Origins of the Chordate Central Nervous System: Insights from Hemichordates

2.02 Origins of the Chordate Central Nervous System: Insights from Hemichordates C J Lowe, University of Chicago, Chicago, IL, USA ª 2007 Elsevier Inc. All rights reserved.

2.02.1 2.02.2 2.02.3 2.02.4 2.02.5 2.02.6 2.02.7 2.02.8

Introduction Deuterostome Phylogeny and Hemichordate Biology Morphological Characteristics of the Hemichordate Nervous System Proposed Morphological Homology between Vertebrate Central Nervous System and Hemichordate Nervous System Molecular Patterning Events in the Anteroposterior Patterning of the Hemichordate Nervous System Evolutionary Interpretations of the Molecular Data from Hemichordates Life History Considerations Future Directions

Glossary amphioxus

direct developer hox genes

indirect developer larvacean life history

Belongs to the cephalochordates, and is the most basally branching node of the chordates. Animals that form an adult body plan from the embryo without an intervening larval stages. Homeodomain transcription factors arranged in clusters in the genome that have conserved roles in patterning a range of axial elements in animals. Animals that employ a discrete feeding larval stage before metamorphosis into an adult. A nonvertebrate chordate closely related to ascidians. The variety of developmental and behavioral strategies utilized by animals and plants to maximize reproductive success.

2.02.1 Introduction The origin of the chordate body plan and its unique nervous system has been debated for over a century. The early part of the twentieth century was a particularly active period of speculation, when many of the most influential hypotheses were proposed, but in the latter part of the century the issue stagnated through a lack of new data. The early evolution of deuterostomes has proven to be a particularly problematic node to reconstruct, and remains largely unresolved (Gee, 1996; Lowe et al., 2003; see Evolution of the Deuterostome Central Nervous System: An Intercalation of Developmental Patterning Processes with Cellular Specification Processes, Gene Expression in the Honeybee Mushroom Body and

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Its Gene Orthologues). There are a variety of factors that contribute to difficulties in addressing this issue, but probably the two most challenging obstacles have been, and continue to be, the uncertainty of deuterostome relationships and the large morphological disparity between extant deuterostome phyla. This has been compounded by a poor fossil record and, by the end of the 1970s, the issue of chordate origins had essentially reached an impasse. The last 20 years have seen a surge in new data with the advent of molecular systematics and molecular developmental genetics. These new data are radically beginning to reshape both our understanding of comparative body plan specification and development, but also, just as importantly, the phylogenetic relationships between the deuterstome phyla (Field et al., 1988; Lake, 1990; Adoutte et al., 2000). Deuterostome phylogeny is currently a very active area of research and will likely be largely resolved at the level of the relationships between the major phyla, as more basal deuterostome genomic data sets become available for more comprehensive molecular phylogenetic analysis. The application of comparative molecular genetics to characterize the development of poorly described groups at critical phylogenetic positions, within the deuterostomes, is beginning to enhance our understanding of the early steps in deuterostome evolutionary history (Holland, 2002; Lowe et al., 2003). We are currently amidst a new wave of discovery in deuterostome evolution as many of the phylogenetically key genomes are now either sequenced or are currently being sequenced. A recent, radical revision to the phylogenetic relationships based on a large genomic data set suggests that there are still plenty of surprises ahead (Delsuc et al., 2006).

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Origins of the Chordate Central Nervous System: Insights from Hemichordates

In this article, I focus on the insights gained into the origins and evolution of the vertebrate nervous system by the characterization of early development of the nervous system of a bilateral phylum closely related to vertebrates, the hemichordates. The nervous system has been characterized as barely more complicated than that of cnidarians (Bullock and Horridge, 1965) yet the expression of a large suite of transcription factors with critical and conserved roles in anteroposterior patterning and brain regionalization in vertebrates shows unprecedented similarities in relative expression topology in the ectoderm of the developing embryos of hemichordates (Lowe et al., 2003). I discuss the implications of this data for the understanding of early deuterostome evolution and the evolution of vertebrate brains.

2.02.2 Deuterostome Phylogeny and Hemichordate Biology The phylum Hemichordata is a bilateral phylum of animals, the sister group of echinoderms, and closely related to the chordates. Along with the newly reclassified marine worm, Xenoturbella bockii, these four groups constitute the deuterostomes (Figure 1). Vertebrates Cephalochordates Tunicates Echinoderms Harramanids Pterobranchs

Hemichordates

Ptychoderids Figure 1 Deuterostome relationships based on 18S RNA. After Cameron, C. B., Garey, J. R., Swalla, B. J. 2000. Evolution of the chordate body plan: New insights from phylogenetic analyses of deuterostome phyla. Proc. Natl. Acad. Sci. USA 97, 4469–4474.

