Origin and evolution of the neural crest: A hypothetical reconstruction of its evolutionary history

Develop. Growth Differ. (2001) 43, 509–520

Introduction

Molecular biological methodologies have been appliedto developmental biology with great success over thelast decade. Developmental systems for morphologicstructures can now be described in terms of gene functions as a result of the tremendous progress in molecular developmental biology, which has made theevolution of morphology a more accessible problem.One of the salient questions of evolutionary biology ishow new structures or new cell types evolve, particu-larly with regard to genetic development.

The invertebrate to vertebrate transition was one of the most important steps in the course of human evolution, because many new cell types and tissuesemerged at this stage. One example is the acquisitionof vertebrae, and hence the name, by ancestral verte-

brates. The evolution of vertebrae was accompaniedby the evolution of new cell types; that is, chondrocytesand osteocytes.

Another new cell type that emerged in ancestralvertebrates was the neural crest. Neural crest cellsarise at the boundary between the neural plate and theepidermis and migrate out, differentiating into fates thatinclude pigment cells, peripheral neurons and skeletaltissue, depending on the final destination (LeDouarin& Kalcheim 1999). Hall (2000) has proposed that theneural crest be regarded as a fourth germ layer,because these properties of pluripotency and cellmigration are comparable to those of mesoderm.

The neural crest has attracted the interest of evo-lutionary biologists because of its intimate relationshipwith the development of complex craniofacial structure,a characteristic of vertebrates, as well as representinga new cell type. Gans and Northcutt (1983) listed 20 new characteristics that have emerged during vertebrate evolution. Most of these are found in thehead and relate to food detection and ingestion, whichwas essential for the transition from the filter-feeding

*Author to whom all correspondence should be addressed.Email: [email protected] 21 April 2001; revised 28 May 2001; accepted

28 May 2001.

Review

Origin and evolution of the neural crest:A hypothetical reconstruction of its evolutionaryhistory

Hiroshi Wada*Seto Marine Biological Laboratory, Kyoto University, 459 Shirahama, Nishimuro-gun, Wakayama 649-2211, Japan.

The neural crest has long been regarded as one of the key novelties in vertebrate evolutionary history. Indeed,the vertebrate characteristic of a finely patterned craniofacial structure is intimately related to the neural crest.It has been thought that protochordates lacked neural crest counterparts. However, recent identification andcharacterization of protochordate genes such as Pax3/7, Dlx and BMP family members challenge this idea,because their expression patterns suggest remarkable similarity between the vertebrate neural crest and theascidian dorsal midline epidermis, which gives rise to both epidermal cells and sensory neurons. The presentpaper proposes that the neural crest is not a novel vertebrate cell population, but may have originated fromthe protochordate dorsal midline epidermis. Therefore, the evolution of the vertebrate neural crest should bereconsidered in terms of new cell properties such as pluripotency, delamination–migration and the carriage of an anteroposterior positional value, key innovations leading to development of the complex craniofacial structure in vertebrates. Molecular evolutionary events involved in the acquisitions of these new cell proper-ties are also discussed. Genome duplications during early vertebrate evolution may have played an importantrole in allowing delamination of the neural crest cells. The new regulatory mechanism of Hox genes in the neuralcrest is postulated to have developed through the acquisition of new roles by coactivators involved in retinoicacid signaling.

Key words: amphioxus, ascidian, evolution, neural crest.

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protochordate state to that of the active predator. They proposed that many features of the vertebrate headderived from the neural crest and placodes. Regardlessof whether one agrees with the detailed scenario ofGans and Northcutt (1983), the impact of the neuralcrest on the evolution of vertebrates is obvious, especi-

ally on craniofacial structure. In the present article I will review our current understanding of the origin and evolution of vertebrate neural crest, together withsome speculative ideas. Evolutionary biology deals with history; thus, at times we need to speculate much more than in experimental sciences. Hypotheses

Fig. 1. Phylogenetic relationshipof deuterostomes. The centralnervous system is shown in blue,and the peripheral nervous system in red. The phylogenetictree is based on Wada and Satoh(1994), Turbeville et al. (1994) and Castresana et al. (1998). Theschematic illustration and distri-bution of peripheral nerves of the acornworm (hemichordate)are adopted from Knight-Jones(1952).

