RESEARCH ARTICLE

Conserved RARE Localization in AmphioxusHox Clusters and Implications for Hox CodeEvolution in the Vertebrate Neural CrestHiroshi Wada,1,2,5* Hector Escriva,3 Shicui Zhang,4 and Vincent Laudet3

The Hox code in the neural crest cells plays an important role in the development of the complexcraniofacial structures that are characteristic of vertebrates. Previously, 3� AmphiHox1 flanking region hasbeen shown to drive gene expression in neural tubes and neural crest cells in a retinoic acid (RA)-dependentmanner. In the present study, we found that the DR5-type RA response elements located at the 3�AmphiHox1 flanking region of Branchiostoma floridae are necessary and sufficient to express reportergenes in both the neural tube and neural crest cells of chick embryos, specifically at the post-otic level. TheDR5 at the 3� flanking region of chick Hoxb1 is also capable of driving the same expression in chick embryos.We found that AmphiHox3 possesses a DR5-type RARE in its 5� flanking region, and this drives an expressionpattern similar to the RARE element found in the 3� flanking region of AmphiHox1. Therefore, the locationof these DR5-type RAREs is conserved in amphioxus and vertebrate Hox clusters. Our findings demonstratethat conserved RAREs mediate RA-dependent regulation of Hox genes in amphioxus and vertebrates, andin vertebrates this drives expression of Hox genes in both neural crest and neural tube. This suggests thatHox expression in vertebrate neural crest cells has evolved via the co-option of a pre-existing regulatorypathway that primitively regulated neural tube (and possibly epidermal) Hox expression. DevelopmentalDynamics 235:1522–1531, 2006. © 2006 Wiley-Liss, Inc.

Key words: amphioxus; Hox; retinoic acid; RARE; neural crest; evolution

Accepted 30 January 2006

INTRODUCTION

A complex craniofacial structure is avertebrate characteristic that accom-panied the evolution of neural crestcells (Gans and Northcutt, 1983;Shimeld and Holland, 2000). Thesecells play a central role in the differ-entiation of the pharyngeal arches by

producing connective tissues of a spe-cific morphology that depends on theanterior-posterior origin of neuralcrest cells. It has been believed thatneural crest cells perform an instruc-tive role, by determining the identityof each pharyngeal arch (LeDouarinand Kalcheim, 1999; Noden, 1986).This idea is consistent with the pres-

ence of a Hox code in the neural crestcells and is further supported by thefact that there is abnormal develop-ment of pharyngeal arches in knock-out mice with Hox genes (Chisaka andCapecchi, 1991; Chisaka et al., 1992;Lufkin et al., 1991; Rijli et al., 1993).However, recent studies have sug-gested that endodermal tissue in the

1Seto Marine Biological Laboratory, FSERC, Kyoto University, Wakayama, Japan2Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan3UMR 5161 du CNRS, INRA LA 1237, Laboratoire de Biologie Moleculaire de la Cellule, IFR128 BioSciences Lyon-Gerland, Ecole NormaleSuperieure de Lyon, Lyon, France4Department of Marine Biology, Ocean University of Qingdao, Qingdao, P. R. China5PRESTO, JST, Kawaguchi, JapanGrant sponsor: CNRS; Grant sponsor: MENRT; Grant sponsor: Association pour la Recherche contre le Cancer (ARC); Grant sponsor: NissanScience Foundation; Grant sponsor: Kato Memorial Bioscience Foundation; Grant sponsor: Ministry of Education, Science, Sports andCulture, Japan; Grant numbers: 12026219, 13045020, 14034228.*Correspondence to: Hiroshi Wada, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba305-8572 Japan. E-mail: 98champ@msg.biglobe.ne.jp

DOI 10.1002/dvdy.20730Published online 14 March 2006 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 235:1522–1531, 2006

© 2006 Wiley-Liss, Inc.

branchial region also plays an impor-tant role in pharyngeal arch pattern-ing. Veitch et al. (1999) showed thatregionalization occurs within individ-ual pharyngeal arches even in the ab-sence of the neural crest cells. More-over, Couly et al. (2002) found thatgrafting small pieces of endoderm al-tered skeletal development in a man-ner that depended on the axial level ofthe graft. However, this only occurredwhen they grafted endoderm onto thepharyngeal arch, where neural crestcells do not express Hox. Collectively,these observations suggest that pha-ryngeal arch patterning is governedby signals from both the neural crestcells and endoderm (Santagati and Ri-jli, 2003).

