The Evolution of Nervous System Patterning: Insights from Sea Urchin Development Lynne M

REVIEW 3613

The evolution of nervous system patterning: insights from sea urchin development Lynne M. Angerer1,*, Shunsuke Yaguchi2, Robert C. Angerer1 and Robert D. Burke3

Summary Box 1. Glossary Recent studies of the sea urchin embryo have elucidated the Aboral ectoderm. Ectoderm on the dorsal side of the embryo and mechanisms that localize and pattern its nervous system. These a small region anterior to the anus on the ventral side that together studies have revealed the presence of two overlapping regions form a squamous epithelium. of neurogenic potential at the beginning of embryogenesis, Anterior neuroectoderm (ANE). Ectoderm that is derived from each of which becomes progressively restricted by separate, yet the animal pole region of the egg, has the potential to produce linked, signals, including Wnt and subsequently Nodal and nerves, and is restricted by canonical Wnt signaling-dependent BMP. These signals act to specify and localize the embryonic processes to the anterior end of the embryo. Ascidians. A class of urochordates, also known as sea squirts, that neural fields – the anterior neuroectoderm and the more are marine filter-feeders. posterior ciliary band neuroectoderm – during development. Bilaterians. Organisms with bilateral symmetry that arose from pre- Here, we review these conserved nervous system patterning bilaterians and include all the remaining animal phyla divided into signals and consider how the relationships between them two major groups: the protostomes and deuterostomes. might have changed during deuterostome evolution. Centralized nervous system. A system of nerves that is restricted to specific regions of ectoderm, such as the dorsal nerve cords of Key words: Wnt, TGF, Nodal, BMP, Dorsal-ventral axis, Anterior- vertebrates and ventral nerve cords of arthropods, or the ANE and posterior axis, Lefty, Chordin, Six3, FoxQ2, Ciliary band, Apical CBE of sea urchin embryos. organ, Ectoderm patterning, Neural development, Neural Cephalochordates. One of the three chordate subphyla, along patterning with vertebrates and urochordates. A modern representative is amphioxus, or lancelet. Ciliary band ectoderm (CBE). Ectoderm that has the potential to Introduction produce nerves and is restricted by TGF signaling to a narrow band Rapid progress has been made in the last five years in understanding of cells (CB) between ventral and dorsal ectoderm. the mechanisms that govern ectoderm, and hence nervous system, Cnidarians. A sister group to the bilateria. Includes corals, sea patterning in sea urchin embryos. These findings have revealed that anemones, jellyfish and hydroids. a fascinating cascade of Wnt-dependent pathways functions during Ctenophores. Commonly known as comb jellies, a pre-bilaterian sea urchin development to regulate Nodal and bone morphogenetic phylum of marine animals that use groups of cilia (‘combs’) to swim. protein (BMP) signaling. Together, these restrict the neurogenic Diffuse nervous system. A system of nerves that is distributed capacity that is initially present throughout the entire embryo to throughout the ectoderm and can form a network of specific locations, generating first neuroectoderm at the anterior end interconnected neurons, such as is found in pre-bilaterians. Deuterostome. Organisms in which the blastopore becomes the of the embryo [the anterior neuroectoderm (ANE), see Glossary, Box anus and a second opening forms the mouth; coeloms develop as 1] and then, within and along a band of ciliated ectoderm, the ciliary hollow outgrowths of the gut. Deuterostomes mentioned here band ectoderm (the CBE, see Glossary, Box 1). These neuroectoderm include echinoderms (sea urchins), hemichordates (Saccoglossus territories are established at different times during development by kowalevskii, Ptychodera flava), cephalochordates (amphioxus), molecularly distinct signaling mechanisms in sea urchin embryos and urochordates (ascidians) and vertebrates (fish, frog, chick, mouse). neural patterning occurs in the absence of any complex tissue layer Hemichordates. A phylum of marine deuterostomes, considered movements or interactions, factors that facilitate a clearer to be a sister group of the echinoderms. understanding of how embryonic nervous systems are shaped by Pre-bilateria. Organisms with radial symmetry; extant members these signals. Importantly, because the sea urchin shares a common include cnidarians, ctenophores and sponges. Protostomes. Organisms in which the blastopore becomes the ancestor with the chordates, analyses of the mechanisms that pattern mouth and coeloms develop from solid masses of mesodermal cells. the sea urchin nervous system can reveal the shared and derived Protostomes mentioned here include ecdysozoans (flies) and features of chordate neural patterning, providing us with insights into lophotrochozoans (polychaete annelids and snails). the evolution of deuterostome (see Glossary, Box 1) nervous systems. Urbilaterian. Refers to the hypothetical last common ancestor of The organization of the nervous system is a defining feature of bilaterians. an animal body plan. Vertebrates and flies possess a centralized nervous system (see Glossary, Box 1), whereas a more primitive and diffuse nervous system (see Glossary, Box 1) is characteristic of pre-bilaterians (see Glossary, Box 1), including cnidarians (see Glossary, Box 1) (Marlow et al., 2009) and ctenophores (see Glossary, Box 1). Transforming growth factor  (TGF) signaling 1National Institute for Dental and Craniofacial Research, National Institutes of centralizes the nervous system along the dorsal side in vertebrates Health, Bethesda, MD 20892, USA. 2Shimoda Marine Research Center, University of and along the ventral side in flies, suggesting that the common Tsukuba, Shimoda 415-0025, Japan. 3Biochemistry/Microbiology Department, bilaterian (see Glossary, Box 1) ancestor had a centralized system University of Victoria, Victoria V3P 5C2, Canada. that was sculpted by the same patterning mechanism (Arendt et al.,

*Author for correspondence ([email protected]) 2008; Denes et al., 2007; Mizutani and Bier, 2008). DEVELOPMENT 3614 REVIEW Development 138 (17)