Fig. 2. Gene expression patterns of HrEpiA, HrBMPa and HrPax-37. (A) Schematic illustration of an ascidian tail-bud embryo (lateral view) indicating six epidermal territories. The dorsal midline epidermis is shown in orange. Numbering of the epidermal territories follows that used in Ishida et al. (1996). (B) Expression of HrEpiA (Ishida et al. 1996) in a tail-bud embryo (dorsal view; anterior to the left). Expression is exclusive of the dorsal midline epidermis. (C) Expression of HrBMPa in a tail-bud embryo (dorsalview; anterior to the left). Expression is observed in the dorsal midline epidermis (Miya et al. 1996) and thus is complementary to thatof HrEpiA. (D–G) Expression of HrPax-37 in the gastrula (D,E; lateral view) and neurula stages (F,G; dorsal view). HrPax-37 expres-sion at the gastrula stage is observed both in cells fated to the dorsal midline epidermis and in those fated to the dorsal neural tube,but is observed only in dorsal midline epidermis at the neurula stage (Wada et al. 1996a).

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have played and continue to play an important role inthe progress of evolutionary biology; therefore, thispaper presents speculations and includes suggestionson how they can be tested.

Brief overview of protochordate neural structure

Vertebrates belong to the phylum Chordata, whichincludes two protochordate groups: cephalochordates,represented by amphioxus, and urochordates, repre-sented by ascidians. Chordates are a member ofdeuterostomes in which two other phyla, Echino-dermata and Hemichordata, are included (Fig. 1).Protochordates (cephalochordates plus urochordates)share a similar body plan with vertebrates as well asseveral characteristics, such as pharyngeal gill slits,notochord and a dorsal tubular nervous system.Although protochordate neural tube structures are relatively simple, the basic structure and significantparts of the genetic mechanisms are conserved in protochordates and vertebrates (Holland & Chen 2001;Wada & Satoh 2001). Protochordates have an anteriorneural tube region where Otx genes are expressed,which is homologous to the chordate forebrain and mid-brain (Wada et al. 1996b; Williams & Holland 1996). Theposterior part of the protochordate neural tube containsdichotomous regions that are comparable to the verte-brate hindbrain and spinal cord (Jackman et al. 2000;Wada & Satoh 2001), although the presence of acounterpart for the vertebrate isthmic organizer remainsa matter of debate (Wada et al. 1998; Kozmik et al.1999; Wada & Satoh 2001). Differentiation of the proto-chordate and vertebrate neural tubes along the dorso-ventral axis appears to be controlled by similar geneticsystems (Shimeld 1999a; Wada & Satoh 2001). Ventralexpression of the hedgehog (hh) gene is observed inamphioxus (Shimeld 1999b), and bone morphogeneticprotein (BMP) genes in the ascidian are expressed inepidermis abutting the neural tube (Miya et al. 1996,1997); however, the contributions of ascidian hh oramphioxus BMP remain to be established.

Despite conservation of the basic dorsoventral patterning system, protochordates are believed to lack definitive neural crest. Although neither ascidiansnor amphioxus possess cells that fit the traditional definition of neural crest (i.e. cells that migrate out oftheir original location in the boundary between theneural tissue and epidermis), recent studies haveshown the existence of protochordate genes homol-ogous to those involved in vertebrate neural crest differentiation (Holland et al. 1996; Baker & Bronner-Fraser 1997; Holland & Holland 1998; Shimeld &Holland 2000; Wada & Satoh 2001).

Tetralogy

Before going on to discuss the origin of the neural crest,the genome duplications that may have occurred inearly stages of vertebrate evolution will be reviewedbriefly. Vertebrates often have two to four genes homol-ogous to invertebrate counterpart genes. These areusually explained as a consequence of two rounds ofgenome duplication occurring in ancestral vertebrates;a feature called ‘tetralogy’ (Holland et al. 1994; Spring1997), although there has been some opposition to thisidea (Martin 2001). Early in the process, duplicatedgenes had redundant functions and some of the dupli-cated genes were lost relatively easily. Thus, in manycases, vertebrates have not retained a full complementof four genes, keeping only two or three counterpartgenes. The supernumerary genes are believed to havestood as raw materials for the evolution of new celltypes and structures (Holland et al. 1994; Holland &Garcia-Fernàndez 1996). Pax2, Pax5 and Pax8 havea single counterpart in invertebrates and are con-sidered a result of genome duplication in ancestral vertebrates (Wada et al. 1998). Of these, Pax5 hasa specific role in B-cell differentiation (Enver 1999), aprocess in which Pax2 or Pax8 are not involved. Thisis an example of recruitment of one of the duplicatedgenes for a new function. In some cases, the duplicatedcounterparts share a function in a vertebrate-specificstructure; for example, both Pax1 and Pax9 areinvolved in sclerotome differentiation (Müller et al.1996). Ascidians and amphioxus have single homologsof Pax1 and Pax9, which function in pharyngeal gill differentiation (Holland et al. 1995; Ogasawara et al.1999). Because vertebrate Pax1 and Pax9 are bothexpressed in the pharyngeal gills (Müller et al. 1996),an ancestral function of the Pax1/9 probably involvespharyngeal gill differentiation. It is possible that theancestral gene of Pax1 and Pax9 acquired a new rolein sclerotome differentiation before genome duplication,which may have been essential for vertebral evolution.Genetic acquisition of new expression sites or newfunctions likely played an important role in morphologicevolution in the early evolution of vertebrates, withoutregard to the contribution made by gene duplication.