Traditionally, it was assumed thatascidians and amphioxus lack a neu-ral crest because no cells migrate fromthe neural tube during embryogene-sis. However, a growing body of evi-dence suggests that the epidermisabutting the neural tube has a patternof gene expression resembling the ver-tebrate neural crest (e.g., Dll andPax3/7) (Holland et al., 1996; Wada etal., 1997). Therefore, it would appearthat the neural crest originated fromthe dorsal midline epidermis, withsome contributions from the dorsalneural tube (Meulemans and Bron-ner-Fraser, 2004; Wada, 2001). Wada(2001) postulated that the true verte-brate neural crest evolved via the ac-quisition of two novel cell properties:elaboration of the capacity to migrateand acquisition of anterior-posterior(AP) positional information. However,recent observations suggest there is amigratory cell population resemblingneural crest cells in the urochordateEcteinascidia turbinata (Jeffery et al.,2004). Therefore, migration may notbe a feature novel to the vertebrateneural crest cells, although the homol-ogy between migrating cells of E. tur-binata and the vertebrate neural crestcells is arguable. With regard to APpositional information, the evolutionof the vertebrate Hox code was a cru-cial event in the evolution of verte-brate craniofacial structure. Althoughurochordates seem to have lost thetypical colinearity between genomicorganization and expression pattern(Ikuta et al., 2004), amphioxus retainsa single Hox gene cluster organiza-tion (Garcia-Fernandez and Holland,

1994). Colinear Hox gene expressionwas observed in the neural tube andepidermis of amphioxus (Schubert etal., 2004; Wada et al., 1999). Schubertet al. (2005) also indicated that Hox1is involved in pharyngeal region ante-rior-posterior patterning. In the ascid-ian Ciona intestinalis, a Hox gene ex-pression pattern was observed thatrespects the anterior-posterior axis inthe neural tube, epidermis, andendoderm (Ikuta et al., 2004). There-fore, the Hox code operates in the ec-toderm and endoderm of both ascid-ians and amphioxus.

It is reasonable to assume that theHox code in the vertebrate neuralcrest cells originated in the ectoderm,either the neural tube or epidermis.Therefore, we can hypothesize threepossible and not exclusive scenariosfor the Hox code origin in the neuralcrest cells. First, the epidermal ex-pression of Hox genes in amphioxussuggests that the regulatory machin-ery for the vertebrate neural crestcells originated from the epidermis ofa chordate ancestor (Schubert et al.,2004) (Fig. 1A). This may be most con-sistent with the idea that the neuralcrest originated from dorsal epidermalcells of an ancestral chordate (Wada,2001; Meulemans and Bronner-Fraser, 2004). In addition, AP2 is in-volved in mouse Hoxa2 neural crestexpression (Maconochie et al., 1999).AP2 homologues are expressed in epi-dermal cells of amphioxus (Meule-mans and Bronner-Fraser, 2002), al-though it is not clear whether AP2 isrequired for the expression of theirHox genes in the epidermis. Second, itis also possible that neural crest ex-pression evolved when ancestral ver-tebrates acquired novel regulatorysystems (Fig. 1B). The observationthat the anterior expression limits ofcertain vertebrate Hox genes in theneural crest cells differ from the limits

within the neural tube supports bothof these scenarios. For example,Hoxa2 is expressed in the neural tubeup to rhombomere (r) 2, but neuralcrest cells migrating from r2 do notexpress Hoxa2 (Prince and Lumsden,1994; Tumpel et al., 2002). Finally,the third possible scenario involvesthe fact that the dorsal neural tubeand neural crest share a common pre-cursor (Fig. 1C) (Bronner-Fraser andFraser, 1988, 1989; Collazo et al.,1993; Serbedzija et al., 1994). Hox ex-pression might have evolved solely be-cause the neural crest inherited theHox expression encoded in the neuraltube or neural plate. This is consistentwith the fact that the Hoxb1 autoreg-ulatory element is sufficient for neuralcrest expression (Popperl et al., 1995).In this case, the difference betweenthe anterior expression limit in theneural tube and crest cells might re-sult from different maintenance sys-tems for the two cell populations(Trainor et al., 2002).

To examine the molecular mecha-nisms that underlie Hox code evolu-tion in the vertebrate neural crestcells, Manzanares et al. (2000) studiedthe genomic DNA regulatory potentialof amphioxus (Branchiostoma flori-dae) in mouse and chick embryos.They found that element 1A, a 2.5-kbgenomic DNA fragment from the 3�AmphiHox1 flanking region, coulddrive reporter gene expression in boththe vertebrate neural tube and crestcells, while element 3B, a 6-kbgenomic DNA from the 5� AmphiHox3flanking region, drives reporter geneexpression only in the neural tube. Inthis study, we further investigated thecis-regulatory activity of elements 1Aand 3B of Branchiostoma floridae. Inparticular, we focused on whether theregulatory elements involved in neu-ral crest expression differ from thosein the neural tube. We found that the

Fig. 1. Possible evolutionary scenarios for the origin of the Hox code of the vertebrate neuralcrest. A: Scenario 1: Neural crest expression was inherited from epidermal expression in ancestralchordates. B: Scenario 2: A novel cis-regulatory system evolved for vertebrate neural crestexpression. C: Scenario 3: The neural crest Hox code evolved simply through transfer of the neuraltube Hox code to the neural crest when cells migrated from the neural tube.