By contrast, a great deal of progress has been made in recent years in elucidating the mechanisms that pattern the neural and non-neural ectoderm territories in embryos of the sea urchin, which mM represents another basal deuterostome. Although a sister phylum of the hemichordates, the echinoderms have routinely been excluded from studies of the origins of the chordate nervous system, largely because their adult body plans lack apparent AP polarity and are thought to be highly derived. The nervous system of echinoderms has also been said to be diffuse throughout D E F development. However, sea urchin embryos clearly have AP as m well as DV polarity, with nerves organized into highly restricted m patterns within the ectoderm, as is the case for centralized nervous a m systems (Fig. 1). Importantly, the creation of these localized LR neuroectoderm domains is now understood at a mechanistic level, owing to recent functional studies. a Here, we review the signals and mechanisms that act to specify and localize the anterior and ciliary band neural ectoderm fields (the ANE and the CBE) during sea urchin embryo development. Key Animal plate Ciliary band Serotonergic neurons We also compare these neural patterning mechanisms with those Ventral ectoderm Blastoporal endoderm- adjacent/dorsal ectoderm used by vertebrates, highlighting the shared features and signaling pathways utilized. Finally, based on these comparisons, we discuss the evolution of these neural patterning mechanisms and propose Fig. 1. The organization of the sea urchin embryo nervous that, although the same mechanisms might have been used system. (A-C)Sea urchin larvae showing the position of neurons (magenta) and serotonergic neurons (green). (A)Ventral/posterior view. throughout evolution, the regulatory relationships between these Bilateral symmetry is indicated by the dashed line; the left (L) and right signaling mechanisms have changed. (R) sides of the larva are marked. Cell nuclei are labeled in blue. (B)Lateral view indicating the embryonic AP and DV axes (see Box 2 for The primary (AP) and secondary (DV) axes of the explanation). (C)Ventral view, indicating the position of the mouth (m). sea urchin embryo Several examples of cell bodies are indicated (arrows). Scale bar: 20m. Because the patterning of ectoderm into neural and non-neural fields (D-F)Schematic representations of the larvae shown in A-C, in the sea urchin embryo is intimately connected to the initial highlighting regions of the ectoderm and the positions of the anterior mechanisms that control cell fate specification along the body axes, neuroectoderm containing the animal plate (red), the ciliary band we begin with a brief review of these axes and their developmental neuroectoderm (green), mouth (m) and anus (a). properties. An explanation for the axial designations used in this review and a comparison of these designations with other commonly used nomenclatures for these axes are provided in Box 2. The fact that nervous systems are localized to either the dorsal (as in vertebrates) or ventral (as in flies) side of embryos, with The ectoderm, endoderm and mesoderm are arrayed along the position of the mouth defining the ventral side, sparked an the AP axis interesting and strongly debated hypothesis that the dorsal- The developmental capacities of the animal and vegetal halves of ventral (DV) axis inverted during evolution (De Robertis and the fertilized sea urchin egg differ, implying that the animal-vegetal Sasai, 1996; Gerhart, 2000), which led investigators to examine (AV) axis is established during oogenesis; isolated animal halves evolutionarily more basal organisms, such as ascidians and the become permanent blastulae in which many neurons develop, hemichordates (see Glossary, Box 1). Attention was focused on whereas the vegetal halves often form complete embryos the Saccoglossus kowalevskii hemichordate embryo, which was (Hörstadius, 1973; Wikramanayake and Klein, 1997; Yaguchi et al., considered to represent a good basal model for chordate nervous 2006). The egg AV axis corresponds to the primary axis of the system patterning because its anterior-posterior (AP) gene embryo, which extends from the animal pole of the embryo to the expression patterns are the same as those observed in chordate blastopore (see Box 2), which is the prospective anus of the larva. embryos (Lowe et al., 2003). However, the nervous system of The prospective ectoderm, endoderm and mesoderm are arrayed Saccoglossus embryos is, in part, diffuse rather than centralized, along this axis (see Box 2). The mouth forms as a secondary and nerve development was found to be insensitive to changes opening which, along with bilateral symmetry (Fig. 1A), is a in BMP signaling, in contrast to other organisms (Lowe et al., hallmark feature of deuterostomes. 2006). Although part of the nervous system of the Saccoglossus embryo is distributed throughout the epidermis, other Posterior signals are required for mesoderm and components are localized along both the dorsal and the ventral endoderm specification midlines (Bullock and Horridge, 1965; Knight-Jones, 1952; In the last decade, a paradigm has emerged that early canonical Nomaksteinsky et al., 2009). These localization patterns are also Wnt signaling establishes AP polarity in bilaterians and marks the found in the nervous system of another enteropneust site of gastrulation (Petersen and Reddien, 2009). In sea urchin hemichordate adult, Ptychodera flava, as judged by the embryos, canonical Wnt signaling also plays a role in specifying expression patterns of several neural marker genes mesoderm and endoderm fates in blastomeres derived from the (Nomaksteinsky et al., 2009). However, very little is understood vegetal regions of the egg and, based on this, the vegetal pole of about the molecular mechanisms that pattern the nervous the sea urchin embryo can be considered analogous to the posterior

systems of hemichordate embryos. of vertebrate embryos (see Box 2). DEVELOPMENT Development 138 (17) REVIEW 3615

In sea urchins, canonical Wnt signaling is first detectable as a wave of nuclear -catenin that begins at the 16-cell stage in the Box 2. A note on axis designations most posterior cells, which are called the micromeres, and passes A Embryo B Larva during the next two cleavage stages through concentric tiers of Primary axis A A progressively less posterior cells that will form mesoderm, endoderm and a small part of the ectoderm (Logan et al., 1999; Wikramanayake et al., 1998) (Fig. 2A). Micromeres are sufficient Ectoderm Secondary V to induce endomesoderm because, when transplanted to the anterior axiss D end, they induce these tissues in presumptive ectoderm (Hörstadius, VDNodal BMP2/4BMP2/4

