The evolution of nervous system centralization

It is currently unknown when and in what form the central nervous system (CNS) in Bilateria first appeared, and how it further evolved in the different bilaterian phyla. To find out, a series of recent molecular studies have compared neu- rodevelopment in slowly evolving deuterostome and protostome invertebrates such as the enter- opneust hemichordate Saccoglossus and the poly- chaete annelid Platynereis. These studies focus on the spatially different activation and, when access- ible, function of genes that set up the molecular anatomy of the neuroectoderm and specify neu- ron types that emerge from distinct molecular coordinates. Complex similarities are detected that reveal aspects of neurodevelopment that most likely already occurred in a similar man- ner in the last common ancestor of the bilaterians, Urbilateria. Using this approach, different aspects of the molecular architecture of the urbilaterian nervous system are being reconstructed and are yielding insight into the degree of centralization that was in place in the bilaterian ancestors.


7.1 Introduction

Surprisingly little is known about the evolutionary origin of the CNS. It is not known when CNSs first appeared in animal evolution nor what their ini- tial structure and function was. It is also unclear whether the CNSs of vertebrates and invertebrates trace back to a common CNS precursor (Arendt and Nübler-Jung, 1999) or whether they have inde- pendent evolutionary origins (Holland, 2003; Lowe et al., 2003). This chapter addresses the questions
of when and in what form the CNS first came into existence, and how it further evolved in different animal phyla. To track the evolutionary transi- tion from ‘diffuse’ to ‘centralized’ in the evolution of the bilaterian nervous system (Figure 7.1) we first define these terms. We then explain what the study of bilaterian neurodevelopment can reveal about this transition. Specifically, we focus on the role of Decapentaplegic (Dpp) signalling in trigger- ing neurogenesis in a polarized manner along the dorsoventral body axis. We then outline the conserved mediolateral molecular anatomy of the bilaterian neuroectoderm (Figure 7.2 and Plate 5) and pinpoint a set of conserved neuron types that develop from corresponding regions (Figure 7.3 and Plate 6). We finally discuss the significance of these data for reconstructing the urbilaterian ner- vous system.


7.1.1 What is a CNS?

In physiological terms, a CNS integrates and proc- esses sensory information coming from the per- iphery, and initiates body-wide responses via neurosecretion into the body fluid or via direct stimulation of the body musculature. Anatomically, a CNS is a delimited nervous tissue that comprises distinct agglomerations of functionally specialized neurons (nuclei) interconnected by axon tracts (neuropil). The CNS may be subdivided into separ- ate parts (ganglia) and it connects to the periphery via nerves. A CNS thus defined is found in vari- ous shapes and degrees of complexity in differ- ent animal phyla, including vertebrates and many


Figure 7.1 Different degrees of centralization in metazoan brains. (a) Centralized nervous system of an oligochaete worm. (b) Nerve net of a cnidarian polyp representing a typical non-
centralized nervous system. Schematized drawings modified from
Bullock and Horridge (1965).



invertebrates such as echinoderms, arthropods,
nematodes, molluscs, and annelids (Figure 7.1a).
In contrast, a diffuse nervous system receives
sensory input and processes locomotor or neurose-
cretory output only locally, without central integra-
tion. This is achieved by the direct interconnection
of sensory neurons and effector neurons (Westfall
et al., 2002). A diffuse nervous system is present in
the body wall epithelium of adult cnidarians, for
example (Figure 7.1b).
Even though these definitions are straightfor-
ward, the categorization of some animal nervous
systems remains ambiguous (Miljkovic-Licina et al.,
2004). For example, some cnidarian medusae pos-
sess an elaborate nerve ring around their central
opening (manubrium) in addition to their diffuse
nerve net (Mackie, 2004). This nerve ring reflects
a considerable degree of centralization. Also, the
nervous system of deuterostome enteropneusts
exhibits aspects of both central and diffuse organ-
ization (reviewed and discussed in Holland, 2003).
On the one hand, enteropneusts have axons tracts
that run along the longitudinal body axis and show
a strong concentration of neurons in the anterior
part of the body, reflecting nervous integration.
On the other hand, enteropneusts have a ‘nerve
net’ interconnecting the cell bodies, dendrites, and
axons of sensory neurons, interneurons, and motor neurons, and neurons are embedded in the epider- mis, as indicative of a diffuse system, rather than forming an anatomically distinct structure (Lowe et al., 2003).
Given the vast differences in nervous system organization in Bilateria, what can we learn about the urbilaterian nervous system from comparative studies? So far, insight has been limited and propos- als about the complexity and shape of the urbilat- erian nervous system range from ‘diffuse’ (Mineta et al., 2003; Lowe et al., 2006) to ‘centralized’ (Denes et al., 2007). Assuming a diffuse urbilaterian nervous system would imply independent centralization events at least in protostomes and deuterostomes (Holland, 2003; Lowe et al., 2006). Assuming a cen- tralized urbilaterian nervous system, on the other hand, would imply secondary simplification of the nervous system of enteropneusts and of many other invertebrate groups (Denes et al., 2007). These two conflicting hypotheses can now be tested. If cen- tralization occurred independently in protostomes and deuterostomes we would expect the neurode- velopment and molecular architecture of their CNSs to be generally divergent. If instead centralization pre-dated Bilateria, this should be reflected by simi- larities in neurodevelopment and CNS molecular architecture between the bilaterian superphyla.