Deuterostome phylogeny has been revised many times over the past few years, which has played a major role in refining hypotheses on early deuterostome evolution and the origin of chordates. The first major reorganization occurred with the advent of molecular systematics and resulted in the reclassification of five phyla into the protostomes, leaving only three deuterostome phyla (Field et al., 1988; Lake, 1990) in a topology that has generally been well supported by subsequent studies (Adoutte et al., 2000; Cameron et al., 2000; Bromham and Degnan, 1999). Recently, an obscure, rare, and morphologically unremarkable marine worm, Xenoturbella, was reclassified into the deuterostomes with an as yet uncertain phylogenetic position within the lineage. The phylogenetic relationships are continuing to be refined, with some potential for yet more dramatic reorganization as new genomic data sets are generated and phylogenetic methods continue to improve. A new study utilizing new, large genomic data sets from amphioxus, tunicates, and sea urchin challenges the topology established from 18S RNA studies (Delsuc et al., 2006). These results support the sister taxon status of tunicates and vertebrates and are statistically robust. Even more surprising is the grouping of amphioxus with echinoderms (Figure 2b). This node has weaker support, and the analysis lacks sequence data from hemichordates and Xenoturbella. If this topology holds up to future analysis, it has sweeping consequences for our understanding of chordate origins and nervous system evolution as it implies the ancestral deuterostome already possessed the fundamental features of the chordate body plan such as somites, notochord, gill slits, and the dorsal centralized nervous system. Of all the nonchordate deuterostome phyla, the hemichordates are the most promising for addressing issues of chordate origins and the early evolution of the deuterostomes (Lowe et al., 2003).

Vertebrates

Vertebrates

Cephalochordates

Tunicates

Tunicates Cephalochordates Hemichordates Echinoderms (a)

Echinoderms

(b)

Figure 2 Deuterostome relationships from different molecular data sets. a, 18S RNA (Cameron et al., 2000). b, 146 genes gathered from the genome of a range of deuterostome organisms (Delsuc et al., 2006).

Origins of the Chordate Central Nervous System: Insights from Hemichordates

While basal chordates such as amphioxus and ascidians have been key for understanding the diversification of the basic chordate body plan, understanding the origins of the chordate body plan can only be addressed by using outgroups. There are limited options within the deuterostomes for informative outgroups, and the issue of divergent morphologies plays a large role in guiding appropriate choices. The most familiar deuterostome outside chordates is the echinoderms. While a fascinating group, their adult body plan is highly derived (Lowe and Wray, 1997) and difficult to compare directly with the chordate body plan. Xenoturbella is rare and poorly characterized so far, which leaves the hemichordates as the only other bilaterian phylum closely related to chordates. Zoological descriptions of hemichordates by Morgan (1894) and Bateson (1885, 1886) recognized their promise for addressing issues of chordate origins and the origin of the chordate central nervous system. As a result of proposed morphological affinities with the chordates, they were originally classified as a subphylum of the chordates and it was not until the 1940s that they were reclassified into their own phylum (Hyman, 1940). The phylum is divided into two major classes: the pterobranchs and the enteropneusts. The pterobranchs are poorly characterized, in part due to their sparse distribution (having only been described in a few specific locations) and their small size. Their life history is largely colonial, with a few exceptions, and they are all direct developers, each zooid producing only small numbers of oocytes. The enteropneusts are all solitary marine worms, with a broad biogeographic range and habitats from the intertidal down to deep waters and hydrothermal vents. They range in size from a few centimeters up to 2m and form U-shaped burrows, feeding by ingesting particles and filtering phytoplankton from the seawater. The phylogenetic relationships of the various groups within the hemichordates are currently uncertain, but are beginning to be resolved by molecular analyses (Cameron et al., 2000; Winchell et al., 2002). Currently, phylogenies based on 18S and 28S RNA phylogenies conflict in their placement of the pterobranchs (Winchell et al., 2002). The topology shown in Figure 1 is based on 18S data. Of the two lineages of enteropneust worms, there is a clear division in life history strategy: in the Harramanid lineage, all species are characterized by direct development, whereas in the other lineage that includes the Ptychoderids, all species have

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complex life cycles with a prolonged larval period (Cameron et al., 2000; Lowe et al., 2004). The continued interest in this group is largely a result of proposed morphological affinities of these worms to the chordates. Figure 3b shows the characteristics of the external and internal hemichordate body plan, and Figure 3a shows a photomicrograph of an early juvenile of the Harramanid Saccoglossus kowalevskii. The animal has a classic deuterostome tripartite body plan with an anterior proboscis used for burrowing and feeding, a collar or mesosome, and trunk or metasome. The mouth opens on the ventral side between the collar and proboscis. The metasome or trunk is very long in the adult. Gill slits are another key feature and are located in the anterodorsal region of this body region and occur paired, in large numbers in adults. From a morphological perspective, the gill slits resemble those of amphioxus quite closely and both morphological analysis and molecular analyses suggest that they may represent true homologues (Ogasawara et al., 1999; Okai et al., 2000; Lowe et al., 2003; Rychel et al., 2006). An anterior projection from the gut called the stomochord supports the heart–kidney complex and has been classically compared to the notochord. However, more recently, both morphological and molecular studies have failed to find support for this homology (Ogasawara et al., 1999; Peterson et al., 1999; Okai et al., 2000; Lowe et al., 2003; Ruppert, 2005).