RARE OF AMPHIOXUS HOX 1523

DR5-type RA response elements lo-cated at the 3� AmphiHox1 flankingregion are necessary and sufficient forreporter gene expression in both theneural tube and crest of vertebrates.Therefore, the cis-regulatory elementsfor neural tubes and crest cells are notseparable. We confirmed that DR5from chick Hoxb1 is also capable ofdriving the same expression. We alsofound that AmphiHox3 possesses aDR5-type RARE in its 5� flanking re-gion, indicating that the location ofthe DR5-type RARE is conserved inamphioxus and vertebrate Hox clus-ters. The Hox code origin in the verte-brate neural crest cells can be ex-plained partly by RA control systemtransfer from a neural tube of an an-cestral chordate to the neural tubeand crest in the vertebrate lineage,and partly by novel regulatory sys-tems, such as AP2, and an inhibitorysignal from the isthmus.

RESULTS

Mapping cis-Elements inElement 1A

To test whether neural crest expres-sion regulatory elements differ fromthose for the neural tube, we electro-porated deletion constructs of the Am-phiHox1 element 1A from Branchios-toma floridae genome. Using deletionassays from both the 5� and 3� ends ofelement 1A, we observed that a DR5type (direct repeats spaced by five nu-cleotides) RARE (DR5-1A) is neces-sary for reporter expression in bothchick neural tubes and crest cells (Fig.2). When electroporating constructs1A1, 1A2, 1A6, and 1A7, we observeda strong lac-Z signal in both the neu-ral tube and neural crest cells, and thesignal was restricted to the post-oticlevel (posterior to the boundary be-tween r6 and r7). We analyzed morethan ten embryos for each construct,and all the embryos tested showed thesame staining pattern. Occasionally,we observed some patchy anteriorstaining; however, this staining wasmuch weaker than that in the post-otic level, and easy to distinguish. Inconstruct 1A3, the 5� region upstreamfrom DR5-1A was truncated, but theDR5-1A sequence driving reporter ex-pression was intact in both the neuraltube and neural crest cells. In con-

trast, construct 1A4, which lost halfthe DR5 sites, lacked reporter activity(Fig. 2B). Similarly, construct 1A8,which lost most of the 3� region down-stream from DR5-1A, but in which theintact DR5-1A was retained, was suf-ficient to drive the expression. Butfurther deletion prevented the activity(1A9; Fig. 2B). Simple deletion ofDR5-1A (1A�dr5) completely abol-ished the reporter expression.

To test whether DR5-1A is sufficientfor gene expression, we assessed activ-ity of two tandemly linked DR5-1A el-ements in the reporter vector (see Ex-perimental Procedures section). Asshown in Figure 3A, DR5-1A was suf-ficient for reporter gene expression inboth the neural tube and crest cells(21/21 were positive for both the neu-ral tube and crest cells). We markedcells that received the DR5-1A re-

porter construct by co-electroporatingthe DR5-1A reporter construct with aGFP expression vector driven by aCMV-IE enhancer (pCAGGS-GFP)(Niwa et al., 1991; Momose et al.,1999). The co-electroporation experi-ment confirmed that DR5-1A-drivenlac-Z was expressed only posterior tothe otic vesicle (Fig. 3A) even thoughneural tube and crest cells had beenelectroporated both anterior and pos-terior to the otic vesicle (Fig. 3D).Therefore, GFP-positive cells anteriorto the otic vesicle received a reporterconstruct, but reporter lac-Z was notactivated within these cells.

The progeny of individual cellswithin the dorsal neural tube contrib-ute to both the neural crest and tube(Bronner-Fraser and Fraser, 1988,1989; Collazo et al., 1993; Serbedzijaet al., 1994). Since it is possible to

Fig. 2. Transgenic analyses of the 3� AmphiHox1 flanking region. A: Genomic fragments used fortransgenic analyses in chick embryos. B: Nucleotide sequence of DR5-type retinoic acid (RA)response elements (DR5-1A, boxed) and surrounding regions. 1A3 and 1A8 contain DR5-1A anddrive reporter expression in both the neural tube and crest. In contrast, 1A4, which contains onlya half site of RAR/RXR binding sequence, and 1A9, which lacks DR5-1A, do not drive reporterexpression. Deleting DR5-1A from element1A1 (1A�dr5) results in no expression in the neural tubeor crest.

1524 WADA ET AL.

detect lac-Z even after transcriptionceases, the lac-Z reporter enzymaticactivity detected in the neural crestmight have originated from the lac-Zexpression that occurred before cellswere committed to the neural tube orcrest. We tested this possibility by ex-amining lac-Z reporter gene tran-scription. As shown in Figure 3B,C,E,and F, we detected lac-Z mRNA inboth the neural tube and migratingneural crest cells. Since in situ hybrid-ization detects the presence of bothelectroporated DNA and lac-Z mRNA(arrows in Fig. 3B), we arrested stain-ing before DNA signal emergence.Since the late signal occurred in a pat-tern identical to the control GFP sig-nal driven by the CMV-IE enhancer, ithad to be from the DNA. The in situhybridization signal was clear at thepost-otic level in both of these regions(neural tube: 15/15; neural crest cells:12/15, Fig. 3B,C,E,F). DR5-1A ap-pears to have cis-regulatory activitythat can drive reporter gene expres-sion in the neural tube and crest cells,and this activity is restricted to thepost-otic level. The weakness of lac-Ztranscription in neural crest cells com-pared with the signal in the neuraltube suggests that the maintenance oftranscription requires additional cis-elements.