7.2 Nervous system centralization—the evo-devo approach

A key strategy to unravelling the degree of central- ization that was in place in the urbilaterian nervous system is the comparison of CNS development between protostome and deuterostome groups. However, depending on the amount of evolution- ary change these groups have accumulated, their neurodevelopment will be more or less informative about ancestral characteristics of nervous system centralization in Bilateria. Ancestral features will be most apparent in the neurodevelopment of spe- cies that have changed relatively little during evo- lution and will be modified to a larger extent in faster-evolving species (Raible et al., 2005). Distinct aspects of neurodevelopment are currently under study in a broad range of protostome and deuteros- tome model species:

E V OL UTION OF NE R V OU S S Y STE M C E NTR A LI Z A TION 67



1. Polarized distribution of neuronal precursors with respect to the main body axes. One important aspect of nervous system centralization is the early devel- opmental segregation of the ectoderm into a ‘non- neural’ and a ‘neural’ portion, the neuroectoderm. In bilaterians, the neuroectoderm is located anteri- orly where the brain and associated sensory organs develop, and on the ‘neural’ trunk side which is ventral in most invertebrates and dorsal in verte- brates due to dorsoventral axis inversion (Arendt and Nübler-Jung, 1994; De Robertis and Sasai, 1996; Lowe et al., 2006). What are the signals that polar- ize the bilaterian ectoderm, and to what extent are they comparable between phyla?
2. Subdivision of the neural anlage into regions (‘molecu- lar anatomy’). Another aspect of nervous system centralization amenable to comparative studies is how the developing nervous system relates to the
‘molecular anatomy’ of the body. Bilaterians have in common an early subdivision of the developing embryo (or larva) into regions of distinct molecular identities (St Johnston and Nusslein-Volhard, 1992; Arendt and Nübler-Jung, 1996; Lowe et al., 2003; Schlosser and Ahrens, 2004; Yu et al., 2007; Lowe,
2008). These are referred to as ‘molecular anat- omy’ and can be used as a molecular map. A simi- lar molecular anatomy of the CNS anlage at early developmental stages has been considered a good indication of CNS homology (Arendt and Nübler- Jung, 1996; Lichtneckert and Reichert, 2005). Note, however, that structures that develop from cor- responding regions in two species are not neces- sarily homologous (Lowe et al., 2003; Lowe, 2008). How similar is the molecular anatomy between species, of the whole body, and of the developing CNS in particular, and what is the significance of conserved expression regions for our understand- ing of CNS evolution?
3. Spatial segregation of neuron types in the CNS. Nervous system centralization not only implies local concentration of neurons but also their func- tional and spatial segregation and interrelation (‘operational centralization’). This is exemplified by Herrick’s longitudinal neuron columns in the ver- tebrate spinal cord, which comprise distinct sets of motor- and interneuron types. With the recent progress in the identification of conserved neuron
types by molecular fingerprint comparisons
(Arendt and Nübler-Jung, 1999; Thor and Thomas,
2002; Arendt et al., 2004), and using the conserved
molecular anatomies as universal molecular maps,
the localization and spatial segregation of neu-
ron types can now be compared between remote
bilaterians (Denes et al., 2007; Sprecher et al., 2007;
Tessmar-Raible et al., 2007). To what extent had
neuron types already been spatially arranged in
Urbilateria, and what does this tell about the ances-
tral state of nervous system centralization?