1973; Minokawa and Amemiya, 1999; Ransick and Davidson, Endoderm 1995). Four known micromere signals, all of which depend on Mesoderm cWnt canonical Wnt signaling, are known to exist. These include Delta, an activator of the Notch signaling pathway (Sherwood and P McClay, 2001; Sweet et al., 2002), ActivinB (Sethi et al., 2009), P Wnt8 (Minokawa et al., 2005; Smith et al., 2008; Smith et al., Classically, the vertebrate egg axis has been referred to as the animal- 2007; Wikramanayake et al., 2004) and an undefined fourth signal vegetal (AV) axis; the embryo develops primarily from the animal half, that downregulates the transcription factor SoxB1, which is a whereas the vegetal half contains most of the yolk. However, the negative regulator of canonical Wnt signaling in mesoderm tissues primary axis of an early vertebrate (e.g. Xenopus) embryo does not (Oliveri et al., 2003; Sethi et al., 2009). The most upstream align with the egg AV axis and is instead referred to as the anterior- component of the canonical Wnt signaling pathway to be identified posterior (AP) axis, with the head marking the anterior and the thus far is Dishevelled (Dsh), which is highly concentrated in blastopore marking the posterior. By contrast, in sea urchins the vegetal egg cortex (Byrum et al., 2009; Leonard and Ettensohn, blastopore forms in the region corresponding to the egg vegetal pole, 2007; Weitzel et al., 2004). Elucidation of the importance of leading most investigators to use AV for both the egg and embryonic canonical Wnt signaling in endomesoderm development and primary axes. Because the early molecular mechanisms that pattern gastrulation led to large-scale screens to identify and construct the sea urchin embryos along this axis (A; canonical Wnt, blue triangle) are similar to those operating along the vertebrate AP axis, in this gene regulatory networks controlling these processes (Davidson, review we refer to the sea urchin embryonic axis as primary or AP. 2009; Davidson et al., 2002; Oliveri et al., 2008). The most commonly used designation for the orthogonal, secondary axis of the sea urchin embryo has been oral-aboral. However, again The secondary (DV) axis is patterned by Nodal and BMP2/4 because the mechanisms used for patterning this axis (A; Nodal, The zygotic expression of Nodal, a member of the TGF yellow arrow; BMP2/4, blue arrow) are similar to those that pattern superfamily, is both necessary and sufficient to specify ventral the DV axes of other deuterostomes, we refer to this axis as dorsal- structures and to initiate cell fate specification along the secondary ventral (DV), with the dorsal and ventral surfaces corresponding (DV) axis of sea urchin embryos (Duboc et al., 2004). During late approximately to the aboral and oral tissue territories. As the embryo cleavage stages, nodal transcription is restricted to the presumptive develops to the pluteus larva stage, the gut bends to form the mouth ventral ectoderm (see Box 2), where it upregulates its own and skeletogenesis supports expansion of the dorsal ectoderm, creating the longest dimension of the larva, which has been referred expression and that of four key genes – bmp2/4, chordin, lefty and to as the AP axis. However, this axis does not align with the earlier gsc – all of which play major roles in cell fate specification and signaling axes (B; green and pink arrows) that establish the body plan patterning along the secondary axis (Angerer et al., 2001; Angerer and the organization of the embryonic nervous system. Therefore, et al., 2000; Bradham et al., 2009; Duboc et al., 2004; Lapraz et al., here we use only the early AP and DV axial designations to compare 2009; Saudemont et al., 2010) (Fig. 2B). The mechanism that the mechanisms that control the positioning of the nervous systems controls the spatially restricted expression of nodal is not yet in sea urchin and vertebrate embryos. completely understood, but might be linked to a maternal redox gradient along the secondary axis (Coffman et al., 2004). In the unfertilized egg, mitochondria are more abundant on the future confined to within several cell diameters of its initial expression ventral side, and when they are artificially transplanted to the site. In the sea urchin embryo this is in ectoderm on the ventral side prospective dorsal side the secondary axis often is reversed (Yaguchi et al., 2007), restricted there presumably because Lefty (Coffman et al., 2009). In addition, the redox-sensitive kinase p38 diffuses further than Nodal and effectively interferes with Nodal is required for secondary axis polarity (Bradham and McClay, autoregulation (Bolouri and Davidson, 2009; Duboc et al., 2008) 2006) and cis-regulatory studies have identified several cis (Fig. 2B). elements within the nodal promoter that may bind transcription BMP2/4, another member of the TGF superfamily, is factors, the orthologs of which are known to be redox sensitive functionally polarized along the secondary axis of sea urchins, as (Nam et al., 2007; Range et al., 2007). However, redox-sensitive in all other bilaterians (Lapraz et al., 2009). It is transcribed only activation of a transcription factor required for nodal transcription on the ventral side of embryos under the control of Nodal, as is the in the sea urchin embryo has not yet been demonstrated. Two other TGF antagonist Chordin (Angerer et al., 2000; Bradham et al., ubiquitous factors required to initiate nodal expression are Univin, 2009; Duboc and Lepage, 2006; Duboc et al., 2004). However, a member of the growth differentiation factor (GDF) subfamily of BMP2/4 functions to specify aboral ectoderm (see Glossary, Box TGFs, and the transcription factor SoxB1 (Range et al., 2007). As 1) on the opposite side of the embryo (Angerer et al., 2000; in other organisms, strong autoregulation of nodal is thought to Bradham et al., 2009; Duboc et al., 2004; Lapraz et al., 2009). amplify a small initial asymmetry to lock down its localized BMP2/4 has been shown to be part of a relay mechanism from expression, which is then maintained by Lefty, a direct Nodal target Nodal to the dorsal side, as cell-autonomous activation of the and Nodal signaling antagonist (Duboc et al., 2008; Meno et al., Nodal signaling pathway in only one blastomere of an 8-cell

1996; Thisse and Thisse, 1999). Consequently, Nodal function is embryo results in the transcription of bmp2/4, which is sufficient DEVELOPMENT 3616 REVIEW Development 138 (17)

A B signature of the sea urchin ANE is similar to that of vertebrate A A Nodal anterior neuroectoderm. After ANE restriction, most of the remaining ectoderm retains a regulatory state that can support the

Lefty BMP2/4 development of posterior neuroectoderm. However, this neuroectoderm is subsequently restricted to a narrow strip of cells Lefty BMP2/4 (the ciliary band or CB) as the CBE. The expression of Hnf6, the Chordin earliest known CBE marker gene, indicates that the CB is specified Chordin by the end of the mesenchyme blastula stage at the border between ventral and dorsal ectoderm tissues, which are diverted to an Wnt epidermal fate by Nodal and BMP2/4, respectively, as described P P above. V D C D Canonical Wnt-dependent processes position the ANE at A Six3 A the animal pole FoxQ2 Nodal When canonical Wnt signaling in sea urchin embryos is blocked, either by removing the posterior half of the cleavage-stage embryo

BMP2/4 or by inhibiting -catenin nuclearization, a remarkable phenotype X is produced: the embryos consist almost entirely of ANE that X contains many serotonergic neurons, a neural cell type that in normal three-day embryos develops only in the ANE (Yaguchi et cWnt cWnt al., 2006). This observation suggested that Wnt signaling represses ANE specification in posterior ectoderm and led to genome-wide P P screens for candidate ANE regulatory genes that are repressed, V D V D rather than activated, by canonical Wnt signaling in this ectoderm Key Animal plate Pre-ciliary band ectoderm Endoderm/mesoderm (Wei et al., 2006; Wei et al., 2009). This resulted in the discovery (ciliary band in B) that the transcription factor Six3 is necessary to drive ANE fate Ventral ectoderm Dorsal ectoderm Blastoporal endoderm- adjacent ectoderm and is sufficient to greatly expand this domain throughout the embryo (Wei et al., 2009). The transcription of six3 and other early Fig. 2. Signals that specify cell fate along the sea urchin ANE factors is initially activated broadly in the cleavage-stage developmental axes. (A)Beginning at the 16-cell stage, a wave of embryo, presumably by maternally encoded factors, and is canonical Wnt signaling (blue arrow) passes through successive tiers to subsequently repressed by canonical Wnt-dependent mechanisms specify mesoderm and endoderm during subsequent cleavages. (Wei et al., 2009; Yaguchi et al., 2008) at the early blastula stage Anterior neuroectoderm (ANE) fate is eliminated from the remainder of in all regions except the ANE (Fig. 2C). Six3 is required for the the ectoderm, much of which has a pre-ciliary band neuroectoderm formation of all ANE nerves and also for the expression of a large (CBE) fate. (B)TGF signaling specifies ectodermal fates along the DV number of downstream early ANE regulatory genes, many of axis, beginning with Nodal, which specifies ventral ectoderm. Nodal is which have orthologs that are expressed in the vertebrate forebrain necessary for the subsequent expression of BMP2/4, which specifies (Wei et al., 2009). Thus, much of the regulatory apparatus of the dorsal ectoderm and ectoderm adjacent to the blastopore endoderm. sea urchin embryo ANE corresponds to that of the anterior Nodal upregulates its own expression and induces the expression of Lefty (a Nodal antagonist) and Chordin (a BMP antagonist), which ectoderm of higher vertebrates, further supporting the view that together protect the ciliary band (green strip) from the epidermis- the sea urchin primary axis is functionally analogous to the AP promoting influences of Nodal and BMP2/4. (C)Wnt-dependent axes of other embryos. Furthermore, like the vertebrate forebrain, processes determine the anterior position of the ANE (red) through an the ANE expresses several Six3-dependent orthologs of known unknown intermediate process ‘X’. Mutual antagonism between canonical Wnt antagonists, and Six3 misexpression can suppress canonical Wnt-dependent and Six3-dependent processes is thought to Wnt ligand transcription (Wei et al., 2009). Thus, canonical Wnt determine the border of the ANE. (D)Canonical Wnt signals support signaling and Six3 affect ANE development in reciprocal ways: the elimination of FoxQ2 from the ectoderm except at the anterior end misexpression of stabilized -catenin or loss of Six3 eliminates of the embryo, probably through the same intermediate process ‘X’ as nerves, whereas misexpression of Six3 or loss of canonical Wnt in C. This allows Nodal signaling to increase through autoregulation to signaling greatly expands the ANE and increases the number of levels sufficient to initiate DV axis patterning. cWnt, canonical Wnt. nerves (Wei et al., 2009; Yaguchi et al., 2006). These studies suggest that the balance between Six3-dependent and canonical Wnt-dependent processes regulates the size of the ANE domain to rescue patterning along the entire secondary axis (Lapraz et al., (Fig. 2C). 2009). BMP2/4 signaling is blocked on the ventral side, where it is inhibited by Chordin (Lapraz et al., 2009; Saudemont et al., 2010), Nodal and BMP2/4 signaling position the CBE and moves to the dorsal side, possibly aided by Chordin, as has The CB is specified as a 4- to 5-cell-wide strip of cells that is been shown in other embryos (Shimmi et al., 2005). carved out, in part, by converting regions of the dorsal and ventral ectoderm to an epidermal fate (Duboc et al., 2008; Duboc et al., Positioning the ANE and the CBE 2004; Lapraz et al., 2009; Saudemont et al., 2010). The earliest Canonical Wnt signals not only specify mesoderm and endoderm molecular evidence of CB formation is the expression of the tissues along the AP axis and promote gastrulation at the posterior transcription factor Hnf6 (or onecut) within the presumptive CB end of the embryo, but they are also required to restrict the domain during mesenchyme blastula stages (Otim et al., 2004; Poustka et