2.02.3 Morphological Characteristics of the Hemichordate Nervous System Our understanding of hemichordate nervous system organization and structure is based heavily on studies that all date back to before the 1960s. The first person to describe the nervous system of the hemichordates was Spengel (1877). His description of the major elements and organization of the enteropneust nervous system has been borne out by further detailed analysis in the 1940s and 1950s, notably by Bullock (1945), whose reinvestigated the general organization of balanoglossids in his classic paper. In addition, he investigated the giant axon system in 1944 (Bullock, 1944) and the general function of the nervous system in 1940 (Bullock, 1940). The last comprehensive study of the nervous system of this group was by Knight-Jones (1952), further developing the work of Bullock. Silen (1950) also contributed to the body of work, but some of his data and interpretations have been questioned by both Knight-Jones and Bullock. While the basic descriptions are clearly established, there remain

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Origins of the Chordate Central Nervous System: Insights from Hemichordates

Prosome/proboscis

Mesosome/ collar

Mouth

Metasome/trunk

Gill slits

Postanal tail

Anus

(a) Trunk/metasome

Collar/mesosome

Proboscis/prosome

Kidney Mouth Gill slits (b)

Dorsal vessel Ventral vessel

Stomocord

Heart

Figure 3 Structure and organization of the enteropneust hemichordate body plan. a, A 13-day-old Saccoglossus kowalevskii juvenile. b, Model of the anterior-most region of an enteropneust. Blue, ectodermal derivatives; yellow, endodermal derivatives; and red, mesodermal derivatives.

many unresolved issues in the structure and function of the nervous systems that would benefit greatly from a molecular characterization. The major organizational feature of the nervous system is a basiepithelial plexus: there is no central nervous system. Cell bodies are scattered throughout the epithelia of the body, with a few exceptions, such as the intestine and gill slits. A mat of axons is spread out along the basement membrane of the epithelia, which is thickened in certain areas of the ectoderm, such as at the base of the proboscis, along the anterodorsal region of the body in the mesosome, and in both the dorsal and ventral midlines of the metasome (Figure 4). In the proboscis ectoderm, there is a particularly dense concentration of nerve cells that have been proposed to be primarily sensory (Figure 4b; Bullock, 1945; Knight-Jones, 1952). Around the

base of the proboscis, there is a particularly dense aggregation of cell bodies. There do not generally seem to be any true sensory organs, but rather individual sensory neurons. One exception is possibly the ciliary organ at the base of the ventral proboscis (Brambell and Cole, 1939). The nerve plexus in the proboscis is particularly thick and axons are organized into lateral and longitudinal tracts that are thought to funnel back to the dorsal peduncle that connects to the collar, passing axons down to the rest of the body (Knight-Jones, 1952). The plexus of axons is particularly thick in this region around the base of the proboscis and forms the so-called anterior nerve ring. Figure 4a shows an updated version of the classic picture from Knight-Jones of the organization of the nerve plexus in hemichordates. A low-magnification confocal Z-series of a 13-dayold juvenile of S. kowalevskii following

Origins of the Chordate Central Nervous System: Insights from Hemichordates n

r

fn

dp

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A Proboscis

c

l

a

Collar

5 mm

v

D

V Trunk

d

Anti5HT (serotonin) Antisynaptotagmin P

(a)

(b)

Figure 4 General organization of the nervous system of enteropneusts. a, Drawing from Knight-Jones (1952) of the nerve plexus of Saccoglossus cambriensis. d, dorsal cord; v, ventral cord; r, prebranchial nerve ring; c, collar; n, neurocord; fn, fan-shaped thickening of nerve fiber layer; l, ciliary organ; a, anterior nerve ring; dp, dorsal concentration of fibers on the proboscis. b, Confocal micrograph of 13-day-old juvenile of S. kowalevskii. Immunocytochemistry of antibodies against serotonin and synaptotagmin (monoclonal antibody courtesy of Bob Burke, University of Victoria). A, anterior; P, posterior; D, dorsal; V, ventral. a, Reproduced from KnightJones, E. 1952. On the nervous system of Saccoglossus cambriensis (Enteropneusta). Philos. Trans. R. Soc. Lond. B: Biol. Sci. 236, 315–354, with permission from The Royal Society. b, Lowe (unpublished).

immunocytochemistry against serotonin and synaptotagmin shows the broad distribution of cell bodies in the ectoderm, particularly densely packed in the anterior ectoderm, collar, and a dense mat of axons, particularly prominent in the base of the proboscis and throughout the proboscis ectoderm, lining the basement membrane (unpublished data). Probably the most well-known aspect of hemichordate anatomy is the mid-dorsal region of the dorsal cord, or collar cord, which is internalized into a hollow tube of epithelium in some species within the Ptychoderidae and in one species of the Spengeliidae. However, in the other major enteropneust lineage, the Harramanids, there is no contiguous hollow tube, but scattered blind lacunae (Bullock and Horridge, 1965; Nieuwenhuys, 2002; Ruppert, 2005; Figure 5a). This structure has been widely compared to the dorsal cord of chordates due to the superficial similarities to the hollow ciliated nerve cord, and similarities in its morphogenesis with neurulation in vertebrates (Morgan, 1891). However, the similarities have generally been overemphasized as it seems to be more of a conducting tract rather than processing center (Ruppert, 2005), as evidenced by both ultrastructural (Dilly et al., 1970) and physiological data (Cameron and Mackie, 1996). Another striking feature of the dorsal cord is the presence of giant axons (Figure 5b). The number is quite variable between different species, but they are always associated with the dorsal cord and are unipolar cells from 15 to 40mm in diameter. The cell bodies project

their axons across the midline and continue posteriorly within the collar cord. It is not known where the axons finally project: Bullock (1945) proposed that they innervate the ventrolateral muscles of the trunk and suspected that their primary function is to elicit a rapid contraction of the ventrolateral musculature. In the metasome, the third body region, the most prominent features are the ventral and dorsal nerve cords, which are both thickenings of the nerve plexus. The dorsal cord is contiguous with the collar cord and projects down the entire length of the metasome. The ventral cord is comparatively much thicker, and more cell bodies are associated with the ventral cord, but both cords are interpreted as being through axon tracts. They seem to play a role in the rapid retreat of the animals following anterior stimulation (Knight-Jones, 1952; Bullock and Horridge, 1965). The collar cord seems to play a more minor role than the ventral cord in this response. Ruppert has proposed that the collar cord may be more associated with innervation of the collar musculature that is involved in retraction of the proboscis into the collar, thus sealing the mouth (Ruppert, 2005). From these studies, there remain some quite fundamental and important questions about the organization of the nervous system. There are still discrepancies between the major texts in terms of the extent of neural plexus and the density of the cell bodies at various regions of the body (Bullock and