7.2.1 Central nervous systems develop from the non-Dpp body side

In all bilaterian animals investigated (with the exception of the nematodes) the bone morpho- genetic protein (Bmp) signalling system sets up tis- sue polarity along the dorsoventral axis (Mizutani et al., 2005; Lowe et al., 2006; Levine and Brivanlou,
2007; Yu et al., 2007). The Bmp system pre-dates the emergence of the bilaterian CNS (Matus et al.,
2006a; Rentzsch et al., 2006) and was thus in place to be adapted for nervous system centralization, i.e. for the differential distribution of neuronal precur- sors along this axis. How similar is the role of Bmp signalling with respect to nervous system central- ization in various bilaterians?
Whenever a CNS is present, it develops from
the non-Bmp body side, in insects (Mizutani et al.,
2005, 2006), vertebrates (Sasai et al., 1995; Levine
and Brivanlou, 2007), amphioxus (Yu et al., 2007),
and also annelids (Denes et al., 2007). Also, in early
vertebrate (Harland and Gerhart, 1997) and fly
development (Mizutani et al., 2006) the antineuro-
genic activity of Bmps sets the limit of the neuroec-
toderm. These findings first suggested that Bmp
signalling had an ancient role in the overall restric-
tion of neurogenesis to the neural body side (e.g.
Padgett et al., 1993). Yet, this simple notion was not
supported by recent additional comparative data:
in enteropneusts (Lowe et al., 2006) and in poly-
chaetes (Denes et al., 2007), the pan-neural marker
elav is not downregulated by exogenously applied
BMP4. How can we reconcile these findings?
The available data are consistent with a refined
evolutionary scenario, which assumes that in early

68 AN I M AL EV O L UTI O N



bilaterians the antineurogenic effect of Bmp sig- nalling was on specific sets of motor neurons (and interneurons) only, restricting them to the neural body side, while there was a positive effect on the formation of sensory neurons that do not form part of the CNS proper (Rusten et al., 2002). In line with this, Bmp signalling has been shown to trig- ger formation of the peripheral sensory neurons at later developmental stages, at the neural plate border and adjacent lateral placodes in the ver- tebrates (Schlosser and Ahrens, 2004), and in the lateral ‘epidermal’ ectoderm in Drosophila (Rusten et al., 2002). In annelids, the types of sensory neu- rons characterized so far arise from the lateral and dorsal sides as opposed to motor- and interneu- rons that form from the ventral body side (Denes et al., 2007); indeed, exogenous BMP4 strongly upregulates the sensory marker atonal, consistent with a conserved role of Dpp/BMP in the specifi- cation of peripheral sensory neurons (Denes et al.,
2007). Even in enteropneusts, where post-mitotic neurons are spread all around the circumference of the trunk (Lowe et al., 2003), the distribution of motor neuron, interneuron, and sensory neu- ron precursors may not be uniform (Lowe et al.,
2006). For example, there is a small population of putative motorneurons in the ventral ectoderm (expressing conserved motor neuron markers) and motor neurons are reported to be enriched in the ventral axon tract. A more in-depth ana- lysis of the role of Bmp signalling and of other signalling systems active along the dorsoventral axis will elucidate a possible conservation of neu- ron type segregation in annelid and enteropneust neurodevelopment.
Our revised scenario—that the ancestral role of Bmp signalling was to promote sensory neu- ron over motor neuron fates, rather than a general antineurogenic effect—fits well with the actual dis- tribution of motor and sensory neurons in many invertebrates, where it appears to be the rule rather than the exception that sensory neurons emerge outside of the neuroectoderm on the ‘non-neural’ (=‘Dpp/Bmp’) body side. If this were indeed an ancestral bilaterian trait this would imply that a certain degree of centralization was present in Urbilateria (i.e. the sorting out of motor versus sen- sory neurons along the secondary body axis).
7.2.2 A conserved pattern of mediolateral regions extending from head to trunk