of ANE to the opposite end of the embryo. The regulatory protein al., 2004), several hours after Nodal and BMP2/4 signaling have DEVELOPMENT Development 138 (17) REVIEW 3617 begun, as indicated by phospho-Smad2/3 and phospho-Smad1/5 continues to be expressed ectopically throughout the expanded accumulation, respectively (Bergeron et al., 2010; Lapraz et al., ANE. If it is also removed from these embryos, nodal expression 2009; Yaguchi et al., 2007). When Nodal (and consequently and secondary patterning are restored, allowing the formation of a BMP2/4) signals are eliminated, the expression domain of Hnf6 CB (Yaguchi et al., 2008). This double-repression mechanism of and those of many other genes that are normally restricted to the canonical Wnt signaling downregulating FoxQ2, which suppresses CB expand into the presumptive ventral and dorsal ectoderm nodal, ensures that, in the absence of any embryonic signaling, the (Bradham et al., 2009; Duboc et al., 2004; Yaguchi et al., 2010a; ANE fate is downregulated in posterior ectoderm precursors before Yaguchi et al., 2006; Saudemont et al., 2010), suggesting that the they are subject to secondary axis patterning. In the normal regulatory state in the absence of TGF signaling supports a CB- embryo, FoxQ2 retains its ability to suppress Nodal autoregulation like fate, as originally proposed by Lepage and colleagues (Duboc in the ANE, thereby helping to maintain its neuroectodermal fate. et al., 2004). Similarly, when only BMP2/4 signaling is eliminated, the expression of CBE markers expands into the dorsal ectoderm, Nervous system development within the ANE and but not into the ventral ectoderm where Nodal signaling remains CBE territories active (Lapraz et al., 2009; Yaguchi et al., 2010a; Saudemont et al., The specification of the localized neuroectoderm territories, the 2010). The altered expression of CBE markers in response to these ANE and CBE, is completed in the blastula stages (18-27 hours). perturbations is not observed in the most posterior ectoderm Neuronal precursors are present during late blastula and gastrula adjacent to the blastopore (Saudemont et al., 2010; Yaguchi et al., stages and appear to differentiate and become functional in early 2010a). Because this region of ectoderm derives from posterior larval stages (55-96 hours). As in flies and vertebrates, the blastomeres, in which canonical Wnt signaling is active during regulation of neuronal differentiation within the sea urchin ANE cleavage stages, it is likely to be regulated, at least in part, by and CBE territories involves Delta-Notch-mediated lateral distinct signals. inhibition (Wei et al., 2011). Not all of the cell types within the The Nodal and BMP signaling antagonists Lefty and Chordin are ANE and CBE have been defined. The ANE contains serotonergic thought to prevent conversion of the CBE to epidermal fates. In and non-serotonergic neurons that express Synaptotagmin B and Lefty morphants, Nodal expression expands, ventralizing the some cells that produce long, immotile cilia (Hörstadius, 1939), embryo and eliminating the CBE except in the ANE (Duboc et al., which might have sensory function, whereas the CBE is 2008; Saudemont et al., 2010; Yaguchi et al., 2010a). Conversely, predominantly composed of specialized ciliated cells and functions in Lefty-misexpressing embryos, the CBE is radialized, as is also as a swimming and feeding organ (Strathmann, 1975). observed in Nodal morphants. In Chordin morphants, the secondary axis patterning mechanism is also compromised because Nerves within the ANE elevated, ectopic BMP2/4 signaling on the ventral side interferes The ANE territory is currently thought to consist of two parts: a with Nodal expression, causing an expanded CBE phenotype central disk called the animal plate and a surrounding torus of cells. (Lapraz et al., 2009; Saudemont et al., 2010). By contrast, in The first nerves to form are the serotonergic neurons, which appear Chordin-misexpressing embryos, as in embryos lacking BMP after gastrulation is complete (Bisgrove and Burke, 1986; Yaguchi signaling (Lapraz et al., 2009; Yaguchi et al., 2010a), the CBE et al., 2000) (Fig. 3A, hatched green ovals). Serotonergic neurons expands to the dorsal side of the embryo. Thus, Nodal and BMP are restricted to the dorsal margin of the animal plate as a inhibit CBE development, whereas Lefty and Chordin support it by consequence of Nodal-mediated suppression of their development inhibiting the inhibitors of CBE development. An interesting, but on the ventral side (Yaguchi et al., 2007) (Fig. 2B, Fig. 3B). Later, yet unanswered, question is how these diffusible signals and additional non-serotonergic nerves expressing Synaptotagmin B antagonists precisely control the position and the width of the CB (Burke et al., 2006) appear around the animal plate and form axons in the normal embryo. and dendrites. In summary, the ANE is established first at the animal pole The expression of ANE marker genes during blastula stages during cleavage stages by a balance of opposing canonical Wnt- indicates that the anterior ectoderm is already molecularly patterned dependent and Six3-dependent processes, whereas the CB is during gastrulation. At the mesenchyme blastula/early gastrula stage, established later at the border between the ventral and dorsal six3 transcripts accumulate primarily at the edge of the animal plate ectoderm by factors that antagonize Nodal and BMP signaling. In where serotonergic and other non-serotonergic neurons will develop, the sea urchin embryo these territories are created at separate times whereas foxQ2 is expressed in the central animal plate region. Six3 and places by distinct mechanisms – properties that have facilitated is necessary for development of the thickened columnar epithelial dissection of the mechanisms driving the development of each. structure of the ANE and for the development of all neurons that develop there (Wei et al., 2009). FoxQ2 is also necessary for Coordination of ANE and CBE positioning mechanisms serotonergic neuron development, as well as for the expression of the The canonical Wnt signaling mechanism that positions the ANE is transcription factor Nk2.1 (Yaguchi et al., 2008), which in turn distinct from the TGF-based mechanism that positions the CBE, controls the expression of a novel, ankryn repeat-containing protein but these mechanisms are connected in a very interesting way. (AnkAT) that is required for the formation of apical tuft cilia (Dunn When canonical Wnt signaling is blocked, secondary axis polarity et al., 2007; Yaguchi et al., 2010b). At the late mesenchyme blastula is also lost. As shown previously (Yaguchi et al., 2008), the loss of stage, cells expressing delta appear in the animal plate (Lapraz et al., this polarity results from a disruption in Nodal autoregulatory 2009; Rottinger et al., 2006; Saudemont et al., 2010; Walton et al., amplification, a key process operating at the top of the secondary 2006) and neuron number is controlled by lateral inhibition (Wei et axis regulatory system. Surprisingly, this blockade requires FoxQ2, al., 2011). All animal plate cells express Hnf6 (Poustka et al., 2004; a transcription factor involved in ANE development (Fig. 2D), Yaguchi et al., 2006) and neuronal 3-tubulin (Duboc et al., 2004; which is initially expressed throughout the anterior half of the Casano et al., 1996). Thus, the ANE is specified by Six3-dependent cleavage-stage embryo and is then progressively restricted to the regulatory factors and behaves like a neural epithelium, with many