DR5 in the 3� FlankingRegion of Chick Hoxb1

There have been several studies ofRARE cis-regulatory activity of themouse RAR-� gene in mice and ze-brafish. These studies indicated thatRARE of RAR-� drives gene expres-sion primarily in the posterior hind-brain, anterior spinal cord, and retina(Mendelsohn et al., 1991; Rossant etal., 1991; Balkan et al., 1992; Perez-Edwards et al., 2001). Although weakreporter gene expression was ob-served in zebrafish branchial arches,no expression was reported in migrat-ing neural crest cells (Perz-Edwardset al., 2001). Since there are no clearstatements made about the absence ofsignals in migrating neural crest cells,it remains possible that the relativelyweak expression in neural crest cellswas overlooked in the above studies.Therefore, we tested whether DR5from the 3� flanking region of chickHoxb1 (Langston et al., 1997) can

drive similar expression in the neuraltube and crest cells. Figure 4A clearlyindicates that chick DR5 drives re-porter expression in both the neuraltube and neural crest cells, and thisexpression is restricted to the post-oticlevel (10/10 are positive in both theneural tube and crest cells), as seen inthe DR5-1A from AmphiHox1. We de-tected in situ hybridization for lac-ZmRNA in both the neural tube andcrest cells (neural tube: 12/12; neuralcrest cells: 8/12, Fig. 4B,C). As seen forthe DR5-1A from AmphiHox1, how-ever, transcription seems to be re-duced in neural crest cells. This sug-gests that although RARE canactivate the initiation of Hox1 tran-scription, its maintenance may re-quire additional cis-elements.

Mapping cis-Elements inElement 3B

Manzanares et al. (2000) showed thatelement 3B in the 5� flanking region ofAmphiHox3 also drives reporter ex-pression in the neural tube. However,while element 1A drives expression inboth the neural tube and crest cells,element 3B only drives reporter ex-pression in the neural tube. Thus, wemapped cis-elements from element 3B(Fig. 5A). Although the cis-regulatoryactivity of element 3B is weaker thanthat of element 1A in chick embryos,reporter expression is restricted to thepost-otic level in the neural tube in asimilar way to element 1A. In contrastto Manzanares et al. (2000), we de-tected lac-Z expression in neural crestcells as well as in the neural tube(11/13 were positive in both the neuraltube and crest cells, Fig. 5C). Occa-sionally, embryos only showed a GFPsignal in the ventral neural tube. Weprobably did not observe a lac-Z signalin neural crest cells of these embryosbecause reporter DNA was not electro-porated into the neural crest. Thismay explain why the lac-Z signal inthe neural crest cells was missing inManzanares et al. (2000). Deletion as-says from both the 5� and 3� ends ofelement 3B also indicated that a0.3-kb genomic region containing aDR5-type RARE (DR5-3B) is neces-sary for reporter expression in boththe neural tube and crest cells (Fig.5A). Deletion of DR5-3B sequencefrom construct 3B1 or constructs 3B3:

3B1�dr5, and 3B3�dr5, respectively(Fig. 5A, B), did not completely abol-ish reporter expression, although itdid drastically reduce the signal (Fig.5D). The presence of weak cis-ele-ments around DR5-3B sequence couldexplain this; indeed, we found RAREconsensus sequences at the 3� flank-ing side of DR5-3B (Fig. 5B). Never-theless, DR5-3B is obviously responsi-ble for most reporter expression byelement 3B.

Electrophoresis MobilityShift Assay (EMSA)

Recently, Yu et al. (2004) pioneeredthe production of transient transgenicamphioxus embryos using microinjec-tion. However, there are still sometechnical limitations to the productionand use of transgenic amphioxus: am-phioxus eggs can only be obtained in afew places worldwide, and even if itwere possible to control spawning eas-ily, it would still be limited to twomonths of the year (Fuentes et al.,2004). Consequently, we decided touse an electrophoresis mobility shiftassay (EMSA) to test whether the cis-element is really functional in am-phioxus. The EMSA indicated thatDR5 sequences from both element 1Aand element 3B can specifically com-pete in a concentration-dependentmanner for interaction between a con-sensus DR5 sequence and amphioxusRAR and RXR recombinant proteins(Fig. 6) (Escriva et al., 2002). Sinceboth DR5s exhibited specific bindingto the amphioxus RAR/RXR het-erodimeric complex, they are likelyfunctional in the amphioxus genome.Since the cis-regulatory activity ofDR5-3B is weaker than that of DR5-1A, we tested whether DR5-1A andDR5-3B exhibited different affinitiesfor binding the amphioxus RAR/RXRheterodimer. However, we could notdetect a significant difference in theaffinity of the two DR5 elements forthe amphioxus recombinant RAR/RXR complex (Fig. 6).