To estimate the complexity of the urbilaterian CNS, we need to know the complexity of the underlying molecular anatomy that was in place in Urbilateria. Although comparative studies have addressed this for both the antero-posterior (Slack et al., 1993; Schilling and Knight, 2001) as well as for the mediolateral (dorsoventral = neural/non- neural) axes (Cornell and Ohlen, 2000), our focus here is on mediolateral patterning. Previous com- parisons of the molecular anatomy of the insect and vertebrate neuroectoderm had revealed a similar mediolateral sequence of nk2.2+, gsx+, and msx+ neurogenic domains (reviewed in Arendt and Nübler-Jung, 1999, and Cheesman et al., 2004) that also extends into the brain anlage (Urbach and Technau, 2003a,b; Sprecher et al., 2007). Notably, in the developing forebrain, medial nk2.2 expression is complemented by the medial expression of its sister gene, nk2.1 (Zaffran et al., 2000). Nk6 genes also play a conserved role in mediolateral pattern- ing because the neuroectodermal expression of the Drosophila orthologue shows medial restriction as observed in the vertebrates (Cheesman et al., 2004). Our recent work on the mediolateral anatomy of the developing annelid nerve cord has revealed an even higher degree of conservation in mediolateral patterning (Figure 7.2). In addition to the previously detected protostome–deuterostome similarities, we find that annelids and vertebrates share a pax6+ column at similar mediolateral level that likewise extends up to the forebrain (violet in Figure 7.2; see Plate 5) (Denes et al., 2007). In both groups the med- ial portion of the pax6+ column overlaps the nk6+ column (yellow in Figure 7.2). In addition to this, annelids and vertebrates share a lateral pax3/7+ column (green in Figure 7.2; note that this gene is expressed strictly segmentally in the Drosophila neuroectoderm; Davis et al., 2005). Our data also revealed that the positioning of the gsx+ column is more variable than initially assumed and the vertebrate dbx+ interneuron columns are prob- ably vertebrate-specific evolutionary acquisitions
(Denes et al., 2007).
The conservation of mediolateral columns
between vertebrates, annelids, and (to a lesser

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7.2.3 Conserved neuron types develop from similar mediolateral progenitor domains











Figure 7.2 Comparison of mediolateral neurogenic columns across Bilateria. Expression of nk2.2/nk2.1) (orange; Shimamura et al., 1995), Nk6 (yellow; Rubenstein et al., 1998), Pax6 (violet; Mastick et al., 1997; Urbach and Technau, 2003a,b), gooseberry/ Pax3/7 (green; Matsunaga et al., 2001; Puelles et al., 2003),
and msh/Msx (blue; Shimeld et al., 1996) orthologues in the neuroectoderm of Drosophila, Platynereis, and mouse (left to right) at pre-differentiation stages. The Drosophila (left) and
Platynereis (centre) schematics represent ventral views, the mouse (right) is a dorsal view with the neural tube unfolded into a neural plate for better comparison. Neurogenic columns are demarcated by expression boundaries and represent cells with a unique combination of transcription factors. All expression patterns are symmetrical but are shown on one side only for clarity. (See also Plate 5.)



extent) insects is in stark contrast to the situation in enteropneusts, where similar columns have not been observed, with the exception of the dorsal dll+ column and the ventral midline column (Lowe et al., 2006; Lowe, 2008).
Two conclusions can be drawn. First, if the complex molecular mediolateral anatomy shared between annelids and vertebrates is indeed due to evolutionary conservation—and this notion seems inescapable given the overall complexity of this pattern (Figure 7.2)—it must have been pre- sent in Urbilateria. The immediate question then arises: what was the difference in developmen- tal fate between these regions in Urbilateria? One plausible scenario is that these regions gave rise to distinct and segregated ancestral neuron types, as will be discussed in the next section. Second, these findings suggest that the mediolateral molecular anatomy in enteropneusts is secondarily simpli- fied (Denes et al., 2007), consistent with the notion of evolutionary loss in a slowly evolving species (see discussions in Lowe et al., 2006, and Denes et al., 2007).

In insects and vertebrates, neuron types emerging from the medial nk2.2+ column pioneer the medial longi- tudinal fascicles as well as peripheral nerves (Arendt and Nübler-Jung, 1999, and references therein). Among these, the neuron populations that send out ascending and descending projections in the vertebrate hind- brain are serotonergic and they modulate spontan- eous locomotor activity (Briscoe et al., 1999; Schmidt and Jordan, 2000; Pattyn et al., 2003). In Platynereis, serotonergic neurons likewise emerge from the med- ial nk2.2 columns and pioneer the longitudinal tracts and segmental nerves (red in Figure 7.3; see Plate 6) (Denes et al., 2007). One type of serotonergic neuron also emerges from the nk2.1+ brain regions, as evi- denced for Platynereis and fish (Tessmar-Raible et al.,
2007) as well as sea urchin (Takacs et al., 2004).
The nk2.1+ region in the developing forebrain of
vertebrates and annelids gives rise to another con-
served neuron type: early differentiating neurose-
cretory cells that synthesize the highly conserved
neuropeptide arg-vasotocin/neurophysin (orange
in Figure 7.3). These cells form in the vicinity of
ciliated photoreceptor cells in the brain that share
the expression of rx and of c-opsin orthologues in
vertebrates and annelids (white in Figure 7.3) and
of molecular clock cells positive for bmal/cycle
(green in Figure 7.3) (Arendt et al., 2004).
Somatic motor neurons exhibit the same tran-
scription factor signature (hb9+, lim3+, islet-1/2+)
in insects, nematodes, and vertebrates (Thor and
Thomas, 2002). In the vertebrates, these neurons
are cholinergic and emerge from the pax+, nk6+
progenitor domain (violet in Figure 7.3) (Ericson
et al., 1997). We found that the same is true for
Platynereis, where the first cholinergic motor neu-
rons that innervate the longitudinal musculature
have the same transcription factor signature and
emerge from the pax6+, nk6+ column (Denes et al.,
2007; AD, GJ and DA, unpublished).
Taken together, these data identify a considerable
number of conserved neuron types that emerge
from similar molecular coordinates in annelids
and vertebrates. Obviously, this comparison is far
from complete and awaits further characterization
and localization of neuron types in both taxa.