Origin of the neural crest in protochordates

Interaction between the neural plate and epidermis,together with signals from paraxial mesoderm, isimportant for the differentiation of the vertebrate neuralcrest (Selleck & Bronner-Fraser 1995; LaBonne &Bronner-Fraser 1999). Therefore, the neural crest is likely to have evolved in the region straddling the border between the neural plate and epidermis, notonly because the neural crest occupies a homologous

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position, but also because this position is essential for its differentiation. In this section, the cells at theboundary between the neural plate and epidermis inprotochordates are examined first, then gene expres-sion patterns in those cells for which vertebratehomologs are involved in neural crest formation arecompared.

The boundary between the neural plate and epidermis of protochordates

Protochordate surface ectoderm is believed to be relatively uniform, except for the presence of some epidermal sensory cells found in both ascidians andamphioxus, and the anterior adhesive papillae ofascidians. In the course of examining epidermis-specific gene expression, Ishida et al. (1996) found that ascidian surface ectoderm can be subdivided intosix regions according to the sets of genes expressed(Fig. 2A,B). Of these, five regions are characterized asfields where epidermal peripheral neurons differentiate.Two of these regions correspond to the dorsal midlineepidermis (regions 1 and 2 in Fig. 2A) and one to theventral midline (region 5 in Fig. 2A). Two others corres-pond to regions where adhesive papilla and associatedneurons differentiate (regions 3 and 4 in Fig. 2A). Thedorsal midline populations abut the neural tube andthus are topologically homologous to the neural crest.Several cells from the dorsal midline population differ-entiate into primary sensory neurons, which are prob-ably mechanosensory neurons with ciliary processesextending into the larval tunic (Torrence & Cloney 1982).Thus, ascidian larvae have a population of cells in theboundary between the neural tube and epidermis from

which both neuronal cells and epidermal cells differ-entiate; however, little is still known about how each dorsal midline epidermal cell chooses one of the twofates. This striking similarity between the dorsal mid-line epidermis and the neural crest brings up the tantalizing possibility that the vertebrate neural crestmay have originated from the dorsal midline epidermisof protochordates (Fig. 3).

In amphioxus, the dorsal midline epidermal cells alsoshow similarity to the neural crest. Neurulation inamphioxus proceeds differently from that in ascidiansor vertebrates and intercellular contact between theepidermis and neural plate is disrupted before theamphioxus neural plate rolls up (Fig. 4; Holland et al.1996). The epidermal sheets from either side of theplate migrate across the dorsal surface by means oflamellipodia, so that epidermis surrounds the entireembryo prior to closure of the neural plate (Fig. 4;Holland et al. 1996). These migrating dorsal midline epi-dermal cells with lamellipodia are marked by expres-sion of the Dlx gene (Holland et al. 1996), suggestingthat the dorsal midline epidermal cells of the amphioxusare a distinct population that display a similar migratoryproperty to cells in the neural crest. However, inamphioxus, sensory epidermal neurons are rather dispersed, not concentrated at the dorsal midline epidermis (Lacalli & Hou 1999). Details of this differ-ence are discussed later.