Cis-Regulatory Activity ofElements 1A and 3B inAscidian Embryos

In contrast to amphioxus embryos, itis possible to obtain ascidian (Cionaintestinalis) embryos easily at any

RARE OF AMPHIOXUS HOX 1525

time of the year, and it is possible toobtain a large number of transgenicembryos using electroporation (Corboet al., 1997). Therefore, we surveyedcis-regulatory activities of amphioxusgenomic fragments in the 3� end of theamphioxus Hox cluster, including ele-ments 1A and 3B. Except for element2B, which is described elsewhere(Wada et al., 2005), none of the DNAfragments analyzed by Manzanares etal. (2000) appeared to drive reporterexpression in Ciona embryos. Sincethe human globin promoter can drivespecific expression in Ciona whencombined with element 2B or a 100-bpgenomic DNA fragment of the 3� Am-phiHox2 flanking region (part of ele-ment 2B: Wada et al., 2005), this in-ability cannot be explained by thefunctional inability of the basal pro-moter. In addition, DR5 from element1A does not drive expression in Cionawhen combined with either the hu-man �-globin promoter or CiHox1 pro-moter (data not shown).

DISCUSSION

Conservation of RARE inChordate Hox Clusters

Marshall et al. (1996) showed that RAregulates transcription of several ver-tebrate Hox genes, especially anteriorHox genes located in the 3� region ofthe clusters. In most cases, RA regu-lation is mediated directly through aRARE. Interestingly, Mainguy et al.(2003) showed that RARE locationswere conserved in the four vertebrateHox clusters, suggesting that theseRAREs were present in a unique an-cestor of these Hox complexes. Theseconserved RAREs were found in the 3�flanking region of Hox1, 5� flankingregion of Hox3, and both sides of Hox4.In addition, the RARE nucleotide se-quences in each cluster location werehighly conserved (Mainguy et al.,2003; Table 1). In this study, we iden-tified RAREs in amphioxus in the 3�Hox1 flanking region and 5� Hox3flanking region, and these are func-tional during vertebrate embryogene-sis. These results are consistent withprevious observations that Amphi-Hox1 and AmphiHox3 expression aresensitive to treatment with ectopic RAor an RA antagonist (Schubert et al.,2004). Since these RAREs can specifi-

cally interact with a recombinantRAR/RXR amphioxus complex, theyare likely to be functional during am-phioxus embryogenesis. Therefore, itappears that the RAREs in the 3�flanking region of Hox1 and 5� flank-ing region of Hox3 are conserved inboth vertebrate and amphioxus Hoxclusters, and they were likely presentin the Hox cluster of an ancestralchordate. Chambeyron and Bickmore,(2004) suggested that RA is involvedin chromatin de-condensation and thenuclear reorganization of vertebrateHox. The conserved nature of RAREsin amphioxus Hox clusters suggeststhat RAs regulate amphioxus Hoxgenes in a similar manner.

In contrast to the conservation ofvertebrate RARE sequences, the cor-responding amphioxus RAREs havedistinct nucleotide sequences of con-sensus binding sites (Table 1). It maybe worth noting that the nucleotidesequence of DR5-3B is more similar tothe conserved sequence of RARE inthe 3� flanking region of vertebrateHox4, although the DR5-3B locatesabout 40 kb away from AmphiHox4(Garcia-Fernandez and Holland,1994). Since it has been suggestedthat nucleotide differences of consen-sus binding sequences of transcrip-tional factors may be involved in thefine regulation of gene expression(Stathopoulos and Levine, 2002), thedistinct nucleotide sequence of theRAREs for each amphioxus Hox genemay contribute to fine Hox gene tran-scriptional regulation. Indeed, switch-ing RARE sequences in mouse Hoxgenes resulted in distinct reporter ex-pression in the neural tube (Gould etal., 1998; Nolte et al., 2003). Since thecis-regulatory activity of DR5-3B isweaker than that of DR5-1A, wetested whether DR5-1A and DR5-3Bexhibited different affinities for bind-ing the amphioxus RAR/RXR het-erodimer. However, we did not detectsignificant differences between them.Escriva et al. (2002) found that TR2/4can compete with RARE for cis regu-lation. Differential affinities to TR2/4may explain differences in the ante-rior expression boundary. As Cham-beyron and Bickmore (2004) sug-gested, colinear expression of Hoxgenes may be controlled at a higherlevel, such as chromosome organiza-tion, although no information is yet

available on the chromatin regula-tions in amphioxus.