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Origins of the Chordate Central Nervous System: Insights from Hemichordates

IV pn

Proboscis

dp

I neurocor

V

fn

II

III

an

d

d

Mesocoel Giant nerve cell bodies Prebrancial nerve ring 0.5 mm r

l a

Dorsal cord

v

(a)

(b)

Figure 5 Nervous system of the enteropneust collar/mesosome. a, Drawing of a sagittal section of the enteropneust collar. a, anterior nerve ring; d, dorsal cord; pn, posterior neuropore; an, anterior neuropore; fn, fan-shaped thickening of the nerve fiber layer; v, ventral cord; r, prebranchial nerve ring; dp, dorsal concentration of fibers on the proboscis. b, Dorsal view of collar showing the arrangement of the giant axons. a, Reproduced from Knight-Jones, E. 1952. On the nervous system of Saccoglossus cambriensis (Enteropneusta). Philos. Trans. R. Soc. Lond. B: Biol. Sci. 236, 315–354, with permission from The Royal Society. b, Redrawn from Bullock, T. H. and Horridge, G. A. 1965. Structure and Function in the Nervous Systems of Invertebrates. W. H. Freeman.

Horridge, 1965). Of fundamental importance is to what extent the nerve plexus and cell bodies show functional differentiation. The classical papers have been unable to distinguish between motor neurons and interneurons, and there is disagreement over which of the ectodermal cells represent neurons, and which ones are glandular or more characteristic of epidermis. The extent to which the plexus shows differentiation along the anteroposterior and dorsoventral axis is also still an open question. Perhaps the most interesting question is whether a greater characterization of the development and differentiation of the nervous system of this group will shed light on the early evolution of the deuterostome nervous system and the evolutionary origins of the chordate central nervous system. Our understanding of the function and differentiation of this nervous system will greatly benefit from the application of basic molecular biological techniques, such as neuron back-filling to map the distribution of cell bodies and where they project their axons; and immunocytochemistry with antibodies raised against conserved neural markers and neurotransmitters to determine the extent of neural differentiation in the nerve plexus.

2.02.4 Proposed Morphological Homology between Vertebrate Central Nervous System and Hemichordate Nervous System As discussed in the previous section, the classic papers on hemichordate development and evolution have emphasized the critical phylogenetic position of

the hemichordates and their relevance for understanding the early evolution of the chordate body plan. Morphological studies have proposed hypotheses on the possible homologies of the enteropneust cords to the dorsal nervous system of chordates. The majority of the papers focus on the possible homology of the dorsal nervous system of chordates with the dorsal collar cord of the enteropneusts. However, more recently an alternative proposal was made that the ventral cord is homologous to the arthropod ventral cord, and the dorsal cord and proboscis plexus homologous to the dorsoanterior brain of arthropods (Nubler-Jung and Arendt, 1996). This would imply that the ventral side of hemichordates is homologous to the dorsal side of chordates. In all of these proposals, both molecular and morphological, the focus of comparisons has been on the axon cords. However, these cords are not the information-processing centers of animals with central nervous systems, but conduction tracts. Both Bullock (1945) and Ruppert (2005) favor homoplasy over homology when comparing the dorsal cord directly with chordates, and argue that the mere presence of a hollow tube is an unreliable character, having evolved independently in several lineages, such as in the cerebral ganglion of some ectoproct bryozoans and in ophiuroids. Other authors have reached the same conclusion (Nieuwenhuys, 2002). Knight-Jones (1952) supports cord homology and argues that the dorsal cord represents a degenerate structure, disagreeing with Bullock. In the next section, I will argue that the fundamental element of relevant comparison between chordates and hemichordates is not the two cords, but the entire basiepithelial nervous system.

Origins of the Chordate Central Nervous System: Insights from Hemichordates

2.02.5 Molecular Patterning Events in the Anteroposterior Patterning of the Hemichordate Nervous System Molecular genetic studies carried out over the past 25 years have established some remarkable similarities in the suite of developmental genes involved in patterning both the dorsoventral and anteroposterior axis of chordates and arthropods and likely all bilaterian phyla (Krumlauf et al., 1993; Arendt and Nubler-Jung, 1994; Finkelstein and Boncinelli, 1994; De Robertis and Sasai, 1996; Lowe et al., 2003; Lichtneckert and Reichert, 2005). Many of the most striking similarities revealed have been during the patterning of the central nervous systems of these two groups (Figure 6). So similar are the relative expression domains of the orthologues of a range of transcription factors along the anteroposterior axis in the developing nervous system of flies and vertebrates that some authors have concluded that the ancestor of bilaterians must have already possessed a complex central nervous system, with an anterior brain already organized into a tripartite structure (Lichtneckert and Reichert, 2005). The plausibility of the arguments depends on this suite of genes being correlated with the development of central nervous systems. However, there has been little comprehensive study of neural specification of any phyla without a highly developed and centralized nervous system. Without comparative data sets in other phyla with more diffuse nervous systems, the hypothesis is difficult to test. The interpretation of this molecular genetic data set is clearly at odds with the classical zoological view of how nervous system evolution has occurred during the evolution of the bilaterian nervous system (Holland, 2003). The current molecular phylogeny of bilaterians

Hindbrain Midbrain

Spinal cord

Chordate Forebrain

plg /un gbx /5/8 2 pax

otx/otd

hox domain

Protocerebrum

Drosophila

Deuterocerebrum Tritocerebrum

Figure 6 Conserved expression domains of transcription factors between vertebrates and chordates in the developing central nervous system.