Protochordate homologs of genes involved in thedifferentiation of the neural crest

The BMP genes, which belong to the transforminggrowth factor (TGF)-� superfamily, have been proposed

Fig. 3. Hypothetical evolution ofthe neural crest.


to be involved in the induction of the neural crest. SomeBMP functions during very early development are toallow differentiation of the ventral phenotype, promoteblood and kidney differentiation in the mesoderm andpromote epidermal cell differentiation through sup-pression of neural differentiation in the ectoderm.Neural induction occurs via inhibition of BMP activity(reviewed by Hogan 1996). After neural induction, theexpression of BMP4 and BMP7 is upregulated in theboundary between the neural plate and the surround-ing epidermis, where neural crest cells differentiate(Liem et al. 1995). The neural crest is induced throughthe interaction between the epidermis and the neuralplate, and arises from both epidermis and neural plate(Selleck & Bronner-Fraser 1995). BMP4 and BMP7 havebeen shown to be able to substitute the activity of the epidermis in inducing neural crest from the neuralplate (Liem et al. 1995). BMPa, an ascidian homologof BMP5-8 (including BMP7), displays a expressionpattern similar to vertebrate BMP7 (Miya et al. 1996).Expression begins predominantly in ectodermal cellsat the gastrula stage. The expression of BMPa isupregulated in the dorsal midline epidermal cells of the neurula later in the differentiation process (Fig. 2C;Miya et al. 1996) and is consistent with the idea thatthe neural crest originates in the dorsal midline epi-dermis. BMPb, the ascidian homolog of vertebrateBMP2/4, is also expressed in dorsal midline epidermalcells (Miya et al. 1996). Thus, a similar genetic mechan-ism may operate for differentiation in both the ascidiandorsal midline epidermis and the vertebrate neural

crest. However, amphioxus BMP is not upregulated inthe dorsal midline epidermis, although it is expressedin epidermal cells of the gastrula (Panopoulou et al.1998).

Several transcription factors are involved in differ-entiation of the vertebrate neural crest. The Pax3 mutantmouse gene, Splotch, produces a defect in neural crestdifferentiation (Franz 1993) and the closely related Pax7also causes defects in the cephalic neural crest(Mansouri et al. 1996). Pax3 and Pax7 expression is firstdetected throughout the entire neural plate. Sub-sequently, their expression is downregulated in the ventral neural tube and maintained in the dorsal neuraltube and neural crest cells (Goulding et al. 1993). Pax3and Pax7 have a single counterpart gene in proto-chordates. HrPax-37, the ascidian homolog of Pax3and Pax7, exhibits a pattern of expression similar to itsvertebrate counterparts (Wada et al. 1996a, 1997). It isexpressed both in cells destined to form the dorsal part of the neural tube and in those destined to formdorsal midline epidermis (Fig. 2D,E). This pattern iscomparable with that of vertebrate genes in the dorsalneural tube and neural crest. Just prior to closure ofthe neural tube, HrPax-37 expression is maintainedsolely in the dorsal midline epidermis (Fig. 2F,G, Wadaet al. 1996a, 1997). The amphioxus homolog of Pax3/7is expressed in the lateral part of the neural plate, whichsubsequently occupies the dorsal part of the neuraltube and is also found in the somitic mesoderm,although no expression is detected in the epidermis(Holland et al. 1999).

Fig. 4. Neurulation of the amphi-oxus embryo. (A) Schematic illus-tration of amphioxus neurulation.The neural plate–neural tube isshown in gray. (B) Dorsal view ofa neurula embryo (anterior is tothe bottom left). Epidermis isbeginning to overgrow the neuralplate (np). (C) Close view of cellsat the edge of the epidermal sheet(top left). An arrowhead indicateslamellipodia from epidermal cells.(B,C) Reproduced from Holland et al. (1996), with permission ofNick Holland and the Company of Biologists Ltd.

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Dlx genes also stand as markers for the neural crest(Robinson & Mahon 1994). AmphiDll, the amphioxushomolog of Dlx, is expressed in the dorsal neural tubeand in epidermis adjacent to the neural plate (Hollandet al. 1996). An ascidian homolog of Dlx is alsoexpressed in the dorsal midline epidermis (Wada et al.1999b).

Snail and slug are two closely related zinc fingergenes that are among the most widely used markersof the neural crest (Essex et al. 1993; Mayor et al. 1995).Slug is necessary for the induction and subsequentmigration of neural crest cells (LaBonne & Bronner-Fraser 2000) and its overexpression leads to expan-sion of the neural crest cell population (LaBonne &Bronner-Fraser 1998). Snail and slug have one counter-part in protochordates (Corbo et al. 1997; Langelandet al. 1998). The ascidian counterpart gene is expres-sed in the dorsal part of the anterior neural tube, fromwhich pigment cells differentiate. The posterior neuraltube of ascidian larvae consists of four rows of cells.At this level, the snail/slug homolog is expressed pre-dominantly in the lateral row (Corbo et al. 1997). InCiona intestinalis, a species of ascidian, expression is restricted to the lateral row (Corbo et al. 1997). Inanother species of ascidian, Halocynthia roretzi, thisgene is expressed weakly in the dorsal row (Wada &Saiga 1999). The snail gene is not expressed in ascid-ian epidermis (Corbo et al. 1997; Wada & Saiga 1999).The amphioxus homolog of the snail/slug gene isexpressed in the dorsal region of the neural tube, butnot in the epidermis (Langeland et al. 1998).