In contrast to aspects of RARE con-servation between amphioxus andvertebrates (i.e., chromosome organi-zation, interchangeability), RAREscannot drive expression in embryo-genesis of the ascidian Ciona intesti-nalis. Since the human �-globin pro-moter can drive specific expressionwhen combined with element 2B ofamphioxus Hox (Wada et al., 2005),the functional inability of the basalpromoter (the human �-globin pro-moter) cannot explain this inability inCiona. Ciona Hox1 expression re-sponds to exogenous RA (Nagatomoand Fujiwara, 2003), so it is likelythat RA is also involved in transcrip-tional regulation of Ciona Hox genes.RA may regulate Ciona Hox genesthrough a distinct mechanism. In-deed, the 3� CiHox1 flanking region isvery short; another gene (cDNA Clus-ter03704, AK113647, which shows ho-

Fig. 3. Reporter expression driven by the DR5-type RARE from the 3� AmphiHox1 flanking re-gion. A: Histological detection of lac-Z activity.B,C: In situ hybridization for lac-Z mRNA intransgenic embryos. D–F: GFP signal driven bya CMV-IE enhancer in the embryos shown inA–C, respectively. Lac-Z enzymatic activity andmRNA expression was observed at HH stage13–14, stage 12–13, respectively. Most of thecells that received electroporated plasmidsproduced a GFP signal. Note that we observedGFP signals beyond the otic vesicle level inneural tube and neural crest cells, particularlythose from rhombomere 4. Signal from electro-porated DNAs were observed in B (arrows). Ineach panel, arrowheads indicate the otic vesi-cle; nc: neural crest cells. All embryos are ori-ented as anterior to the left.Fig. 4. Reporter expression driven by DR5-type RARE from the 3� flanking region of chickHox1. A: Histological detection of lac-Z activity.B,C: In situ hybridization for lac-Z mRNA intransgenic embryos. Note that some anteriorepidermal cells (A and C) are positive for lac-ZmRNA. We found that the basal promoter (thehuman �-globin promoter) can drive strongbackground expression in epidermal cells evenwithout DR5. This anterior epidermal expres-sion is thus due to non-specific activity of thebasal promoter. D–F: GFP signal in the em-bryos shown in A–C, respectively. Lac-Z enzy-matic activity and mRNA expression was ob-served at HH stage 13–14, stage 12–13,respectively. Note that we observed GFP sig-nals beyond the otic vesicle level in neural tubeand neural crest cells, particularly those fromrhombomere 4. In each panel, arrowheads rep-resent the otic vesicle; nc: neural crest cells. Allembryos are oriented as anterior to the left.

1526 WADA ET AL.

Fig. 3.

Fig. 4.

Fig. 5. Transgenic analyses of the 5� Amphi-Hox3 flanking region. A,B: Genomic fragmentsused for transgenic analyses in chick embryos.DR-5 type RARE (DR5-3B) is boxed, and twoother single RARE consensus sequences areunderlined. C: Histological detection of lac-Zreporter expression driven by 3B4. Patchy sig-nal in anterior hindbrain is non-specific stainingthat is not reproduced in other embryos. D:Lac-Z expression driven by 3B3�dr5. E,F: GFPsignal in the embryos shown in C, D, respec-tively. Lac-Z enzymatic activity was observed atHH stage 13–14. Arrowheads indicate the oticvesicle; nc: neural crest cells. All embryos areoriented as anterior to the left.

RARE OF AMPHIOXUS HOX 1527

mology to mouse F-box and leucine-richrepeat protein 15 protein [Fbxl15]) islocated just 1.5 kb from the 3� end ofCiHox1. We found no sequence similarto RARE in the region. Rather, the 5�upstream region and second intron ofCiona Hox1 has cis-regulatory activityfor epidermis and neural tube reporterexpression, respectively, and the re-porter expression is sensitive to exoge-nous RA (Wada et al., unpublisheddata). We think the unique regulatorymechanisms of Ciona Hox by RA is asecondarily derived state, which mayhave to do with the fact that clusterorganization in Ciona Hox genes wasdisrupted. It seems unlikely that theRAREs in the 3� Hox1 flanking regionand in 5� Hox3 flanking region haveevolved independently in amphioxus

and vertebrates. As far as we know,there is no evidence that RA is involvedin Hox gene regulation of echinodermsor hemichordates. Our preliminarysearch for RARE in sea urchin Hox clus-ter identified a putative DR5-typeRARE (CGTTCA-n5- TGTTCA) in 3.3kb downstream from homeodomain cod-ing reagion of Hox1 (http://www.hg-sc.bcm.tmc.edu/blast/?organism�Spurpuratus), althoughwe are not certain whether it is in-volved in Hox transcriptional regula-tion.