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most parsimoniously supports a reconstruction of a bilaterian ancestor with a modest nervous system organized as a basiepithlial nervous net, and not the complex, highly regionalized and centralized nervous system, as suggested by the molecular genetic data set. We undertook a large comparative study to investigate the role of the transcription factors with conserved roles in anteroposterior patterning of arthropods and chordates during the development of the hemichordate S. kowalevskii. The study had two aims: (1) to test the hypothesis of a bilaterian nervous system evolution and (2) to investigate the relationship between the chordate and hemichordate nervous systems. Clearly these suites of genes are very useful axial markers in bilaterian phyla, and their relative expression domains can act as a basic means of comparing body plans of quite distantly related phyla with divergent morphologies (Lowe et al., 2003). S. kowalevskii belongs to the Harramanids, which are all characterized by direct development (Figure 1): the adult body plan is formed directly from the egg without any intervening larval stages. We first investigated the observations of Bullock that the entire ectoderm of this animal is neurogeneic without contiguous areas of non-neurogenic ectoderm as in arthropods and chordates. The first group of genes described are all markers of neural differentiation, such as sox1/2/3, musashi, and elv. All of these genes are early markers of neural plate in vertebrates: the first two are early markers of proliferating neural precursors while elv is a marker of differentiating neurons (Kim and Baker, 1993; Kaneko et al., 2000; Sasai, 2001). All three orthologues of these genes are expressed throughout the ectoderm of the developing embryos of S. kowalevskii from very early stages of gastrulation through to all stages examined. The expression of these genes begins broadly throughout the ectoderm, with expression uniform at early developmental stages. At later stages, expression in the prosome and mesosome is stronger than in the metasome. This observation generally correlates with the distribution of cell bodies described by Bullock (1945). The remaining characterization of the expression of developmental transcriptional regulators describes the expression of genes involved in the anteroposterio patterning of the vertebrate neural plate. Twenty-two transcription factors with conserved roles in patterning the anteroposterior neuraxis of vertebrates were examined. The surprising result from the study was that the relative expression patterns of these genes in developing embryos of S. kowalevskii are highly correlated

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Origins of the Chordate Central Nervous System: Insights from Hemichordates

Telencephalon/prosome Midbrain/ Hindbrain-spinal cord Postanal tail mesosome-anterior posterior metasome metasome otx, pax6, emx, lim 1/5 gbx,hox1,2,3,4,5,6/7,7/8 hox9/10, dlx, nk2-1, bf-1 otp, barh, dbx en, tll 11/13a,b,c rx, six3, vax

Figure 7 Model showing the similarities of expression domains between S. kowalevskii and vertebrates for conserved transcription factors with critical roles in patterning the brain and central nervous system of chordates.

when compared to those in vertebrates (Figure 7). The expression domains, however, were not restricted to either the dorsal or ventral side of the developing embryo, but were expressed in concentric rings in the ectoderm, reflecting the organization of the basiepithelial nerve net. The general domains of expression can be divided into three main fields. In the first, genes that are generally markers of vertebrate forebrain development (retinal homeobox (rx), six3, nk2-1, brain factor-1 (bf-1), distalless (dlx), and ventral anterior homeobox (vax)) are expressed uniquely in the developing proboscis ectoderm. In the second region, genes that are predominantly markers of the forebrain and midbrain show correspondingly further posterior domains of expression in the hemichordate embryo; their expression is detected in the more caudal region of the proboscis ectoderm and into the collar/anterior mesosome (pax6, tailless (tll), emx, barH, dorsal brain homeobox (dbx), lim1/5, iroquois (irx), orthodenticle (otx), and engrailed (en)). Again, their expression is detected in concentric rings in the ectoderm in patterns that approximate their relative expression domains in the midbrain and into the anterior hindbrain of vertebrates. Moving even further posterior in the vertebrate brain, into the hindbrain and spinal cord, in the most anterior domain at the midbrain–hindbrain boundary (MHB), gbx defines the limit of the midbrain and antagonizes the expression of otx, which is expressed rostrally. The mutual antagonism of these two transcription factors is involved in placing