ANE. In embryos lacking canonical Wnt signaling, FoxQ2 cells having neural potential. DEVELOPMENT 3618 REVIEW Development 138 (17)

A Ventral/posterior view B Lateral view position of the CBE, which has neurogenic potential, but not the number of nerves that develop within it. In addition, TGF signaling appears to regulate the structure and connectivity of m CB neurons (Yaguchi et al., 2010a). These signaling mechanisms appear to be the same as those used by chordate embryos to m shape their neuroectoderm territories. a A four-step model for nervous system development in sea urchin embryos Based on the studies discussed above, a four-step model for patterning the nervous system of sea urchin embryos is presented a in Fig. 4 and is summarized below. First, zygotic gene expression establishes a pre-ANE gene Key regulatory state throughout the embryonic ectoderm. Initially, the Animal plate Ciliary band Serotonergic neurons early ectoderm displays an ANE bias established by the early broad Ventral ectoderm Blastoporal endoderm- Ciliary band neurons zygotic transcription of six3 and other genes in the ANE gene adjacent/dorsal ectoderm regulatory network. Second, canonical Wnt-mediated signaling from posterior blastomeres downregulates the expression of six3, foxQ2 Fig. 3. The structure of neuroectodermal territories. and other members of the ANE regulatory network, progressively Ventral/posterior (A) and lateral (B) views of a three-day old sea urchin restricting the expression of these genes to the definitive ANE larva. At this stage, the ANE is composed of the animal plate (red) and domain. An additional consequence is that FoxQ2 is removed from a surrounding torus of cells. Within the animal plate are cells with long, all but the anterior ectoderm, allowing nodal autoregulation to occur. immotile cilia that constitute the apical tuft (black lines in B). Serotonergic neurons (hatched green ovals) develop at the dorsal edge Third, Nodal upregulation activates the secondary axial patterning of the animal plate. Neural precursors in the region of the ciliary band mechanism and, together with BMP, suppresses neurogenesis in the are indicated by pink ovals. The positions of the mouth (m) and anus (a) ventral and dorsal ectoderm, thus respecifying these tissues as are indicated. epidermis. A strip of cells between these regions retains the early neuroectoderm specification of the CBE, presumably through the activities of Lefty and Chordin, which suppress Nodal and BMP2/4 Nerves within the CB function in this region. Fourth, nerves develop in the ANE and CBE, The CBE also contains scattered delta-expressing cells and cells that regions that are protected from canonical Wnt and TGF signals. express orthologs of genes that encode neural markers in other The two themes of this model are that the embryo starts with two embryos (Saudemont et al., 2010). Several groups of cells first regulatory layers of broad overlapping neurogenic potential (the appear during gastrulation near the CB, beneath the ventral ectoderm ANE and the CBE), each of which is sequentially downregulated and the lateral sections of the CB, inside the dorsal ectoderm (Fig. by successive and distinct signals. Protection from signaling 3A). Subsequently, ~40-50 regularly spaced neural cell bodies appears to be the principal mechanism that results in restricted connected by processes form a highly structured chain along the regions that differentiate as neuroepithelia. ventral side of the CB (Fig. 1C, Fig. 3B). Axons of these neurons are either bundled in the central tract or they project towards and beneath Deuterostome neural system patterning: shared the dorsal ectoderm (Nakajima et al., 2004) (Fig. 1, Fig. 3B). How mechanisms these CB nerves connect to each other or to nerves in the ANE is The pre-signaling neuroectodermal regulatory states of early unknown. In addition, how the early CB-associated neurons respond embryonic cells and the signaling mechanisms that specify neural to TGF signals is unclear, but in the absence of these signals the versus non-neural fates in the sea urchin are remarkably similar to scattered nerves that develop within the expanded CBE do not form those observed in other deuterostome embryos. The highly ordered interconnecting bundled axonal tracts (Yaguchi et al., 2010a). In localized arrangement of nerves in sea urchin embryos strongly addition, Nodal and BMP signals might play a later role in regulating reinforces the recent findings that hemichordate nervous systems neural identity, as has been found in insects and vertebrates (Mizutani are more centralized (Nomaksteinsky et al., 2009) than previously et al., 2006; Rusten et al., 2002). thought (Lowe et al., 2003). Centralization mechanisms also exist The distribution of nerves within the CBE is controlled by the in the protostome (see Glossary, Box 1) polychaete annelid positioning of the CBE, as shown by Nodal and BMP2/4 Platynereis dumerilii (Denes et al., 2007), supporting their perturbations that alter the position, size and shape of the CBE existence in the urbilaterian ancestor (see Glossary, Box 1). Below, (Bradham et al., 2009; Lapraz et al., 2009; Saudemont et al., we highlight the mechanisms of neural patterning that are shared 2010; Yaguchi et al., 2010a). The observation that ectopic among deuterostomes. neurons produced under a variety of experimental conditions [e.g. in Nodal morphants or following inhibition of BMP Patterning of the nervous system along the AP axis of signaling (Saudemont et al., 2010; Yaguchi et al., 2010a)] remain deuterostomes employs an ancient Wnt-based set of largely confined to the CBE strongly suggests that the CBE signals provides an environment conducive to neural differentiation. Embryonic cells become anterior neuroectoderm unless When the Nodal or BMP2/4 signaling pathways are blocked by instructed otherwise interfering with components that function cell-autonomously In the absence of all known zygotic signaling (i.e. in the absence (e.g. their receptors and Smads), the distribution, but not the of canonical Wnt), virtually the entire sea urchin embryo assumes number, of nerves changes in proportion to the size of the CBE. an ANE fate (Yaguchi et al., 2006). Similarly, in Xenopus embryos,