Evolution of the New HoxCode in Vertebrates

The present study demonstrated thatAmphiHox1 and chick Hox1 RAREs

are sufficient to activate reporter geneexpression in both the neural tube andcrest cells posterior to the otic vesicle.This suggests that Hox1 gene expres-sion in the neural crest cells reflects atransfer of regulatory informationfrom the neural tube. Since element3B can also drive expression in boththe neural tube and crest cells, it islikely that the transfer of regulatoryinformation to neural crest cells is ap-plicable to Hox genes controlled byRA. Although no RARE has beenfound for vertebrate Hox2 (Mainguy etal., 2003), cross-regulation betweenHox genes is responsible for Hoxb2 ex-pression (Ferretti et al., 2000). In ad-dition, mutation in the 3� Hoxa1 DR5affects Hoxa2 expression (Dupe et al.,1997). Therefore, a transfer of regula-tory information from the neural tubemight explain the evolution of ante-rior Hox gene neural crest expression.However, traditionally it has been be-lieved that Hox gene expression in theneural crest cells is regulated inde-pendently from the neural tube. Twoobservations support this idea. First,the anterior boundaries of some Hoxgenes in the neural crest cells differfrom those in the neural tube: for ex-ample, Hoxa2 is expressed up to levelr2, but not in the neural crest cellsmigrating out of r2 (Prince and Lums-den, 1994). Trainor et al. (2002)claimed that this absence of Hox ex-pression from r2 crest cells is due to arepressive isthmus signal. AlthoughHoxa2 expression is activated both inthe neural tube and neural crest of r2,the absence of Hoxa2 expression fromr2 crest cells is due to a neural crest–specific repressive signal. Second,there are some neural crest–specificcis-elements of Hox expression. Hoxa2possesses r4 crest–specific cis-ele-ments regulated by AP2 and otherfactors (Maconochie et al., 1999).However, Tumpel et al. (2002) demon-strated that the AP2 consensus bind-ing sequence is found only in mam-mals, and not in chick or shark Hoxa2.Therefore, AP2 involvement in neuralcrest development may have been ac-quired later in vertebrate evolution,specifically after the mammalian lin-eage diverged from reptiles. Further-more, auto- or cross-regulatory ele-ments can activate Hoxb1 and Hoxb2expression in the r4 neural tube andcrest cells (Popperl et al., 1995; Fer-

Fig. 6. Electrophoretic mobility shift assay. A: We synthesized AmphiRAR and AmphiRXR in vitroand allowed them to bind to a 32P-labeled consensus DR5 probe (CGA TTT GAG GTC ACC AGGAGG TCA CAC AGT) in the EMSA (lines 2–15). Lane 1, unprogrammed (pSG5) reticulocytes, wasa control. We added increasing amounts of unlabelled oligonucleotides corresponding to the sameDR5 consensus element, DR5 from element 1A of AmphiHox1, and DR5 from element 3B fromAmphiHox3 (over the probe) as competitors. We also added a non-specific element (NS) in a molarexcess of 100� (over the probe) as a competitor control. We repeated the experiment three timesand quantified the retarded bands on a Storm 860 (Molecular Dynamics) apparatus (bottom). B,C: Aregression plot of binding percentage versus competitor concentration shows that there were nosignificant differences in affinity between the different DR5 elements used.

TABLE 1. Comparison of the Nucleotide Sequence of DR5 Type RARE

3� of vertebrate Hox1 GGTTCA (n5) AGTTCA3� of amphioxus Hox1 GGGTCA (n5) CGGTCA5� of vertebrate Hox3 GGTTCA (n5) AGTTCA5� of amphioxus Hox3 GGGTCA (n5) AGGACA3� of vertebrate Hox4 (A/G)GTTCA (n5) AGGACA

1528 WADA ET AL.

retti et al., 2000). This is consistentwith initial Hox activation in the neu-ral crest and tube being regulated bythe same system. Therefore, the initi-ation of the Hox code in the neuralcrest was set up by the RA-responsesystem that originated from the RA-responsive system in the neural tubeof an ancestral chordate. This Hoxcode is still plastic, and thus needed tobe maintained by AP2 or auto- orcross-regulatory systems. In addition,because of the plasticity, expression ofsome genes can still be inhibited bythe signal from ithsmus. Those main-tanance and inhibitory signals may benovel systems of vertebrates.

In order to determine the evolutionof the neural crest Hox code, we mustalso find out how DR5 elements areinvolved in transcriptional regulationof amphioxus Hox. Schubert et al.(2004) demonstrated that Amphi-Hox1, 3, and 4 expression are sensi-tive to exogenous RA in both the neu-ral tube and epidermis. We arecurrently investigating how RAREsare involved in expression of the neu-ral tube and epidermis of ascidian andamphioxus embryos. We have con-firmed that in Ciona, epidermal ex-pression is regulated by a cis-regula-tory element distinct from those forneural tube expression (Wada et al.,unpublished data).

In conclusion, we propose that thenew neural crest Hox code evolved us-ing a combination of Scenarios 2 and 3(Fig. 7). It may be partly explained bytransference of the RA response sys-tem of a chordate ancestor to verte-brate neural tissues that included thedevelopmental progenitors of the neu-ral tube and crest. Therefore, the ini-tial Hox code was set in neural tissuebefore lineage segregation occurredbetween the neural tube and crest(Scenario 3: Fig. 1C). However, theneural crest expression needs to be

maintained, and is further refined incertain vertebrate lineages by addi-tional regulatory systems, such as au-to- or cross regulatory system, AP-2,and inhibitory signals from the isth-mus (Scenario 2: Fig. 1B) (Trainor etal., 2002). Assuming that ancestralchordates possess distinct cis-regula-tory elements for epidermis from thatfor neural tube, as in present ascid-ians (Wada et al., unpublished data),cis elements for neural tube expres-sion may have been primarily respon-sible for the innovation of neural crestHox code, because the initial Hox codewas set in neural tissue before lineagesegregation occurred between theneural tube and crest. However, thecis-regulatory mechanisms of Hoxgene expression in epidermis of ascid-ians and amphioxus must be analyzedto determine the contributions fromthe factors explained in Scenario 1.