These comparisons of gene expression patterns arenot completely consistent with the idea that the neuralcrest originates in the dorsal midline epidermis: expres-sion patterns differ significantly between ascidians andamphioxus, as summarized in Table 1. The amphioxusBMP2/4 and Pax3/7 homologs are not expressed in the dorsal midline epidermis (Panopoulou et al. 1998;Holland et al. 1999), possibly correlating with the scattered distribution of the sensory neuronal cells in amphioxus (Lacalli & Hou 1999), while the ascidiansensory neurons are restricted in several fields(Fig. 1A). However, the strong similarities in the patterns

of gene activity in the dorsal midline epidermis of ascidians and the vertebrate neural crest are unlikelyto be the result of evolutionary convergence. In addi-tion, Dll is expressed in amphioxus dorsal midline epi-dermis (Holland et al. 1996). Lack of dorsal expressionof amphioxus BMP2/4 and Pax3/7 is described most parsimoniously as a secondary loss that took placeafter the divergence of vertebrates and amphioxus.However, in order to accept this hypothesis of neuralcrest origin in the dorsal midline epidermis, somereasonable explanation for the amphioxus exceptionshould be made.

Another point for consideration is whether the dorsal midline epidermis is the sole origin of the neuralcrest, or whether the dorsal neural tube also contributesto its development. Ascidian Pax3/7 and amphioxus Dll genes are also expressed in the dorsal part of the neural tube (Holland et al. 1996; Wada et al.1996a, 1997). Amphioxus Pax3/7 and the snail/sluggenes of both ascidians and amphioxus are onlyexpressed in the dorsal neural tube (Corbo et al. 1997;Langeland et al. 1998; Holland et al. 1999; Wada &Saiga 1999). Langeland et al. (1998) suggest that the neural crest originated in the dorsal part of theneural tube, while Holland et al. (1996) suggest that its origin may be found in both the dorsal midline epidermis and the dorsal part of the neural tube. How-ever, it may be inappropriate to look for the origin of the vertebrate neural crest in a distinct cell popu-lation. Cell lineage analyses of cells in the vertebrateneural fold reveal that a single cell can give rise to neuronal cells, neural crest cells and epidermal cells(Bronner-Fraser & Fraser 1988, 1989; Garcia-Castro &Bronner-Fraser 1999). Moreover, because neural crestcells generate a wide diversity of cell types andbecause neural crest cells in the cephalic region havea quite distinct character from those in trunk region, itis not certain whether the neural crest has a single origin (Shimeld & Holland 2000). The main pointstressed here is that the boundary between the neuraltube and epidermis in protochordates is less welldefined than has been believed in the past. In addi-tion, there are cells that show gene activity and cell

Table 1. Expression of neural crest marker genes in protochordates

Ascidian AmphioxusDorsal neural tube Dorsal midline epidermis Dorsal neural tube Dorsal midline epidermis

BMP2/4 – + – –*Pax3/7 + + + –Dlx – + + +Snail +† – + –

*Earliest expression is observed in whole epidermis, including the future dorsal midline epidermis. †In Ciona intestinalis, the dorsalneural tube expression is only in the anterior part (Corbo et al. 1997). See text for details.


properties comparable to the vertebrate neural crest.Thus, they perhaps originate the neural crest, possiblywith some contribution from the dorsal neural tube (Fig. 3). Because we know that the interaction betweenneural tissue and the epidermis performs a central rolefor neural crest induction (Selleck & Bronner-Fraser1995), we can ask whether a similar interaction is neces-sary for cell differentiation or specific gene expressionin the dorsal midline epidermis of protochordates. Thishypothesis can also be tested by comparing the genetic mechanisms for differentiation of the neuralcrest and dorsal midline epidermis in greater detail.