the MHB (Li and Joyner, 2001; Rhinn et al., 2005). In hemichordates, there is a large degree of overlap in gbx and otx expression, suggesting that these genes may not have similar regulatory interactions in this group. Posterior to gbx is the expression of hox genes in the hindbrain and spinal cord. As has been well characterized, hox cluster members in the 39 region of the cluster are expressed more anteriorly during embryonic development than cluster members located more 59. A similar expression relationship is found in hemichordates, with hox1 expressed just below the first gill slit and the remaining cluster members expressed increasingly more caudally in the ectoderm. This is a consistent pattern for hox3, 4, 6/7, 7/8, and 11/13c. All the remaining hox members have been characterized (hox 2, 5, 9/ 10, and hox11/13a, b) and all members exhibit colinearity in the same way that other bilaterian groups do (unpublished data). The most posterior members of the hox cluster, hox11/13, a, b, and c are all expressed in the ventral postanal tail of the late juvenile (Figure 7). The extent to which vertebrates and hemichordates share patterning similarities is work in progress and is clearly critical for establishing the early patterning systems of the deuterostome ancestor. My lab is investigating the role of signaling molecules with conserved roles in early neural patterning in vertebrates during the early development of hemichordates. The expression of the transcription factors described in the previous section is downstream of early patterning from signaling factors immediately following neural induction in vertebrate development. By the end of gastrulation, the neural plate has already become subdivided into the precursors of the fore-, mid-, and hindbrain (Rhinn et al., 2006). Immediately following neural induction, the entire neural plate expresses otx2 that in later stages becomes restricted to the fore- and midbrain. This suggests that, at neural induction, the entire neural plate is initially fated as anterior (Gamse and Sive, 2000). Posteriorization by transformation of anterior to posterior fates has been postulated to be the primary mechanism to achieve neural partitioning with candidate signaling from wnt (McGrew et al., 1995; Fekany-Lee et al., 2000; Kiecker and Niehrs, 2001), fgf (Kengaku and Okamoto, 1993; Lamb and Harland, 1995), and retinoic acid (RA) (Durston et al., 1989; Sive et al., 1990; Conlon, 1995), which are all present in the posterior region of the embryo. Following gastrulation, after the initial global action of wnts, fgfs, and RA, local signaling centers are set up in the neural plate that further refine the anteroposterior patterning established from earlier signals. These

Origins of the Chordate Central Nervous System: Insights from Hemichordates

centers are the anterior neural ridge at the far rostral tip of the neural plate (Shimamura and Rubenstein, 1997; Houart et al., 2002), and the zona limitans intrathalamica at the boundary of prosomeres 2 and 3 (Kiecker and Lumsden, 2004). Further caudally is the MHB or isthmus, which maintains distinct cell fates between the midbrain and hindbrain (Wurst and Bally-Cuif, 2001; Raible and Brand, 2004; Nakamura et al., 2005; Rhinn et al., 2005), and at the far posterior is the tail organizer (Agathon et al., 2003). Is there any evidence of either early action of wnts, fgfs, and RA in posteriorization of hemichordate ectoderm and nervous system? Is there evidence of signaling centers homologous to those found in vertebrate neural plate? The evidence for a role of fgf, wnt, and RA in global posteriorization is mixed outside vertebrates. RA shows the strongest evidence for a conserved role in posteriorization by experiments carried out in both ascidians and amphioxus (Hinman and Degnan, 2000; Matsumoto et al., 2004; Schubert et al., 2004, 2005). There is strong evidence of a posterior organizing center in amphioxus, and weaker evidence in ascidians, but there is ample evidence to propose an ancestral posterior signaling center homologous to the tail organizer in vertebrates (Holland, 2002). However, the role of fgf in posteriorization is not well established outside the vertebrates. There are currently no fgf data from amphioxus, and data from ascidians showing some localized expression of fgf8/17/18 in a region that loosely resembles an MHB (Imai et al., 2002) but no functional data have yet been described. Beyond chordates, the evolutionary origins of these signaling factors in ectodermal patterning are not well characterized. RA metabolism has no published account outside chordates, and the role of wnts and fgfs in Drosophila does not support a conserved role in neural patterning homologous to that of vertebrates. The origins of the MHB are equally uncertain: outside vertebrates there is weak evidence for the presence of an MHB-like patterning cassette based on the profile of gene expression (Tallafuss and Bally-Cuif, 2002). Amphioxus perhaps shows the least amount of similarities to vertebrates and lacks many critical markers of this boundary (Takahashi, 2005). Within the urochordates, Ciona intestinalis exhibits the most likely candidate for an MHB. However, between species of ascidians, the domains shift quite significantly, and in the larvacean there is no good evidence for such a region (Can˜estro et al., 2005). Hemichordates, as an outgroup of chordates, offer an opportunity to investigate the origins of vertebrate neural patterning centers. We have some preliminary molecular

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evidence that the evolutionary origins of wnts, fgfs, and RA may be far more ancient than previously anticipated (Gerhart et al., 2005, unpublished data). The localization of the expression of certain fgfs, wnts, and wnt antagonists in the ectoderm of the developing embryo of S. kowalevskii, when mapped onto the existing transcriptional map of conserved anteroposterior genes, shows remarkable topological similarities with the localization of their vertebrate orthologues during neural plate regionalization (unpublished data). From our expressed sequence tag (EST) data we have also cloned many members of RA metabolism, such as the receptors RXR and RAR, the enzyme Raldh2 that metabolizes retinol to produce RA (Conlon, 1995), and the enzyme cyp26 that breaks down RA (Ray et al., 1997). Functional data will be required to test whether the early patterning events, following the induction of neural plate, have more ancient deuterostome origins than previously anticipated, but the data so far are very promising.