This implies that TGF signaling primarily controls the size and when early signals that promote non-neural ectodermal fates are DEVELOPMENT Development 138 (17) REVIEW 3619

A Egg B Cleavage C Embryo D Larva Nodal cWnt BMP2/4

V D

0 hour 8 hour 24 hour 48 hour (fertilized egg) (16-cell) (blastula) (prism)

Key Pre-ciliary band ectoderm Animal plate (ciliary band in C and D) Endoderm/mesoderm Ventral ectoderm Dorsal ectoderm Endoderm-adjacent ectoderm

Serotonergic neuron precursor in C Differentiated serotonergic neuron in D Ciliary band neuron precursor in C Differentiated ciliary band neuron in D

Fig. 4. A four-step model for specification and organization of the sea urchin embryo nervous system. (A)In the first step, an anterior neuroectoderm regulatory state (red) is present throughout the egg and much of the embryo during early cleavage stages. (B)In the second step, which occurs during very early blastula stages, this state is eliminated by canonical Wnt (cWnt)-dependent signals from all but the anterior neuroectoderm, revealing a ciliary band-like neuroectoderm (green) that contains scattered neural precursors (light pink circles). (C)In the third step, which occurs during the mesenchyme blastula/early gastrula stages, Nodal and BMP2/4 signals convert ventral and dorsal ectoderm to non-neural ectoderm except in the anterior neuroectoderm (red) and ciliary band (green), which are protected from these signals. (D)During the fourth and final step, by which point the embryo has transitioned into a larva, CBE and ANE neural progenitors differentiate. The timeline indicates hours post- fertilization and embryonic stages. eliminated, the entire embryo expresses Sox2 (Reversade and De FoxQ2 (Yaguchi et al., 2010b), to the anterior end of the embryo. Robertis, 2005; Reversade et al., 2005), a transcription factor that The same canonical Wnt-mediated restriction has recently been supports the neural precursor state in mouse embryos (Bylund et described in Saccoglossus (Darras et al., 2011). FoxQ2 is also al., 2003; Graham et al., 2003). Other anterior neural markers are expressed at the anterior end of the embryo of the cephalochordate expressed in the early medial epiblast cells of chick embryos (see Glossary, Box 1) amphioxus (Branchiostoma floridae) (Yu et (reviewed by Wilson and Houart, 2004), in the epiblast of mouse al., 2002), raising the possibility that a similar mechanism also embryos (reviewed by Levine and Brivanlou, 2007) and in mouse operates in these organisms. Amazingly, this process also occurs in embryonic stem cells in the absence of signals that promote non- the hydrozoan cnidarian Clytia hemisphaerica: FoxQ2 is expressed neural ectodermal fates (Tropepe et al., 2001; Vallier et al., 2004). where the apical tuft forms, at the pole opposite to canonical Wnt Thus, it is likely that in vertebrates, as in sea urchin embryos, ANE signaling (Momose et al., 2008), which is where gastrulation fate is not induced but rather is driven by early maternal regulatory occurs (Wikramanayake et al., 2003). Further, as in sea urchin factors. embryos, the size of the FoxQ2 expression domain in Clytia is regulated by canonical Wnt signaling (Momose et al., 2008). This Wnt signaling defines the posterior pole and restricts the indicates that canonical Wnt-restricted anterior expression of ANE to the opposite pole FoxQ2 predates the cnidarian-bilaterian split. As discussed recently by others (Niehrs, 2010; Petersen and The canonical Wnt-dependent restriction of expression of other Reddien, 2009), canonical Wnt signaling is an ancient function that genes, such as six3, to the ANE of sea urchin embryos or to the operates at the posterior end of nearly all bilaterian embryos. anterior neural plate and forebrain of vertebrate embryos is Similarly, in sea urchin embryos a gradient of nuclear -catenin conserved (Braun et al., 2003; Lagutin et al., 2003; Wei et al., emanates from posterior blastomeres during cleavage stages and is 2009). Furthermore, an AP gradient of nuclear -catenin has been necessary to specify mesoderm and endoderm (Logan et al., 1999; demonstrated in the presumptive Xenopus neural plate ectoderm Wikramanayake et al., 1998). At the opposite end of vertebrate and during gastrulation, around the time when the AP and DV axes sea urchin embryos, the tissues that develop are of anterior begin to separate (Kiecker and Niehrs, 2001). Thus, anterior character. Whereas suppression of canonical Wnt signaling neuroectoderm fate requires low levels of canonical Wnt signaling anteriorizes all three germ layers in vertebrates, this process is in both sea urchin and vertebrate embryos. known thus far to affect only the ectoderm in sea urchin embryos, as blocking canonical Wnt completely abrogates endomesoderm Roles of canonical Wnt and TGF signaling in positioning development. Thus, since the ectoderm at the anterior end of sea anterior neuroectoderm urchin embryos has many properties of anterior neuroectoderm, In the sea urchin embryo, canonical Wnt-mediated suppression and as canonical Wnt signaling functions at the opposite end in of ANE fate occurs in non-ANE ectoderm during cleavage these embryos, the primary axis of the sea urchin resembles the AP stages by a mechanism that has not yet been defined. It requires axis of other embryos. neither Nodal nor BMP2/4, nor their antagonists (Wei et al., The canonical Wnt-dependent remodeling of a broad 2009). When canonical Wnt signaling is blocked, the entire neuroectoderm territory is an ancient function. In sea urchin embryo assumes ANE fate. By contrast, in vertebrate embryos,