EXPERIMENTALPROCEDURES

Construction of Plasmids

We tested the activity of cis-actingregulatory elements of genomic DNAfrom amphioxus (Branchiostoma flori-dae) in a reporter expression vectorthat contained an SV40 polyadenyla-tion signal and the minimal human�-globin promoter linked to a bacterialbeta-galactosidase gene (lac-Z) (Man-zanares et al., 2000). Deletion con-structs were made either using Exo-nuclease III and mung bean nuclease,or by amplifying truncated fragmentsusing specific primers. We producedtwo tandemly linked DR5 from ele-ment 1A and from chick Hoxa1 (Lang-ston et al., 1997) using the followingoligonucleotides, and inserted theminto the above-mentioned lac-Z ex-pression vector and complementaryoligonucleotide: the DR5 from element

1A was 5�-ggaagcttGGGTCATCTAT-CGGTCAggGGGTCATCTATCGGT-CAagcttcc-3� and the DR5 from chickHoxb1 was 5�-ggctcgagCAGGTTCA-CACAAAGTTCAGccGGTTCACACA-AAGTTCAGCaagcttcc-3� (sequencesfrom the amphioxus genome are under-lined, and RAR/RXR binding consensussequences are shown in bold. Lowercasecharacters are sequences inserted forrestriction digestion or spacing).

Electroporation

We electroporated chick embryos, asdescribed by Nakamura et al. (2000).Briefly, this involved injecting 1mg/ml plasmid DNA into the closedneural tube at the eight- to ten-somitestage (Hamburger and Hamilton [HH]stage 9–10; 33–36 hr), and applyingfive electric pulses (20-mV squarepulses at 50-ms intervals) throughelectrodes spaced 4 mm apart; weused an Electrosquareporater T820(BTX, Genetronics) during this pro-cess. Embryos were processed forlac-Z staining or in situ hybridizationafter being cultured for 12–18 hr fol-lowing electroporation (HH stage 12–14; 45–50 hr). In situ hybridization ofchick embryos followed the process asdetailed by Nieto et al. (1996). Wetested more than ten embryos for eachconstruct in the reporter assays inchick embryos. Ciona electroporationessentially followed the method de-scribed by Corbo et al. (1997): a 50-mVsquare electric pulse was applied for20 ms in a 4-mm-wide cuvette usingthe Electrosquareporater T820 (BTX,Genetronics).

Electrophoretic MobilityShift Assay (EMSA)

We translated proteins (AmphiRARand AmphiRXR) in vitro using a TNTreticulocyte lysate kit (Promega, Mad-ison, WI) and allowed them to bind toa 32P-end-labeled 30-bp oligonucleo-tide in a reaction buffer containing10% glycerol, 10 mM HEPES, 30 mMKCl, 4 mM spermidine, 0.1 mMEDTA, 0.25 mM dithiothreitol (DTT),and 1 mM Na2HPO4 (pH 7.9). Weadded single-stranded salmon spermDNA (1 �g) and poly(dI-dC) (0.4 �g).The reaction products were run on a5% native acrylamide gel. Where indi-cated, a 10- to 100-fold (10, 25, 50, and

Fig. 7. Schematic illustration of the evolutionary scenarios for the Hox code origin in the verte-brate neural crest. The RA response system of a chordate ancestor was transferred to vertebrateneural tissues, including developmental progenitors of the neural tube and crest. However, neuralcrest expression was needed to be maintained by an auto- or cross-regulatory system and AP2,and was further refined by additional regulatory systems, such as inhibitory signals from theisthmus.

RARE OF AMPHIOXUS HOX 1529

100) molar excess of 30-bp unlabeledoligonucleotides (a consensus DR5,the AmphiHox1 DR5, the AmphiHox3DR5, and a non-specific probe) wereadded as competitors. To confirm thatAmphiRAR and AmphiRXR proteinswere properly produced in the in vitrosystem, we added [35S]methionine tothe translation mixture in parallelside-reactions of protein analyses,which we performed under standardconditions.

ACKNOWLEDGMENTSWe thank Yoshiko Takahashi for herkind instruction of chick electropora-tion and for providing us withpCAGGS-GFP. We also thank RobbKrumlauf for providing us reporterconstructs with �-globin minimal pro-moter; Seb Shimeld, Linda Holland,and Michael Schubert for criticalreading of the manuscript; and MiguelManzanares, Nobue Itasaki, and Pe-ter Holland for their comments on aprevious version of the manuscript.This work is supported by the CNRS,MENRT, and Association pour la Re-cherche contre le Cancer (ARC) toV.L., and by grants from Nissan Sci-ence Foundation, Kato Memorial Bio-science Foundation, and Grants-in-Aid from the Ministry of Education,Science, Sports and Culture, Japan(12026219, 13045020, 14034228) toH.W.

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