolution of bilaterian nervous system. Traditionally, it had been assumed that the dorsoventral axes of bilaterians were homologous and directly comparable, and that the dorsal central nervous system of chordates and the ventral central nervous system of protostomes were not homologous. Given that the axes inversion occurred in the chordate lineage, the central nervous systems of chordates and protostomes are directly comparable (Arendt & Nübler-Jung, 1994; Nübler-Jung & Arendt, 1994). This scenario dates back to the early 19th century when Geoffroy-Saint-Hilaire compared the dorsal nervous system of vertebrates and the ventral nervous system of arthropods, although these two structures were not considered the result of evolution in the pre-evolutionary period. Findings of highly conserved patterning mechanisms of the central nervous system have also supported the hypothesis that the nervous system centralization predates the diversification of deuterostomes and protostomes (Denes et al., 2007; Tomer, Denes, Tessmar-Raible, & Arendt, 2010). In this scenario, the body plan of chordates is compared with that of protostome adults, rather than that of larvae.
As mentioned above, molecular developmental biology of hemichordates sheds new light on the debate about the origin of the central nervous system in chordates. Domains where neurons are concentrated in the dorsal and ventral midlines of adult enteropneusts contribute to the hemichordates nervous system becoming somehow centralized (Nomaksteinsky et al., 2009). If the dorsoventral axis inversion is taken into account, the ventral nerve cord occupies the corresponding position of the dorsal neural tube. However, the conserved mediolateral patterning of the central nervous system reported in chordates and protostomes is not found in the ventral nerve cord of hemichordates. Furthermore, anteroposterior patterning genes of the chordate central nervous system are expressed in the whole ectoderm in the same anteroposterior manner, rather than being restricted to the nerve cord (Aronowicz & Lowe, 2006; Lowe et al., 2003; Pani et al., 2012). The morphogenesis of the collar cord is similar to that of neurulation; however, the dorsally situated collar cord is topologically incompatible with the neural tube. For these reasons, both the ancestral state of the nervous system of deuterostomes and the evolutionary origin of the central nervous system of chordates are still under controversy. Compared with well-studied chordates and protostomes nervous systems, information on the hemichordate nervous system is still limited. Especially, neurophysiological aspects of hemichordates are poorly understood. Comprehensive studies involving differentiation, patterning, cell type distribution, anatomy, and function of larvae and adults of both enteropneusts and pterobranchs should provide important information about nervous system centralization and the evolutionary origin of the central nervous system of chordates.
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