70 AN I M AL EV O L UTI O N





Figure 7.3 Conserved neural cell types in annelids and vertebrates. The neuron types emerging from homologous regions in the molecular coordinate systems in annelids and vertebrates and expressing orthologous effector genes are marked with the same colour. Homologous cell types include the molecular clock cells positive for bmal (dark green), ciliary photoreceptors positive for c-opsin and rx (white), rhabdomeric photoreceptors positive for r-opsin, atonal, and pax6 (yellow), vasotocinergic cells positive for nk2.1, rx, and otp (orange), serotonergic cells positive for nk2.1/ nk2.2 (red), cholinergic motor neurons positive for pax6, nk6, and hb9 (violet), interneurons positive for dbx (pink), as well as trunk sensory cells positive for atonal and msh (light blue). (See also Plate 6.)



As for the peripheral nervous system, we have so far identified and compared rhabdomeric photo- receptor cells in annelids and retinal ganglion cells in vertebrates (yellow in Figure 7.3) that form from the eye anlage in both species (dashed circles in Figure 7.3). In the trunk we found some conserved sensory neuron types that emerge from similar lat- eral molecular coordinates in annelids and verte- brates (blue in Figure 7.3) (ath+ or trpv+) (Denes et al., 2007); this comparison is ongoing.


7.3 Reconstructing the urbilaterian nervous system

In conclusion, the comparison of neurodevelop- ment in protostome and deuterostome animal models reveals a conserved molecular architecture
of considerable complexity that was inherited from the Urbilateria. Beginning with a diffuse nerve net with homogeneously distributed neuron types, a first segregation of motor and sensory neurons occurred along the dorsoventral axis in the line of evolution leading to the bilaterians. This involved Bmp signalling and possibly other signalling cas- cades. These signals established a refined medi- olateral molecular anatomy, involving at least four longitudinal neurogenic regions with distinct molecular identities (nk2.2+/nk6+, pax6+/nk6+, pax6+/pax3/7+, msx+/pax3/7+; Figure 7.2) that gave rise to spatially segregated neurons. Among these were medial serotonergic neurons, intermediate cholinergic motor neurons, some sort of interneu- rons and lateral sensory neurons (Figure 7.3) (Denes et al., 2007). These neuron types presum- ably controlled ancestral locomotor patterns such as undulatory swimming and/or peristalsis. In the head region, specialized light-sensitive cell types evolved, integrating different kinds of photic input to set the molecular clock and to control neurose- cretory and motor output (Tessmar-Raible et al.,
2007). While this already reflects a considerable degree of nervous system centralization that pre- sumably was in place in Urbilateria, a renewed push in research combining developmental genet- ics with classical neuroethology in slowly evolving protostomes and deuterostomes will be needed to refine and complete this picture.


7.4 Acknowledgements

We thank an anonymous reviewer for very valu- able comments. This work was supported by grants from the Marine Genomics Europe Network of Excellence [NoE-MGE (DA), GOCE-04–505403 (DA and FR)], fellowships of the Boehringer Ingelheim Foundation and of the Marie Curie RTN ZOONET [MRTN-CT-2004–005624 (KT-R)], and the Deutsche Forschungsgemeinschaft (Deep Metazoan Phylo- geny; DA:Ar387/1–1 and HH: Ha4443/1–1). AD was supported by a Louis Jeantet Foundation fellowship.