embryos, it restricts FoxQ2 and the apical tuft, which depends on canonical Wnt signaling results in localized production of BMP DEVELOPMENT 3620 REVIEW Development 138 (17) signaling antagonists, which protect the neuroectoderm Nervous system positioning along the DV axes of regulatory state in the dorsal ectoderm. Thus, when canonical deuterostomes relies on TGF signals Wnt signaling is blocked in vertebrate embryos, they completely Much of the ectoderm adopts a CBE fate unless instructed lack neuroectoderm and are strongly ventralized (Heasman et al., otherwise by Nodal and BMP2/4 2000). In addition, canonical Wnt signal activity posteriorizes Nodal and BMP2/4 carve out the CB by diverting ectoderm on the the neuroectoderm except where it is protected by Wnt ventral and dorsal sides of the embryo to a non-neural epidermal fate. antagonists (reviewed by Yamaguchi, 2001; Houart et al., 2002). This process shares some properties with neuroectoderm patterning These two processes, canonical Wnt-dependent creation of a mechanisms along the DV axes of other bilaterian embryos. It neural plate by antagonizing BMP signaling and posteriorization operates along an axis orthogonal to the canonical Wnt system, by high levels of canonical Wnt activity in posterior regions of which specifies cell fates along the AP axis. Although nodal, lefty, the neural plate, occur during gastrulation. If both BMP signaling bmp2/4 and chordin are all transcribed on the ventral side of the sea and canonical Wnt signaling are blocked, then the underlying urchin embryo, BMP2/4 signaling occurs only on the side opposite neuroectoderm regulatory state can be detected and it is anterior to that of nodal and chordin expression, as is seen in vertebrate and in character, as posteriorizing signals are lacking (see Reversade arthropod embryos. As discussed above, most of the widespread et al., 2005). Thus, in both vertebrate and sea urchin embryos, latent bias toward neuroectoderm and neural structures in vertebrate there is a basal, probably maternally driven, ANE regulatory embryos is suppressed by early BMP signaling and maintained state that is restricted to anterior ectoderm regions by canonical where antagonistic activities prevent this signaling. In addition, Wnt signaling. Where the differences lie is in the regulatory fibroblast growth factor (FGF) signaling and inhibition of canonical relationships between canonical Wnt and TGF signaling Wnt signaling promote neural development by reducing the half-life activities. In sea urchin embryos, the production of Nodal and of the BMP effector protein Smad (Fuentealba et al., 2007), and, in BMP2/4 (and their respective antagonists) depends on canonical Xenopus embryos, this is required in addition to secreted antagonists Wnt signaling, whereas in the vertebrate embryo this is only the for neural development (Pera et al., 2003). Although it is clear that case for the BMP antagonists. Furthermore, in sea urchin downregulation of BMP2/4 signals is important for neuroectoderm embryos, the creation of the more posterior CBE via TGF development in the sea urchin embryo, whether FGF signaling also signaling depends on the prior restriction of the ANE to the plays a role in this process is not yet known. anterior end of the embryo, whereas in vertebrate embryos these Although antagonism of BMP signaling is considered to be the are independent events that may occur at approximately the same primary mechanism that prevents ectoderm from differentiating as time. Thus, the development of non-neural and neural ectoderm epidermis in higher deuterostomes, Nodal signaling also overrides territories in sea urchin and vertebrate embryos make use of the an early neuroectoderm regulatory state. In Xenopus, continued same signaling pathways, but these are deployed differently as a suppression of effectors downstream of both Nodal and BMP result of the different regulatory linkages among them. signaling is required for neural induction (Chang and Harland, 2007), and, in zebrafish and mice, neural fates have been shown to emerge Some regulatory properties of the sea urchin embryo ANE in presumptive endomesoderm cells in the absence Nodal signaling are conserved (Camus et al., 2006; Feldman et al., 2000; Schier and Talbot, 2001). The ANE regulatory properties of the sea urchin embryo are similar to those described for mouse embryos. In both mouse and DV axis patterning mechanisms are connected to different sea urchins, Six3 functions near the top of the ANE regulatory upstream polarizing mechanisms networks (Lagutin et al., 2003; Lavado et al., 2008; Wei et al., Although the antagonism of TGF signaling is employed in 2009). Furthermore, many of the same transcription factors, positioning the nervous system in virtually all cases that have been including, Rx, Achaete-Scute, Zic2, Ebf3, Fez, Nkx2.1, SoxC examined, this process is connected to distinctly different initial and the Notch ligand Delta (Wei et al., 2009) are expressed in polarizing mechanisms, and the timing and manner in which these the ANE of both mouse and sea urchin embryos. Mutual mechanisms are utilized are highly variable. For example, DV axial antagonism between Six3 and canonical Wnt signals operates in specification in Xenopus is provided by the sperm entry point and both systems. The sea urchin ANE is likely to be protected from by cytoplasmic rotation (Moon and Kimelman, 1998). In flies, it canonical Wnt signals by Six3-dependent Wnt antagonists, such depends on a gradient of Dorsal activity controlled by follicle cell as Dkk1, just as the zebrafish telencephalon is protected by a activities (Steward and Govind, 1993), whereas in sea urchins secreted frizzled-related protein, a Wnt antagonist called Tlc redox asymmetry is thought to be involved (Coffman et al., 2009; (Houart et al., 2002). Coffman et al., 2004; Steward and Govind, 1993). The sea urchin Many of the transcription factors expressed in the sea urchin embryo provides an especially clear example of that connection embryo ANE (Kenny et al., 2003; Wei et al., 2009) are also because both the establishment of the nervous system and the expressed in the ectodermal layer of the cnidarian Nematostella specification of cell fates along the secondary axis can be directly vectensis [e.g. six3, hbn, rx, otx, soxb1, soxb2 (Marlow et al., traced to a single event: the activation of Nodal in the presumptive 2009)]. Some of these are expressed in patterns similar to those ventral ectoderm, possibly dependent on the mitochondrial gradient observed in the sea urchin embryo ANE, consistent with the in the egg. The fact that Nodal controls the expression of BMP2/4, intriguing possibility that the cnidarian ectoderm is similar to the and that Nodal and BMP2/4 then play repressive roles in sea urchin ANE. Both regions resist respecification by canonical positioning the nervous system, make Nodal a key regulator of this Wnt signaling and contain many neurons. An additional process in the sea urchin embryo. provocative hypothesis suggests that most of the body of Hydra, another cnidarian, gave rise to the heads of deuterostomes and Patterns of Nodal expression during evolution protostomes (Meinhardt, 2002). This shared and ancient state of The nodal gene appeared sometime after the cnidarian-bilaterian early ectoderm might thus be part of the default state of early, pre- split in the common ancestor of protostomes and deuterostomes, as

signaling ectoderm in vertebrate embryos as well. it is expressed in echinoderms and in snails (Grande and Patel, DEVELOPMENT Development 138 (17) REVIEW 3621