Evolution of the new cell property of the neuralcrest

Even accepting the presented hypothesis that the origin of the neural crest is in protochordate dorsal midline epidermis, possibly including the dorsal neuraltube, remarkable differences remain between the verte-brate neural crest and its protochordate counterparts;for example, cell migratory behavior and pluripotency.Thus, I propose that the evolution of the vertebrateneural crest was not the birth of a new cell type; rather,it was the acquisition of these new cell properties bythe protochordate precursors (Fig. 3). In addition tomigration and pluripotency, the neural crest acquiredanother important new property: carriage of antero-posterior positional information. The neural crest cellsused to be regarded as playing a pivotal role in pattern-ing of the pharyngeal arch. However, recent studieshave revealed that pharyngeal arch patterning occursat least partially independently of the neural crest cells(Graham & Smith 2001). Even so, because neural crestcontributes to skeletal tissues or cranial ganglia, whichshow a distinct shape or innervation pattern accord-ing to their rhombomeric origin, anteroposterior posi-tional information of the neural crest is important for thedevelopment of the complex craniofacial structure invertebrates.

This formation of a complex craniofacial structure isa characteristic feature of vertebrates. Pluripotencycontributes to this development by allowing for facialskeleton and peripheral nerve differentiation. Trans-mission of anteroposterior positional information,together with cell migratory behavior, contributes to the building of a system that controls the coordinateddifferentiation of several cell types in the pharyngealarches. In this way, the evolution of these new cell prop-erties was a key innovation for the evolution of the complex craniofacial region in vertebrates. Molecularevolutionary processes involved in the acquistion ofthese new properties are suggested in the followingsection.

Pluripotency

The vertebrate neural crest differentiates into severalcell types, including pigment cells, peripheral nervecells inclusive of sensory neurons, peripheral glia andsympathetic neurons. In the cephalic region, it also dif-ferentiates into skeletal and connective tissues. Proto-chordates also possess peripheral nerves and pigmentcells. The first stage of neural crest evolution likelyaffected peripheral nerves, because neural crest cells differentiate at the boundary between the epi-dermis and neural tissue. Indeed, sensory nerves differentiate from the dorsal midline epidermis in ascid-ians (Torrence & Cloney 1982). Interestingly, two BMPgenes and the Dlx homolog in ascidians are expressedin the ventral midline epidermis, where primary sensorycells also differentiate (Miya et al. 1996, 1997; Wada et al. 1999b). This is rather similar to the nerve plexusin hemichordates. Although acornworms have a dis-persed nerve plexus that resembles that of echino-derms, it is concentrated in the dorsal and ventralmidlines of the epidermal layer of the trunk (Fig. 1;Knight-Jones 1952). In contrast, amphioxus has arather scattered peripheral nervous system, with noobservable concentration of the peripheral nerves inthe dorsal midline (Lacalli & Hou 1999). This hypothesisregards ancestral chordates as having peripheralnerves in the dorsal and ventral midline, as seen inascidians and acornworms. Subsequently, the dorsalmidline evolved into the neural crest. Conversely,amphioxus changed its distribution of peripheral nervesand only Dlx retained its dorsal midline expression.

Pigment cells are found in the neural tubes of bothascidians and amphioxus. Two cell types, otoliths andocelli, differentiate in the dorsal part of the ascidianbrain (Nishida 1987). Ascidians and vertebrates shareconserved genetic mechanisms for pigment cell differ-entiation. For example, Mitf regulates tyrosinase expres-sion and subsequent melanization in both ascidiansand vertebrates (I. Yajima & H. Yamamoto, pers.comm., 2001). In addition, the early dorsal neural tubemarker gene HrPax-37 (Fig. 2D–G), when overex-pressed in the ventral neural tube, has been shown todrive ectopic tyrosinase expression (Wada et al. 1997),which may be comparable to the abnormal pigmen-tation pattern seen in the mouse Pax3 mutant Splotch(Franz 1993). The fact that ascidian pigment cells differentiate from the dorsal lineage of the neural tubesupports the idea that the dorsal part of the proto-chordate neural tube may also contribute to the neuralcrest. In addition, it implicates neural crest involvementin pigment cell differentiation at an early stage of evolution. However, amphioxus pigment cells in theneural tube (Hesse ocelli) sit ventrally in the neural tube(Ruppert 1997; S. M. Shimeld, pers. comm., 2001). The

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origin of pigmentation long predates that of the neuralcrest, but our current understanding cannot discernhow or when the genetic machinery for pigmentationbecame associated with the neural crest.