2.02.6 Evolutionary Interpretations of the Molecular Data from Hemichordates The expression of the correlated suite of anteroposterior patterning genes with such conserved domains in the hemichordate ectoderm has many implications for our understanding of the evolution of deuterostome nervous systems, but also for how reliable body-patterning genes can be used for reconstructing ancestral neural anatomies of distantly related groups of animals. Do these data allow us to test hypotheses of morphological homology between hemichordates and other bilaterians? For example, a subset of the study genes from Lowe et al. (2003) has similar relative expression domains between arthropods and chordates. These similarities have been used to support the hypothesis that the ancestor of protostomes and deuterostomes must have already possessed a complicated and central nervous system (Lichtneckert and Reichert, 2005), as was discussed in an earlier section. However, the correlated expression domains of this suite of genes broadly in the ectoderm of hemichordates raises the possibility that this group of genes acts as a conserved regulatory suite for patterning all bilaterian nervous systems. The study establishes that these genes are not uniquely associated with central nervous system development. It is, of course, also possible that hemichordates lost centralization from a deuterostome ancestor with a central nervous system. However, even if this proves to be the case, correlated suites of developmental

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Origins of the Chordate Central Nervous System: Insights from Hemichordates

genes expressed in similar topologies between distantly related groups are clearly deployed to pattern diverse types of neural architectures. Caution should therefore be used about using this suite of genes to test hypotheses of morphological homology of specific neuroanatomies. Clearly the similarities in gene expression between vertebrates and hemichordates does not indicate any sort of morphological homology between regions of the vertebrate brain and the basiepithelial plexus of the hemichordates, so what exactly do these data allow us to compare between groups, and what do they tell us about the early evolution of the chordate nervous system? These data provide, for the first time, a transcriptional rationale or map with which to compare the body plans of vertebrates and hemichordates, and give some critical insights into the deterostome ancestor that gave rise to the chordates. While the details of the morphology of the nervous system (centralized, or diffuse) are difficult to reconstruct from this kind of data, the ancestor clearly used this group of genes to pattern its anteroposterior axis, and likely its nervous system. The various suites of genes involved in patterning the three regions of the vertebrate brain were already present in the deuterostome ancestor. This is a surprising finding based on the studies of similar genes from cephalochordates and urochordates (Takahashi and Holland, 2004; Can˜estro et al., 2005; Takahashi, 2005). Neither of these two groups exhibits the degree of similarity in the relative expression domains between vertebrates and hemichordates, suggesting that they have lost some of the complexity of the ancestral transcriptional network. Work from larvaceans, ascidians, and amphioxus has reached sometimes conflicting conclusions regarding the evolution and early chordate origins of the vertebrate brain. These issues have been extensively reviewed elsewhere (Wada et al., 1998; Wada and Satoh, 2001; Mazet and Shimeld, 2002; Tallafuss and Bally-Cuif, 2002; Takahashi and Holland, 2004; Takahashi, 2005). Given the uncertainties of the neural organization of the deuterostome ancestor, searching for morphological homologues of vertebrate brain regions in the early chordates using gene expression as the primary data could lead to misleading conclusions.

2.02.7 Life History Considerations A discussion of the origins of the chordate nervous system would not be complete without a discussion of life history issues and particularly the potential role of larval forms in the evolution of the chordate dorsal nervous system. As described earlier in this

article, enteropeust hemichordates are divided into two major lineages characterized by divergent developmental strategies (Cameron et al., 2000). Developmental studies have been carried out from species from both lineages (Lowe et al., 2004). While S. kowalevskii is a direct developer, Ptychodera flava is an indirect developer and its early development is characterized by a prolonged larval period in the plankton before metamorphosis into a juvenile many months after fertilization (Tagawa et al., 2001). The early larval development of hemichordates resembles the early larva of many echinoderm species and has been termed dipleurulatype. Study of P. flava allows for the investigation of the development of both the larval and the adult body plan of enteropneusts. One of the most influential and enduring hypotheses on the origin of the chordate nervous system is from Garstang (1894, 1928). He proposed the auricularian hypothesis, which drew directly on the larval similarities between the echinoderms and hemichordates. While the adult body plans of hemichordates and echinoderms are highly divergent, the larval body plans are very similar. The key component of the hypothesis is that Garstang derived the chordate dorsal central nervous system by a dorsal migration of the lateral ciliated bands of an auricularian-type (dipleurula) larva, similar to the early larva of echinoderms and hemichordates. Garstang’s hypothesis has been extremely influential on subsequent work in this area (Gee, 1996). There have been many modifications to the original hypothesis, but the essential elements remain the same: the adult body plan of chordates is derived by transformation of a larval life history stage. There are a range of reviews on the features of Garstang’s original hypothesis (Lacalli, 1995; Nielsen, 1999), and I will only focus on the molecular genetic studies that are relevant to this hypothesis. There have been a series of papers over the past 10 years on the role of body-patterning genes in larval development relevant for testing the auricularian hypothesis (Lowe and Wray, 1997; Harada et al., 2000, 2002; Shoguchi et al., 2000b; Tagawa et al., 2000; Lowe et al., 2002; Taguchi et al., 2002; Poustka et al., 2004). Some of these studies propose molecular support for the auricularian hypothesis and show expression domains for developmental genes with conserved roles in the development of the central nervous system in chordates, as described in an earlier section. In order to argue compellingly for a molecular case to support this hypothesis, there should be evidence that the anteroposterior patterning domains, which show nested in the nervous system of chordates, have