2009). In snails, it functions to specify right-sidedness, assuming Angerer, L. M., Oleksyn, D. W., Levine, A. M., Li, X., Klein, W. H. and the mouth is ventral. This, along with the fact that BMP2/4 Angerer, R. C. (2001). Sea urchin goosecoid function links fate specification along the animal-vegetal and oral-aboral embryonic axes. Development 128, signaling in sea urchins and BMP2/4 expression in hemichordates 4393-4404. is dorsal (Lowe et al., 2006), suggest that the positioning of major Arendt, D., Denes, A. S., Jekely, G. and Tessmar-Raible, K. (2008). The parts of the nervous system in basal deuterostomes should be evolution of nervous system centralization. Philos. Trans. R. Soc. Lond. B Biol. Sci. ventral. This is not the case in sea urchin embryos, largely because 363, 1523-1528. Bergeron, K. F., Xu, X. and Brandhorst, B. P. (2010). Oral-aboral patterning and Nodal specifies that region as non-neural ventral ectoderm. During gastrulation of sea urchin embryos depend on sulfated glycosaminoglycans. deuterostome evolution the position of Nodal signaling and its Mech. Dev. 128, 71-89. relative importance in nervous system positioning have changed. Bisgrove, B. W. and Burke, R. D. (1986). Development of serotongic neurons in embryos of the sea urchin, Strongylocentrotus purpuratus. Dev. Growth Differ. In vertebrate embryos, the primary function of Nodal is in 28, 569-574. specifying mesoderm and endoderm rather than ectoderm, whereas Bolouri, H. and Davidson, E. H. (2009). The gene regulatory network basis of in the cephalochordate amphioxus (Onai et al., 2010; Yu et al., the ‘community effect’, and analysis of a sea urchin embryo example. Dev. Biol. 2007) it supports dorsal/anterior fates in both mesoderm and 340, 170-178. Bradham, C. A. and McClay, D. R. (2006). p38 MAPK is essential for secondary ectoderm (Onai et al., 2010). Although the wide variations in the axis specification and patterning in sea urchin embryos. Development 133, 21- patterns of Nodal expression in the ectoderm among deuterostomes 32. make it impossible to clearly identify ancestral versus derived Bradham, C. A., Oikonomou, C., Kuhn, A., Core, A. B., Modell, J. W., McClay, D. R. and Poustka, A. J. (2009). Chordin is required for neural but not axial traits, studies on Nodal function in sea urchin embryos demonstrate development in sea urchin embryos. Dev. Biol. 328, 221-233. that it plays a crucial role in determining non-neural fates and, as Braun, M. M., Etheridge, A., Bernard, A., Robertson, C. P. and Roelink, H. a consequence, the organization of the nervous system. (2003). Wnt signaling is required at distinct stages of development for the induction of the posterior forebrain. Development 130, 5579-5587. Bullock, T. and Horridge, G. (1965). Hemichordata. Structure and Function of the Conclusions Nervous System of Invertebrates, pp. 1567-1577. San Francisco: W. H. Freeman. In sea urchin embryos, the positioning of the ANE and the CBE Burke, R. D., Osborne, L., Wang, D., Murabe, N., Yaguchi, S. and Nakajima, within a broad neuroectoderm territory is controlled by successive Y. (2006). Neuron-specific expression of a synaptotagmin gene in the sea urchin Strongylocentrotus purpuratus. J. Comp. Neurol. 496, 244-251. primary and secondary axis patterning mechanisms. The regulatory Bylund, M., Andersson, E., Novitch, B. G. and Muhr, J. (2003). Vertebrate links between these signaling mechanisms allow their functions to neurogenesis is counteracted by Sox1-3 activity. Nat. Neurosci. 6, 1162-1168. be separated in time – Nodal expression does not become Byrum, C. A., Xu, R., Bince, J. M., McClay, D. R. and Wikramanayake, A. H. (2009). Blocking Dishevelled signaling in the noncanonical Wnt pathway in sea established until the ANE is restricted to the anterior end of the urchins disrupts endoderm formation and spiculogenesis, but not secondary embryo by canonical Wnt-dependent processes, and BMP2/4 mesoderm formation. Dev. Dyn. 238, 1649-1665. expression does not begin without Nodal. By contrast, the Camus, A., Perea-Gomez, A., Moreau, A. and Collignon, J. (2006). Absence of regulatory scheme in vertebrate embryos compresses these events Nodal signaling promotes precocious neural differentiation in the mouse embryo. Dev. Biol. 295, 743-755. temporally and spatially, in part because BMP expression initially Casano, C., Ragusa, M., Cutrera, M., Costa, S. and Gianguzza, F. (1996). occurs broadly and independently of canonical Wnt and Nodal. As Spatial expression of alpha and beta tubulin genes in the late embryogenesis of a result, the positioning of the nervous system dorsally in higher the sea urchin Paracentrotus lividus. Int. J. Dev. Biol. 40, 1033-1041. Chang, C. and Harland, R. M. (2007). Neural induction requires continued deuterostomes or ventrally in protostomes depends heavily on the suppression of both Smad1 and Smad2 signals during gastrulation. localized production of activities that suppress BMP signaling. Development 134, 3861-3872. Although the signals and their antagonists are the same, the Coffman, J. A., McCarthy, J. J., Dickey-Sims, C. and Robertson, A. J. (2004). regulatory connections between the Wnt and BMP pathways are Oral-aboral axis specification in the sea urchin embryo. II. Mitochondrial distribution and redox state contribute to establishing polarity in different in the embryos of sea urchins and vertebrates, yielding Strongylocentrotus purpuratus. Dev. Biol. 273, 160-171. distinct neural versus non-neural patterns. The neural patterning Coffman, J. A., Coluccio, A., Planchart, A. and Robertson, A. J. (2009). Oral- mechanisms in the common ancestor of echinoderms and other aboral axis specification in the sea urchin embryo III. Role of mitochondrial redox signaling via H2O2. Dev. Biol. 330, 123-130. deuterostomes cannot be determined, but a clearer understanding Darras, S., Gerhart, J., Terasaki, M., Kirschner, M. and Lowe, C. J. (2011). of the evolution of extant deuterostomes can be obtained by {beta}-Catenin specifies the endomesoderm and defines the posterior organizer detailed comparisons of diverse embryos. Importantly, it is of the hemichordate Saccoglossus kowalevskii. Development 138, 959-970. becoming evident that to understand their role in neural patterning Davidson, E. H. (2009). Network design principles from the sea urchin embryo. Curr. Opin. Genet. Dev. 19, 535-540. we must not simply determine the expression patterns of Wnt and Davidson, E. H., Rast, J. P., Oliveri, P., Ransick, A., Calestani, C., Yuh, C. H., TGF signaling pathway components, but also test their regulatory Minokawa, T., Amore, G., Hinman, V., Arenas-Mena, C. et al. (2002). A relationships, as has been done in the sea urchin embryo. provisional regulatory gene network for specification of endomesoderm in the sea urchin embryo. Dev. Biol. 246, 162-190. De Robertis, E. M. and Sasai, Y. (1996). A common plan for dorsoventral Acknowledgements patterning in Bilateria. Nature 380, 37-40. We thank Adi Sethi, Diane Adams and Ryan Range for discussions and Denes, A. S., Jekely, G., Steinmetz, P. R., Raible, F., Snyman, H., especially Zheng Wei for experimental and bioinformatic contributions. Prud’homme, B., Ferrier, D. E., Balavoine, G. and Arendt, D. (2007). Support for this work was provided by the Division of Intramural Research in Molecular architecture of annelid nerve cord supports common origin of nervous the National Institute for Dental and Craniofacial Research, National Institutes system centralization in bilateria. Cell 129, 277-288. of Health (L.M.A. and R.C.A.), Special Coordination Funds for Promoting Duboc, V. and Lepage, T. (2006). A conserved role for the nodal signaling Science and Technology of the Ministry of Education, Culture, Sports, Science pathway in the establishment of dorso-ventral and left-right axes in and Technology of the Japanese Government (MEXT), by a Grant-in Aid for deuterostomes. J. Exp. Zool. B Mol. Dev. Evol. 310, 41-53. Young Scientists (Start-up) (S.Y.) and NSERC (R.D.B.). Deposited in PMC for Duboc, V., Rottinger, E., Besnardeau, L. and Lepage, T. (2004). Nodal and release after 12 months. BMP2/4 signaling organizes the oral-aboral axis of the sea urchin embryo. Dev. Cell 6, 397-410. 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