Amphioxus has a visceral arch skeleton; however,because its chemical composition is not well under-stood, its homology with the vertebrate pharyngealskeleton remains unclear (Holland & Chen 2001). Moreknowledge of protochordate homologs of genes, suchas Cbfa1 or scleraxis, is needed in order to examinethe relationships between the neural crest and skeleto-genesis.

Shimeld and Holland (2000) suggest that the firststep in neural crest evolution may have been the origin of a specific dorsal neural cell population thatcontributed to sensory processing; subsequently, thedownstream genetic pathways for pigmentation andconnective tissue were recruited in neural crest differ-entiation.

Migration

Amphioxus dorsal midline epidermal cells displaymigration during neurulation, as described previously.However, these cells do not delaminate and the migra-tion proceeds as a sheet of cells. Thus, delaminationis unique to the vertebrate neural crest. For this pro-cess, the switching of cell adhesion molecules is essen-tial. Cadherin6 and N-cadherin are expressed inpremigratory neural crest cells and these cell adhesionmolecules must be downregulated in order for neuralcrest cells to delaminate. Subsequently, distinct celladhesion molecules, such as cadherin7, are expressedin some neural crest cells (Nakagawa & Takeichi 1995,1998). Examination of the molecular evolutionary his-tories of the genes encoding cell adhesion moleculesprovides a clue as to how this delamination processevolved. Cadherin6 and cadherin7 are closely relatedgenes, which were derived from a single ancestralprotochordate gene (Levi et al. 1997; H. Wada, unpubl.data, 2001). Because these two genes likely evolvedduring the genome duplications of ancestral verte-brates, the genome duplications may have beenessential for the evolution of the genetic machinery ofneural crest delamination. Similarly, the rhoB gene,which encodes a small G-protein and is upregulatedby the BMP gene in the premigratory neural crest, isessential for delamination of the neural crest (Liu &Jessel 1998). Vertebrates contain three types of rhogenes (rhoA, B and C), of which only rhoB is involvedin delamination (Liu & Jessel 1998). These three geneshave a single counterpart in protochordates and geneduplication must have also been essential for one ofthe rho genes to be co-opted to the delamination

process (Wada, unpubl. data, 2001). The histories ofthe cadherin and rho genes suggest that the geneticmachinery of delamination evolved through recruitmentof duplicated and thus expendable genes.

Cells at the boundary between the larval neural tubeand epidermis in ascidians do not migrate; however,during postmetamorphic development some cells in thenervous system may delaminate and migrate (i.e. fromthe dorsal strand of the ascidian adult nervous system;Baker & Bronner-Fraser 1997). Whether this behavioris related to the vertebrate neural crest is unclear.Based on the hypothesis that gene duplications were essential for the evolution of the neural crestdelamination genetic system, this process may not becontrolled by a system homologous with the neuralcrest.

Anteroposterior positional information

Anteroposterior positional information carried by theneural crest is important for development of the verte-brate craniofacial region. This information is encodedin the pattern of Hox gene expression (Hunt et al. 1991),but the Hox expression pattern in the neural crest isnot simply transferred from the neural tube. The anteriorlimit of Hoxa-2 expression reaches to rhombomere 2,although neural crest cells that migrate from rhombo-mere 2 quickly inactivate expression of Hoxa-2 (Prince& Lumsden 1994). In addition, cis-regulatory analysesreveal that neural crest expression is, at least partially,regulated independently from that of the neural tube(Maconochie et al. 1999). In contrast, the Hox code inascidians and amphioxus is restricted to the neuraltube (Holland et al. 1992; Katsuyama et al. 1995; Giontiet al. 1998; Locascio et al. 1999; Wada et al. 1999a).Amphioxus Hox genes are expressed in the mesoderm,but this expression is always confined to the posteriorregion (Holland et al. 1992; Wada et al. 1999a): no Hoxcode is observed in the dorsal midline epidermis.These observations indicate that the evolution of thenew regulatory system for the neural crest was essen-tial to confer the capacity to carry anteroposterior posi-tional information.

Manzanares et al. (2000) challenge this molecularevolutionary background of Hox regulatory mechan-isms. They tested putative enhancers of amphioxusHox genes in mice and chickens by performing trans-genic assays. They identified four separate DNA elements with reproducible enhancer activities in trans-genic mice in the genomic region around AmphiHox1–3spanning ~30 kb. The element designated as 1A, whichsits 3� downstream of AmphiHox1, drives expressionin the neural tube with an anterior limit in the posteriorhindbrain, which is reminiscent of expression driven by


the mouse endogenous Hox1 enhancer. Interestingly,element 1A also drives expression in the neural crest.Expression in both the neural crest and neural tube is dependent on retinoic acid (RA) signaling, asdemonstrated in an inhibitory experiment using a dominant negative RA receptor as well as by inductionof enhanced reporter expression by exogenous RA.This is consistent with the observation that exogenousRA affects AmphiHox1 expression (Holland & Holland1996). Element 3B, which is located 5� upstream of Hox3, drives expression in the neural tube, with itsanterior limit at the boundary between rhombomeres 6and 7. Signaling by RA also controls this activity; however, element 3B interestingly does not driveexpression in the neural crest.