Origins of the Chordate Central Nervous System: Insights from Hemichordates

similarly correlated domains during larval development. Not finding such domains does not disprove the auricularian hypothesis, but finding a correspondingly complex set of domains during the adult life history stage would argue quite strongly against Garstang’s theory (Lowe et al., 2003; Haag, 2005, 2006). Of the genes so far studied, there is the most comparative information on otx, which has an anterior localization during the development of the vertebrate nervous system and likely plays a conserved role in the determination of the anterior region of all bilaterians (Finkelstein and Boncinelli, 1994). The expression of otx during the development of larvae shows a general association with the development of ciliated bands. There is not, however, any particular anterior bias, with quite a variation in detailed expression domains between larval types of hemichordate (Harada et al., 2000) and holothuroid development (Lowe and Wray, 1997; Shoguchi et al., 2000b; Lowe et al., 2002). t-brain is a T-box gene with a largely conserved anterior domain of expression in the development of the forebrain of vertebrates (Bulfone et al., 1995; Mione et al., 2001). Tagawa et al. (2000) investigated the expression of the hemichordate orthologue of t-brain during early larval development in P. flava and detected its expression in the apical organ, at the very anterior tip of the larval ectoderm. They discussed the evolutionary implications of the data and raised the possibility of homology between the apical organ and vertebrate forebrain. However, in a similar study on asteroid larval development, whose early development resembles that of P. flava quite closely, there is no such expression detected in the apical organ, and it is confined to the blastopore and endoderm (Shoguchi et al., 2000a). How does one resolve the variability in the expression domains of conserved developmental genes in similar larval morphologies? It is hard to draw firm conclusions when there is such variability in gene expression domains. The same observation is made when Dlx expression is examined across echinoderm larval types (Lowe et al., 2002). There is quite a range of expression domains of Dlx throughout three different classes of echinoderms, and between different developmental modes during development. Expression in a range of holothuroid larval types is associated with cilia and ciliated bands. However, expression of Dlx is not detected in the ciliated bands of asteroid or echinoid larvae, and, unlike vertebrates, is not expressed anteriorly. Of particular note in this study was that, while larval domains of Dlx expression varied extensively, the expression domains during

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early adult body patterning is conservative, and is initially expressed at the onset of the adult body plan as the hydrocoel first begins to form five pouches – the first sign of the radial symmetry of the adult body plan. Perhaps the most telling study is of the expression of hox genes during the development of echinoid larvae (Arenas-Mena et al., 2000). Very few hox genes are expressed during early development of the larva, and those that are, are expressed in a lineage-specific fashion and not in nested domains, as found in most other bilaterian groups. The first evidence of the expression of hox genes in a co-linear manner is late in larval development during the formation of the adult body plan, and expression is detected in the mesoderm rather than the ectoderm. While a full characterization of body-patterning genes during the development of larval body plans is still lacking, the current evidence supporting larval origins of the chordate body plan is weak . Evidence from Lowe et al. (2003) in hemichordate adult patterning, and from limited data during adult body patterning of echinoderms (Morris and Byrne, 2005), support the alternative view that the most fruitful approach for understanding the early origins of the chordate body plan will come from the adult life history stage and not larvae (Lowe et al., 2003; Haag, 2005, 2006). However, it is important to reiterate here that the search for morphological homologies between chordate central nervous system and either life history stage using molecular markers is unlikely to be informative, and even misleading, if gene expression is the only criterion to assess hypotheses of homology. Even similarities in suites of correlated expression domains, as found in Lowe et al. (2003), only allow for a reconstruction of the regulatory map that an ancestor must have utilized in early development. Additional data in the form of morphological or fossil data are also required to make strong conclusions about homology.

2.02.8 Future Directions It seems likely that the story of the origins of chordates and the murky history of early deuterostome evolution is going to remain a topic of hot debate in the near future. The most significant advances will likely occur in the next few years, and most of the progress will be directly due to the completion in sequencing of several key genomes. The sea urchin and amphioxus genomes are close to completion and the genome for S. kowalevskii is also timetabled for completion this year. The phylogenetic issues raised in Delsuc et al. (2006) will begin to be

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Origins of the Chordate Central Nervous System: Insights from Hemichordates

resolved one way or another with increased phylogenetic sampling to include additional hemichordates, echinoderms, and Xenoturbella. This study clearly shows how interpretation of the molecular genetic data is only as good as the phylogeny used to map the characters. One of the largest gaps in our understanding of body patterning in the deuterostomes is that of the echinoderms. There have only been a handful of studies investigating body patterning of the adult echinoderms, and mention of echinoderms in discussion of deuterostome body plans generally dismisses them as being too derived to give informative data for reconstructing early deuterostome evolution. It is still possible that the highly divergent symmetry system of echinoderms, and their odd morphologies, are still regulated by the conserved ectodermal transcriptional network as described in this review. Further work will be required to test this hypothesis. Ongoing research in my lab is investigating the similarities in nervous system patterning between vertebrates and hemichordates, and this work will help establish the extent to which transcriptional and signaling elements of vertebrate brain patterning were established early in deuterostome evolutionary history, and which ones evolved much later during chordate diversification.

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Further Reading Delsuc, F., Brinkmann, H., Chourrout, D., and Philippe, H. 2006. Tunicates and not cephalochordates are the closest living relatives of vertebrates. Nature 439, 965–968. Gerhart, J., Lowe, C., and Kirschner, M. 2005. Hemichordates and the origin of chordates. Curr. Opin. Genet. Dev. 15, 461–467. Holland, N. D. 2003. Early central nervous system evolution: An era of skin brains? Nat. Rev. Neurosci. 4, 617–627. Lichtneckert, R. and Reichert, H. 2005. Insights into the urbilaterian brain: Conserved genetic patterning mechanisms in insect and vertebrate brain development. Heredity 94, 465–477. Lowe, C. J., Wu, M., Salic, A., et al. 2003. Anteroposterior patterning in hemichordates and the origins of the chordate nervous system. Cell 113, 853–865.