These results indicate that newly acquired Hoxregulation in the neural crest had been achievedthrough incorporation of the RA signaling system by the neural crest. However, this did not appear to beachieved by the simple addition of new functions in theneural crest for retinoic acid receptor (RAR) or retinoidX receptor (RXR); otherwise, element 3B should alsodrive expression in the neural crest. Rather, theseresults can be interpreted as that the activities of ele-ments 1A and 3A are regulated through the RA respon-sive element (RARE) and additional cis elements. It hasbeen shown that some of the vertebrate RARE need tobe accompanied by additional cis elements for theirfunctions (Ogura & Evans 1995a,b). Three models canaccount for these results (Fig. 5). In the first case,coactivator interaction with element 1A (coactivator Bin Fig. 5) drives expression in both the neural tube and

neural crest, while coactivator interaction with element3A (coactivator A in Fig. 5) only drives expression in the neural tube. In this case, it appears that neural crest expression of Hox genes was acquired by co-opting coactivator B for neural crest regulation.Alternatively, element 1A may contain a distinct RAREplus coactivator-binding site for the neural crest andneural tube, respectively. Elements 1A and 3B interactwith the same coactivator (coactivator A) for neural tubeexpression and the other RARE and cis elements forcoactivator B of element 1A may function for neuralcrest expression. Coactivator B may have its originalfunction in the neural tube, because the amphioxus Hoxgene is thought to only operate at that site. Alternatively,because Hox1 is expressed with a clear anteropost-erior boundary in the epidermis of both amphioxus andascidians (Katsuyama et al. 1995; Wada et al. 1999a),it may function in the epidermis. In either case, recruit-ment of the additional coactivator (coactivator B in Fig. C) must have been essential for the evolution ofneural crest expression. The earlier results might also be explained in that element 3B contains a neuralcrest-specific repressor (factor C; Fig, 5C). In this case,we need to ask which kind of roles the neural crest specific repressor (factor C) in amphioxus would play.

Conclusions

A remarkable number of genes are expressed in similar patterns in the vertebrate neural crest and the dorsal midline epidermis of protochordates. Theoccurrence of these patterns is most pronounced in the

Fig. 5. Activities of putativeamphioxus Hox enhancers in vertebrates. In these models, Hox genes have acquired neuralcrest expression by co-option of the hypothetical coactivator B.

518 H. Wada

ascidian dorsal midline epidermis, which differentiatesinto both epidermal cells and sensory neuronal cells.These observations suggest that the origin of the neuralcrest may be in the dorsal midline epidermis, possiblyincluding the dorsal neural tube. Thus, the vertebrateneural crest is likely not a new cell population. The evolution of the neural crest should be re-evaluated and looked at as a series of acquisitions of new cellproperties, which include pluripotency, delamination–migration and carriage of anteroposterior positionalinformation. These novel cell properties were essentialfor the evolution of the finely patterned craniofacialstructure, which is a defining vertebrate feature.Molecular evolutionary events that were required forthese new cell properties are currently under study.Genome duplications during early vertebrate evolutionmay have been essential for the evolution of the neuralcrest cell delamination process. Acquisition of a newrole by a coactivator that functions in cooperation withretinoic acid signaling may have achieved a new regu-latory mechanism of Hox genes in the neural crest.

Acknowledgements

I thank Clare Baker, Nobue Itasaki, Linda Holland andNick Holland for critical reading and helpful commentson the manuscript. I also thank Seb Shimeld, IchiroYajima and Hiroaki Yamamoto for communicating dataprior to publication and Nori Satoh for providing probesof in situ hybridization. I also thank anonymous review-ers for their instructive suggestions. Part of the workpresented here was supported by a Grant-in-Aid forScientific Research from the Ministry of Education,Science, Sports and Culture of Japan, and NissanScience Foundation.

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Origin and evolution of the neural crest: A hypothetical reconstruction of its evolutionary history